Biology subjects

Zhang, W.

Publications and source records attributed to Zhang, W..

At least 1,099 records · Page 61Linked to original sources

bioRxiv2018

Isonitrile Formation by a Non-heme Iron(II)-dependent Oxidase/Decarboxylase

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, members of which are critical for the virulence of pathogenic mycobacteria.

biochemistry↗

bioRxiv2018

Structure-guided disruption of pseudopilus tip inhibits Type II secretion in Pseudomonas aeruginosa

Pseudomonas aeruginosa utilizes the Type II secretion system (T2SS) to translocate a wide range of large, structured protein virulence factors through the periplasm to the extracellular environment for infection. In the T2SS, five pseudopilins assemble into the pseudopilus that acts as a piston to extrude exoproteins out of cells. Through structure determination of the pseudopilin complexes of XcpVWX and XcpVW and function analysis, we have confirmed that two minor pseudopilins, XcpV and XcpW, constitute a core complex indispensable to the pseudopilus tip. The absence of either XcpV or -W resulted in the non-functional T2SS. Our small-angle X-ray scattering experiment for the first time revealed the architecture of the entire pseudopilus tip and established the working model. Based on the interaction interface of complexes, we have developed inhibitory peptides. The structure-based peptides not only disrupted of the XcpVW core complex and the entire pseudopilus tip in vitro but also inhibited the T2SS in vivo. More importantly, these peptides effectively reduced the virulence of P. aeruginosa towards Caenorhabditis elegans.

microbiology↗

bioRxiv2018

Structures of filaments from Pick’s disease reveal a novel tau protein fold

The ordered assembly of tau protein into abnormal filamentous inclusions underlies many human neurodegenerative diseases1. Tau assemblies appear to spread through specific neural networks in each disease2, with short filaments having the greatest seeding activity3. The abundance of tau inclusions strongly correlates with disease symptoms4. Six tau isoforms are expressed in normal adult human brain - three isoforms with four microtubule-binding repeats each (4R tau) and three isoforms lacking the second repeat (3R tau)1. In various diseases, tau filaments can be composed of either 3R tau or 4R tau, or of both 3R and 4R tau. They have distinct cellular and neuroanatomical distributions5, with morphological and biochemical differences suggesting that they may be able to adopt disease-specific molecular conformations6,7. Such conformers may give rise to different neuropathological phenotypes8,9, reminiscent of prion strains10. However, the underlying structures are not known. Using electron cryo-microscopy (cryo-EM), we recently reported the structures of tau filaments from Alzheimers disease, which contain both 3R and 4R tau11. Here we have determined the structures of tau filaments from Picks disease, a neurodegenerative disorder characterised by frontotemporal dementia. They consist of residues K254-F378 of 3R tau, which are folded differently when compared to tau in Alzheimers disease filaments, establishing the existence of conformers of assembled tau. The Pick fold explains the selective incorporation of 3R tau in Pick bodies and the differences in phosphorylation relative to the tau filaments of Alzheimers disease. Our findings show how tau can adopt distinct folds in human brain in different diseases, an essential step for understanding the formation and propagation of molecular conformers.

biophysics↗

bioRxiv2018

Probing the Acyl Carrier Protein-Enzyme Interactions within Terminal Alkyne Biosynthetic Machinery

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.\n\nTopical HeadingBiomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food

bioengineering↗

bioRxiv2018

Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

Activation of the NLRP3 inflammasome is a key process of host immune response, the first line of defense against cellular stresses and pathogen infections. However, excessive inflammasome activation damages the hosts, and thus it must be precisely controlled. The mechanism underlying the repression of systematic inflammasome activation remains largely unknown. This study reveals that CUL1, a key component of the SCF E3 ligase, plays a critical role in regulation of the inflammasome. CUL1 suppresses the inflammasome activation in HEK293T cells, inhibits endogenous NLRP3 in macrophages, and represses inflammatory responses in C57BL/6 mice. Under normal physiological conditions, CUL1 interacts with NLRP3 to disrupt the inflammasome assembly, and catalyzes NLRP3 ubiquitination to repress the inflammasome activation. In response to inflammatory stimuli, CUL1 disassociates from NLRP3 to release the repression of NLRP3 inflammasome activation. This work reveals a distinct mechanism underlying the repression of inflammasome activation under physiological conditions and the induction of inflammasome activation in response to inflammatory stimuli, and thus provides insights into the prevention and treatment of infectious and inflammatory diseases.

immunology↗

bioRxiv2018

Checkpoint signaling abrogation after cell cycle reentry reveals that differentiated neurons are mitotic cells

Mitotic activity associated to neuron cell-death instead of cell-division is reported in neurodegenerative diseases. However, why mitotic activity can take place in supposedly postmitotic neurons and how it is associated to cell-death remains largely unexplained. To address these questions, we have studied the response of primary neurons to oncogenic deregulation using a fusion protein based on truncated Cyclin E and Cdk2. Oncogenic Cyclin E/Cdk2 elicits mitotic checkpoint signaling, resulting in cell-cycle arrest and cell-death. However, as in mitotic cells, checkpoint suppression enables oncogenic cell-cycle progression and neuronal division. Further, neurons actively adapt to the cell-cycle by losing and reforming the axon initial segment, which integrates synaptic inputs to sustain action potentials. We conclude that neurons are mitotic cells in a reversible quiescent-like state, which is falsely portrayed as irreversible by mitotic checkpoints. In extension, neuronal death in lieu of cell-division reflects oncosuppressive checkpoint signaling.

neuroscience↗

bioRxiv2018

Whole-exome sequencing identified rare variants associated with body length and girth in cattle

Body measurements can be used in determining body size to monitor the cattle growth and examine the response to selection. Despite efforts putting into the identification of common genetic variants, the mechanism understanding of the rare variation in complex traits about body size and growth remains limited. Here, we firstly performed GWAS study for body measurement traits in Simmental cattle, however there were no SNPs exceeding significant level associated with body measurements. To further investigate the mechanism of growth traits in beef cattle, we conducted whole exome analysis of 20 cattle with phenotypic differences on body girth and length, representing the first systematic exploration of rare variants on body measurements in cattle. By carrying out a three-phase process of the variant calling and filtering, a sum of 1158, 1151, 1267, and 1303 rare variants were identified in four phenotypic groups of two growth traits, higher/ lower body girth (BG_H and BG_L) and higher/lower body length (BL_H and BL_L) respectively. The subsequent functional enrichment analysis revealed that these rare variants distributed in 886 genes associated with collagen formation and organelle organization, indicating the importance of collagen formation and organelle organization for body size growth in cattle. The integrative network construction distinguished 62 and 66 genes with different co-expression patterns associated with higher and lower phenotypic groups of body measurements respectively, and the two sub-networks were distinct. Gene ontology and pathway annotation further showed that all shared genes in phenotypic differences participate in many biological processes related to the growth and development of the organism. Together, these findings provide a deep insight into rare genetic variants of growth traits in cattle and this will have a promising application in animal breeding.

genetics↗

bioRxiv2018

Crystallographic observation of nonenzymatic RNA primer extension

The importance of genome replication has inspired detailed crystallographic studies of enzymatic DNA/RNA polymerization. In contrast, the mechanism of nonenzymatic polymerization is less well understood, despite its critical role in the origin of life. Here we report the direct observation of nonenzymatic RNA primer extension through time-resolved crystallography. We soaked crystals of an RNA primer-template-dGMP complex with guanosine-5'-phosphoro-2-aminoimidazolide for increasing times. At early times we see the activated ribonucleotides bound to the template, followed by formation of the imidazolium-bridged dinucleotide intermediate. At later times, we see a new phosphodiester bond forming between the primer and the incoming nucleotide. The intermediate is pre-organized because of the constraints of base-pairing with the template and hydrogen bonding between the imidazole amino group and both flanking phosphates. Our results provide atomic-resolution insight into the mechanism of nonenzymatic primer extension, and set the stage for further structural dissection and optimization of the RNA copying process.

biochemistry↗

bioRxiv2018

Role of anterograde motor Kif5b in clathrin-coated vesicle uncoating and clathrin-mediated endocytosis

Kif5b-driven anterograde transport and clathrin-mediated endocytosis (CME) are responsible for opposite intracellular trafficking, contributing to plasma membrane homeostasis. However, whether and how the two trafficking processes coordinate remain unclear. Here, we show that Kif5b directly interacts with clathrin heavy chain (CHC) at a region close to that for uncoating catalyst (Hsc70) and preferentially localizes on large clathrin-coated vesicles (CCVs). Uncoating in vitro is decreased for CCVs from the cortex of kif5b conditional knockout (mutant) mouse and facilitated by adding CHC-binding Kif5b fragments, while cell peripheral distribution of CHC or Hsc70 keeps unaffected by Kif5b depletion. Furthermore, cellular entry of vesicular stomatitis virus that internalized into large CCV is inhibited in cells by Kif5b depletion or introducing a dominant-negative Kif5b fragment. These findings showed a new role of Kif5b in CCV uncoating and CME, indicating Kif5b as a molecular knot connecting anterograde transport to CME.

cell biology↗

bioRxiv2018

Identification of novel mutations associated with clofazimine resistance in Mycobacterium abscessus

Mycobacterium abscessus (Mab) is a major non-tuberculous mycobacterial (NTM) pathogen responsible for about 80% of all pulmonary infections caused by rapidly growing mycobacteria. Clofazimine is an effective drug active against Mab and shows synergistic activity when given with amikacin, but the mechanism of resistance to clofazimine in Mab is unknown.\n\nObjectiveTo investigate the molecular basis of clofazimine resistance in Mab.\n\nMethodsWe isolated 29 Mab mutants resistant to clofazimine, and subjected them to whole genome sequencing and Sanger sequencing to identify possible mutations associated with clofazimine resistance.\n\nResultsMutations in MAB_2299c gene which encodes possible transcriptional regulatory protein were identified in 23 of the 29 clofazimine-resistant mutants. In addition, 6 mutations in MAB_1483 were found in 21 of the 29 mutants, and one mutation in MAB_0540 was found in 16 of the 29 mutants. Mutations in MAB_0416c, MAB_4099c, MAB_2613, MAB_0409, MAB_1426 were also associated with clofazimine resistance in less frequency. Two identical mutations which are likely to be polymorphisms unrelated to clofazimine resistance were found in MAB_4605c and MAB_4323 in 13 mutants.\n\nConclusionMutations in MAB_2299c, MAB_1483, and MAB_0540 are the major mechanisms of clofazimine resistance in Mab. Future studies are needed to address the role of the identified mutations in clofazimine resistance in Mab, and our findings have implications for developing a rapid molecular test for detecting clofazimine resistance in this organism.

microbiology↗

bioRxiv2018

The homoeologous genes for the Rec8-like meiotic cohesin in wheat: structure, function, and evolutionary implication

The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiosis. Here, we cloned two Rec8-like homoeologous genes (homoeoalleles) from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by comparative analysis. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1. Therefore, the Rec8 homoeoalleles from the subgenomes A and D may be functionally more active than the one from the subgenome B in wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in the polyploid, and imply the distinction of the wheat subgenome B from other subgenomes in the origin and evolution.\n\nHIGHLIGHTThis work revealed the structural and expression patterns of the Rec8-like homoeologous genes in polyploid wheat, implying a unique origin and evolutionary route of the wheat B subgenome.

genetics↗

bioRxiv2018

Identification of Variable and Joining germline genes and alleles for Rhesus macaque from B-cell receptor repertoires

The Rhesus macaque is a valuable preclinical animal model to estimate vaccine effectiveness, and is also important for understanding antibody maturation and B-cell repertoire evolution responding to vaccination; however, incomplete mapping of rhesus immunoglobulin germline genes hinders the research efforts. To address this deficiency, we sequenced B-cell receptor (BCR) repertoires of 75 India Rhesus macaques. Using a bioinformatic method that has been validated with BCR repertoire analysis of three human donors, we were able to infer rhesus Variable (V) and Joint(J) germline alleles, identifying a total of 122 V and 20 J germline alleles. Importantly, 91 V and 13 J alleles were novel, and 40 V and 13 J genes were found at a novel genome region that has not been previously recorded. The novelty of these newly identified alleles was supported by two observations. Firstly, 50 V and 5 J novel alleles were observed in whole genome sequencing data of 10 Rhesus macaques. Secondly, using alignment reference including the novel alleles, the mutation rate of rearranged repertoires was significant declined in 9 other irrelevant samples, and all our identified novel V and J alleles were 100% identity mapped by rearranged repertoire data. These newly identified novel alleles, along with previous reported alleles, provide an important reference for future investigations of rhesus immune repertoire evolution, in response to vaccination or infection. In addition, the method outlined in our study offered an example to future efforts in identifying novel immunoglobulin alleles.

immunology↗

bioRxiv2018

SlARF10, an auxin response factor, is required for chlorophyll and sugar accumulation during tomato fruit development

Tomato green fruits photosynthesis contributes to fruit growth and carbon economy. Tomato auxin response factor 10 (SlARF10) is one of the members of ARF family. Our results showed that SlARF10 locates in the nucleus and has no transcriptional activity. SlARF10 was expressed in various tomato tissues, but highly expressed in green fruit. Up-regulation of SlARF10 produced dark green phenotype of fruits, whereas down-regulation of SlARF10 had light green phenotype. Autofluorescence and chlorophyll content analysis confirmed the phenotypes, which indicated that SlARF10 plays an important role in chlorophyll accumulation in tomato fruits. Up-regulation of SlARF10 increased the photochemical potential in tomato leaves and fruits. Furthermore, the SlARF10 up-regulating lines displayed improved accumulation of starch in fruits, whereas SlARF10 suppressed lines had inhibited starch accumulation. Up-regulation of SlARF10 increased the expression of AGPases, the starch biosynthesis genes. SlARF10 up-regulating lines had increased accumulation of SlGLK1 and SlGLK2 transcripts in fruits. The promoter sequence of SlGLK1 gene had two conserved ARF binding sites. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in fruits. Our study provided more insight on the link between auxin signaling, chloroplastic activity and sugar metabolism during the development of tomato fruits.\n\nAbbreviations\n\nHighlightSlARF10 played an important role in the chlorophyll accumulation and photosynthesis in tomato fruits. SlARF10 was involved in starch accumulation by controlling the expression of starch synthesis related enzyme genes. SlARF10 may regulate the expression of SlGLK1, thus controlling chlorophyll accumulation, photosynthesis rates and sugars synthesis in tomato fruits.

plant biology↗

bioRxiv2018

Identification of novel mutations in LprG (rv1411c), rv0521, rv3630, rv0010c, ppsC, cyp128 associated with pyrazinoic acid/pyrazinamide resistance in Mycobacterium tuberculosis

There is currently considerable interest in understanding the mechanisms of action of pyrazinamide (PZA), a critical frontline tuberculosis (TB) drug that plays a unique role in shortening TB therapy due to its unique activity against Mycobacterium tuberculosis persisters that are not killed by other TB drugs.1,2 Despite the importance of PZA in the treatment of both drug susceptible and drug-resistant TB and its simple structure, its mechanisms of action are complex and are not well understood.1,2 PZA is a prodrug that is converted to the active form pyrazinoic acid (POA) by nicotinamidase/pyrazinamidase (PZase) encoded by the pncA gene,3 whose mutation is the most common mechanism of PZA resistance in M. tuberculosis.3-5 However, some low level PZA-resistant strains (MIC=200-300 g/ml, pH6.0) do not have mutations in the pncA gene.5,6 Recent studies have identified rpsA, which encodes the ribosomal protein S1 involved in both translation and trans-translation process, as a target of PZA,7 where its mutations are associated with PZA resistance from clinical isolates. In addition, mutations in panD encoding aspartate decarboxylase were identified as a new mechanism of PZA resistance from in vitro mutants resistant to PZA and the PanD protein was found to be another target of PZA.8,9 panD mutations were initially found in mutants resistant to PZA 8 and then in mutants resistant to POA. 9,10 It is worth noting that previously POA-resistant mutants could not be isolated at acid pH which is required for higher activity of PZA against M. tuberculosis. However, we were able to successfully isolate POA-resistant mutants with high POA concentrations at close to neutral pH (pH 6.8),9 which led to discovery of new genes involved in POA and PZA resistance. For example, clpC1, which was also isolated from mutants resistant to PZA,11 was identified in mutants resistant to POA.12

microbiology↗

bioRxiv2017

3.7 A cryo-EM structure of the core Centromere Binding Factor 3 complex

The Centromere Binding Factor 3 (CBF3) complex binds the third Centromere DNA Element in organisms with point centromeres, such as S. cerevisiae. It is the only essential centromere binding complex as it facilitates genetic specification of point centromeres. It is therefore the most fundamental complex of the kinetochore in these organisms and its association with centromere DNA allows association of all other kinetochore components. We have determined the atomic structure of the core complex of CBF3, comprising 3 of its 4 components, using cryo-EM. The architecture of the complex is 'U'-shaped, with a deep, strongly basic channel that is narrow at one end and wide at the other. Combining our structure and in vitro assays, we present a model for its association with centromere DNA.

molecular biology↗

bioRxiv2017

Highly tamoxifen-inducible principal-cell-specific Cre mice with complete fidelity in cell specificity and no leakiness

An ideal inducible system should be cell-specific and have absolute no background recombination without induction (i.e. no leakiness), a high recombination rate after induction, and complete fidelity in cell specificity (i.e. restricted recombination exclusively in cells where the driver gene is expressed). However, such an ideal mouse model remains unavailable for collecting duct research. Here, we report a mouse model that meets these criteria. In this model, a cassette expressing ERT2CreERT2 (ECE) is inserted at the ATG of the endogenous Aqp2 locus to disrupt Aqp2 function and to express ECE under the control of the Aqp2 promoter. The resulting allele is named Aqp2ECE. There was no indication of a significant impact of disruption of a copy of Aqp2 on renal function and blood pressure control in adult Aqp2ECE/+ heterozygotes. Without tamoxifen, Aqp2ECE did not activate a Cre-dependent red fluorescence protein (RFP) reporter in adult kidneys. A single injection of tamoxifen (2 mg) to adult mice enables Aqp2ECE to induce robust RFP expression in the whole kidney 24h post injection, with the highest recombination efficiency of 95% in the inner medulla. All RFP-labeled cells expressed principal cell markers (Aqp2 & Aqp3), but not intercalated cell markers (V-ATPase B1B2, and carbonic anhydrase II). Hence, Aqp2ECE confers principal cell-specific tamoxifen-inducible recombination with absolute no leakiness, high inducibility, and complete fidelity in cell specificity, which should be an important tool for temporospatial control of target genes in the principal cells and for Aqp2+ lineage tracing in adult mice.

genetics↗

bioRxiv2017

DARPP-32 and t-DARPP promote lung cancer growth through IKKα-dependent cell migration and Akt/Erk-mediated cell survival

Lung cancer is the leading cause of cancer-related death worldwide. In this study, we demonstrate that elevated expression of dopamine and cyclic adenosine monophosphate-regulated phosphoprotein, Mr 32000 (DARPP-32) and its truncated splice variant t-DARPP promotes lung tumor growth, while abrogation of DARPP-32 expression in human non-small cell lung cancer (NSCLC) cells reduces tumor growth in orthotopic mouse models. We observe a novel physical interaction between DARPP-32 and inhibitory kappa B kinase- (IKK) that promotes NSCLC cell migration through non-canonical nuclear factor kappa-light-chain-enhancer of activated B cells 2 (NF-{kappa}B2) signaling. Bioinformatics analysis of 513 lung adenocarcinoma patients reveals elevated t-DARPP isoform expression is associated with poor overall survival. Histopathological investigation of 62 human lung adenocarcinoma tissues also showed that t-DARPP expression is elevated with increasing tumor (T) stage. Our data suggest that DARPP-32 is a negative prognostic marker associated with increasing stages of NSCLC and may represent a novel therapeutic target.

cancer biology↗

bioRxiv2017

Identification of residue pairing in interacting β-strands from a predicted residue contact map

Despite the rapid progress of protein residue contact prediction, predicted residue contact maps frequently contain many errors. However, information of residue pairing in {beta} strands could be extracted from a noisy contact map, due to the presence of characteristic contact patterns in {beta}-{beta} interactions. This information may benefit the tertiary structure prediction of mainly {beta} proteins. In this work, we introduce a novel ridge-detection-based {beta}-{beta} contact predictor, RDb2C, to identify residue pairing in {beta} strands from any predicted residue contact map. The algorithm adopts ridge detection, a well-developed technique in computer image processing, to capture consecutive residue contacts, and then utilizes a novel multi-stage random forest framework to integrate the ridge information and additional features for prediction. Starting from the predicted contact map of CCMpred, RDb2C remarkably outperforms all state-of-the-art methods on two conventional test sets of {beta} proteins (BetaSheet916 and BetaSheet1452), and achieves F1-scores of ~62% and ~76% at the residue level and strand level, respectively. Taking the prediction of the more advanced RaptorX-Contact as input, RDb2C achieves impressively higher performance, with F1-scores reaching ~76% and ~86% at the residue level and strand level, respectively. According to our tests on 61 mainly {beta} proteins, improvement in the {beta}-{beta} contact prediction can further ameliorate the structural prediction.\n\nAvailability: All source data and codes are available at http://166.111.152.91/Downloads.html or at the GitHub address of https://github.com/wzmao/RDb2C.\n\nAuthor summaryDue to the topological complexity, mainly {beta} proteins are challenging targets in protein structure prediction. Knowledge of the pairing between {beta} strands, especially the residue pairing pattern, can greatly facilitate the tertiary structure prediction of mainly {beta} proteins. In this work, we developed a novel algorithm to identify the residue pairing in {beta} strands from a predicted residue contact map. This method adopts the ridge detection technique to capture the characteristic pattern of {beta}-{beta} interactions from the map and then utilizes a multi-stage random forest framework to predict {beta}-{beta} contacts at the residue level. According to our tests, our method could effectively improve the prediction of {beta}-{beta} contacts even from a highly noisy contact map. Moreover, the refined {beta}-{beta} contact information could effectively improve the structural modeling of mainly {beta} proteins.

bioinformatics↗