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  • PEGylation-based strategy to identify pathways involved in the activation of apoptotic BAX protein
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-24
    Yu-Jing Lan; Yu-Ting Wang; Chien-Lun Hung; Yun-Wei Chiang
    更新日期:2020-01-24
  • Interactions between PAMAM dendrimers and DOPC lipid multilayers: Membrane thinning and structural disorder
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-24
    Laura J. Fox; Anna Slastanova; Nicolas Taylor; Magdalena Wlodek; Oier Bikondoa; Robert M. Richardson; Wuge H. Briscoe
    更新日期:2020-01-24
  • A novel neuronal organoid model mimicking glioblastoma (GBM) features from induced pluripotent stem cells (iPSC)
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-21
    Jin Wook Hwang; Julien Loisel-Duwattez; Christophe Desterke; Theodoros Latsis; Sarah Pagliaro; Frank Griscelli; Annelise Bennaceur-Griscelli; Ali G. Turhan

    Background Current experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. Methods We have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). Results Herein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. General significance This study shows the feasibility of generating “off-the shelf” neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.

    更新日期:2020-01-22
  • Why do water molecules around small hydrophobic solutes form stronger hydrogen bonds than in the bulk?
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-21
    Franci Merzel; Franc Avbelj

    Molecular solutes are known to have a strong effect on the structural and dynamical properties of the surrounding water. In our recent study (PNAS, 114, 322 (2017)) we have identified the presence of strengthened water hydrogen bonds near hydrophobic solutes by using both IR spectroscopy and ab-initio molecular dynamics simulations. The water molecules involved in the enhanced hydrogen bonding have been shown to display extensive structural ordering and restricted mobility. We observed that an individual pair of water molecules can make stronger hydrogen bond to each other if it is not surrounded by intercalating water molecules. Here we present compelling simulation results which unravel a simple mechanistic picture of the emergence of the hydrogen bond (HB) strengthening around solvated methane. We show explicitly that actual absence of water molecules within the excluded volume due to the hydrophobic molecule assures smaller residual torque on neighboring water molecules enabling the formation of stronger HBs between them.

    更新日期:2020-01-21
  • Directly targeting glutathione peroxidase 4 may be more effective than disrupting glutathione on ferroptosis-based cancer therapy
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-18
    Yunpeng Wei; Huanhuan Lv; Atik Badshah Shaikh; Wei Han; Hongjie Hou; Zhihao Zhang; Shenghang Wang; Peng Shang

    Background Cancer is one of the major threats to human health and current cancer therapies have been unsuccessful in eradicating it. Ferroptosis is characterized by iron-dependence and lipid hydroperoxides accumulation, and its primary mechanism involves the suppression of system Xc−-GSH (glutathione)-GPX4 (glutathione peroxidase 4) axis. Co-incidentally, cancer cells are also metabolically characterized by iron addiction and ROS tolerance, which makes them vulnerable to ferroptosis. This may provide a new tactic for cancer therapy. Scope of review The general features and mechanisms of ferroptosis, and the basis that makes cancer cells vulnerable to ferroptosis are described. Further, we emphatically discussed that disrupting GSH may not be ideal for triggering ferroptosis of cancer cells in vivo, but directly inhibiting GPX4 and its compensatory members could be more effective. Finally, the various approaches to directly inhibit GPX4 without disturbing GSH were described. Major conclusions Targeting system Xc− or GSH may not effectively trigger cancer cells' ferroptosis in vivo the existence of other compensatory pathways. However, directly targeting GPX4 and its compensatory members without disrupting GSH may be more effective to induce ferroptosis in cancer cells in vivo, as GPX4 is essential in preventing ferroptosis. General significance Cancer is a severe threat to human health. Ferroptosis-based cancer therapy strategies are promising, but how to effectively induce ferroptosis in cancer cells in vivo is still a question without clear answers. Thus, the viewpoints raised in this review may provide some references and different perspectives for researchers working on ferroptosis-based cancer therapy.

    更新日期:2020-01-21
  • The lac repressor hinge helix in context: The effect of the DNA binding domain and symmetry
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-17
    Danielle Seckfort; Gillian C. Lynch; B. Montgomery Pettitt
    更新日期:2020-01-17
  • Rigidity of protein structure revealed by incoherent neutron scattering
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-17
    Hiroshi Nakagawa; Mikio Kataoka

    The rigidity and flexibility of a protein is reflected in its structural dynamics. Studies on protein dynamics often focus on flexibility and softness; this review focuses on protein structural rigidity. The extent of rigidity can be assessed experimentally with incoherent neutron scattering; a method that is complementary to molecular dynamics simulation. This experimental technique can provide information about protein dynamics in timescales of pico- to nanoseconds and at spatial scales of nanometers; these dynamics can help quantify the rigidity of a protein by indices such as force constant, Boson peak, dynamical transition, and dynamical heterogeneity. These indicators also reflect the rigidity of a protein's secondary and tertiary structures. In addition, the indices reveal how rigidity is influenced by different environmental parameters, such as hydration, temperature, pressure, and protein-protein interactions. Hydration affects both rigidity and softness more than other environmental factors. Interestingly, hydration affects harmonic and anharmonic motions in opposite ways. This difference is probably due to the protein's dynamic coupling with water molecules via hydrogen bonding.

    更新日期:2020-01-17
  • Prediction of peptide binding to MHC using machine learning with sequence and structure-based feature sets
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-16
    Michelle P. Aranha; Catherine Spooner; Omar Demerdash; Bogdan Czejdo; Jeremy C. Smith; Julie C. Mitchell

    Selecting peptides that bind strongly to the major histocompatibility complex (MHC) for inclusion in a vaccine has therapeutic potential for infections and tumors. Machine learning models trained on sequence data exist for peptide:MHC (p:MHC) binding predictions. Here, we train support vector machine classifier (SVMC) models on physicochemical sequence-based and structure-based descriptor sets to predict peptide binding to a well-studied model mouse MHC I allele, H-2Db. Recursive feature elimination and two-way forward feature selection were also performed. Although low on sensitivity compared to the current state-of-the-art algorithms, models based on physicochemical descriptor sets achieve specificity and precision comparable to the most popular sequence-based algorithms. The best-performing model is a hybrid descriptor set containing both sequence-based and structure-based descriptors. Interestingly, close to half of the physicochemical sequence-based descriptors remaining in the hybrid model were properties of the anchor positions, residues 5 and 9 in the peptide sequence. In contrast, residues flanking position 5 make little to no residue-specific contribution to the binding affinity prediction. The results suggest that machine-learned models incorporating both sequence-based descriptors and structural data may provide information on specific physicochemical properties determining binding affinities.

    更新日期:2020-01-17
  • Identification of novel RNA design candidates by clustering the extended RNA-as-graphs library
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-16
    Swati Jain; Qiyao Zhu; Amiel S.P. Paz; Tamar Schlick

    Background: We re-evaluate our RNA-As-Graphs clustering approach, using our expanded graph library and new RNA structures, to identify potential RNA-like topologies for design. Our coarse-grained approach represents RNA secondary structures as tree and dual graphs, with vertices and edges corresponding to RNA helices and loops. The graph theoretical framework facilitates graph enumeration, partitioning, and clustering approaches to study RNA structure and its applications. Methods: Clustering graph topologies based on features derived from graph Laplacian matrices and known RNA structures allows us to classify topologies into ‘existing’ or hypothetical, and the latter into, ‘RNA-like’ or ‘non RNA-like’ topologies. Here we update our list of existing tree graph topologies and RAG-3D database of atomic fragments to include newly determined RNA structures. We then use linear and quadratic regression, optionally with dimensionality reduction, to derive graph features and apply several clustering algorithms on our tree-graph library and recently expanded dual-graph library to classify them into the three groups. Results: The unsupervised PAM and K-means clustering approaches correctly classify 72–77% of all existing graph topologies and 75–82% of newly added ones as RNA-like. For supervised k-NN clustering, the cross-validation accuracy ranges from 57 to 81%. Conclusions: Using linear regression with unsupervised clustering, or quadratic regression with supervised clustering, provides better accuracies than supervised/linear clustering. All accuracies are better than random, especially for newly added existing topologies, thus lending credibility to our approach. General Significance: Our updated RAG-3D database and motif classification by clustering present new RNA substructures and RNA-like motifs as novel design candidates.

    更新日期:2020-01-17
  • Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-15
    Albert Liclican; Loredana Serafini; Weimei Xing; Gregg Czerwieniec; Bart Steiner; Ting Wang; Katherine M. Brendza; Justin D. Lutz; Kathleen S. Keegan; Adrian S. Ray; Brian E. Schultz; Roman Sakowicz; Joy Y. Feng

    Background Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. Methods Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. Results Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M−1 s−1 for BTK with selectivity against important off-targets. Conclusions For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. General significance This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

    更新日期:2020-01-15
  • Enhanced cell selectivity of hybrid peptides with potential antimicrobial activity and immunomodulatory effect
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-15
    Xiaokang Miao; Tianxiong Zhou; Jingying Zhang; Jingjie Xu; Xiaomin Guo; Hui Hu; Xiaowei Zhang; Mingning Hu; Jingyi Li; Wenle Yang; Junqiu Xie; Zhaoqing Xu; Lingyun Mou

    Background Hybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP. Methods Two repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1–8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated. Results Hybrid peptides KFSP1–4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression. Conclusion Hybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.

    更新日期:2020-01-15
  • Metformin attenuates adhesion between cancer and endothelial cells in chronic hyperglycemia by recovery of the endothelial glycocalyx barrier
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-15
    Marta Targosz-Korecka; Katarzyna Ewa Malek-Zietek; Damian Kloska; Zenon Rajfur; Ewa Łucja Stepien; Anna Grochot-Przeczek; Marek Szymonski

    Background Epidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized. Methods Methods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression. Results SCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1. Conclusion Our results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells. General significance Metformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.

    更新日期:2020-01-15
  • Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-13
    Matías Valenzuela Álvarez; Luciana Gutiérrez; Jerónimo Auzmendi; Alejandro Correa; Alberto Lazarowski; Marcela F. Bolontrade

    Background Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastasis at the moment of diagnosis. Methods Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. Results Our results suggest that metastatic OS cells posses a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. Conclusions All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. General significance Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

    更新日期:2020-01-13
  • Adaptation of striated muscles to Wolframin deficiency in mice: Alterations in cellular bioenergetics
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-11
    Kersti Tepp; Marju Puurand; Natalja Timohhina; Jekaterina Aid-Vanakova; Indrek Reile; Igor Shevchuk; Vladimir Chekulayev; Margus Eimre; Nadežda Peet; Lumme Kadaja; Kalju Paju; Tuuli Käämbre

    Background Wolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. Methods Alterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. Results Oxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. Conclusions Wfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. General significance This work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance.

    更新日期:2020-01-13
  • Ezrin interacts with L-periaxin by the “head to head and tail to tail” mode and influences the location of L-periaxin in Schwann cell RSC96
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-10
    Tao Guo; Lei Zhang; Hong Xiao; Yan Yang; Yawei Shi

    In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.

    更新日期:2020-01-10
  • Structural characterization and polymorphism analysis of the NS2B-NS3 protease from the 2017 Brazilian circulating strain of Yellow Fever virus
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-10
    Gabriela Dias Noske; Victor Oliveira Gawriljuk; Rafaela Sachetto Fernandes; Nathalia Dias Furtado; Myrna Cristina Bonaldo; Glaucius Oliva; Andre Schutzer Godoy
    更新日期:2020-01-10
  • 更新日期:2020-01-04
  • Characterization of Macrophage Galactose-type Lectin (MGL) ligands in colorectal cancer cell lines
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    Martina Pirro; Yoann Rombouts; Alexandre Stella; Olivier Neyrolles; Odile Burlet-Schiltz; Sandra J. van Vliet; Arnoud H. de Ru; Yassene Mohammed; Manfred Wuhrer; Peter A. van Veelen; Paul J. Hensbergen

    Background The Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GalNAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor disease-free survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive. Methods Using a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics. Results HCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope. Conclusions We have identified cell surface MGL-ligands on CRC cell lines. General significance Advances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC.

    更新日期:2020-01-04
  • Kinetic and structural analysis of Escherichia coli phosphoenolpyruvate carboxykinase mutants
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    AkosiereremS. Sokaribo; Brian A.A. Novakovski; Julien Cotelesage; Aaron P. White; David Sanders; Hughes Goldie

    Background Phosphoenolpyruvate carboxykinase (PEPCK) is a metabolic enzyme in the gluconeogenesis pathway, where it catalyzes the reversible conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) and CO2. The substrates for Escherichia coli PEPCK are OAA and MgATP, with Mn2+ acting as a cofactor. Analysis of PEPCK structures have revealed amino acid residues involved in substrate/cofactor coordination during catalysis. Methods Key residues involved in coordinating the different substrates and cofactor bound to E. coli PEPCK were mutated. Purified mutant enzymes were used for kinetic assays. The structure of some mutant enzymes were determined using X-ray crystallography. Results Mutation of residues D269 and H232, which comprise part of the coordination sphere of Mn2+, reduced kcat by 14-fold, and significantly increased the Km values for Mn2+ and OAA. Mutation of K254 a key residue in the P-loop motif that interacts with MgATP, significantly elevated the Km value for MgATP and reduced kcat. R65 and R333 are key residues that interacts with OAA. The R65Q and R333Q mutations significantly increased the Km value for OAA and reduced kcat respectively. Conclusions Our results show that mutation of residues involved in coordinating OAA, MgATP and Mn2+ significantly reduce PEPCK activity. K254 plays an important role in phosphoryl transfer, while R333 is involved in both OAA decarboxylation and phosphoryl transfer by E. coli PEPCK. General significance In higher organisms including humans, PEPCK helps to regulate blood glucose levels, hence PEPCK is a potential drug target for patients with non-insulin dependent diabetes mellitus.

    更新日期:2020-01-04
  • Fibrillar conformation of an apolipoprotein A-I variant involved in amyloidosis and atherosclerosis
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    Romina A. Gisonno; Eduardo D. Prieto; Juan P. Gorgojo; Lucrecia M. Curto; M. Eugenia Rodriguez; Silvana A. Rosú; Gisela M. Gaddi; Gabriela S. Finarelli; M. Fernanda Cortez; Guillermo R. Schinella; M. Alejandra Tricerri; Nahuel A. Ramella

    Background Different protein conformations may be involved in the development of clinical manifestations associated with human amyloidosis. Although a fibrillar conformation is usually the signature of damage in the tissues of patients, it is not clear whether this species is per se the cause or the consequence of the disease. Hereditary amyloidosis due to variants of apolipoprotein A-I (apoA-I) with a substitution of a single amino acid is characterized by the presence of fibrillar protein within the lesions. Thus mutations result in increased protein aggregation. Here we set up to characterize the folding of a natural variant with a mutation leading to a deletion at position107 (apoA-I Lys107–0). Patients carrying this variant show amyloidosis and severe atherosclerosis. Methods We oxidized this variant under controlled concentrations of hydrogen peroxide and analyzed the structure obtained after 30-day incubation by fluorescence, circular dichroism and microscopy approaches. Neutrophils activation was characterized by confocal microscopy. Results We obtained a high yield of well-defined stable fibrillar structures of apoA-I Lys107–0. In an in vitro neutrophils system, we were able to detect the induction of Neutrophils Extracellular Traps when we incubated with oxidized apoA-I variants. This effect was exacerbated by the fibrillar structure of oxidized Lys 107–0. Conclusions We conclude that a pro-inflammatory microenvironment could result in the formation of aggregation-prone species, which, in addition may induce a positive feed-back in the activation of an inflammatory response. General significance These events may explain a close association between amyloidosis due to apoA-I Lys107–0 and atherosclerosis.

    更新日期:2020-01-04
  • Genetic analysis of oxidative and endoplasmic reticulum stress responses induced by cobalt toxicity in budding yeast
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    Yun-ying Zhao; Chun-lei Cao; Ying-li Liu; Jing Wang; Shi-yun Li; Jie Li; Yu Deng
    更新日期:2020-01-04
  • Four countries for science
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    Jeremy C. Smith

    A personal retrospect is given on four decades in science in four countries - the UK, France, Germany and the USA. Some historical observations are made together with a time transect through scientific systems and institutions in the four countries.

    更新日期:2020-01-04
  • A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence
    BBA Gen. Subj. (IF 3.681) Pub Date : 2020-01-03
    Maria Victoria Barcarolo; Betiana S. Garavaglia; Natalia Gottig; Eduardo A. Ceccarelli; Daniela L. Catalano-Dupuy; Jorgelina Ottado
    更新日期:2020-01-04
  • Profiling of isomer-specific IgG N-glycosylation in cohort of Chinese colorectal cancer patients
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-27
    Si Liu; Zhiwen Huang; Qiwei Zhang; Yang Fu; Liming Cheng; Bi-Feng Liu; Xin Liu
    更新日期:2019-12-27
  • The transition between active and inactive conformations of Abl kinase studied by rock climbing and Milestoning
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-27
    Brajesh Narayan; Arman Fathizadeh; Clark Templeton; Peng He; Shima Arasteh; Ron Elber; Nicolae-Viorel Buchete; Ron M. Levy
    更新日期:2019-12-27
  • Region-specific upregulation of HNK-1 glycan in the PRMT1-deficient brain
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-27
    Misuzu Hashimoto; Tetsuya Hirata; Chizuko Yonekawa; Kaho Takeichi; Akiyoshi Fukamizu; Tsutomu Nakagawa; Yasuhiko Kizuka

    Background Brains express structurally unique glycans, including human natural killer-1 (HNK-1), which participate in development and high-order functions. However, the regulatory mechanisms of expression of these brain-specific glycans are largely unknown. We examined whether arginine methylation, another type of protein modification essential for neural development, impacts the expression of various glycans in the developing brain. Methods We analyzed several types of glycans, including the HNK-1 epitope, in the cerebellum and cerebral cortex from mice with nervous system-specific knockout of protein arginine methyltransferase 1 (PRMT1). We also analyzed the expression levels of glycosyltransferases responsible for HNK-1 and of HNK-1 carrier glycoproteins by quantitative RT-PCR and western blotting. Results Among several glycans, expression of HNK-1 glycan was strikingly upregulated in the PRMT1-deficient cerebellum. Furthermore, such upregulation was found in the cerebellum but not in the cerebral cortex. Regarding the mechanisms, we demonstrated that the mRNA level and activity of the responsible glycosyltransferase (B3gat1) were elevated in the knockout cerebellum. We also showed that the expression of HNK-1 carrier glycoproteins such as neural cell adhesion molecule (NCAM), L1 and AMPA receptor subunit GluA2 were also increased in the PRMT1-deficient cerebellum. Conclusions Loss of arginine methylation leads to an increase in HNK-1 glycan in the developing cerebellum but not in the cerebral cortex via upregulation of the biosynthetic enzyme and carrier glycoproteins. General significance PRMT1 is a novel regulator of HNK-1 glycan production in the cerebellum. Mechanisms involving crosstalk between glycosylation and arginine methylation are suggested to occur.

    更新日期:2019-12-27
  • Lyn regulates creatine uptake in an imatinib-resistant CML cell line
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-24
    Denis O. Okumu; Lucas J. Aponte-Collazo; Brian J. Dewar; Nathan J. Cox; Michael P. East; Katherine Tech; Ian M. McDonald; Andrey P. Tikunov; Ekhson Holmuhamedov; Jeffrey M. Macdonald; Lee M. Graves

    Background Imatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. Methods Stable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. Results Inhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. Conclusions These data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. General significance These studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

    更新日期:2019-12-25
  • Dehydrin ERD14 activates glutathione transferase Phi9 in Arabidopsis thaliana under osmotic stress
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-20
    Phuong N. Nguyen; Maria-Armineh Tossounian; Denes S. Kovacs; Tran T. Thu; Benoit Stijlemans; Didier Vertommen; Jarne Pauwels; Kris Gevaert; Geert Angenon; Joris Messens; Peter Tompa

    Background Fully intrinsically disordered plant dehydrin ERD14 can protect enzymes via its chaperone-like activity, but it was not formally linked with enzymes of the plant redox system yet. This is of particular interest, as the level of H2O2 in Arabidopsis plants increases during osmotic stress, which can be counteracted by overexpression of ERD14. Methods The proteomic mass-spectrometry analysis of stressed plants was performed to find the candidates affected by ERD14. With cross-linking, microscale thermophoresis, and active-site titration kinetics, the interaction and influence of ERD14 on the function of two target proteins: glutathione transferase Phi9 and catalase was examined. Results Under osmotic stress, redox enzymes, specifically the glutathione transferase Phi enzymes, are upregulated. Using microscale thermophoresis, we showed that ERD14 directly interacts with GSTF9 with a KD of ~25 μM. ERD14 activates the inactive GSTF9 molecules, protects GSTF9 from oxidation, and can also increases the activity of the enzyme. Aside from GSTF9, we found that ERD14 can also interact with catalase, an important cellular H2O2 scavenging enzyme, with a KD of ~0.13 μM, and protects it from dehydration-induced loss of activity. Conclusions We propose that fully intrinsically disordered dehydrin ERD14 might protect and even activate redox enzymes, helping plants to survive oxidative stress under dehydration conditions. General significance ERD14 has a direct effect on the activity of redox enzymes.

    更新日期:2019-12-21
  • Mitochondrial glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase abrogate p53 induced apoptosis in a yeast model: Possible implications for apoptosis resistance in cancer cells
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-17
    Archana Kumari Redhu; Jayadeva Paike Bhat

    Background Escape from apoptosis is an important hallmark of tumor progression and drug resistance in cancer cells. It is well demonstrated that over-expression of human wtp53 in Saccharomyces cerevisiae induces apoptosis by directly targeting the mitochondria. In this study, we showed that how S.cerevisiae escaped from p53 induced apoptosis in the presence of a fermentable carbon source (sucrose), but not on non-fermentable carbon source (glycerol). Methods Mitochondrial fractions from yeast cultures grown in the presence of sucrose or glycerol with and without p53 expression were fractionated and analyzed by LC-MS/MS. Differentially expressed proteins were studied and detailed biochemical analysis for selected proteins was performed.The effect of mitochondrial HXK-2 over-expression induced by p53 in sucrose grown cells on cell survival was evaluated using gene deletion/tagging, co-localisation and mitochondrial ROS detection. Results We observe that mitochondria isolated from p53 over-expressing cells accumulate Pentose phosphate Pathway (PPP) enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) which led to enhanced mitochondrial NADPH production only when cells are cultured in sucrose but not glycerol. In contrast, mitochondria isolated from Δhxk2 p53 over-expressing cells grown in sucrose did not accumulate G6PDH and 6PGDH and resulted in defective growth. Conclusions Enhanced association of HXK2 with the mitochondria with the concomitant accumulation of G6PDG and 6PGDH results in increased NADPH that scavenges ROS and provides resistance to apoptosis. General significance Given the extensive similarity of aerobic glycolysis between humans and yeast, the phenomena described here could as well be responsible for the escape of apoptosis in cancer cells.

    更新日期:2019-12-18
  • Assessment of cytotoxic and genotoxic potentials of a mononuclear Fe(II) Schiff base complex with photocatalytic activity in Trigonella
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-06
    Kalyan Mahapatra, Ayon Kanti Ghosh, Sayanti De, Noyel Ghosh, Pritam Sadhukhan, Sharmistha Chatterjee, Rajarshi Ghosh, Parames C. Sil, Sujit Roy
    更新日期:2019-12-07
  • Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-05
    Marc Sprague-Piercy, Eric Wong, Kyle W. Roskamp, Joseph Fakhoury, J. Alfredo Freites, Douglas J. Tobias, Rachel W. Martin

    Background The eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin, which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant (γS-G18V). The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. Methods Molecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. Results γS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. Conclusions αB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. General significance Human αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.

    更新日期:2019-12-05
  • Ligand binding and activation of UTP-activated G protein-coupled P2Y2 and P2Y4 receptors elucidated by mutagenesis, pharmacological and computational studies
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-12-05
    Isaac Y. Attah, Alexander Neumann, Haneen Al-Hroub, Muhammad Rafehi, Younis Baqi, Vigneshwaran Namasivayam, Christa E. Müller
    更新日期:2019-12-05
  • Heat shock factor 4 regulates lysosome activity by modulating the αB-crystallin-ATP6V1A-mTOR complex in ocular lens
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-29
    Xiukun Cui, Ruiping Feng, Jungai Wang, Chunxiao Du, Xiahui Pi, Danling Chen, Jing Li, Hui Li, Jun Zhang, Jing Zhang, Hongmei Mu, Fengyan Zhang, Mugen Liu, Yanzhong Hu

    Background Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear. Methods HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation. Results HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues. Conclusion HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization. General significance This study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.

    更新日期:2019-11-29
  • The role of isoaspartate in fibrillation and its prevention by Protein-L-isoaspartyl methyltransferase
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-28
    Tanaya Chatterjee, Gaurav Das, Barun K. Chatterjee, Jesmita Dhar, Surajit Ghosh, Pinak Chakrabarti
    更新日期:2019-11-28
  • BBE31 from the Lyme disease agent Borrelia burgdorferi, known to play an important role in successful colonization of the mammalian host, shows the ability to bind glutathione
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-27
    Kalvis Brangulis, Inara Akopjana, Ivars Petrovskis, Andris Kazaks, Diana Zelencova, Atis Jekabsons, Kristaps Jaudzems, Kaspars Tars
    更新日期:2019-11-28
  • 更新日期:2019-11-28
  • 更新日期:2019-11-28
  • The Protein Disulfide Isomerase Family: from proteostasis to pathogenesis
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-04-12
    Motonori Matsusaki, Shingo Kanemura, Misaki Kinoshita, Young-Ho Lee, Kenji Inaba, Masaki Okumura
    更新日期:2019-11-18
  • Molecular dynamics in cells: A neutron view
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-15
    Giuseppe (Joseph) Zaccai

    Experiments to characterize intracellular molecular dynamics in vivo are discussed following a description of the incoherent neutron scattering method. Work reviewed includes water diffusion in bacteria, archaea, red blood cells, brain cells and cancer cells, and the role of proteome molecular dynamics in adaptation to physiological temperature and pressure, and in response to low salt stress in an extremophile. A brief discussion of the potential links between neutron scattering results and MD simulations on in-cell dynamics concludes the review.

    更新日期:2019-11-15
  • Effects of charge contrast and composition on microgel formation and interactions with bacteria-mimicking liposomes
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-15
    Bruno C. Borro, Marie S. Toussaint, Saskia Bucciarelli, Martin Malmsten
    更新日期:2019-11-15
  • Knocking out histone methyltransferase PRMT1 leads to stalled tadpole development and lethality in Xenopus tropicalis
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-15
    Yuki Shibata, Morihiro Okada, Thomas C. Miller, Yun-Bo Shi
    更新日期:2019-11-15
  • Lipid peroxidation is involved in calcium dependent upregulation of mitochondrial metabolism in skeletal muscle
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-15
    Afnan Saleh Al-Menhali, Sameem Banu, Plamena R. Angelova, Andrei Barcaru, Peter Horvatovich, Andrey Y. Abramov, Morana Jaganjac

    Background: Skeletal muscle cells continuously generate reactive oxygen species (ROS). Excessive ROS can affect lipids resulting in lipid peroxidation (LPO). Here we investigated the effects of myotube intracellular calcium-induced signaling eliciting contractions on the LPO induction and the impact of LPO-product 4-hydroxynonenal (4-HNE) on physiology/pathology of myotubes using C2C12 myoblasts. Methods: C2C12 myoblasts were differentiated into myotubes, stimulated with caffeine and analyzed for the induction of LPO and formation of 4-HNE protein adducts. Further effects of 4-HNE on mitochondrial bioenergetics, NADH level, mitochondrial density and expression of mitochondrial metabolism genes were determined. Results: Short and long-term caffeine stimulation of myotubes promoted superoxide production, LPO and formation of 4-HNE protein adducts. Furthermore, low 4-HNE concentrations had no effect on myotube viability and cellular redox homeostasis, while concentrations from 10 μM and above reduced myotube viability and significantly disrupted homeostasis. A time and dose-dependent 4-HNE effect on superoxide production and mitochondrial NADH-autofluorescence was observed. Finally, 4-HNE had strong impact on maximal respiration, spare respiratory capacity, ATP production, coupling efficiency of mitochondria and mitochondrial density. Conclusion: Data presented in this work make evident for the first time that pathological 4-HNE levels elicit damaging effects on skeletal muscle cells while acute exposure to physiological 4-HNE induces transient adaptation. General significance: This work suggests an important role of 4-HNE on the regulation of myotube's mitochondrial metabolism and cellular energy production. It further signifies the importance of skeletal muscle cells hormesis in response to acute stress in order to maintain essential biological functions.

    更新日期:2019-11-15
  • 更新日期:2019-11-15
  • Active probing of the mechanical properties of biological and synthetic vesicles
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-14
    Melissa C. Piontek, Rafael B. Lira, Wouter H. Roos
    更新日期:2019-11-14
  • Unveiling the structural features of nonnative trimers of human superoxide dismutase 1
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-14
    Wei-Chih Chao, Jyh-Feng Lu, Jinn-Shyan Wang, Tzu-Hsuan Chiang, Li-Ju Lin, Yao-Lin Lee, Pi-Tai Chou
    更新日期:2019-11-14
  • Metabolic fingerprinting reveals extensive consequences of GLS hyperactivity
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-14
    Lynne Rumping, Mia L. Pras-Raves, Johan Gerrits, Yuen Fung Tang, Marcel A. Willemsen, Roderick H.J. Houwen, Gijs van Haaften, Peter M. van Hasselt, Nanda M. Verhoeven-Duif, Judith J.M. Jans

    Background High glutaminase (GLS;EC3.5.1.2) activity is an important pathophysiological phenomenon in tumorigenesis and metabolic disease. Insight into the metabolic consequences of high GLS activity contributes to the understanding of the pathophysiology of both oncogenic pathways and inborn errors of glutamate metabolism. Glutaminase catalyzes the conversion of glutamine into glutamate, thereby interconnecting many metabolic pathways. Methods We developed a HEK293-based cell-model that enables tuning of GLS activity by combining the expression of a hypermorphic GLS variant with incremental GLS inhibition. The metabolic consequences of increasing GLS activity were studied by metabolic profiling using Direct-Infusion High-Resolution Mass-Spectrometry (DI-HRMS). Results and conclusions Of 12,437 detected features [m/z], 109 features corresponding to endogenously relevant metabolites were significantly affected by high GLS activity. As expected, these included strongly decreased glutamine and increased glutamate levels. Additionally, increased levels of tricarboxylic acid (TCA) intermediates with a truncation of the TCA cycle at the level of citrate were detected as well as increased metabolites of transamination reactions, proline and ornithine synthesis and GABA metabolism. Levels of asparagine and nucleotide metabolites showed the same dependence on GLS activity as glutamine. Of the nucleotides, especially metabolites of the pyrimidine thymine metabolism were negatively impacted by high GLS activity, which is remarkable since their synthesis depend both on aspartate (product of glutamate) and glutamine levels. Metabolites of the glutathione synthesizing γ-glutamyl-cycle were either decreased or unaffected. General significance By providing a metabolic fingerprint of increasing GLS activity, this study shows the large impact of high glutaminase activity on the cellular metabolome.

    更新日期:2019-11-14
  • Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-14
    Marcela Franco Mineiro, Eliziane de Souza Patricio, Álbert Souza Peixoto, Thaís Larissa Silva Araujo, Railmara Pereira da Silva, Ana Iochabel Soares Moretti, Filipe Silva Lima, Francisco Rafael Martins Laurindo, Flavia Carla Meotti

    Background Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. Methods Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. Results Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M−1 s−1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. Conclusions Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. General significance These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

    更新日期:2019-11-14
  • Melatonin interacts with repeat domain of Tau to mediate disaggregation of paired helical filaments
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-09
    Abhishek Ankur Balmik, Rashmi Das, Abha Dangi, Nalini Vijay Gorantla, Udaya Kiran Marelli, Subashchandrabose Chinnathambi

    Tau is the major neuronal protein involved in the stabilization of microtubule assembly. In Alzheimer's disease, Tau self assembles to form intracellular protein aggregates which are toxic to cells. Various methods have been tried and tested to restrain the aggregation of Tau. Most of the agents tested for this purpose have limitations in their effectiveness and availability to neuronal cells. We have tested melatonin, a neurohormone secreted by pineal gland and a well-known anti-oxidant, for its ability to interact with the repeat domain of Tau using ITC and NMR. In aggregation inhibition and disaggregation studies of repeat Tau, melatonin was found to modulate the aggregation propensity of repeat Tau at a concentration of 5000 μM and was more effective in dissolving preformed aggregates rather than acting as an aggregation inhibitor. However, there were no major conformational changes in Tau in presence of melatonin as observed in CD spectroscopy. On the basis of our findings, we are proposing a mechanism by which melatonin can interact with the repeat domain of Tau and exhibit its disaggregation effect.

    更新日期:2019-11-11
  • Bayesian inference for parameter estimation in lactoferrin-mediated iron transport across blood-brain barrier
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-11-01
    Aminul Islam Khan, Jin Liu, Prashanta Dutta

    Background In neurodegenerative diseases such as Alzheimer's and Parkinson's, excessive irons as well as lactoferrin (Lf), but not transferrin (Tf), have been found in and around the affected regions of the brain. These evidences suggest that lactoferrin plays a critical role during neurodegenerative diseases, although Lf-mediated iron transport across blood-brain barrier (BBB) is negligible compared to that of transferrin in normal condition. However, the kinetics of lactoferrins and lactoferrin-mediated iron transport are still unknown. Method To determine the kinetic rate constants of lactoferrin-mediated iron transport through BBB, a mass-action based ordinary differential equation model has been presented. A Bayesian framework is developed to estimate the kinetic rate parameters from posterior probability density functions. The iron transport across BBB is studied by considering both Lf- and Tf-mediated pathways for both normal and pathologic conditions. Results Using the point estimates of kinetic parameters, our model can effectively reproduce the experimental data of iron transport through BBB endothelial cells. The robustness of the model and parameter estimation process are further verified by perturbation of kinetic parameters. Our results show that surge in high-affinity receptor density increases lactoferrin as well as iron in the brain. Conclusions Due to the lack of a feedback loop such as iron regulatory proteins (IRPs) for lactoferrin, iron can transport to the brain continuously, which might increase brain iron to pathological levels and can contribute to neurodegeneration. General significance This study provides an improved understanding of presence of lactoferrin and iron in the brain during neurodegenerative diseases.

    更新日期:2019-11-01
  • How does the mood stabilizer lithium bind ATP, the energy currency of the cell
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-31
    A. Haimovich, A. Goldbourt
    更新日期:2019-11-01
  • Omeprazole as a potent activator of human cytosolic aldehyde dehydrogenase ALDH1A1
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-31
    Luis Francisco Calleja, Javier Alejandro Belmont-Díaz, Oscar Medina-Contreras, Héctor Quezada, Belem Yoval-Sánchez, Jesús Campos-García, José Salud Rodríguez-Zavala

    Background Accumulation of lipid aldehydes plays a key role in the etiology of human diseases where high levels of oxidative stress are generated. In this regard, activation of aldehyde dehydrogenases (ALDHs) prevents oxidative tissue damage during ischemia-reperfusion processes. Although omeprazole is used to reduce stomach gastric acid production, in the present work this drug is described as the most potent activator of human ALDH1A1 reported yet. Methods Docking analysis was performed to predict the interactions of omeprazole with the enzyme. Recombinant human ALDH1A1 was used to assess the effect of omeprazole on the kinetic properties. Temperature treatment and mass spectrometry were conducted to address the nature of binding of the activator to the enzyme. Finally, the effect of omeprazole was evaluated in an in vivo model of oxidative stress, using E. coli cells expressing the human ALDH1A1. Results Omeprazole interacted with the aldehyde binding site, increasing 4–6 fold the activity of human ALDH1A1, modified the kinetic properties, altering the order of binding of substrates and release of products, and protected the enzyme from inactivation by lipid aldehydes. Furthermore, omeprazole protected E. coli cells over-expressing ALDH1A1 from the effects of oxidative stress generated by H2O2 exposure, reducing the levels of lipid aldehydes and preserving ALDH activity. Conclusion Omeprazole can be repositioned as a potent activator of human ALDH1A1 and may be proposed for its use in therapeutic strategies, to attenuate the damage generated during oxidative stress events occurring in different human pathologies.

    更新日期:2019-11-01
  • Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-31
    Michihiro Suga, Atsuhiro Shimada, Fusamichi Akita, Jian-Ren Shen, Takehiko Tosha, Hiroshi Sugimoto

    Background The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. Scope of review Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusions XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significance XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing ‘live’ protein molecules.

    更新日期:2019-11-01
  • 更新日期:2019-11-01
  • Characterization of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidases from Borrelia burgdorferi: Antibiotic targets for Lyme disease
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-31
    Kenneth A. Cornell, Reece J. Knippel, Gerald R. Cortright, Meghan Fonken, Christian Guerrero, Amy R. Hall, Kristen A. Mitchell, John H. Thurston, Patrick Erstad, Aoxiang Tao, Dong Xu, Nikhat Parveen

    Background Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. Methods Here we examine three unique 5′-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. Results The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5′-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5′-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3–0.4 μg/mL against cultured B. burgdorferi cells. Conclusion B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. General significance The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.

    更新日期:2019-11-01
  • How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-31
    Tanja J. Kutzner, Alonso M. Higuero, Martina Süßmair, Jürgen Kopitz, Michael Hingar, Natalia Díez-Revuelta, Gabriel García Caballero, Herbert Kaltner, Ingo Lindner, José Abad-Rodríguez, Dietmar Reusch, Hans-Joachim Gabius

    Background Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route. Methods Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator. Results Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1. Conclusions Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation. General significance Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.

    更新日期:2019-11-01
  • Spatial arrangement of LD motif-interacting residues on focal adhesion targeting domain of Focal Adhesion Kinase determine domain-motif interaction affinity and specificity
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-30
    Anjali Bansal Gupta, Somsubhro Mukherjee, Catherine Pan Quirong, Adrian Velazquez-Campoy, J. Sivaraman, Boon Chuan Low

    Background Leucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. Methods Various point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. Results This led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 μM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 μM vs 63 μM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10−5 μm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10−3 μm2/s). Conclusions We observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. General significance Our study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.

    更新日期:2019-11-01
  • ROS directly activates transforming growth factor β type 1 receptor signalling in human vascular smooth muscle cells
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-30
    Raafat Mohamed, Yingnan Cao, Rizwana Afroz, Suowen Xu, Hang Ta, Michael Barras, Wenhua Zheng, Peter J. Little, Danielle Kamato
    更新日期:2019-11-01
  • Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-30
    Saleem Yousuf Bhat, Insaf Ahmed Qureshi

    Background M20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues. Methods PepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods. Results PepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P1 position. Peptidomimetics amastatin and actinonin occupied S1 pocket by competing with the substrate for binding to active site and inhibited PepT potently while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme. Conclusions PepT preferentially hydrolyzes oligopeptides carrying alanine at P1 position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions. General significance This study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.

    更新日期:2019-11-01
  • Histone acetylation landscape in S. cerevisiae nhp6ab mutants reflects altered glucose metabolism
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-30
    Durano Diletta, Di Felice Francesca, Caldarelli Federica, Lukacs Andrea, D. Alfonso Anna, Saliola Michele, Sciubba Fabio, Miccheli Alfredo, Zambelli Federico, Pavesi Giulio, Bianchi Marco Emilio, Camilloni Giorgio
    更新日期:2019-11-01
  • Structural basis for the design of allosteric inhibitors of the Aurora kinase A enzyme in the cancer chemotherapy
    BBA Gen. Subj. (IF 3.681) Pub Date : 2019-10-30
    Valéria Barbosa de Souza, Daniel Fábio Kawano

    Aurora kinases are essential enzymes for the control of cell cycle. The specific role of aurora kinase A (AURKA) is the regulation of spindle assembly and stability by promoting centrosome maturation and separation. Because AURKA is an essential protein for the normal occurrence of the cycle, mutations and deregulations in the activities of this protein are associated with several cancers. The kinase activity of AURKA is controlled by autocatalytic phosphorylation, which is facilitated after binding to a regulator protein, the Target Protein for Xenopuskinesin-like protein 2 (TPX2). Scope of review This review highlights the physiological and pathophysiological properties of AURKA, the structure of the AURKA/TPX2 complex and the main structural features that can be explored for the design of selective AURKA inhibitors. Major conclusions The design of selective AURKA inhibitors remains as a challenge as most of the currently available inhibitors target only the ATP binding cleft and are nonselective among kinases. However, by exploring the inactive form of the kinase, researchers get access to an adjacent hydrophobic pocket, allowing the design of more selective inhibitors. Additionally, the possibility of designing potent allosteric AURKA inhibitors look very promising from the clinical perspective, since it tends to yield the most selective class of compounds. General significance Herein we detailed the binding modes of the most selective AURKA inhibitors currently reported. We believe this will aid researchers in defining the structural patterns necessary for selective AURKA inhibition, guiding the design of more potent compounds to be therapeutically explored in cancer patients.

    更新日期:2019-11-01
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