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  • Diacylglycerol kinase δ and sphingomyelin synthase–related protein functionally interact via their sterile α motif domains
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Chiaki Murakami, Fumi Hoshino, Hiromichi Sakai, Yasuhiro Hayashi, Atsushi Yamashita, Fumio Sakane

    The δ isozyme of diacylglycerol kinase (DGKδ) plays critical roles in lipid signaling by converting diacylglycerol (DG) to phosphatidic acid (PA). We previously demonstrated that DGKδ preferably phosphorylates palmitic acid (16:0)- and/or palmitoleic acid (16:1)-containing DG molecular species, but not arachidonic acid (20:4)-containing DG species, which are recognized as DGK substrates derived from phosphatidylinositol turnover, in high glucose-stimulated myoblasts. However, little is known about where these DG molecular species come from. DGKδ and two DG-generating enzymes, sphingomyelin synthase (SMS) 1 and SMS-related protein (SMSr), contain a sterile α motif domain (SAMD). In the present study, we found that SMSr-SAMD, but not SMS1-SAMD, co-immunoprecipitates with DGKδ-SAMD. Full-length DGKδ co-precipitated with full-length SMSr more strongly than with SMS1. However, SAMD-deleted variants of SMSr and DGKδ interacted only weakly with full-length DGKδ and SMSr, respectively. These results strongly suggested that DGKδ interacts with SMSr through their respective SAMDs. To determine the functional outcomes of the relationship between DGKδ and SMSr, we used LC-MS/MS to investigate whether overexpression of DGKδ and/or SMSr in COS-7 cells alters the levels of PA species. We found that SMSr overexpression significantly enhances the production of 16:0- or 16:1-containing PA species such as 14:0/16:0-, 16:0/16:0-, 16:0/18:1-, and/or 16:1/18:1-PA in DGKδ-overexpressing COS-7 cells. Moreover, SMSr enhanced DGKδ activity via their SAMDs in vitro. Taken together, these results strongly suggest that SMSr is a candidate DG-providing enzyme upstream of DGKδ and that the two enzymes represent a new pathway independent of phosphatidylinositol turnover.

    更新日期:2020-01-26
  • High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Yves A Muller, Sigrun Häge, Sewar Alkhashrom, Tobias Höllriegl, Sebastian Weigert, Simon Dolles, Kerstin Hof, Sascha A Walzer, Claudia Egerer-Sieber, Marcus Conrad, Stephanie Holst, Josephine Lösing, Eric Sonntag, Heinrich Sticht, Jutta Eichler, Manfred Marschall

    Herpesviruses uniquely express two essential nuclear egress–regulating proteins forming a heterodimeric basic structure of the nuclear-egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein–Barr virus (EBV) BFRF1-BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approx. 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.

    更新日期:2020-01-26
  • Targeting viperin to the mitochondrion inhibits the thiolase activity of the trifunctional enzyme complex
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Arti B. Dumbrepatil, Kelcie A. Zegalia, Keerthi Sajja, Robert T. Kennedy, E. Neil G. Marsh

    Understanding the mechanisms by which viruses evade host-cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus (HCMV) co-opts the antiviral radical SAM enzyme, viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), to enhance viral infectivity. This process involves translocation of viperin to the mitochondrion where it binds the β-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes the thiolysis of β-ketoacyl-CoA esters as part of fatty acid β-oxidation. Here, we investigated how the interaction between these two enzymes alters their activities and affects cellular ATP levels. Experiments with purified enzymes indicated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin leading to the synthesis of the antiviral nucleotide 3ʹ-deoxy-3′,4ʹ-didehydro-CTP. Measurements of enzyme activities in lysates prepared from transfected HEK 293T cells expressing these enzymes mirrored the findings obtained with purified enzymes. Thus, localizing viperin to the mitochondria decreased thiolase activity and co-expression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB is retro-translocated out of mitochondria and degraded, providing an additional mechanism by which viperin reduces HADHB activity. Targeting viperin to the mitochondria decreased cellular ATP levels by > 50 %, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which HCMV subverts viperin; they also provide a biochemical rationale for viperin’s recently discovered role in regulating thermogenesis in adipose tissues.

    更新日期:2020-01-26
  • Improved workflow for mass spectrometry-based metabolomics analysis of the heart
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Douglas A. Andres, Lyndsay E. A. Young, Sudhakar Veeranki, Tara R. Hawkinson, Bryana M. Levitan, Daheng He, Chi Wang, Jonathan Satin, Ramon C. Sun

    MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GCMS-based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion), and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol treatment (ISO)) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as liquid chromatography or capillary electrophoresis.

    更新日期:2020-01-26
  • Uncovering the activities, biological roles, and regulation of bacterial cell wall hydrolases and tailoring enzymes
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Truc Do, Julia E Page, Suzanne Walker

    Bacteria account for a thousand-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are crosslinked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall tailoring enzymes that regulate glycan strand length, degree of crosslinking, and addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

    更新日期:2020-01-24
  • Metformin lowers glucose 6-phosphate in hepatocytes by activation of glycolysis downstream of glucose phosphorylation
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Tabassum Moonira, Shruti S. Chachra, Brian E. Ford, Silvia Marin, Ahmed Alshawi, Natasha S. Adam-Primus, Catherine Arden, Ziad H. Al-Oanzi, Marc Foretz, Benoit Viollet, Marta Cascante, Loranne Agius

    The chronic effects of metformin on liver gluconeogenesis involve repression of the G6pc gene, which is regulated by the Carbohydrate response element binding protein through raised cellular intermediates of glucose metabolism. In this study we determined the candidate mechanisms by which metformin lowers glucose 6-phosphate (G6P) in mouse and rat hepatocytes challenged with high glucose or gluconeogenic precursors. Cell metformin loads in the therapeutic range lowered cell G6P but not ATP and decreased G6pc mRNA at high glucose. The G6P lowering by metformin was mimicked by a Complex 1 inhibitor (rotenone), an uncoupler (dinitrophenol) and by overexpression of mGPDH, which lowers glycerol 3-phosphate and G6P and also mimics the G6pc repression by metformin. In contrast, direct allosteric activators of AMPK (A-769662, 991, C13) had opposite effects from metformin on glycolysis, gluconeogenesis and cell G6P. The G6P lowering by metformin which also occurs in hepatocytes from AMPK-knock-out mice, is best explained by allosteric regulation of phosphofructokinase-1 (PFK-1) and/or fructose bisphosphatase-1, as supported by: increased metabolism of [3-3H]glucose relative to [2-3H]glucose; an increase in lactate m2/m1 isotopologue ratio from [1,2-13C2]glucose, by lowering of glycerol 3-phosphate an allosteric inhibitor of phosphofructokinase-1 and by marked G6P elevation by selective inhibition of phosphofructokinase-1 but not by a more reduced cytoplasmic NADH/NAD redox state. We conclude that therapeutically relevant doses of metformin lower G6P in hepatocytes challenged with high glucose by stimulation of glycolysis by an AMPK-independent mechanism through changes in allosteric effectors of PFK-1 and fructose bisphosphatase-1, including AMP, inorganic phosphate and glycerol 3-phosphate.

    更新日期:2020-01-24
  • Optimization of sortase A ligation for flexible engineering of complex protein systems
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Jess Li, Yue Zhang, Olivier Soubias, Domarin Khago, Fa-an Chao, Yifei Li, Katherine Shaw, R. Andrew Byrd

    Engineering and bio-conjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multi-domain protein may be complicated by undesirable side reactions to non-involved domains. Furthermore, the advantages of segmental (or domain specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods – expressed protein ligation (EPL), protein trans-splicing (PTS), and native chemical ligation) – each have specific limitations. Here, we evaluated the use of different variants of Staphylococcal aureus sortase A (SrtA) for a range of ligation reactions and demonstrate that conditions are readily can be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.

    更新日期:2020-01-24
  • Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Yanxiang Meng, Campbell R. Sheen, Nicholas J. Magon, Mark B. Hampton, Renwick C.J. Dobson

    During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo. We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC–MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78–X–X–Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins.

    更新日期:2020-01-24
  • Pulse-chase SILAC–based analyses reveal selective over-synthesis and rapid turnover of mitochondrial protein components of respiratory complexes
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Daniel F. Bogenhagen, John D Haley

    Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes), with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F1/Fo ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI, i.e. NADH dehydrogenase, is far less efficient, with dramatic over-synthesis of numerous proteins, particularly in the matrix-exposed N- and Q-domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol–cytochrome C reductase Rieske iron–sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.

    更新日期:2020-01-24
  • A synthetic heparinoid blocks tau aggregate cell uptake and amplification
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Barbara E Stopschinski, Talitha L Thomas, Sourena Nadji, Eric Darvish, Linfeng Fan, Brandon B. Holmes, Anuja R Modi, Jordan Finnell, Omar M Kashmer, Sandi Estill-Terpack, Hilda Mirbaha, Hung S Luu, Marc I Diamond

    Tau aggregation underlies neurodegeneration in Alzheimer’s disease and related tauopathies. We and others have proposed that transcellular propagation of pathology is mediated by tau prions, which are ordered protein assemblies that faithfully replicate in vivo and cause specific biological effects. The prion model predicts the release of aggregates from a first-order cell and subsequent uptake into a second-order cell. The assemblies then serve as templates for their own replication, a process termed “seeding.” We have previously observed that heparan sulfate proteoglycans (HSPGs) on the cell surface mediate the cellular uptake of tau aggregates. This interaction is blocked by heparin, a sulfated glycosaminoglycan. Indeed, heparin-like molecules, or heparinoids, have previously been proposed as a treatment for PrP prion disorders. However, heparin is not ideal for managing chronic neurodegeneration, as it is difficult to synthesize in defined sizes, may have poor brain penetration because of its negative charge, and is a powerful anticoagulant. Therefore, we sought to generate an oligosaccharide that would bind tau and block its cellular uptake and seeding, without exhibiting anticoagulation activity. We created a compound, SN7-13, from pentasaccharide units and tested it in a range of assays that measured direct binding of tau to glycosaminoglycans, and inhibition of tau uptake and seeding in cells. SN7- 13 does not inhibit coagulation, binds tau with low nanomolar affinity, and inhibits cellular tau aggregate propagation similarly to standard porcine heparin. This synthetic heparinoid could facilitate the development of agents to treat tauopathy.

    更新日期:2020-01-24
  • Intracellular vesicle clusters are organelles that synthesize extracellular vesicle-associated cargo proteins
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-23
    Chelsea M Winters, Ly Q. Hong-Brown, Hui-Ling Chiang

    Extracellular vesicles (EVs) play important roles in cell–cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles—called intracellular vesicle clusters (IVCs)—serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

    更新日期:2020-01-24
  • Structure of the RAD9-RAD1-HUS1 checkpoint clamp bound to RHINO sheds light on the other side of the DNA clamp
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Kodai Hara, Nao Iida, Ryota Tamafune, Eiji Ohashi, Hitomi Sakurai, Yoshinobu Ishikawa, Asami Hishiki, Hiroshi Hashimoto

    DNA clamp, a highly conserved ring-shaped protein, binds dsDNA within its central pore. Also, DNA clamp interacts with various nuclear proteins on its front, thereby stimulating their enzymatic activities and biological functions. It has been assumed that the DNA clamp is a functionally single-faced ring from bacteria to humans. Here, we report the crystal structure of the heterotrimeric RAD9-RAD1-HUS1 (9-1-1) checkpoint clamp bound to a peptide of RHINO, a recently identified cancer-related protein that interacts with 9-1-1 and promotes activation of the DNA damage checkpoint. This is the first structure of 9-1-1 bound to its partner. The structure reveals that RHINO is unexpectedly bound to the edge and around the back of the 9-1-1 ring through specific interactions with the RAD1 subunit of 9-1-1. Our finding indicates that 9-1-1 is a functionally double-faced DNA clamp.

    更新日期:2020-01-24
  • The balancing act of R-loop biology: The good, the bad, and the ugly
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Youssef A. Hegazy, Chrishan M. Fernando, Elizabeth J. Tran

    An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop–mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo. It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.

    更新日期:2020-01-24
  • The structural basis of T-cell receptor (TCR) activation: An enduring enigma
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Roy A. Mariuzza, Pragati Agnihotri, John Orban

    T cells are critical for protective immune responses to pathogens and tumors. The T-cell receptor (TCR)–CD3 complex is composed of a diverse αβ TCR heterodimer noncovalently associated with the invariant CD3 dimers CD3ϵγ, CD3ϵδ, and CD3ζζ. The TCR mediates recognition of antigenic peptides bound to MHC molecules (pMHC), whereas the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T-cell signaling pathways, the mechanism whereby TCR engagement by pMHC is first communicated to the CD3 signaling apparatus, a process termed early T-cell activation, remains largely a mystery. In this review, we examine the molecular basis for TCR activation in light of the recently determined cryoEM structure of a complete TCR–CD3 complex. This structure provides an unprecedented opportunity to assess various signaling models that have been proposed for the TCR. We review evidence from single-molecule and structural studies for force-induced conformational changes in the TCR–CD3 complex, for dynamically-driven TCR allostery, and for pMHC-induced structural changes in the transmembrane and cytoplasmic regions of CD3 subunits. We identify major knowledge gaps that must be filled in order to arrive at a comprehensive model of TCR activation that explains, at the molecular level, how pMHC-specific information is transmitted across the T-cell membrane to initiate intracellular signaling. An in-depth understanding of this process will accelerate the rational design of immunotherapeutic agents targeting the TCR–CD3 complex.

    更新日期:2020-01-24
  • Antibody validation for Western blot: By the user, for the user
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Lakshmi Pillai-Kastoori, Sam Heaton, Steve D. Shiflett, Annabelle C. Roberts, Alejandra Solache, Amy R. Schutz-Geschwender

    Well-characterized antibody reagents play a key role in the reproducibility of research findings, and inconsistent antibody performance leads to variability in Western blotting and other immunoassays. The current lack of clear, accepted standards for antibody validation and reporting of experimental details contributes to this problem. Because the performance of primary antibodies is strongly influenced by assay context, recommendations for validation and usage are unique to each type of immunoassay. Practical strategies are proposed for the validation of primary antibody specificity, selectivity, and reproducibility using Western blot analysis. The antibody should produce reproducible results within and between Western blotting experiments and the observed effect confirmed with a complementary or orthogonal method. Routine implementation of standardized antibody validation and reporting in immunoassays such as Western blotting may promote improved reproducibility across the global life sciences community.

    更新日期:2020-01-24
  • Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Shasha Gong, Xiaoqiong Wang, Feilong Meng, Limei Cui, Qiuzi Yi, Qiong Zhao, Xiaohui Cang, Zhiyi Cai, Jun Qin Mo, Yong Liang, Min-Xin Guan

    The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis. The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNAHis structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, HARS2 overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

    更新日期:2020-01-24
  • The protein tyrosine phosphatase RPTPζ/phosphacan is critical for perineuronal net structure
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Geoffrey J. Eill, Ashis Sinha, Markus Morawski, Mariano S. Viapiano, Russell T. Matthews

    Perineuronal nets (PNNs) are conspicuous neuron-specific substructures within the extracellular matrix of the central nervous system that have generated an explosion of interest over the last decade. These reticulated structures appear to surround synapses on the cell bodies of a subset of the neurons in the central nervous system and play key roles in both developmental and adult-brain plasticity. Despite the interest in these structures and compelling demonstrations of their importance in regulating plasticity, their precise functional mechanisms remain elusive. The limited mechanistic understanding of PNNs is primarily because of an incomplete knowledge of their molecular composition and structure and a failure to identify PNN-specific targets. Thus, it has been challenging to precisely manipulate PNNs to rigorously investigate their function. Here, using mouse models and neuronal cultures, we demonstrate a role of receptor protein tyrosine phosphatase zeta (RPTPζ) in PNN structure. We found that in the absence of RPTPζ, the reticular structure of PNNs is lost and phenocopies the PNN structural abnormalities observed in tenascin-R knockout brains. Furthermore, we biochemically analyzed the contribution of RPTPζ to PNN formation and structure, which enabled us to generate a more detailed model for PNNs. We provide evidence for two distinct kinds of interactions of PNN components with the neuronal surface, one dependent on RPTPζ and the other requiring the glycosaminoglycan hyaluronan. We propose that these findings offer important insight into PNN structure and lay important groundwork for future strategies to specifically disrupt PNNs to precisely dissect their function.

    更新日期:2020-01-24
  • Distinct alterations of gut morphology and microbiota characterize accelerated diabetes onset in nonobese diabetic mice
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Marie-Christine Simon, Anna Lena Reinbeck, Corinna Wessel, Julia Heindirk, Tomas Jelenik, Kirti Kaul, Juan Arreguin-Cano, Alexander Strom, Michael Blaut, Fredrik Bäckhed, Volker Burkart, Michael Roden

    The rising prevalence of type 1 diabetes (T1D) over the past decades has been linked to lifestyle changes, but the underlying mechanisms are largely unknown. Recent findings point to gut-associated mechanisms in the control of T1D pathogenesis. In nonobese diabetic (NOD) mice, a model of T1D, diabetes development accelerates after deletion of the Toll-like receptor 4 (TLR4). We hypothesized that altered intestinal functions contribute to metabolic alterations, which favor accelerated diabetes development in TLR4-deficient (TLR4−/−) NOD mice. In 70–90-day-old normoglycemic (prediabetic) female NOD TLR4+/+ and NOD TLR4−/− mice, gut morphology and microbiome composition were analyzed. Parameters of lipid metabolism, glucose homeostasis, and mitochondrial respiratory activity were measured in vivo and ex vivo. Compared with NOD TLR4+/+ mice, NOD TLR4−/− animals showed lower muscle mass of the small intestine, higher abundance of Bacteroidetes, and lower Firmicutes in the large intestine, along with lower levels of circulating short-chain fatty acids (SCFA). These changes are associated with higher body weight, hyperlipidemia, and severe insulin and glucose intolerance, all occurring before the onset of diabetes. These mice also exhibited insulin resistance–related abnormalities of energy metabolism, such as lower total respiratory exchange rates and higher hepatic oxidative capacity. Distinct alterations of gut morphology and microbiota composition associated with reduction of circulating SCFA may contribute to metabolic disorders promoting the progression of insulin-deficient diabetes/T1D development.

    更新日期:2020-01-24
  • Cultivation at high osmotic pressure confers ubiquinone 8–independent protection of respiration on Escherichia coli
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Laura Tempelhagen, Anita Ayer, Doreen E. Culham, Roland Stocker, Janet M. Wood

    Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli. In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.

    更新日期:2020-01-24
  • Disruption of hepatic small heterodimer partner induces dissociation of steatosis and inflammation in experimental nonalcoholic steatohepatitis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Nancy Magee, An Zou, Priyanka Ghosh, Forkan Ahamed, Don Delker, Yuxia Zhang

    Nonalcoholic steatohepatitis (NASH) is a leading cause of chronic liver disease worldwide and is characterized by steatosis, inflammation, and fibrosis. The molecular mechanisms underlying NASH development remain obscure. The nuclear receptor small heterodimer partner (Shp) plays a complex role in lipid metabolism and inflammation. Here, we sought to determine SHP's role in regulating steatosis and inflammation in NASH. Shp deletion in murine hepatocytes (ShpHep−/−) resulted in massive infiltration of macrophages and CD4+ T cells in the liver. ShpHep−/− mice developed reduced steatosis, but surprisingly increased hepatic inflammation and fibrosis after being fed a high-fat, -cholesterol, and -fructose (HFCF) diet. RNA-Seq analysis revealed that pathways involved in inflammation and fibrosis are significantly activated in the liver of ShpHep−/− mice fed a chow diet. After having been fed the HFCF diet, WT mice displayed up-regulated peroxisome proliferator-activated receptor γ (Pparg) signaling in the liver; however, this response was completely abolished in the ShpHep−/− mice. In contrast, livers of ShpHep−/− mice had consistent NF-κB activation. To further characterize the role of Shp specifically in the transition of steatosis to NASH, mice were fed the HFCF diet for 4 weeks, followed by Shp deletion. Surprisingly, Shp deletion after steatosis development exacerbated hepatic inflammation and fibrosis without affecting liver steatosis. Together, our results indicate that, depending on NASH stage, hepatic Shp plays an opposing role in steatosis and inflammation. Mechanistically, Shp deletion in hepatocytes activated NF-κB and impaired Pparg activation, leading to the dissociation of steatosis, inflammation, and fibrosis in NASH development.

    更新日期:2020-01-24
  • The terminal sialic acid of stage-specific embryonic antigen-4 has a crucial role in binding to a cancer-targeting antibody
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Caroline Soliman, Jia Xin Chua, Mireille Vankemmelbeke, Richard S. McIntosh, Andrew J. Guy, Ian Spendlove, Lindy G. Durrant, Paul A. Ramsland

    Cancer remains a leading cause of morbidity and mortality worldwide, requiring ongoing development of targeted therapeutics such as monoclonal antibodies. Carbohydrates on embryonic cells are often highly expressed in cancer and are therefore attractive targets for antibodies. Stage-specific embryonic antigen-4 (SSEA-4) is one such glycolipid target expressed in many cancers, including breast and ovarian carcinomas. Here, we defined the structural basis for recognition of SSEA-4 by a novel monospecific chimeric antibody (ch28/11). Five X-ray structures of ch28/11 Fab complexes with the SSEA-4 glycan headgroup, determined at 1.5–2.7 Å resolutions, displayed highly similar three-dimensional structures indicating a stable binding mode. The structures also revealed that by adopting a horseshoe-shaped conformation in a deep groove, the glycan headgroup likely sits flat against the membrane to allow the antibody to interact with SSEA-4 on cancer cells. Moreover, we found that the terminal sialic acid of SSEA-4 plays a dominant role in dictating the exquisite specificity of the ch28/11 antibody. This observation was further supported by molecular dynamics simulations of the ch28/11-glycan complex, which show that SSEA-4 is stabilized by its terminal sialic acid, unlike SSEA-3, which lacks this sialic acid modification. These high-resolution views of how a glycolipid interacts with an antibody may help to advance a new class of cancer-targeting immunotherapy.

    更新日期:2020-01-24
  • Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Ishfaq Ahmed, Badal Chandra Roy, Laxmi Uma Maheswar Rao Jakkula, Dharmalingam Subramaniam, Prasad Dandawate, Shrikant Anant, Venkatesh Sampath, Shahid Umar

    Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor–related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in β-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20–34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and β-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased β-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.

    更新日期:2020-01-24
  • The chloroplast metalloproteases VAR2 and EGY1 act synergistically to regulate chloroplast development in Arabidopsis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Yafei Qi, Xiaomin Wang, Pei Lei, Huimin Li, Liru Yan, Jun Zhao, Jingjing Meng, Jingxia Shao, Lijun An, Fei Yu, Xiayan Liu

    Chloroplast development and photosynthesis require the proper assembly and turnover of photosynthetic protein complexes. Chloroplasts harbor a repertoire of proteases to facilitate proteostasis and development. We have previously used an Arabidopsis leaf variegation mutant, yellow variegated2 (var2), defective in thylakoid FtsH protease complexes, as a tool to dissect the genetic regulation of chloroplast development. Here, we report a new genetic enhancer mutant of var2, enhancer of variegation3–1 (evr3–1). We confirm that EVR3 encodes a chloroplast metalloprotease, reported previously as ethylene-dependent gravitropism-deficient and yellow-green1 (EGY1)/ammonium overly sensitive1 (AMOS1). We observed that mutations in EVR3/EGY1/AMOS1 cause more severe leaf variegation in var2–5 and synthetic lethality in var2–4. Using a modified blue-native PAGE system, we reveal abnormal accumulations of photosystem I, photosystem II, and light-harvesting antenna complexes in EVR3/EGY1/AMOS1 mutants. Moreover, we discover distinct roles of VAR2 and EVR3/EGY1/AMOS1 in the turnover of photosystem II reaction center under high light stress. In summary, our findings indicate that two chloroplast metalloproteases, VAR2/AtFtsH2 and EVR3/EGY1/AMOS1, function coordinately to regulate chloroplast development and reveal new roles of EVR3/EGY1/AMOS1 in regulating chloroplast proteostasis in Arabidopsis.

    更新日期:2020-01-24
  • Structural insights into the catalytic mechanism of lovastatin hydrolase
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Yajing Liang, Xuefeng Lu

    The lovastatin hydrolase PcEST from the fungus Penicillium chrysogenum exhibits enormous potential for industrial-scale applications in single-step production of monacolin J, the key precursor for synthesis of the cholesterol-lowering drug simvastatin. This enzyme specifically and efficiently catalyzes the conversion of lovastatin to monacolin J but cannot hydrolyze simvastatin. Understanding the catalytic mechanism and the structure–function relationship of PcEST is therefore important for further lovastatin hydrolase screening, engineering, and commercial applications. Here, we solved four X-ray crystal structures, including apo PcEST (2.3 Å), PcEST in complex with monacolin J (2.48 Å), PcEST complexed with the substrate analog simvastatin (2.4 Å), and an inactivated PcEST variant (S57A) with the lovastatin substrate (2.3 Å). Structure-based biochemical analyses and mutagenesis assays revealed that the Ser57 (nucleophile)–Tyr170 (general base)–Lys60 (general acid) catalytic triad, the hydrogen-bond network (Trp344 and Tyr127) around the active site, and the specific substrate-binding tunnel together determine efficient and specific lovastatin hydrolysis by PcEST. Moreover, steric effects on nucleophilic attack caused by the 2′,2-dimethybutyryl group of simvastatin resulted in no activity of PcEST on simvastatin. On the basis of structural comparisons, we propose several indicators to define lovastatin esterases. Furthermore, using structure-guided enzyme engineering, we developed a PcEST variant, D106A, having improved solubility and thermostability, suggesting a promising application of this variant in industrial processes. To our knowledge, this is the first report describing the mechanism and structure–function relationship of lovastatin hydrolase and providing insights that may guide rapid screening and engineering of additional lovastatin esterase variants.

    更新日期:2020-01-24
  • SMARCAD1-mediated recruitment of the DNA mismatch repair protein MutLα to MutSα on damaged chromatin induces apoptosis in human cells
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Yukimasa Takeishi, Ryosuke Fujikane, Mihoko Rikitake, Yuko Obayashi, Mutsuo Sekiguchi, Masumi Hidaka

    The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.

    更新日期:2020-01-24
  • C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Andreia Albuquerque-Wendt, Damien Jacot, Nicolas Dos Santos Pacheco, Carla Seegers, Patricia Zarnovican, Falk F. R. Buettner, Hans Bakker, Dominique Soldati-Favre, Françoise H. Routier

    C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase–deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

    更新日期:2020-01-24
  • A 49-residue sequence motif in the C terminus of Nav1.9 regulates trafficking of the channel to the plasma membrane
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Daria V. Sizova, Jianying Huang, Elizabeth J. Akin, Mark Estacion, Carolina Gomis-Perez, Stephen G. Waxman, Sulayman D. Dib-Hajj

    Genetic and functional studies have confirmed an important role for the voltage-gated sodium channel Nav1.9 in human pain disorders. However, low functional expression of Nav1.9 in heterologous systems (e.g. in human embryonic kidney 293 (HEK293) cells) has hampered studies of its biophysical and pharmacological properties and the development of high-throughput assays for drug development targeting this channel. The mechanistic basis for the low level of Nav1.9 currents in heterologous expression systems is not understood. Here, we implemented a multidisciplinary approach to investigate the mechanisms that govern functional Nav1.9 expression. Recombinant expression of a series of Nav1.9-Nav1.7 C-terminal chimeras in HEK293 cells identified a 49-amino-acid-long motif in the C terminus of the two channels that regulates expression levels of these chimeras. We confirmed the critical role of this motif in the context of a full-length channel chimera, Nav1.9-Ct49aaNav1.7, which displayed significantly increased current density in HEK293 cells while largely retaining the characteristic Nav1.9-gating properties. High-resolution live microscopy indicated that the newly identified C-terminal motif dramatically increases the number of channels on the plasma membrane of HEK293 cells. Molecular modeling results suggested that this motif is exposed on the cytoplasmic face of the folded C terminus, where it might interact with other channel partners. These findings reveal that a 49-residue-long motif in Nav1.9 regulates channel trafficking to the plasma membrane.

    更新日期:2020-01-24
  • Phosphatidylinositol 3,4-bisphosphate synthesis and turnover are spatially segregated in the endocytic pathway
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Haibin Wang, Dinah Loerke, Caroline Bruns, Rainer Müller, Philipp-Alexander Koch, Dmytro Puchkov, Carsten Schultz, Volker Haucke

    Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.

    更新日期:2020-01-24
  • Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Mukul Sherekar, Sae-Won Han, Rodolfo Ghirlando, Simon Messing, Matthew Drew, Dana Rabara, Timothy Waybright, Puneet Juneja, Hugh O'Neill, Christopher B. Stanley, Debsindhu Bhowmik, Arvind Ramanathan, Sriram Subramaniam, Dwight V. Nissley, William Gillette, Frank McCormick, Dominic Esposito

    Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.

    更新日期:2020-01-24
  • The zebrafish NLRP3 inflammasome has functional roles in ASC-dependent interleukin-1β maturation and gasdermin E–mediated pyroptosis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Jiang-Yuan Li, Yue-Yi Wang, Tong Shao, Dong-Dong Fan, Ai-Fu Lin, Li-Xin Xiang, Jian-Zhong Shao

    The NLR family pyrin domain containing 3 (NLRP3) inflammasome is one of the best-characterized inflammasomes in humans and other mammals. However, knowledge about the NLRP3 inflammasome in nonmammalian species remains limited. Here, we report the molecular and functional identification of an NLRP3 homolog (DrNLRP3) in a zebrafish (Danio rerio) model. We found that DrNLRP3's overall structural architecture was shared with mammalian NLRP3s. It initiates a classical inflammasome assembly for zebrafish inflammatory caspase (DrCaspase-A/-B) activation and interleukin 1β (DrIL-1β) maturation in an apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC)-dependent manner, in which DrNLRP3 organizes DrASC into a filament that recruits DrCaspase-A/-B by homotypic pyrin domain (PYD)–PYD interactions. DrCaspase-A/-B activation in the DrNLRP3 inflammasome occurred in two steps, with DrCaspase-A being activated first and DrCaspase-B second. DrNLRP3 also directly activated full-length DrCaspase-B and elicited cell pyroptosis in a gasdermin E (GSDME)-dependent but ASC-independent manner. These two events were tightly coordinated by DrNLRP3 to ensure efficient IL-1β secretion for the initiation of host innate immunity. By knocking down DrNLRP3 in zebrafish embryos and generating a DrASC-knockout (DrASC−/−) fish clone, we characterized the function of the DrNLRP3 inflammasome in anti-bacterial immunity in vivo. The results of our study disclosed the origin of the NLRP3 inflammasome in teleost fish, providing a cross-species understanding of the evolutionary history of inflammasomes. Our findings also indicate that the NLRP3 inflammasome may coordinate inflammatory cytokine processing and secretion through a GSDME-mediated pyroptotic pathway, uncovering a previously unrecognized regulatory function of NLRP3 in both inflammation and cell pyroptosis.

    更新日期:2020-01-24
  • Correction: Defining α-synuclein species responsible for Parkinson's disease phenotypes in mice.
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Jessica M. Froula, Marta Castellana-Cruz, Nadia M. Anabtawi, José D. Camino, Serene W. Chen, Drake R. Thrasher, Jennifer Freire, Allen A. Yazdi, Sheila Fleming, Christopher M. Dobson, Janet R. Kumita, Nunilo Cremades, Laura A. Volpicelli-Daley

    VOLUME 294 (2019) PAGES 10392–10406In the published article, there was an inadvertent error in Fig. 5. The panel for the retrobeads (red) and p-α-synuclein inclusions (green) in the cortex at 1 week post-injection was the same as 2 weeks post-injection. The 1-week post-injection panel was incorrect and has been corrected. There are no phosphorylated α-synuclein inclusions or retrotracer beads present in the cortex (or amygdala or SNc) 1 week post-injection.jbc;295/4/1142/F5F1F5Figure 5.

    更新日期:2020-01-24
  • Activation of sphingosine 1-phosphate receptor 2 attenuates chemotherapy-induced neuropathy
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Wei Wang, Ping Xiang, Wee Siong Chew, Federico Torta, Aishwarya Bandla, Violeta Lopez, Wei Lun Seow, Brenda Wan Shing Lam, Jing Kai Chang, Peiyan Wong, Kanokporn Chayaburakul, Wei-Yi Ong, Markus R. Wenk, Raghav Sundar, Deron R. Herr

    Platinum-based therapeutics are used to manage many forms of cancer, but frequently result in peripheral neuropathy. Currently, the only option available to attenuate chemotherapy-induced neuropathy is to limit or discontinue this treatment. Sphingosine 1-phosphate (S1P) is a lipid-based signaling molecule involved in neuroinflammatory processes by interacting with its five cognate receptors: S1P1–5. In this study, using a combination of drug pharmacodynamic analysis in human study participants, disease modeling in rodents, and cell-based assays, we examined whether S1P signaling may represent a potential target in the treatment of chemotherapy-induced neuropathy. To this end, we first investigated the effects of platinum-based drugs on plasma S1P levels in human cancer patients. Our analysis revealed that oxaliplatin treatment specifically increases one S1P species, d16:1 S1P, in these patients. Although d16:1 S1P is an S1P2 agonist, it has lower potency than the most abundant S1P species (d18:1 S1P). Therefore, as d16:1 S1P concentration increases, it is likely to disproportionately activate proinflammatory S1P1 signaling, shifting the balance away from S1P2. We further show that a selective S1P2 agonist, CYM-5478, reduces allodynia in a rat model of cisplatin-induced neuropathy and attenuates the associated inflammatory processes in the dorsal root ganglia, likely by activating stress-response proteins, including ATF3 and HO-1. Cumulatively, the findings of our study suggest that the development of a specific S1P2 agonist may represent a promising therapeutic approach for the management of chemotherapy-induced neuropathy.

    更新日期:2020-01-24
  • The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-24
    Alexander S. Jureka, Alex B. Kleinpeter, Jennifer L. Tipper, Kevin S. Harrod, Chad M. Petit

    A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

    更新日期:2020-01-24
  • Sulphated and sialylated N-glycans in the echinoderm Holothuria atra reflect its marine habitat and phylogeny
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-22
    Jorick Vanbeselaere, Chunsheng Jin, Barbara Eckmair, Iain B. H. Wilson, Katharina Paschinger

    Amongst the earliest deuterostomes, the echinoderms are an evolutionary important group of ancient marine animals. Within this phylum, the holothuroids (sea cucumbers) are known to produce a wide range of glycoconjugate biopolymers with apparent benefits to health; therefore, they are of economic and culinary interest throughout the world. Other than their highly modified glycosaminoglycans (e.g. fucosylated chondroitin sulphate and fucoidan), nothing is known about their protein-linked glycosylation. Here, we used multi-step N-glycan fractionation to efficiently separate the anionic and neutral N-glycans before analyzing the N-glycans of the black sea cucumber (Holothuria atra) by MS in combination with enzymatic and chemical treatments. These analyses showed the presence of various fucosylated, phosphorylated, sialylated and multiply sulphated moieties as modifications of oligomannosidic, hybrid and complex-type N-glycans. The high degree of sulphation and fucosylation parallels the modifications previously observed on holothuroid glycosaminoglycans. Compatible with its phylogenetic position, H. atra not only expresses vertebrate motifs such as sulpho- and sialyl-Lewis A epitopes, but displays a high degree of anionic substitution of its glycans as observed in other marine invertebrates. Thus, as for other echinoderms, the phylum- and order-specific aspects of this species’ N-glycosylation reveal both invertebrate- and vertebrate-like features.

    更新日期:2020-01-23
  • The architecture of the diaminobutyrate acetyltransferase active site provides mechanistic insight into the biosynthesis of the chemical chaperone ectoine
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-22
    Alexandra A Richter, Stefanie Kobus, Laura Czech, Astrid Hoeppner, Jan Zarzycki, Tobias J. Erb, Lukas Lauterbach, Jeroen S Dickschat, Erhard Bremer, Sander H. J. Smits

    Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by L-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl CoA to EctB-formed L-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-L-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-ADABA. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) for (i) its apo form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both with CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

    更新日期:2020-01-23
  • Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-22
    Franco K.K. Li, Federico I. Rosell, Robert T. Gale, Jean-Pierre Simorre, Eric D. Brown, Natalie C.J. Strynadka

    Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell wall is composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR-CpsA-Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections as the target enzymes are implicated in the assembly of many other bacterial cell wall polymers including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU and TagV, in the apo form at 1.6-2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multi-angle light scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors.

    更新日期:2020-01-23
  • Extracellular domains I-II of cell-surface glycoprotein CD44 mediate its trans-homophilic dimerization and tumor cluster aggregation
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-22
    Madoka Kawaguchi, Nurmaa Dashzeveg, Yue Cao, Yuzhi Jia, Xia Liu, Yang Shen, Huiping Liu

    CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning-based computational modeling combined with experimental mutagenesis tests revealed that the extracellular domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 residues in domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro. Importantly, the substitutions in domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer-dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

    更新日期:2020-01-23
  • HLA-DM catalytically enhances peptide dissociation by sensing peptide–MHC class II interactions throughout the peptide binding cleft
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-22
    Eduardo Reyes-Vargas, Adam P. Barker, Zemin Zhou, Xiao He, Peter E. Jensen

    Human leukocyte antigen-DM (HLA-DM) is an integral component of the major histocompatibility complex class II (MHCII) antigen-processing and -presentation pathway. HLA-DM shapes the immune system by differentially catalyzing peptide exchange on MHCII molecules, thereby editing the peptide–MHCII (pMHCII) repertoire by imposing a bias on the foreign and self-derived peptide cargos that are presented on the cell surface for immune surveillance and tolerance induction by CD4+ T cells. To better understand DM selectivity, here we developed a real-time fluorescence anisotropy assay to delineate the pMHCII intrinsic stability, DM-binding affinity, and catalytic turnover, independent kinetic parameters of HLA-DM enzymatic activity. We analyzed prominent pMHCII contacts by differentiating the kinetic parameters in pMHCII homologs, observing that peptide interactions throughout the MHCII binding cleft influence both the rate of peptide dissociation from the DM–pMHCII catalytic complex and the binding affinity of HLA-DM for a pMHCII. We show that the intrinsic stability of a pMHCII linearly correlates with DM catalytic turnover, but is non-linearly correlated with its binding affinity. Surprisingly, interactions at the peptides N-terminus up to and including the MHCII position one (P1) anchor affected the catalytic turnover, suggesting that the active DM–pMHCII catalytic complex operates on pMHCII complexes with full peptide occupancy. Furthermore, interactions at the peptide C-terminus modulated DM-binding affinity, suggesting distal communication between peptide interactions with the MHCII and the DM–pMHCII binding interface. Our results imply an intimate linkage between the DM–pMHCII interface and peptide–MHCII interactions throughout the peptide-binding cleft.

    更新日期:2020-01-23
  • Mechanism underlying autoinducer recognition in the Vibrio cholerae DPO-VqmA quorum-sensing pathway
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-21
    Xiuliang Huang, Olivia P Duddy, Justin E Silpe, Jon E Paczkowski, Jianping Cong, Brad R. Henke, Bonnie L Bassler

    Quorum sensing is a bacterial communication process whereby bacteria produce, release, and detect extracellular signaling molecules called autoinducers to coordinate collective behaviors. In the pathogen Vibrio cholerae, the quorum-sensing autoinducer 3,5-dimethyl-pyrazin-2-ol (DPO) binds the receptor and transcription factor VqmA. The DPO-VqmA complex activates transcription of vqmR, encoding the VqmR small RNA, which represses genes required for biofilm formation and virulence factor production. Here, we show that VqmA is soluble and properly folded, and activates basal-level transcription of its target vqmR in the absence of DPO. VqmA transcriptional activity is increased in response to increasing concentrations of DPO, allowing VqmA to drive the V. cholerae quorum-sensing transition at high cell densities. We solved the DPO-VqmA crystal structure to 2.0 Å resolution and compared it to existing structures to understand the conformational changes VqmA undergoes upon DNA binding. Analysis of DPO analogs showed that a hydroxyl or carbonyl group at the 2’ position is critical for binding to VqmA. The proposed DPO precursor, a linear molecule, N-alanyl-aminoacetone or Ala-AA, also bound and activated VqmA. Results from site-directed mutagenesis and competitive ligand-binding analyses revealed that DPO and Ala-AA occupy the same binding site. In summary, our structure–function analysis identifies key features required for VqmA activation and DNA binding and establishes that, while VqmA binds two different ligands, VqmA does not require a bound ligand for folding or basal transcriptional activity. However, bound ligand is required for maximal activity.

    更新日期:2020-01-22
  • Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin binding protein
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-21
    Daniel R. Sandoval, Alejandro Gomez Toledo, Chelsea D. Painter, Ember M. Tota, M. Osman Sheikh, Alan M.V. West, Martin M. Frank, Lance Wells, Ding Xu, Roy Bicknell, Kevin D. Corbett, Jeffrey D. Esko

    Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate-protein interactions. The identification of membrane heparan-sulfate binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC–MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and, using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire glycan-binding proteins for further study.

    更新日期:2020-01-22
  • A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-21
    Tung O. Chan, Roger S. Armen, Santosh Yadav, Sushrut Shah, Jin Zhang, Brian C. Tiegs, Nikhil Keny, Brian Blumhof, Deepak A Deshpande, Ulrich Rodeck, Raymond B Penn

    Phosphorylation of specific residues in the activation loops of AGC family protein kinases is required for activity of most of these kinases, including the catalytic subunit of protein kinase A (PKAc). Although many phosphorylated AGC kinases are sensitive to phosphatase-mediated dephosphorylation, the PKAc activation loop uniquely resists dephosphorylation, rendering it “constitutively” phosphorylated in cells. Previous biophysical experiments and structural modeling have suggested that the N-terminal myristoylation signal and the C-terminal FxxF motif in PKAc regulate its thermal stability and catalysis. Here, using site-directed mutagenesis, molecular modeling, and in both cell-free and cell-based systems, we demonstrate that substitutions of either the PKAc myristoylation signal or the FxxF motif only modestly reduce phosphorylation and fail to affect PKAc function in cells. However, we observed that these two sites cooperate with an N-terminal FxxW motif to cooperatively establish phosphatase resistance of PKAc while not affecting kinase-dependent phosphorylation of the activation loop. We noted that this tripartite cooperative mechanism of phosphatase resistance is functionally relevant, as demonstrated by changes in morphology, adhesion, and migration of human airway smooth muscle cells transfected with PKAc variants containing amino acid substitutions in in these three sites. These findings establish that three allosteric sites located at the PKAc N and C termini coordinately regulate phosphatase sensitivity of this enzyme. This cooperative mechanism of phosphatase resistance of AGC kinase opens new perspectives toward therapeutic manipulation of kinase signaling in disease.

    更新日期:2020-01-22
  • Hematopoietic progenitor kinase 1 down-regulates the oncogenic receptor tyrosine kinase AXL in pancreatic cancer
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-20
    Xianzhou Song, Hironari Akasaka, Hua Wang, Reza Abbasgholizadeh, Ji-hyun Shin, Fenglin Zang, Jiayi Chen, Craig D Logsdon, Anirban Maitra, Andrew J. Bean, Huamin Wang

    The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells, and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, baflomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest–specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression (P < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC.

    更新日期:2020-01-21
  • Von Hippel–Lindau tumor suppressor (VHL) stimulates TOR signaling by interacting with phosphoinositide 3-kinase (PI3K)
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-20
    Sun-Hong Hwang, Sunhoe Bang, Wonho Kim, Jongkyeong Chung

    Cell growth is positively controlled by phosphoinositide 3-kinase (PI3K)-TOR signaling pathway under conditions of abundant growth factors and nutrients. To discover additional mechanisms that regulate cell growth, here we performed RNAi-based mosaic analyses in the Drosophila fat body, the primary metabolic organ in the fly. Unexpectedly, the knockdown of the Drosophila von Hippel-Lindau (VHL) gene markedly decreased cell size and body size. These cell growth phenotypes induced by VHL loss-of-function were recovered by activation of TOR signaling in Drosophila. Consistent with the genetic interactions between VHL and the signaling components of PI3K-TOR pathway in Drosophila, we observed that VHL loss-of-function in mammalian cells causes decreased phosphorylation of ribosomal protein S6 kinase (S6K) and Akt, which represent the main activities of this pathway. We further demonstrate that VHL activates TOR signaling by directly interacting with the p110 catalytic subunit of PI3K. On the basis of the evolutionarily conserved regulation of PI3K-TOR signaling by VHL observed here, we propose that VHL plays an important role in the regulation and maintenance of proper cell growth in metazoans.

    更新日期:2020-01-21
  • The discovery and maturation of peptide biologics targeting the small G protein Cdc42: a bioblockade for Ras-driven signalling
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-20
    George J. N. Tetley, Natasha P. Murphy, Stephane Bonetto, Gabriela Ivanova-Berndt, Jefferson Revell, Helen R Mott, R. Neil Cooley, Darerca Owen

    Aberrant Ras signalling drives 30% of cancers and inhibition of Rho family small-GTPase signalling has been shown to combat Ras-driven cancers. Here we present the discovery of a 16mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely related small-GTPases. The structure of the tightest binding peptide was solved by NMR and its binding site on Cdc42 determined. Addition of a cell penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signalling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data adds to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.

    更新日期:2020-01-21
  • Receptor-mediated cell entry of Paramyxoviruses: mechanisms, and consequences for tropism and pathogenesis
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Chanakha K. Navaratnarajah, Alex R Generous, Iris Yousaf, Roberto Cattaneo

    Research in the last decade has uncovered many new paramyxoviruses, airborne agents that cause epidemic diseases in animals including humans. Most paramyxoviruses enter epithelial cells of the airway using sialic acid as receptor, and cause only mild disease. However, others cross the epithelial barrier and cause more severe disease. For some of these viruses, the host receptors have been identified, and the mechanisms of cell entry elucidated. The tetrameric attachment proteins of paramyxoviruses have vastly different binding affinities for their cognate receptors, which they contact through different binding surfaces. Nevertheless, all input signals are converted to the same output: conformational changes that trigger refolding of trimeric fusion proteins, and membrane fusion. Experiments with selectively receptor-blinded viruses inoculated into their natural hosts have provided insights into tropism, identifying the cells and tissues that support growth, and revealing the mechanisms of pathogenesis. These analyses also shed light on diabolically elegant mechanisms used by morbilliviruses, including measles virus, to promote massive amplification within the host, followed by efficient aerosolization and rapid spread through host populations. In another paradigm of receptor-facilitated severe disease, henipaviruses, including Nipah and Hendra viruses, use different members of one protein family to cause zoonoses. Specific properties of different paramyxoviruses, like neurotoxicity and immunosuppression, are now understood in the light of receptor specificity. We propose that research on the specific receptors for several newly identified members of the Paramyxoviridae family that may not bind sialic acid is needed to anticipate their zoonotic potential, and generate effective vaccines and antiviral compounds.

    更新日期:2020-01-17
  • DAPLE protein inhibits nucleotide exchange on Gαs and Gαq via the same motif that activates Gαi
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Arthur Marivin, Marcin Maziarz, Jingyi Zhao, Vincent DiGiacomo, Isabel Olmos Calvo, Emily A Mann, Jason Ear, Juan B Blanco-Canosa, Elliott M. Ross, Pradipta Ghosh, Mikel Garcia-Marcos

    Besides being regulated by G protein-coupled receptors, the activity of heterotrimeric G proteins is modulated by many cytoplasmic proteins. GIV/Girdin and DAPLE are the best characterized members of a group of cytoplasmic regulators that contain a Gα-binding-and-activating (GBA) motif and whose dysregulation underlies human diseases, including cancer and birth defects. GBA motif-containing proteins were originally reported to modulate G proteins by binding Gα subunits of the Gi/o family (Gαi) over other families (such as Gs, Gq/11 or G12/13), and promoting nucleotide exchange in vitro. However, some evidence suggests that this is not always the case, as phosphorylation of the GBA motif of GIV promotes its binding to Gαs and inhibits nucleotide exchange. The G protein specificity of DAPLE and how it might affect nucleotide exchange on G proteins besides Gαi remains to be investigated. Here, we show that DAPLE’s GBA motif, in addition to Gαi, binds efficiently to members of the Gs and Gq/11 families (Gαs and Gαq, respectively), but not of the G12/13 family (Gα12) in the absence of post-translational phosphorylation. We pinpointed Met-1669 as the residue in the GBA motif of DAPLE that diverges from that in GIV and enables better binding to Gαs and Gαq. Unlike the nucleotide exchange acceleration observed for Gαi, DAPLE inhibited nucleotide exchange on Gαs and Gαq. These findings indicate that GBA motifs have versatility in their G protein-modulating effect, i.e. they can bind to Gα subunits of different classes, and either stimulate or inhibit nucleotide exchange depending on the G protein subtype.

    更新日期:2020-01-17
  • HYBID and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Hiroyuki Yoshida, Mika Aoki, Aya Komiya, Yoko Endo, Keigo Kawabata, Tomomi Nakamura, Shingo Sakai, Tetsuya Sayo, Yasunori Okada, Yoshito Takahashi

    The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of hyaluronan-binding protein involved in hyaluronan depolymerization (HYBID, aka CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine’s effects on HA synthase (HAS) expression, the molecular sizes of the HAs produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine’s effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ (PKCδ) or PI3K–AKT signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ~2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation.

    更新日期:2020-01-17
  • Loss of voltage-gated proton channel Hv1 decreases insulin secretion and leads to hyperglycemia and glucose intolerance in mice
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Huimin Pang, Xudong Wang, Shiqun Zhao, Wang Xi, Jili Lv, Jiwei Qin, Qing Zhao, Yongzhe Che, Liangyi Chen, Shu Jie Li

    Insulin secretion by pancreatic islet β-cells is regulated by glucose levels and is accompanied by proton generation. The voltage-gated proton channel Hv1 is present in pancreatic β-cells and extremely selective for protons. However, whether Hv1 is involved in insulin secretion is unclear. Here, we demonstrate that Hv1 promotes insulin secretion of pancreatic β-cells and glucose homeostasis. Hv1-deficient mice displayed hyperglycemia and glucose intolerance due to reduced insulin secretion, but retained normal peripheral insulin sensitivity. Moreover, Hv1 loss contributed much more to severe glucose intolerance as the mice got older. The islets of Hv1-deficient and heterozygous mice were markedly deficient in glucose- and K+-induced insulin secretion. In perifusion assays, Hv1 deletion dramatically reduced both the first and second phase of glucose-stimulated insulin secretion (GSIS). Islet insulin and proinsulin contents were reduced, and histological analysis of pancreas slices revealed an accompanying modest reduction of β-cell mass in the Hv1-knockout mice. EM observations also indicated a reduction in insulin granule size, but not granule number or granule docking, in the Hv1-deficient mice. Mechanistically, Hv1 loss limited the capacity of glucose-induced membrane depolarization, accompanying the reduced ability of glucose to raise Ca2+ levels in islets, evidenced by a decreased duration of individual calcium oscillations. Moreover, Hv1 expression was significantly reduced in pancreatic β-cells from streptozotocin-induced diabetic mice, indicating that Hv1 deficiency is associated with β-cell dysfunction and diabetes. We conclude that Hv1 regulates insulin secretion and glucose homeostasis through a mechanism that depends on intracellular Ca2+ levels and membrane depolarization.

    更新日期:2020-01-17
  • Functional Binding of E-selectin to its Ligands is Enhanced by Structural Features Beyond its Lectin Domain
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Fajr A Aleisa, Kosuke Sakashita, Jae Man Lee, Dina B. AbuSamra, Bader Alwan, Shuho Nozue, Muhammad Tehseen, Samir M Hamdan, Satoshi Habuchi, Takahiro Kusakabe, Jasmeen S Merzaban

    Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- or L- selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains.

    更新日期:2020-01-17
  • A transient amphipathic helix in the prodomain of PCSK9 facilitates binding to low-density lipoprotein particles
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-16
    Samantha K. Sarkar, Alexander C.Y. Foo, Angela Matyas, Ikhuosho Asikhia, Tanja Kosenko, Natalie K Goto, Ariela Vergara-Jaque, Thomas A. Lagace

    Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a ligand of low-density lipoprotein receptor (LDLR) that promotes LDLR degradation in late endosomes/lysosomes. In human plasma, 30%–40% of PCSK9 is bound to LDL particles; however, the physiological significance of this interaction remains unknown. LDL binding in vitro requires a disordered N-terminal region in PCSK9’s prodomain. Here, we report that peptides corresponding to a predicted amphipathic α-helix in the prodomain N-terminus adopt helical structure in a membrane-mimetic environment. This effect was greatly enhanced by an R46L substitution representing an atheroprotective PCSK9 loss-of-function mutation. A helix-disrupting proline substitution within the putative α-helical motif in full-length PCSK9 lowered LDL binding affinity >5-fold. Modeling studies suggested that the transient α-helix aligns multiple polar residues to interact with positively charged residues in the C-terminal domain. Gain-of-function PCSK9 mutations associated with familial hypercholesterolemia (FH) and clustered at the predicted interdomain interface (R469W, R496W, and F515L) inhibited LDL binding, which was completely abolished in the case of the R496W variant. These findings shed light on allosteric conformational changes in PCSK9 required for high-affinity binding to LDL particles. Moreover, the initial identification of FH-associated mutations that diminish the PCSK9’s ability to bind LDL reported here support the notion that PCSK9-LDL association in the circulation inhibits PCSK9 activity.

    更新日期:2020-01-17
  • Multiple distinct pathways lead to hyperubiquitylated insoluble TDP-43 protein independent of its translocation into stress granules
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Friederike Hans, Hanna Glasebach, Philipp J. Kahle

    Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress–induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R–like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal–regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.

    更新日期:2020-01-17
  • Triple-cell lineage tracing by a dual reporter on a single allele
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Kuo Liu, Muxue Tang, Hengwei Jin, Qiaozhen Liu, Lingjuan He, Huan Zhu, Xiuxiu Liu, Ximeng Han, Yan Li, Libo Zhang, Juan Tang, Wenjuan Pu, Zan Lv, Haixiao Wang, Hongbin Ji, Bin Zhou

    Genetic lineage tracing is widely used to study organ development and tissue regeneration. Multicolor reporters are a powerful platform for simultaneously tracking discrete cell populations. Here, combining Dre-rox and Cre-loxP systems, we generated a new dual-recombinase reporter system, called Rosa26 traffic light reporter (R26-TLR), to monitor red, green, and yellow fluorescence. Using this new reporter system with the three distinct fluorescent reporters combined on one allele, we found that the readouts of the two recombinases Cre and Dre simultaneously reflect Cre+Dre−, Cre−Dre+, and Cre+Dre+ cell lineages. As proof of principle, we show specific labeling in three distinct progenitor/stem cell populations, including club cells, AT2 cells, and bronchoalveolar stem cells, in Sftpc-DreER;Scgb1a1-CreER;R26-TLR mice. By using this new dual-recombinase reporter system, we simultaneously traced the cell fate of these three distinct cell populations during lung repair and regeneration, providing a more comprehensive picture of stem cell function in distal airway repair and regeneration. We propose that this new reporter system will advance developmental and regenerative research by facilitating a more sophisticated genetic approach to studying in vivo cell fate plasticity.

    更新日期:2020-01-17
  • Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Aarti Krishnan, Joachim Kloehn, Matteo Lunghi, Dominique Soldati-Favre

    The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

    更新日期:2020-01-17
  • How to catch a HIF—the work of Gregg Semenza's lab on hypoxia-inducible factor 1
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Martin J. Spiering

    Molecular oxygen is essential for the viability and function of every cell of the animal body. Because skin, tissues, and organs impede oxygen diffusion directly from the air, molecular oxygen concentrations inside the body often are less than 5% (1), much lower than the 21% in Earth's atmosphere.As multicellular organisms evolved, this steep oxygen gradient necessitated the development of structures such as the circulatory system and of biochemical mechanisms that monitor and control oxygen levels in the body.A major player in sensing potentially harmful drops in cellular oxygen concentrations (hypoxia) is the transcription factor hypoxia-inducible factor 1 (HIF-1).2 HIF-1, and its close relative HIF-2, regulate many genes, including the erythropoietin (EPO) gene, which encodes a hormone that stimulates production of red blood cells (2, 3).One important milestone in uncovering HIF-1's pivotal role in oxygen sensing was its purification and biochemical characterization in the mid-1990s by the lab of Gregg Semenza (Fig. 1), a geneticist at Johns Hopkins University School of Medicine. This work was reported in two JBC papers recognized as Classics here (4, 5).jbc;295/3/715/F1F1F1Figure 1.Gregg Semenza and colleagues isolated and biochemically characterized the HIF-1 protein. Photo courtesy of Johns Hopkins Medicine.“JBC was the first choice,” says Semenza, referring to the publication of the first Classics article. “To me, that was a classic JBC paper.”A few years before this work, Semenza's team had found that hypoxia induces the binding of a nuclear protein to a 50-nucleotide-long enhancer region located in the 3′-flanking region of the EPO gene (2, 3). Although...

    更新日期:2020-01-17
  • The Arabidopsis AtGCD3 protein is a glucosylceramidase that preferentially hydrolyzes long-acyl-chain glucosylceramides
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Guang-Yi Dai, Jian Yin, Kai-En Li, Ding-Kang Chen, Zhe Liu, Fang-Cheng Bi, Chan Rong, Nan Yao

    Cellular membranes contain many lipids, some of which, such as sphingolipids, have important structural and signaling functions. The common sphingolipid glucosylceramide (GlcCer) is present in plants, fungi, and animals. As a major plant sphingolipid, GlcCer is involved in the formation of lipid microdomains, and the regulation of GlcCer is key for acclimation to stress. Although the GlcCer biosynthetic pathway has been elucidated, little is known about GlcCer catabolism, and a plant GlcCer-degrading enzyme (glucosylceramidase (GCD)) has yet to be identified. Here, we identified AtGCD3, one of four Arabidopsis thaliana homologs of human nonlysosomal glucosylceramidase, as a plant GCD. We found that recombinant AtGCD3 has a low Km for the fluorescent lipid C6-NBD GlcCer and preferentially hydrolyzes long acyl-chain GlcCer purified from Arabidopsis leaves. Testing of inhibitors of mammalian glucosylceramidases revealed that a specific inhibitor of human β-glucosidase 2, N-butyldeoxynojirimycin, inhibits AtGCD3 more effectively than does a specific inhibitor of human β-glucosidase 1, conduritol β-epoxide. We also found that Glu-499 and Asp-647 in AtGCD3 are vital for GCD activity. GFP-AtGCD3 fusion proteins mainly localized to the plasma membrane or the endoplasmic reticulum membrane. No obvious growth defects or changes in sphingolipid contents were observed in gcd3 mutants. Our results indicate that AtGCD3 is a plant glucosylceramidase that participates in GlcCer catabolism by preferentially hydrolyzing long-acyl-chain GlcCers.

    更新日期:2020-01-17
  • Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Kieu T. M. Pham, Ziyin Li

    The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei. Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

    更新日期:2020-01-17
  • The structure of the Thermococcus gammatolerans McrB N-terminal domain reveals a new mode of substrate recognition and specificity among McrB homologs
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Christopher J. Hosford, Anthony Q. Bui, Joshua S. Chappie

    McrBC is a two-component, modification-dependent restriction system that cleaves foreign DNA-containing methylated cytosines. Previous crystallographic studies have shown that Escherichia coli McrB uses a base-flipping mechanism to recognize these modified substrates with high affinity. The side chains stabilizing both the flipped base and the distorted duplex are poorly conserved among McrB homologs, suggesting that other mechanisms may exist for binding modified DNA. Here we present the structures of the Thermococcus gammatolerans McrB DNA-binding domain (TgΔ185) both alone and in complex with a methylated DNA substrate at 1.68 and 2.27 Å resolution, respectively. The structures reveal that TgΔ185 consists of a YT521-B homology (YTH) domain, which is commonly found in eukaryotic proteins that bind methylated RNA and is structurally unrelated to the E. coli McrB DNA-binding domain. Structural superposition and co-crystallization further show that TgΔ185 shares a conserved aromatic cage with other YTH domains, which forms the binding pocket for a flipped-out base. Mutational analysis of this aromatic cage supports its role in conferring specificity for the methylated adenines, whereas an extended basic surface present in TgΔ185 facilitates its preferential binding to duplex DNA rather than RNA. Together, these findings establish a new binding mode and specificity among McrB homologs and expand the biological roles of YTH domains.

    更新日期:2020-01-17
  • Protein phosphatase 2A activation as a therapeutic strategy for managing MYC-driven cancers
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Caroline C. Farrington, Eric Yuan, Sahar Mazhar, Sudeh Izadmehr, Lauren Hurst, Brittany L. Allen-Petersen, Mahnaz Janghorban, Eric Chung, Grace Wolczanski, Matthew Galsky, Rosalie Sears, Jaya Sangodkar, Goutham Narla

    The tumor suppressor protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose activity is inhibited in most human cancers. One of the best-characterized PP2A substrates is MYC proto-oncogene basic helix–loop–helix transcription factor (MYC), whose overexpression is commonly associated with aggressive forms of this disease. PP2A directly dephosphorylates MYC, resulting in its degradation. To explore the therapeutic potential of direct PP2A activation in a diverse set of MYC-driven cancers, here we used biochemical assays, recombinant cell lines, gene expression analyses, and immunohistochemistry to evaluate a series of first-in-class small-molecule activators of PP2A (SMAPs) in Burkitt lymphoma, KRAS-driven non–small cell lung cancer, and triple-negative breast cancer. In all tested models of MYC-driven cancer, the SMAP treatment rapidly and persistently inhibited MYC expression through proteasome-mediated degradation, inhibition of MYC transcriptional activity, decreased cancer cell proliferation, and tumor growth inhibition. Importantly, we generated a series of cell lines expressing PP2A-dependent phosphodegron variants of MYC and demonstrated that the antitumorigenic activity of SMAPs depends on MYC degradation. Collectively, the findings presented here indicate a pharmacologically tractable approach to drive MYC degradation by using SMAPs for the management of a broad range of MYC-driven cancers.

    更新日期:2020-01-17
  • Crystal structure of phytochromobilin synthase in complex with biliverdin IXα, a key enzyme in the biosynthesis of phytochrome
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Masakazu Sugishima, Kei Wada, Keiichi Fukuyama, Ken Yamamoto

    Phytochromobilin (PΦB) is a red/far-red light sensory pigment in plant phytochrome. PΦB synthase is a ferredoxin-dependent bilin reductase (FDBR) that catalyzes the site-specific reduction of bilins, which are sensory and photosynthesis pigments, and produces PΦB from biliverdin, a heme-derived linear tetrapyrrole pigment. Here, we determined the crystal structure of tomato PΦB synthase in complex with biliverdin at 1.95 Å resolution. The overall structure of tomato PΦB synthase was similar to those of other FDBRs, except for the addition of a long C-terminal loop and short helices. The structure further revealed that the C-terminal loop is part of the biliverdin-binding pocket and that two basic residues in the C-terminal loop form salt bridges with the propionate groups of biliverdin. This suggested that the C-terminal loop is involved in the interaction with ferredoxin and biliverdin. The configuration of biliverdin bound to tomato PΦB synthase differed from that of biliverdin bound to other FDBRs, and its orientation in PΦB synthase was inverted relative to its orientation in the other FDBRs. Structural and enzymatic analyses disclosed that two aspartic acid residues, Asp-123 and Asp-263, form hydrogen bonds with water molecules and are essential for the site-specific A-ring reduction of biliverdin. On the basis of these observations and enzymatic assays with a V121A PΦB synthase variant, we propose the following mechanistic product release mechanism: PΦB synthase-catalyzed stereospecific reduction produces 2(R)-PΦB, which when bound to PΦB synthase collides with the side chain of Val-121, releasing 2(R)-PΦB from the synthase.

    更新日期:2020-01-17
  • Arabidopsis protein l-ISOASPARTYL METHYLTRANSFERASE repairs isoaspartyl damage to antioxidant enzymes and increases heat and oxidative stress tolerance
    J. Biol. Chem. (IF 4.106) Pub Date : 2020-01-17
    Shraboni Ghosh, Nitin Uttam Kamble, Pooja Verma, Prafull Salvi, Bhanu Prakash Petla, Shweta Roy, Venkateswara Rao, Abhijit Hazra, Vishal Varshney, Harmeet Kaur, Manoj Majee

    Stressful environments accelerate the formation of isoaspartyl (isoAsp) residues in proteins, which detrimentally affect protein structure and function. The enzyme PROTEIN l-ISOASPARTYL METHYLTRANSFERASE (PIMT) repairs other proteins by reverting deleterious isoAsp residues to functional aspartyl residues. PIMT function previously has been elucidated in seeds, but its role in plant survival under stress conditions remains undefined. Herein, we used molecular, biochemical, and genetic approaches, including protein overexpression and knockdown experiments, in Arabidopsis to investigate the role of PIMTs in plant growth and survival during heat and oxidative stresses. We demonstrate that these stresses increase isoAsp accumulation in plant proteins, that PIMT activity is essential for restricting isoAsp accumulation, and that both PIMT1 and PIMT2 play an important role in this restriction and Arabidopsis growth and survival. Moreover, we show that PIMT improves stress tolerance by facilitating efficient reactive oxygen species (ROS) scavenging by protecting the functionality of antioxidant enzymes from isoAsp-mediated damage during stress. Specifically, biochemical and MS/MS analyses revealed that antioxidant enzymes acquire deleterious isoAsp residues during stress, which adversely affect their catalytic activities, and that PIMT repairs the isoAsp residues and thereby restores antioxidant enzyme function. Collectively, our results suggest that the PIMT-mediated protein repair system is an integral part of the stress-tolerance mechanism in plants, in which PIMTs protect antioxidant enzymes that maintain proper ROS homeostasis against isoAsp-mediated damage in stressful environments.

    更新日期:2020-01-17
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