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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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...

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.

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.

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.

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.

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.

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.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4β (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.

The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain–containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.

I have traveled many roads during my career. After spending my first 19 years in Los Angeles, I became somewhat of an academic nomad, studying and/or working in six universities in the United States and three in Sweden. In chronological order, I have a B.A. in Scandinavian languages and literature from UCLA, a Ph.D. in biochemistry from Uppsala University, and an M.S. in toxicology from the Karolinska Institute. I have been in schools of natural science, pharmacy, and medicine and have worked in multiple basic science departments and one clinical department. I have served as a research-track and tenured faculty member, department chair, associate dean, and dean. My research has spanned toxinology, biochemistry, toxicology, and pharmacology. Through all the moves, I have gained much and lost some. For the past 40 years, my interest has been cytochrome P450 structure-function and structure-activity relationships. My lab has focused on CYP2B enzymes using X-ray crystallography, site-directed mutagenesis, deuterium-exchange MS, isothermal titration calorimetry, and computational methods in conjunction with a variety of functional assays. This comprehensive approach has enabled detailed understanding of the structural basis of the remarkable substrate promiscuity of CYP2B enzymes. We also have investigated the mechanisms of CYP3A4 allostery using biophysical and advanced spectroscopic techniques, and discovered a pivotal role of P450-P450 interactions and of multiple-ligand binding. A major goal of this article is to provide lessons that may be useful to scientists in the early and middle stages of their careers and those more senior scientists contemplating an administrative move.

Cytochrome P450 enzymes (P450s) are broadly distributed among living organisms and play crucial roles in natural product biosynthesis, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. P450s are considered as the most versatile biocatalysts in nature because of the vast variety of substrate structures and the types of reactions they catalyze. In particular, P450s can catalyze regio- and stereoselective oxidations of nonactivated C–H bonds in complex organic molecules under mild conditions, making P450s useful biocatalysts in the production of commodity pharmaceuticals, fine or bulk chemicals, bioremediation agents, flavors, and fragrances. Major efforts have been made in engineering improved P450 systems that overcome the inherent limitations of the native enzymes. In this review, we focus on recent progress of different strategies, including protein engineering, redox-partner engineering, substrate engineering, electron source engineering, and P450-mediated metabolic engineering, in efforts to more efficiently produce pharmaceuticals and other chemicals. We also discuss future opportunities for engineering and applications of the P450 systems.

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase–like proteins or MDLs) of the model plant Arabidopsis thaliana. Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS–IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS–IL-1Ra molecules, PAS600–IL-1Ra and PAS800–IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600–IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600–IL-1Ra and PAS800–IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800–IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800–IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600–IL-1Ra, and PAS800-IL-Ra reduced IL-1α–induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans–induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS–IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS–IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

VOLUME 294 (2019) PAGES 3312–3320Due to publisher error, F. Peter Guengerich is listed as the Editor. The correct Editor for this article is Patrick Sung.

VOLUME 294 (2019) PAGES 10708–10719There was an error in the title. The abbreviation “NAPDH” should be “NADPH.”

VOLUME 290 (2015) PAGES 4356–4366The wrong images were used for the long form of Stra6 and RARγ in Fig. 4D. These errors have now been corrected and do not affect the results or conclusions of this work.jbc;295/3/898/F4F1F4Figure 4.

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharyomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1. Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro. Mutations in this region resulted in the temperature and pH-dependent growth phenotype characteristic of rav∆ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE’s role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.

In environments where glucose is limited, some pathogenic bacteria metabolise host derived sialic acid as a nutrient source. N-Acetylmannosamine kinase (NanK) is the second enzyme of the bacterial sialic acid import and degradation pathway and adds phosphate to N-acetylmannosamine using ATP to prime the molecule for future pathway reactions. Sequence alignments reveal that Gram-positive NanK enzymes belong to the Repressor, Open-reading frame, Kinase (ROK) family, but many lack the canonical Zn-binding motif expected for this function, and the sugar-binding ExGH motif is altered to ExGY. As a result, it is unclear how they perform this important reaction. Here, we study the Staphylococcus aureus NanK (SaNanK), which is the first characterisation of a Gram-positive NanK. We report the kinetic activity of SaNanK along with the ligand-free, N-acetylmannosamine-bound and substrate analog N-acetylglucosamine-bound crystal structures (2.33, 2.20 and 2.20 Å resolution, respectively). These demonstrate, in combination with small-angle X-ray scattering, that SaNanK is a dimer that adopts a closed conformation upon substrate binding. Analysis of the ExGY motif reveals that the tyrosine binds to the N-acetyl group to select for the ‘boat’ conformation of N-acetylmannosamine. Moreover, SaNanK has a stacked arginine pair coordinated by negative residues critical for thermal stability and catalysis. These combined elements serve to constrain the active site and orient the substrate in lieu of Zn binding, representing a significant departure from canonical NanK binding. This characterisation provides insight into differences in the ROK family and highlights a novel area for antimicrobial discovery to fight Gram-positive and S. aureus infections.

The calcineurin/nuclear factor of activated T cell (CN/NFAT) signaling pathway plays a critical role in the immune response. Therefore, inhibition of the CN/NFAT pathway is an important target for inflammatory disease. The conserved PxIxIT and LxVP motifs of CN substrates and targeting proteins have been recognized. Based on the affinity ability and inhibitory effect of these docking sequences on CN, we designed a bioactive peptide (named pep3) against the CN/NFAT interaction, which has two binding sites derived from the RCAN1-PxIxIT motif and the NFATc1-LxVP motif. The shortest linker between the two binding sites in pep3 is derived from A238L, a physiological binding partner of CN. Microscale thermophoresis (MST) revealed that pep3 has two docking sites on CN. Pep3 also has the most potent inhibitory effect on CN. It is suggested that pep3 contains an NFATc1-LxVP-substrate recognition motif and RCAN1-PxIxIT-mediated anchoring to CN. Expression of this peptide significantly suppresses CN/NFAT signaling. Cell-permeable 11-arginine-modified pep3 (11R-pep3) blocks the NFAT downstream signaling pathway. Intranasal administration of the 11R-pep3 peptide inhibits airway inflammation in an ovalbumin (OVA)-induced asthma model. Our results suggest that pep3 is promising as an immunosuppressive agent and can be used in topical remedies.

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores toward the end of mitosis. Using cellular, molecular and genetic tools, including fluorescence and electron microscopy, quantitative PCR and RNAi mediated depletion, here; we report that ELYS ortholog (dElys) plays critical roles during Drosophila development. dElys localized to the nuclear rim in interphase cells, but during mitosis, it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid. The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that dElys has conserved structural features, but contains a non-canonical AT-hook like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by dElys during development.

Homeostasis in healthy tissues strongly relies on cell-to-cell adhesion and cell-to-extracellular matrix interactions. For instance, normal epithelial cells maintain tissue structure by adhering to each other and to the extracellular matrix. The proteins that mediate these distinct interactions are collectively called cell adhesion molecules and are divided into four major groups: cadherins, integrins, selectins, and immunoglobulins. They not only physically anchor cells, but also critically integrate signaling between the extracellular microenvironment and cells. These signals include biochemical cues, as adhesion proteins can both act as ligand-activated receptors and activate mechanotransduction triggered by changes in the physical environment. Molecular mechanisms related to cell adhesion signaling have been extensively studied, especially since mutations and changes in expression of these proteins, particularly cadherins and integrins, are frequently associated with diseases ranging from developmental intellectual disability to cancer. In fact, two major hallmarks of cancer, loss of cell-to-cell adhesion and anchorage-independent growth, are both dependent on cell adhesion molecules. Despite many studies elucidating the relationships between malignant transformation and metastasis and cellular adhesion processes, several areas still await exploration. Here, we highlight recently discovered roles of adhesion molecules in collective cancer cell migration and discuss the utility of three-dimensional models in studying cell–cell adhesion. We also describe recent therapeutic approaches targeting adhesion molecules.

Deubiquitinases (DUBs) are vital for the regulation of ubiquitin signals, and both catalytic activity of and target recruitment by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (also known as OTU domain-containing protein 7B (OTUD7B)), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for factor inhibiting HIF1 (FIH1)- and oxygen-dependent asparagine hydroxylation. We found that FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show that UBACez binds Lys11-, Lys48-, Lys63- and Met1-linked ubiquitin chains in vitro, establishing UBACez as a functional ubiquitin-binding domain. Our findings also reveal that the interaction of UBACez with ubiquitin is mediated via a non-canonical surface, and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that Cezanne recruitment to specific target proteins depends on UBACez. Our results indicate that UBACez can indeed fulfil this role as regulatory domain by binding various ubiquitin chain types. They also uncover that this interaction with ubiquitin, and thus with modified substrates, can be modulated by oxygen-dependent asparagine hydroxylation, suggesting that Cezanne is regulated by oxygen levels.

The regulation mechanisms involved in matrix metalloproteinase (MMP) expression and the motility of human endometrial/decidual stromal cells (ESCs/DSCs) during decidualization remain unclear. DSCs show a significant increased cell motility and expression of FOS like 1 (FOSL1) and MMP-1, -2, -9 when compared with ESCs, whereas a lack of decidualization inducers lead to a rapid decrease of FOSL1 and MMP-1, -9 expression in DSCs in vitro. Therefore, we hypothesized that a link exists between decidualization inducers and FOSL1 in the up-regulation of motility during decidualization. Detected based on the response of ESCs/DSCs to different decidualization systems in vitro, we found that progesterone (P4) alone exerted no significant effect and 17β-estradiol (E2) significantly increased cell motility and FOSL1, MMP-1, -9 expression at the mRNA and protein levels, whereas 8-Bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cAMP) significantly decreased cell motility and FOSL1, MMP-9 expression in the presence of P4. In addition, we showed that E2 triggered the phosphorylation of estrogen receptor 1 (ESR1), which could directly bind to the promoter of FOSL1 in ESCs/DSCs. Additionally, we also revealed the silence of ESR1 expression by siRNA abrogated E2-induced FOSL1 expression at the transcript and protein levels. Moreover, the silence of FOSL1 expression by siRNA was able to block E2-induced MMP-1, -9 expression and cell motility in ESCs/DSCs. Taken together, our data suggests that, in addition to its enhancement of the secretory function, the change in MMP expression and cell motility is another component of the decidualization of ESCs/DSCs, including estrogen-dependent MMP-1, -9 expression mediated by E2-ESR1-FOSL1 signaling.

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1–sufficient and –deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK ) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), zeta chain of T-cell receptor–associated protein kinase 70 (ZAP-70), and three MAP kinases (ERK, JNK1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1–induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog–sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1’s inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

Hyaluronan (HA) is one of the most prevalent glycosaminoglycans of the vascular extracellular matrix (ECM). Abnormal HA accumulation within blood vessel walls is associated with tissue inflammation and is prominent in most vascular pathological conditions such as atherosclerosis and restenosis. Hyaluronan synthase 2 (HAS2) is the main HAS enzyme involved in HA synthesis and uses cytosolic UDP-glucuronic acid and UDP-N-acetyl glucosamine as substrates. The synthesis of UDP-glucuronic acid can alter the NAD+:NADH ratio via the enzyme UDP-glucose dehydrogenase, which oxidizes the alcohol group at C6 to the COO− group. Here, we show that HAS2 expression can be modulated by sirtuin 1 (SIRT1), the master metabolic sensor of the cell, belonging to the class of NAD+–dependent deacetylases. Our results revealed that 1) treatments of human aortic smooth muscle cells (AoSMCs) with SIRT1 activators (SRT1720 and resveratrol) inhibit both HAS2 expression and accumulation of pericellular HA coats. 2) Tumor necrosis factor α (TNFα) induced HA-mediated monocyte adhesion and AoSMCs migration, whereas SIRT1 activation prevented immune cell recruitment and cell motility by reducing the expression levels of the receptor for HA-mediated motility, RHAMM, and the HA-binding protein TNF-stimulated gene 6 protein (TSG6). 3) SIRT1 activation prevented nuclear translocation of NF-kB (p65), which, in turn, reduced the levels of HAS2-AS1, a long-non coding RNA that epigenetically controls HAS2 mRNA expression. In conclusion, we demonstrate that both HAS2 expression and HA accumulation by AoSMCs are down-regulated by the metabolic sensor SIRT1.

The homeodomain protein NK2 homeobox 2 (NKX2-2) is a transcription factor that plays a critical role in the control of cell fate specification and differentiation in many tissues. In the developing central nervous system (CNS), this developmentally important transcription factor functions as a transcriptional repressor that governs oligodendrocyte (OL) differentiation and myelin gene expression, but the roles of various NKX2-2 structural domains in this process are unclear. In this study, using in situ hybridization, immunofluorescence, and Co-immunoprecipitation, we determined the structural domains that mediate the repressive functions of murine NKX2-2 and identified the transcriptional co-repressors that interact with it in OL cells. Through in ovo electroporation in embryonic chicken spinal cords, we demonstrate that the N-terminal Tinman (TN) domain and C-terminal (CT) domain synergistically promote OL differentiation by recruiting distinct transcriptional co-repressors, including enhancer of split Groucho 3 (GRG3), histone deacetylase 1 (HDAC1), and DNA methyltransferase 3 α (DNMT3A). We also observed that the NK2-specific (SD) domain suppresses the function of the CT domain in OL differentiation. These findings delineate the distinct NKX2-2 domains and their roles in OL differentiation and suggest that NKX2-2 regulates differentiation by repressing gene expression via multiple co-factors and molecular mechanisms.

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine (PC) by methylating phosphatidylethanolamine using S-adenosylmethionine (SAM) as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase [PEMT], expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method (SCAM). The results of these analyses indicated that Opi3 (as well as PEMT) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases (ICMTs) with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by SCAM, and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

The transferrin receptor (TfR) of the bloodstream form (BSF) of Trypanosoma brucei is a heterodimer comprising glycosylphosphatidylinositol (GPI)-anchored expression site–associated gene 6 (ESAG6 or E6) and soluble ESAG7. Mature E6 has five N-glycans, consisting of three oligomannose and two unprocessed paucimannose structures. Its GPI anchor is modified by addition of 4–6 α-galactose residues. TfR binds tomato lectin (TL), specific for N-acetyllactosamine (LacNAc) repeats, and previous studies have shown increases in transport-dependent E6 size consistent with post-glycan processing in the endoplasmic reticulum. Using pulse-chase radiolabeling, peptide-N-glycosidase F treatment, lectin pulldowns, and exoglycosidase treatment, we have now investigated TfR N-glycan and GPI processing. E6 increased ~5 kDa during maturation, becoming reactive with both TL and Erythrina cristagalli lectin (ECL, terminal LacNAc), indicating synthesis of poly-LacNAc on paucimannose N-glycans. This processing was lost after exoglycosidase treatment and after RNAi-based silencing of TbSTT3A, the oligosaccharyltransferase that transfers paucimannose structures to nascent secretory polypeptides. These results contradict previous structural studies. Minor GPI processing was also observed, consistent with α-galactose addition. However, increasing the spacing between E6 protein and the GPI ω-site (4–7 aa) resulted in extensive post-translational processing of the GPI anchor to a form that was TL/ECL reactive, suggesting addition of LacNAc structures, confirmed by identical assays with BiPNHP, a non-N-glycosylated GPI-anchored reporter. We conclude that BSF trypanosomes can modify GPIs by generating structures reminiscent of those present in insect-stage trypanosomes, and that steric constraints, not stage-specific expression of glycosyltransferases, regulate GPI processing.

Class 3 mutations in B-Raf proto-oncogene, Ser/Thr kinase (BRAF) that result in kinase-impaired or kinase-dead BRAF have the highest mutation frequency in BRAF gene in lung adenocarcinoma. Several studies reported that kinase-dead BRAF variants amplify Mitogen-Activated Protein Kinase (MAPK) signaling by dimerizing with and activating wild-type C-Raf proto-oncogene, Ser/Thr kinase (CRAF). However, the structural and functional principles underlying their activation remain elusive. Herein, using cell biology and various biochemical approaches, we established that variant BRAFD594G, a kinase-dead representative of class 3 mutation-derived BRAF variants, has a higher dimerization potential as compared to wild-type BRAF. Molecular dynamics simulations uncovered that the D594G substitution orients the aC-helix toward the IN position and extends the activation loop within the kinase domain, shifting the equilibrium towards the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. We found that B/CRAF heterodimers are the most thermodynamically stable RAF dimers, suggesting that RAF heterodimers, and not homodimers, are the major player in determining the amplitude of MAPK signaling in cells. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might contribute to longer duration of MAPK pathway signaling in cancer cells. Lastly, we propose that the dimer interface of BRAFD594G:CRAF heterodimer may represent a promising target in the design of novel anticancer therapeutics.

Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by non-bilayer lipids that can influence protein functions by lipid-protein interactions. The non-bilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to developed a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. Is it hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating of the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.

Viruses depend on the host cell translation machinery for their replication and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3) but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Towards this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosomes-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5’ UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral proteins expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels.

PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acyl-phosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty-acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acyl-phosphate synthesis and in the flux through the phosphatidic-acid pathway, we then created mutations and gene fusions that prevent the PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic-acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

The genus Flavivirus in the family Flaviviridae comprises many medically important viruses, such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV). The quest for therapeutic targets to combat flavivirus infections requires a better understanding of the kinetics of virus–host interactions during infections with native viral strains. However, this is precluded by limitations of current cell-based systems for monitoring flavivirus infection in living cells. In the present study, we report the construction of fluorescence activatable sensors to detect the activities of flavivirus NS2B-NS3 serine proteases in living cells. The system consists of GFP-based reporters that become fluorescent upon cleavage by recombinant DENV-2/ZIKV proteases in vitro. A version of this sensor containing the flavivirus internal NS3 cleavage site linker reported the highest fluorescence activation in stably transduced mammalian cells upon DENV-2/ZIKV infection. Moreover, the onset of fluorescence correlated with viral protease activity. A far-red version of this flavivirus sensor had the best signal-to-noise ratio in a fluorescent Dulbecco’s plaque assay, leading to the construction of a multi-reporter platform combining the flavivirus sensor with reporter dyes for detection of chromatin condensation and cell death, enabling studies of viral plaque formation with single-cell resolution. Finally, the application of this platform enabled the study of cell-population kinetics of infection and cell death by DENV-2, ZIKV, and YFV. We anticipate that future studies of viral infection kinetics with this reporter system will enable basic investigations of virus–host interactions and facilitate future applications in antiviral drug research to manage flavivirus infections.

The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N-terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes. To validate our results, we determined the structures of select proteins in the N-degron pathway and compared them to the PDB-deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.

Dental enamel comprises interwoven arrays of extremely long and narrow crystals of carbonated hydroxyapatite called enamel rods. Amelogenin (AMELX) is the predominant extracellular enamel matrix protein and plays an essential role in enamel formation (amelogenesis). Previously, we have demonstrated that full-length AMELX forms higher-order supramolecular assemblies that regulate ordered mineralization in vitro, as observed in enamel rods. Phosphorylation of the sole AMELX phosphorylation site (Ser-16) in vitro greatly enhances its capacity to stabilize amorphous calcium phosphate (ACP), the first mineral phase formed in developing enamel, and prevents apatitic crystal formation. To test our hypothesis that AMELX phosphorylation is critical for amelogenesis, we generated and characterized a hemizygous knock-in (KI) mouse model with a phosphorylation-defective Ser-16 to Ala-16 substitution in AMELX. Using EM analysis, we demonstrate that in the absence of phosphorylated AMELX, KI enamel lacks enamel rods, the hallmark component of mammalian enamel, and, unlike WT enamel, appears to be composed of less organized arrays of shorter crystals oriented normal to the dentinoenamel junction. KI enamel also exhibited hypoplasia and numerous surface defects, whereas heterozygous enamel displayed highly variable mosaic structures with both KI and WT features. Importantly, ACP-to-apatitic crystal transformation occurred significantly faster in KI enamel. Secretory KI ameloblasts also lacked Tomes’ processes, consistent with the absence of enamel rods, and underwent progressive cell pathology throughout enamel development. In conclusion, AMELX phosphorylation plays critical mechanistic roles in regulating ACP phase transformation and enamel crystal growth, and in maintaining ameloblast integrity and function during amelogenesis.

MAX network transcriptional repressor (MNT) is an MXD family transcription factor of the bHLH family. MNT dimerizes with another transcriptional regulator, MYC-associated factor X (MAX), and down-regulates genes by binding to E boxes. MAX also dimerizes with MYC, an oncogenic bHLH transcription factor. Upon E-box binding, the MYC–MAX dimer activates gene expression. MNT also binds to MAX dimerization protein MLX (MLX), and MNT–MLX and MNT-MAX dimers coexist. However, all MNT functions have been attributed to MNT–MAX dimers, and no functions of the MNT–MLX dimer have been described. MNT’s biological role has been linked to its function as a MYC oncogene modulator, but little is known about its regulation. We show here that MNT localizes to the nucleus of MAX-expressing cells and that MNT–MAX dimers bind and repress the MNT promoter, an effect that depends on one of the two E boxes on this promoter. In MAX-deficient cells, MNT was overexpressed and redistributed to the cytoplasm. Interestingly, MNT was required for cell proliferation even in the absence of MAX. We show that in MAX-deficient cells, MNT binds to MLX, but also forms homodimers. RNA-Seq experiments revealed that MNT regulates the expression of several genes even in the absence of MAX, with many of these genes being involved in cell cycle regulation and DNA repair. Of note, MNT–MNT homodimers regulated the transcription of some genes involved in cell proliferation. The tight regulation of MNT and its functionality even without MAX suggest a major role for MNT in cell proliferation.

Lyme disease, also known as Lyme borreliosis, is the most common tick-transmitted disease in the Northern Hemisphere. The disease is caused by the bacterial spirochete Borrelia burgdorferi and other related Borrelia species. One of the many fascinating features of this unique pathogen is an elaborate system for antigenic variation, whereby the sequence of the surface-bound lipoprotein VlsE is continually modified through segmental gene conversion events. This perpetual changing of the guard allows the pathogen to remain one step ahead of the acquired immune response, enabling persistent infection. Accordingly, the vls locus is the most evolutionarily diverse genetic element in Lyme disease–causing borreliae. Small stretches of information are transferred from a series of silent cassettes in the vls locus to generate an expressed mosaic vlsE gene version that contains genetic information from several different silent cassettes, resulting in ∼1040 possible vlsE sequences. Yet, despite its extreme evolutionary flexibility, the locus has rigidly conserved structural features. These include a telomeric location of the vlsE gene, an inverse orientation of vlsE and the silent cassettes, the presence of nearly perfect inverted repeats of ∼100 bp near the 5′ end of vlsE, and an exceedingly high concentration of G runs in vlsE and the silent cassettes. We discuss the possible roles of these evolutionarily conserved features, highlight recent findings from several studies that have used next-generation DNA sequencing to unravel the switching process, and review advances in the development of a mini-vls system for genetic manipulation of the locus.

The catalytic residues of an enzyme comprise the amino acids located in the active center responsible for accelerating the enzyme-catalyzed reaction. These residues lower the activation energy of reactions by performing several catalytic functions. Decades of enzymology research has established general themes regarding the roles of specific residues in these catalytic reactions, but it has been more difficult to explore these roles in a more systematic way. Here, we review the data on the catalytic residues of 648 enzymes, as annotated in the Mechanism and Catalytic Site Atlas (M-CSA), and compare our results with those in previous studies. We structured this analysis around three key properties of the catalytic residues: amino acid type, catalytic function, and sequence conservation in homologous proteins. As expected, we observed that catalysis is mostly accomplished by a small set of residues performing a limited number of catalytic functions. Catalytic residues are typically highly conserved, but to a smaller degree in homologues that perform different reactions or are nonenzymes (pseudoenzymes). Cross-analysis yielded further insights revealing which residues perform particular functions and how often. We obtained more detailed specificity rules for certain functions by identifying the chemical group upon which the residue acts. Finally, we show the mutation tolerance of the catalytic residues based on their roles. The characterization of the catalytic residues, their functions, and conservation, as presented here, is key to understanding the impact of mutations in evolution, disease, and enzyme design. The tools developed for this analysis are available at the M-CSA website and allow for user specific analysis of the same data.

Transfusion of blood, or more commonly red blood cells (RBCs), is integral to health care systems worldwide but requires careful matching of blood types to avoid serious adverse consequences. Of the four main blood types, A, B, AB, and O, only O can be given to any patient. This universal donor O-type blood is crucial for emergency situations where time or resources for typing are limited, so it is often in short supply. A and B blood differ from the O type in the presence of an additional sugar antigen (GalNAc and Gal, respectively) on the core H-antigen found on O-type RBCs. Thus, conversion of A, B, and AB RBCs to O-type RBCs should be achievable by removal of that sugar with an appropriate glycosidase. The first demonstration of a B-to-O conversion by Goldstein in 1982 required massive amounts of enzyme but enabled proof-of-principle transfusions without adverse effects in humans. New α-galactosidases and α-N-acetylgalactosaminidases were identified by screening bacterial libraries in 2007, allowing improved conversion of B and the first useful conversions of A-type RBCs, although under constrained conditions. In 2019, screening of a metagenomic library derived from the feces of an AB donor enabled discovery of a significantly more efficient two-enzyme system, involving a GalNAc deacetylase and a galactosaminidase, for A conversion. This promising system works well both in standard conditions and in whole blood. We discuss remaining challenges and opportunities for the use of such enzymes in blood conversion and organ transplantation.

The diversity of natural products not only fascinates us intellectually, but also provides an armamentarium against the microbes that threaten our health. The increased prevalence of pathogens that are resistant to one or more classes of available medicines continues to be a growing global threat. As drug-resistant pathogens erode the effectiveness of the current reserve of antibiotics and antifungals, methodological advances open additional avenues for discovery of new classes of drugs, as well as novel derivatives of existing (and proven) classes of compounds. In this Thematic Review Series, we aim to provide a snapshot of the current state of the art in natural product discovery. The reviews in this series encapsulate convergent approaches toward the identification of different classes of primary and specialized metabolites, including nonribosomal peptides, polyketides, and ribosomally synthesized and post-translationally modified peptides, from all kingdoms of life. Traction in unraveling new and diverse classes of molecules has come largely from the academic sector, which ensures availability of methods and data sets. Such knowledge is needed to thwart serious threats to human health and calls to mind the proverb praemonitus praemunitus (forewarned is forearmed).

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro. We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI–mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

The envelope glycoproteins (Envs) of HIV-1 are embedded in the cholesterol-rich lipid membrane of the virus. Chemical depletion of cholesterol from HIV-1 particles inactivates their infectivity. We observed that diverse HIV-1 strains exhibit a range of sensitivities to such treatment. Differences in sensitivity to cholesterol depletion could not be explained by variation in Env components known to interact with cholesterol, including the cholesterol-recognition motif and cytoplasmic tail of gp41. Using antibody-binding assays, measurements of virus infectivity, and analyses of lipid membrane order, we found that depletion of cholesterol from HIV-1 particles decreases the conformational stability of Env. It enhances exposure of partially cryptic epitopes on the trimer and increases sensitivity to structure-perturbing treatments such as antibodies and cold denaturation. Substitutions in the cholesterol-interacting motif of gp41 induced similar effects as depletion of cholesterol. Surface-acting agents, which are incorporated into the virus lipid membrane, caused similar effects as disruption of the Env-cholesterol interaction. Furthermore, substitutions in gp120 that increased structural stability of Env (i.e. induced a “closed” conformation of the trimer) increased virus resistance to cholesterol depletion and to the surface-acting agents. Collectively, these results indicate a critical contribution of the viral membrane to the stability of the Env trimer and to neutralization resistance against antibodies. Our findings suggest that the potency of poorly neutralizing antibodies, which are commonly elicited in vaccinated individuals, may be markedly enhanced by altering the lipid composition of the viral membrane.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site–incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

The SIN3 histone-modifying complex regulates the expression of multiple methionine catabolic genes, including SAM synthetase (Sam-S), as well as SAM levels. To further dissect the relationship between methionine catabolism and epigenetic regulation by SIN3, we sought to identify genes and metabolic pathways controlled by SIN3 and SAM synthetase (SAM-S) in Drosophila melanogaster. Using several approaches, including RNAi-mediated gene silencing, RNA-Seq– and quantitative RT-PCR–based transcriptomics, and ultra-high-performance LC-MS/MS– and GC/MS–based metabolomics, we found that, as a global transcriptional regulator, SIN3 impacted a wide range of genes and pathways. In contrast, SAM-S affected only a narrow range of genes and pathways. The expression and levels of additional genes and metabolites, however, were altered in Sin3A+Sam-S dual knockdown cells. This analysis revealed that SIN3 and SAM-S regulate overlapping pathways, many of which involve one-carbon and central carbon metabolisms. In some cases, the factors acted independently; in some others, redundantly; and for a third set, in opposition. Together, these results, obtained from experiments with the chromatin regulator SIN3 and the metabolic enzyme SAM-S, uncover a complex relationship between metabolism and epigenetic regulation.

MicroRNA-122 (miR-122) is highly expressed in hepatocytes, where it plays an important role in regulating cholesterol and fatty acid metabolism, and it is also a host factor required for hepatitis C virus replication. miR-122 is selectively stabilized by 3′ adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2 (also known as PAPD4 or TENT2). However, it is unclear how GLD-2 specifically stabilizes miR-122. Here, we show that QKI7 KH domain-containing RNA binding (QKI-7), one of three isoforms of the QKI proteins, which are members of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, is involved in miR-122 stabilization. QKI down-regulation specifically decreased the steady-state level of mature miR-122, but did not affect the pre-miR-122 level. We also found that QKI-7 uses its C-terminal region to interact with GLD-2 and its QUA2 domain to associate with the RNA-induced silencing complex protein Argonaute 2 (Ago2), indicating that the GLD-2–QKI-7 interaction recruits GLD-2 to Ago2. QKI-7 exhibited specific affinity to miR-122 and significantly promoted GLD-2–mediated 3′ adenylation of miR-122 in vitro. Taken together, our findings indicate that miR-122 binds Ago2–interacting QKI-7, which recruits GLD-2 for 3′ adenylation and stabilization of miR-122.

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

Caloric restriction has been associated with increased life span and reduced aging-related disorders and reduces fibrosis in several diseases. Fibrosis is characterized by deposition of excess fibrous material in tissues and organs and is caused by aging, chronic stress, injury, or disease. Myofibroblasts are fibroblast-like cells that secrete high levels of extracellular matrix proteins, resulting in fibrosis. Histological studies have identified many-fold increases of myofibroblasts in aged organs where myofibroblasts are constantly generated from resident tissue fibroblasts and other cell types. However, it remains unclear how aging increases the generation of myofibroblasts. Here, using mouse models and biochemical assays, we show that sirtuin 6 (SIRT6) deficiency plays a major role in aging-associated transformation of fibroblasts to myofibroblasts, resulting in tissue fibrosis. Our findings suggest that SIRT6-deficient fibroblasts transform spontaneously to myofibroblasts through hyperactivation of transforming growth factor β (TGF-β) signaling in a cell-autonomous manner. Importantly, we noted that SIRT6 haploinsufficiency is sufficient for enhancing myofibroblast generation, leading to multiorgan fibrosis and cardiac dysfunction in mice during aging. Mechanistically, SIRT6 bound to and repressed the expression of key TGF-β signaling genes by deacetylating SMAD family member 3 (SMAD3) and Lys-9 and Lys-56 in histone 3. SIRT6 binding to the promoters of genes in the TGF-β signaling pathway decreased significantly with age and was accompanied by increased binding of SMAD3 to these promoters. Our findings reveal that SIRT6 may be a potential candidate for modulating TGF-β signaling to reduce multiorgan fibrosis during aging and fibrosis-associated diseases.