Biology subjects

Feng, Z.

Publications and source records attributed to Feng, Z..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Cdc13 exhibits dynamic DNA strand exchange in the presence of telomeric DNA

Telomerase is the enzyme that lengthens telomeres and is tightly regulated by a variety of means to maintain genome integrity. Several DNA helicases function at telomeres, and we previously found that the Saccharomyces cerevisiae helicases Hrq1 and Pif1 directly regulate telomerase. To extend these findings, we are investigating the interplay between helicases, single-stranded DNA (ssDNA) binding proteins (ssBPs), and telomerase. The yeast ssBPs Cdc13 and RPA differentially affect Hrq1 and Pif1 helicase activity, and experiments to measure helicase disruption of Cdc13/ssDNA complexes instead revealed that Cdc13 can exchange between substrates. Although other ssBPs display dynamic binding, this was unexpected with Cdc13 due to the reported in vitro stability of the Cdc13/telomeric ssDNA complex. We found that the DNA exchange by Cdc13 occurs rapidly at physiological temperatures, requires telomeric repeat sequence DNA, and is affected by ssDNA length. Cdc13 truncations revealed that the low-affinity binding site (OB1), which is distal from the high-affinity binding site (OB3), is required for this intermolecular dynamic DNA exchange (DDE). We hypothesize that DDE by Cdc13 is the basis for how Cdc13 "moves" at telomeres to alternate between modes where it regulates telomerase activity and assists in telomere replication.

biochemistry↗

bioRxiv2023

Protein tyrosine phosphatase receptor kappa regulates glycolysis and de novo lipogenesis to promote hepatocyte metabolic reprogramming in obesity

Fat accumulation, de novo lipogenesis, and glycolysis are key drivers of hepatocyte reprogramming and the consequent metabolic dysfunction-associated steatotic liver disease (MASLD). Here we report that obesity leads to dysregulated expression of hepatic protein-tyrosine phosphatases (PTPs). PTPRK was found to be increased in steatotic hepatocytes in both humans and mice, and positively correlated with PPAR{gamma}-induced lipogenic signalling. High-fat-fed PTPRK knockout mice displayed reduced weight gain and hepatic fat accumulation. Phosphoproteomic analysis in primary hepatocytes and hepatic metabolomics identified fructose-1,6-bisphosphatase 1 and glycolysis as PTPRK targets in metabolic reprogramming. Silencing PTPRK in hepatoma cell lines resulted in reduced colony-forming ability and PTPRK knockout mice developed smaller tumours after diethylnitrosamine-induced hepatocarcinogenesis. Our study defines a novel role for PTPRK in regulating hepatic glycolysis, lipid metabolism, and tumour development. PTPRK inhibition may provide therapeutic possibilities in obesity-associated liver diseases. HighlightsO_LIHepatic receptor-type PTPs are increased in MASLD C_LIO_LIPTPRK is expressed in hepatocytes and upregulated in obesity C_LIO_LIPTPRK deficiency reduces body fat mass and liver steatosis in diet-induced obesity C_LIO_LIPTPRK regulates hepatic glycolysis and lipogenesis, promoting tumorigenesis C_LI

cancer biology↗

bioRxiv2023

Structural and mechanistic insights into disease-associated endolysosomal exonucleases PLD3 and PLD4

Endolysosomal exonucleases PLD3 and PLD4 (phospholipases D3 and D4) are associated with autoinflammatory and autoimmune diseases. We report structures of these enzymes, and the molecular basis of their catalysis. The structures reveal an intra-chain dimer topology forming a basic active site at the interface. Like other PLD superfamily members, PLD3 and PLD4 carry HxKxxxxD/E motifs and participate in phosphodiester-bond cleavage. The enzymes digest ssDNA and ssRNA in a 5'-to-3' manner and are blocked by 5'-phosphorylation. We captured structures in apo, intermediate, and product states and revealed a link-and-release two-step catalysis. We also unexpectedly demonstrated phosphatase activity via a covalent 3- phosphistidine intermediate. PLD4 contains an extra hydrophobic clamp that stabilizes substrate and could affect oligonucleotide substrate preference and product release. Biochemical and structural analysis of disease-associated mutants of PLD3/4 demonstrated reduced enzyme activity or thermostability and the possible basis for disease association. Furthermore, these findings provide insight into therapeutic design.

biochemistry↗

bioRxiv2023

PDB NextGen Archive: Centralising Access to Integrated Annotations and Enriched Structural Information by the Worldwide Protein Data Bank

The Protein Data Bank (PDB) archive is the global repository for public-domain 3D biomolecular structural information. The archival nature of the PDB presents certain challenges pertaining to updating or adding associated annotations from trusted external biodata resources. While each Worldwide PDB (wwPDB) partner has made best efforts to provide up-to-date external annotations, accessing and integrating information from disparate wwPDB data centers can be an involved process. To address this issue, the wwPDB has established the PDB Next Generation or NextGen Archive, developed to centralize and streamline access to enriched structural annotations from wwPDB partners and trusted external sources. At present, the archive provides mappings between experimentally determined 3D structures of proteins and UniProt amino acid sequences, together with domain annotations from Pfam, SCOP2, and CATH databases, and intra-molecular connectivity information. Since launch, the PDB NextGen Archive has seen substantial user engagement with over 3.5 million data file downloads, ensuring researchers have access to accurate, up-to-date, and easily accessible structural annotations. Database URLhttp://www.wwpdb.org/ftp/pdb-nextgen-archive-site

bioinformatics↗

bioRxiv2023

Distinct functions of EHMT1 and EHMT2 in cancer chemotherapy and immunotherapy

EHTM1 (GLP) and EHMT2 (G9a) are closely related protein lysine methyltransferases often thought to function together as a heterodimer to methylate histone H3 and non-histone substrates in diverse cellular processes including transcriptional regulation, genome methylation, and DNA repair. Here we show that EHMT1/2 inhibitors cause ATM-mediated slowdown of replication fork progression, accumulation of single-stranded replication gaps, emergence of cytosolic DNA, and increased expression of STING. EHMT1/2 inhibition strongly potentiates the efficacy of alkylating chemotherapy and anti-PD-1 immunotherapy in mouse models of tripe negative breast cancer. The effects on DNA replication and alkylating agent sensitivity are largely caused by the loss of EHMT1-mediated methylation of LIG1, whereas the elevated STING expression and remarkable response to immunotherapy appear mainly elicited by the loss of EHMT2 activity. Depletion of UHRF1, a protein known to be associated with EHMT1/2 and LIG1, also induces STING expression, and depletion of either EHMT2 or UHRF1 leads to demethylation of specific CpG sites in the STING1 promoter, suggestive of a distinct EHMT2-UHRF1 axis that regulates DNA methylation and gene transcription. These results highlight distinct functions of the two EHMT paralogs and provide enlightening paradigms and corresponding molecular basis for combination therapies involving alkylating agents and immune checkpoint inhibitors.

cancer biology↗

bioRxiv2023

Identification of Host Restriction Factors Critical for Recombinant AAV Transduction of Polarized Human Airway Epithelium

Recombinant (r)AAV2.5T was selected from the directed evolution of an AAV capsid library in human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequence of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome wide CRISPR gRNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screen identified several genes that are critical for rAAV2.5T transduction in HeLa S3 cells, including previously reported genes KIAA0319L, TM9SF2, VPS51, and VPS54, as well as a novel gene WDR63. We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed WDR63 is an important player in rAAV2.5T transduction of HAE, while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T. SignificanceThe essential steps of a successful gene delivery by rAAV include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

microbiology↗

bioRxiv2023

Nifuroxazide enhances radiotherapy efficacy against hepatocellular carcinoma by upregulating PD-L1 degradation via the ubiquitin-proteasome pathway

Radiation therapy is a primary treatment for hepatocellular carcinoma (HCC), but its effectiveness can be diminished by various factors. The over-expression of PD-L1 has been identified as a critical reason for radiotherapy resistance. Previous studies have demonstrated that nifuroxazide exerts antitumor activity by damaging the Stat3 pathway, but its efficacy against PD-L1 has remained unclear. In this study, we investigated whether nifuroxazide could enhance the efficacy of radiotherapy in HCC by reducing PD-L1 expression. Our results showed that nifuroxazide significantly increased the sensitivity of tumor cells to radiation therapy by inhibiting cell proliferation and migration while increasing apoptosis in vitro. Additionally, nifuroxazide attenuated the up-regulation of PD-L1 expression induced by irradiation, which may be associated with increased degradation of PD-L1 through the ubiquitination-proteasome pathway. Furthermore, nifuroxazide greatly enhanced the efficacy of radiation therapy in H22-bearing mice by inhibiting tumor growth, improving survival, boosting the activation of T lymphocytes, and decelerating the ratios of Treg cells in spleens. Importantly, nifuroxazide limited the increased expression of PD-L1 in tumor tissues induced by radiation therapy. This study confirms, for the first time, that nifuroxazide can augment PD-L1 degradation to improve the efficacy of radiation therapy in HCC-bearing mice.

immunology↗

bioRxiv2023

Structural basis of closed groove scrambling by a TMEM16 protein

Activation of Ca2+-dependent TMEM16 scramblases induces the externalization of phosphatidylserine, a key molecule in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove, and that Ca2+ dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements involved in groove opening and of how lipids reorganize outside the closed groove remain unknown. Using cryogenic electron microscopy, we directly visualize how lipids associate at the closed groove of Ca2+-bound nhTMEM16 in nanodiscs. Functional experiments pinpoint the lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two {pi}-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryoEM structure determination.

biochemistry↗

bioRxiv2023

Melatonin alleviates valproic acid-induced neural tube defects by modulating Src/PI3K/ERK signaling and oxidative stress

Neural tube defects (NTDs) represent a developmental disorder of the nervous system that can lead to significant disability in children and impose substantial social burdens. Valproic acid (VPA), a widely prescribed first-line antiepileptic drug for epilepsy and various neurological conditions, has been associated with a fourfold increase in the risk of NTDs when used during pregnancy. Consequently, urgent efforts are required to identify innovative prevention and treatment approaches for VPA-induced NTDs. Studies have demonstrated that the disruption in the delicate balance between cell proliferation and apoptosis is a crucial factor contributing to NTDs induced by VPA. Encouragingly, our current data reveal that melatonin (MT) exerts significant inhibition on apoptosis while promoting the restoration of neuroepithelial cells proliferation impaired by VPA. Moreover, further investigations demonstrate that MT substantially reduces the incidence of neural tube malformations resulting from VPA exposure, primarily achieved by suppressing apoptosis through the modulation of intracellular reactive oxygen species levels. In addition, the Src/PI3K/ERK signaling pathway appears to play a pivotal role in VPA-induced NTDs, with a significant inhibition observed in the affected samples. Notably, MT treatment successfully reinstates the Src/PI3K/ERK signals, thereby offering a potential underlying mechanism for MTs protective effects against VPA-induced NTDs. In summary, our current study substantiates the considerable protective potential of MT in mitigating VPA-triggered NTDs, thereby offering valuable strategies for the clinical management of VPA-related birth defects.

developmental biology↗

bioRxiv2023

Sorcin-STAT3-Serpin E1/CCL5 axis can be the trigger of pancreatic cancer-associated new-onset diabetes

A rise in blood glucose is the early warning signs of underlying pancreatic cancer (PC), which could be the externalization of genetic events in PC progression. But there is still a vacancy in the field of mechanism research on pancreatic cancer-associated new-onset diabetes (PCAND). Using siRNA-mediated gene knockdown in vitro, we made MIN6 cells incubated with conditioned media from transfected PC cells, and detected its response. Immunological techniques were used to explore the interaction between sorcin and STAT3. Human cytokine array was performed to explore the inflammatory cytokines downstream of sorcin. In the present study, we have identified a PCAND driver gene SRI. In PC cells, sorcin and STAT3 form a positive feedback loop to enhance the transcription of serpin E1 and CCL5, which can impair nearby islet {beta}-cells, likely by activating the p38 pathway. In 88 biopsies, expression of sorcin was elevated in PC tissues, especially so in PCAND patient samples. Furthermore, clinical-SRI gene combination model can better distinguish PCAND from T2DM, and serpin E1 level is higher in the peripheral blood samples from PCAND than T2DM. Thus, Sorcin could be the key driver in PCAND, and figuring out sorcin-STAT3-serpin E1/CCL5 signaling axis can help us better understand the pathogenesis of PCAND and identify potential biomarkers. Statement of significanceThis study mapped out a novel sorcin-STAT3-Serpin E1/CCL5 signaling axis in pancreatic cancer cells, which explains how early pre-symptomatic pancreatic cancer may coincide with new-onset diabetes in some patients.

cancer biology↗

bioRxiv2023

Widespread impact of immunoglobulin V gene allelic polymorphisms on antibody reactivity

The ability of human immune system to generate antibodies to any given antigen can be strongly influenced by immunoglobulin V gene (IGV) allelic polymorphisms. However, previous studies have provided only a limited number of examples. Therefore, the prevalence of this phenomenon has been unclear. By analyzing >1,000 publicly available antibody-antigen structures, we show that many IGV allelic polymorphisms in antibody paratopes are determinants for antibody binding activity. Biolayer interferometry experiment further demonstrates that paratope allelic mutations on both heavy and light chain often abolish antibody binding. We also illustrate the importance of minor IGV allelic variants with low frequency in several broadly neutralizing antibodies to SARS-CoV-2 and influenza virus. Overall, this study not only highlights the pervasive impact of IGV allelic polymorphisms on antibody binding, but also provides mechanistic insights into the variability of antibody repertoires across individuals, which in turn have important implications for vaccine development and antibody discovery.

immunology↗

bioRxiv2023

A deep learning-based drug repurposing screening and validation for anti-SARS-CoV-2 compounds by targeting the cell entry mechanism

The recent outbreak of Corona Virus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a severe threat to the global public health and economy, however, effective drugs to treat COVID-19 are still lacking. Here, we employ a deep learning-based drug repositioning strategy to systematically screen potential anti-SARS-CoV-2 drug candidates that target the cell entry mechanism of SARS-CoV-2 virus from 2,635 FDA-approved drugs and 1,062 active ingredients from Traditional Chinese Medicine herbs. In silico molecular docking analysis validates the interactions between the top compounds and host receptors or viral spike proteins. Using a SARS-CoV-2 pseudovirus system, we further identify several drug candidates including Fostamatinib, Linagliptin, Lysergol and Sophoridine that can effectively block the cell entry of SARS-CoV-2 variants into human lung cells even at a nanomolar scale. These efforts not only illuminate the feasibility of applying deep learning-based drug repositioning for antiviral agents by targeting a specified mechanism, but also provide a valuable resource of promising drug candidates or lead compounds to treat COVID-19.

microbiology↗

bioRxiv2023

Projectome-defined subtypes and modular intra-hypothalamic subnetworks of peptidergic neurons

The hypothalamus plays a vital role in coordinating essential neuroendocrine, autonomic, and somatomotor responses for survival and reproduction. While previous studies have explored population-level projections of hypothalamic neurons, the specific innervation patterns of individual hypothalamic axons remain unclear. To understand the organization of hypothalamic axon projections, we conducted a comprehensive reconstruction of single-cell projectomes from 7,180 mouse hypothalamic neurons expressing specific neuropeptides. Our analysis identified 31 distinct subtypes based on projectome-defined characteristics, with many exhibiting long-range axon collateral projections to multiple brain regions. Notably, these subtypes selectively targeted specific subdomains within downstream areas, either unilaterally or bilaterally. Furthermore, we observed that individual peptidergic neuronal types encompassed multiple projectome-defined subtypes, explaining their diverse functional roles. Additionally, by examining intra-hypothalamic axon projections, we uncovered six modular subnetworks characterized by enriched intramodular connections and distinct preferences for downstream targets. This modular organization of the intra-hypothalamic network likely contributes to the coordinated organization of hypothalamic outputs. In summary, our comprehensive projectome analysis reveals the organizational principles governing hypothalamic axon projections, providing a framework for understanding the neural circuit mechanisms underlying the diverse and coordinated functions of the hypothalamus.

neuroscience↗

bioRxiv2023

Broadly neutralizing antibodies targeting a conserved silent face of spike RBD resist extreme SARS-CoV-2 antigenic drift

Developing broad coronavirus vaccines requires identifying and understanding the molecular basis of broadly neutralizing antibody (bnAb) spike sites. In our previous work, we identified sarbecovirus spike RBD group 1 and 2 bnAbs. We have now shown that many of these bnAbs can still neutralize highly mutated SARS-CoV-2 variants, including the XBB.1.5. Structural studies revealed that group 1 bnAbs use recurrent germline encoded CDRH3 features to interact with a conserved RBD region that overlaps with class 4 bnAb site. Group 2 bnAbs recognize a less well-characterized "site V" on the RBD and destabilize spike trimer. The site V has remained largely unchanged in SARS-CoV- 2 variants and is highly conserved across diverse sarbecoviruses, making it a promising target for broad coronavirus vaccine development. Our findings suggest that targeted vaccine strategies may be needed to induce effective B cell responses to escape resistant subdominant spike RBD bnAb sites.

immunology↗

bioRxiv2023

A novel cell division protein critical for the assembly of the bacterial divisome

Identification of unique essential bacterial genes is important for not only the understanding of their cell biology but also the development of new antimicrobials. Here we report a previously unrecognized core component of the Acinetobacter baumannii divisome. Our results reveal that the protein, termed Aeg1 interacts with multiple cell division proteins, including FtsN, which is required for components of the divisome to localize to the midcell. We demonstrate that the FtsAE202K and FtsBE65A mutants effectively bypassed the need of Aeg1 by A. baumannii, as did the activation variants FtsWM254I and FtsWS274G. Our results suggest that Aeg1 is a cell division protein that arrives at the division site to initiate cell division by recruiting FtsN, which activates FtsQLB and FtsA to induce the septal peptidoglycan synthase FtsWI. The discovery of the new essential cell division protein has provided a new target for the development of antibacterial agents.

biochemistry↗

bioRxiv2023

Cortical gradient perturbation in attention deficit hyperactivity disorder correlates with neurotransmitter-, cell type-specific and chromosome- transcriptomic signatures

Neurofunctional dysregulations in spatially discrete areas or isolated pathways have been suggested as neural markers for attention deficit hyperactivity disorder (ADHD). However, multiscale perspectives into the neurobiological underpins of ADHD spanning multiple biological systems remain sparse. This points to the need of multi-levels of analysis encompassing brain functional organization and its correlation with molecular and cell-specific transcriptional signatures are stressed. Here, we capitalized on diffusion mapping embedding model to derive the functional connectome gradient, and deployed multivariate partial least square (PLS) method to uncover the enrichment of neurotransmitomic, cellular and chromosomal connectome-transcriptional signatures of ADHD. Compared to typical control, ADHD children presented connectopic cortical perturbations in lateral orbito-frontal and superior temporal regions, which had also been validated in another independent sample. This gradient-derived variants in ADHD further aligned spatially with distributions of GABAA/BZ and 5-HT2A receptors and co-varied with genetic transcriptional expression. Cognitive decoding and gene-expression annotation showed the correlates of these variants in memory, emotional regulation and spatial attention. Moreover, the gradient-derived transcriptional signatures of ADHD exhibited enriched expression of oligodendrocyte precursors and endothelial cells, and were mainly involved as variants of chromosome 18, 19 and X. In conclusion, our findings bridged in-vivo neuroimging assessed functional brain organization patterns to a multi-level molecular pathway in ADHD, possibly shedding light on the interrelation of biological systems that may coalesce to the emergence of this disorder.

neuroscience↗

bioRxiv2023

On-grid purification of electron microscopy samples via a 3D-printed flow-cell

While recent advances in cryo-EM, coupled with single particle analysis, have the potential to allow structure determination in a near-native state from vanishingly few individual particles, this vision has yet to be realised in practise. Requirements for particle numbers that currently far exceed the theoretical lower limits, challenges with the practicalities of achieving high concentrations for difficult-to-produce samples, and inadequate sample-dependent imaging conditions, all result in significant bottlenecks preventing routine structure determination using cryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks by developing affinity purification of samples on-grid; at once obviating the need to produce large amounts of protein, as well as more directly controlling the variable, and sample-dependent, process of grid preparation. In this proof-of-concept study, we demonstrate a further practical step towards this paradigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification from raw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-cell device can be interfaced directly with routinely-used laboratory equipment such as liquid chromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16") connectors, and can be used to allow binding of samples to affinity grids in a controlled environment prior to the extensive washing required to remove impurities. Furthermore, by designing a device which can be 3D printed and coupled to routinely used laboratory equipment, we hope to increase the accessibility of the techniques presented herein to researchers working towards single-particle macromolecular structures.

biophysics↗

bioRxiv2023

Empowering Beginners in Bioinformatics with ChatGPT

The impressive conversational and programming abilities of ChatGPT make it an attractive tool for facilitating the education of bioinformatics data analysis for beginners. In this study, we proposed an iterative model to fine-tune instructions for guiding a ChatGPT in generating code for bioinformatics data analysis tasks. We demonstrated the feasibility of the model by applying it to various bioinformatics topics. Additionally, we discussed practical considerations and limitations regarding the use of the model in chatbot-aided bioinformatics education.

bioinformatics↗