Biology subjects

Lin, H.

Publications and source records attributed to Lin, H..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

The emerging antibiotic resistance requires the development of new antibiotics working on novel bacterial targets. Here, we reported an antibiotic discovery workflow by combining the cysteine-reactive compound library phenotypic screening with activity-based protein profiling, which enables the rapid identification of lead compounds as well as new druggable targets in pathogens. Compounds featuring chloromethyl ketone scaffolds exhibited a notably high hit rate against both gram-negative and gram-positive bacterial strains, but not the more commonly used warheads such as acrylamide or chloroacetamide. Target identification of the lead compound, 10-F05, revealed that its primary targets in S. flexneri are FabH Cys112 and MiaA Cys273. We validated the target relevance through biochemical and genetic interactions. Mechanistic studies revealed modification of MiaA by 10-F05 impair substrate tRNA binding, leading to decreased bacterial stress resistance and virulence. Our findings underscore chloromethyl ketone as a novel antibacterial warhead in covalent antibiotic design. The study showcases that combining covalent compound library phenotypic screening with chemoproteomics is an efficient way to identify new drug targets as well as lead compounds, with the potential to open new research directions in drug discovery and chemical biology. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/576576v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8d6420org.highwire.dtl.DTLVardef@a89d44org.highwire.dtl.DTLVardef@1ed0ae2org.highwire.dtl.DTLVardef@1659d3e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Synaptotagmin 7 C2 domains induce membrane curvature stress via electrostatic interactions and the wedge mechanism

0.Synaptotagmin 7 (Syt-7) is part of the synaptotagmin protein family that regulates exocytotic lipid membrane fusion. Among the family, Syt-7 stands out by its membrane binding strength and stabilization of long-lived membrane fusion pores. Given that Syt-7 vesicles form long-lived fusion pores, we hypothesize that its interactions with the membrane stabilize the specific curvatures, thicknesses, and lipid compositions that support a metastable fusion pore. Using all-atom molecular dynamics simulations and FRET-based assays of Syt-7s membrane-binding C2 domains (C2A and C2B), we found that Syt-7 C2 domains sequester anionic lipids, are sensitive to cholesterol, thin membranes, and generate lipid membrane curvature by two competing, but related mechanisms. First, Syt-7 forms strong electrostatic contacts with the membrane, generating negative curvature stress. Second, Syt-7s calcium binding loops embed in the membrane surface, acting as a wedge to thin the membrane and induce positive curvature stress. These curvature mechanisms are linked by the protein insertion depth as well as the resulting protein tilt. Simplified quantitative models of the curvature-generating mechanisms link simulation observables to their membrane-reshaping effectiveness.

biophysics↗

bioRxiv2024

Genomic signatures of the evolution of loss of egg yolk in parasitoid wasps

Trait regression and loss have occurred repeatedly in numerous lineages throughout evolutionary history in response to changing environments. In parasitoid wasps, a mega-diverse group of hymenopteran insects, loss or reduction of yolk in the egg has been reported in many species. This phenotypic change likely evolved as a response to the shift from ectoparasitism to endoparasitism. However, the genetic basis of this trait and the impact of its loss on genome evolution remain poorly understood. Here, we performed a comparative genomic analysis of 64 hymenopteran insects. The conserved insect yolk protein gene vitellogenin (Vg) underwent five independent loss events in four families, involving 23 of the analyzed endoparasitoid species. Whole-genome alignment suggested that Vg loss occurred during genome rearrangement events. Analysis of Vg receptor gene (VgR) loss, selection, and structural variation in lineages lacking Vg demonstrated functional biases in the patterns of gene loss. The ectoparasitism to endoparasitism transition did not appear to be the primary driver of Vg loss or the subsequent VgR evolution. A number of parallel and convergent genomic changes were observed between Vg-loss lineages, including gene family evolution and selection of genes related to transport, development, and metabolism. These changes may have facilitated embryonic development without the yolk in these lineages. Together, these findings reveal the genomic basis underlying a unique trait loss in parasitoid wasps. More broadly, this study enhances our understanding of yolk loss evolution outside the class Mammalia, highlighting a potential evolutionary trend arising from the availability of an alternative nutrient source for embryonic development.

evolutionary biology↗

bioRxiv2023

Exposure and resistance to lantibiotics impact microbiota composition and function

The intestinal microbiota is composed of hundreds of distinct microbial species that interact with each other and their mammalian host. Antibiotic exposure dramatically impacts microbiota compositions and leads to acquisition of antibiotic-resistance genes. Lantibiotics are ribosomally synthesized and post-translationally modified peptides produced by some bacterial strains to inhibit the growth of competing bacteria. Nisin A is a lantibiotic produced by Lactococcus lactis that is commonly added to food products to reduce contamination with Gram-positive pathogens. Little is known, however, about lantibiotic-resistance of commensal bacteria inhabiting the human intestine. Herein, we demonstrate that Nisin A administration to mice alters fecal microbiome compositions and the concentration of taurine-conjugated primary bile acids. Lantibiotic Resistance System genes (LRS) are encoded by lantibiotic-producing bacterial strains but, we show, are also prevalent in microbiomes across human cohorts spanning vastly different lifestyles and 5 continents. Bacterial strains encoding LRS have enhanced in vivo fitness upon dietary exposure to Nisin A but reduced fitness in the absence of lantibiotic pressure. Differential binding of host derived, secreted IgA contributes to fitness discordance between bacterial strains encoding or lacking LRS. Although LRS are associated with mobile genetic elements, sequence comparisons of LRS encoded by distinct bacterial species suggest they have been long-term components of their respective genomes. Our study reveals the prevalence, abundance and physiologic significance of an underappreciated subset of antimicrobial resistance genes encoded by commensal bacterial species constituting the human gut microbiome, and provides insights that will guide development of microbiome augmenting strategies.

microbiology↗

bioRxiv2023

SntB triggers the antioxidant pathways to regulate development and aflatoxin biosynthesis in Aspergillus flavus

The epigenetic reader SntB was identified as an important transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus. However, the underlying molecular mechanism is still unclear. In this study, sntB gene deletion ({Delta}sntB), complementary (Com-sntB), and HA tag fused to sntB (sntB-HA) strains were constructed by using the homologous recombination method, respectively. Our results revealed that deletion of sntB inhibited the processes of mycelia growth, conidial production, sclerotia formation, aflatoxin synthesis, and ability to colonize host, and the defective phenotype of knockout strain {Delta}sntB could be restored in its complementary strain Com-sntB. Chromatin immunoprecipitation sequencing (ChIP-seq) of sntB-HA and WT, and RNA sequencing (RNA-seq) of {Delta}sntB and WT strains revealed that SntB played key roles in oxidative stress response of A. flavus. The function of catC gene (encode a catalase) was further analyzed based on the integration results of ChIP-seq and RNA-seq. In {Delta}sntB strain, the relative expression level of catC was significantly higher than in WT strain, while a secretory lipase encoding gene (G4B84_008359) was down-regulated. Under the stress of oxidant menadione sodium bisulfite (MSB), the deletion of sntB obvious down-regulated the expression level of catC. After deletion of catC gene, the mycelia growth, conidial production, and sclerotia formation were inhibited, while ROS level and aflatoxin production were increased compared to the WT strain. Results also showed that the inhibition rate of MSB to {Delta}catC strain was significantly lower than that of WT group and AFB1 yield of the {Delta}catC strain was significantly decreased than that of WT strain under the stress of MSB. Our study revealed the potential machinery that SntB regulated fungal morphogenesis, mycotoxin anabolism, and fungal virulence through the axle of from SntB to fungal virulence and mycotoxin bio-synthesis, i.e. H3K36me3 modification-SntB-Peroxisomes-Lipid hydrolysis-fungal virulence and mycotoxin bio-synthesis. The results of this study shed light into the SntB mediated transcript regulation pathways of fungal mycotoxin anabolism and virulence, which provided potential strategy for control the contamination of A. flavus and its aflatoxins.

pharmacology and toxicology↗

bioRxiv2023

An evolution-based framework for describing human gut bacteria

The human gut microbiome contains many bacterial strains of the same species ( strain-level variants). Describing strains in a biologically meaningful way rather than purely taxonomically is an important goal but challenging due to the genetic complexity of strain-level variation. Here, we measured patterns of co-evolution across >7,000 strains spanning the bacterial tree-of-life. Using these patterns as a prior for studying hundreds of gut commensal strains that we isolated, sequenced, and metabolically profiled revealed widespread structure beneath the phylogenetic level of species. Defining strains by their co-evolutionary signatures enabled predicting their metabolic phenotypes and engineering consortia from strain genome content alone. Our findings demonstrate a biologically relevant organization to strain-level variation and motivate a new schema for describing bacterial strains based on their evolutionary history. One Sentence SummaryDescribing bacterial strains in the human gut by a statistical model that captures their evolutionary history provides insight into their biology.

systems biology↗

bioRxiv2023

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs

Circular RNAs (circRNAs) are a class of single-stranded, covalently closed RNA that contain a unique back-splice junction (bsj) sequence created by the ligation of their 5 and 3 ends via spliceosome-catalyzed back-splicing. A key step in illuminating the cellular roles of specific circRNAs is via increasing their expression. This is frequently done by transfecting cells with plasmid DNA containing cloned exons from which the circRNA is transcribed, flanked by sequences that promote back-splicing. We observed that commonly used plasmids lead to the production of circRNAs with molecular scars at the circRNA bsj. Stepwise redesign of the cloning vector corrected this problem, ensuring bona fide circRNAs are produced with their natural bsj at high efficiency. The fidelity of circRNAs produced from this new construct was validated by RNA sequencing and also functionally validated. To increase the utility of this modified resource for expressing circRNA, we developed an expanded set of vectors incorporating this design that (i) enables selection with a variety of antibiotics and fluorescent proteins, (ii) employs a range of promoters varying in promoter strength and (iii) generated a complementary set of lentiviral plasmids for difficult-to-transfect cells. These resources provide a novel and versatile toolkit for high-efficiency and scarless overexpression of circular RNAs that fulfill a critical need for the investigation of circRNA function.

molecular biology↗

bioRxiv2023

NLRP3 Cys126 palmitoylation by ZDHHC7 Promotes Inflammasome Activation

NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) mediates inflammasome activation in response to multiple pathogen and damage-associated molecular patterns in macrophages. Hyperactivation of NLRP3 inflammasome contributes to many human chronic inflammatory diseases. Understanding how NLRP3 inflammasome is regulated can potentially provide new strategies to treat inflammatory diseases. Here, we demonstrated that NLRP3 is palmitoylated on Cys126 by palmitoyl-acyltransferase ZDHHC7 in macrophages, which is critical for NLRP3-mediated inflammasome activation. Perturbating NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation, diminishes NLRP3 activation and the consequential Caspase-1 and Gasdermin D (GSDMD) cleavage, and IL-1{beta} and IL-18 secretion in mouse primary macrophages and human macrophages. Furthermore, NLRP3 Cys126 palmitoylation is vital for inflammasome activation in vivo, as Zdhhc7 knockout, pharmacological inhibition, or NLRP3 C126A mutation protects mice from LPS-induced endotoxic shock and monosodium urate (MSU)-induced peritonitis. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN (dTGN), which is indispensable for the recruitment and oligomerization of adaptor protein ASC after inflammasome activation. The activation of NLRP3 by ZDHHC7-mediated Cys126 palmitoylation is different from the previously reported inhibitory effect by ZDHHC12-mediated Cys841 palmitoylation, highlighting the versatile regulatory roles of S-palmitoylation. Therefore, our study identifies a new regulatory mechanism of NLRP3 activation and suggests targeting ZDHHC7 or NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders. HighlightsO_LINLRP3 Cys126 is palmitoylated by ZDHHC7. C_LIO_LIZDHHC7 promotes NLRP3 activation in macrophages, which can be inhibited by ZDHHCs inhibitors, 2-bromopalmitate and MY-D4. C_LIO_LICys126 palmitoylation of NLRP3 is critical for NLRP3 activation. C_LIO_LINLRP3 TGN/dTGN localization depends on ZDHHC7-mediated Cys126 palmitoylation, which is crucial for ASC recruitment and inflammasome assembly. C_LIO_LINLRP3 inflammasome activation by ZDHHC7 differs from ZDHHC12-mediated NLRP3 inhibition. C_LI

immunology↗

bioRxiv2023

LEAFY interacts with WHEAT ORTHOLOG OF APO1 to regulate spikelet number per spike in wheat

In wheat, the transition of the inflorescence meristem to a terminal spikelet (IM[->]TS) determines the spikelet number per spike (SNS), an important yield component. In this study, we demonstrate that the plant-specific transcription factor LEAFY (LFY) physically and genetically interacts with WHEAT ORTHOLOG OF APO1 (WAPO1) to regulate SNS and floret development. Loss-of-function mutations in either or both genes result in significant and similar reductions in SNS, as a result of a reduction in the rate of spikelet meristems formation per day. SNS is also modulated by significant genetic interactions between LFY and SQUAMOSA MADS-box genes VRN1 and FUL2, which promote the IM[->]TS transition. Single-molecule fluorescence in-situ hybridization revealed a down-regulation of LFY and up-regulation of the SQUAMOSA MADS-box genes in the distal part of the developing spike during the IM[->]TS transition, supporting their opposite roles in the regulation of SNS in wheat. Concurrently, the overlap of LFY and WAPO1 transcription domains in the developing spikelets contributes to normal floret development. Understanding the genetic network regulating SNS is a necessary first step to engineer this important agronomic trait. SUMMARY STATEMENTThe plant specific transcription factor LEAFY plays an important role in the regulation of the number of spikelets per spike in wheat.

plant biology↗

bioRxiv2023

Dysregulated neuroimmune interactions and sustained type I interferon signaling after human immunodeficiency virus type 1 infection of human iPSC derived microglia and cerebral organoids

Human immunodeficiency virus type-1 (HIV-1) associated neurocognitive disorder (HAND) affects up to half of HIV-1 positive patients with long term neurological consequences, including dementia. There are no effective therapeutics for HAND because the pathophysiology of HIV-1 induced glial and neuronal functional deficits in humans remains enigmatic. To bridge this knowledge gap, we established a model simulating HIV-1 infection in the central nervous system using human induced pluripotent stem cell (iPSC) derived microglia combined with sliced neocortical organoids. Upon incubation with two replication-competent macrophage-tropic HIV-1 strains (JRFL and YU2), we observed that microglia not only became productively infected but also exhibited inflammatory activation. RNA sequencing revealed a significant and sustained activation of type I interferon signaling pathways. Incorporating microglia into sliced neocortical organoids extended the effects of aberrant type I interferon signaling in a human neural context. Collectively, our results illuminate the role of persistent type I interferon signaling in HIV-1 infected microglial in a human neural model, suggesting its potential significance in the pathogenesis of HAND. Highlights of the workO_LIHIV-1 productively infects iPSC-derived microglia and triggers inflammatory activation. C_LIO_LIHIV-1 infection of microglia results in sustained type I interferon signaling. C_LIO_LIMicroglia infected by HIV-1 incorporate into sliced neocortical organoids with persistent type I interferon signaling and disease risk gene expression. C_LI

neuroscience↗

bioRxiv2023

ROCK1 activates mitochondrial fission leading to oxidative stress and muscle atrophy

Chronic kidney disease (CKD) is often associated with protein-energy wasting (PEW), which is characterized by a reduction in muscle mass and strength. Although mitochondrial dysfunction and oxidative stress have been implicated to play a role in the pathogenesis of muscle wasting, the underlying mechanisms remain unclear. In this study, we used transcriptomics, metabolomics analyses and mouse gene manipulating approaches to investigate the effects of mitochondrial plasticity and oxidative stress on muscle wasting in mouse CKD models. Our results showed that the expression of oxidative stress response genes was increased, and that of oxidative phosphorylation genes was decreased in the muscles of mice with CKD. This was accompanied by reduced oxygen consumption rates, decreased levels of mitochondrial electron transport chain proteins, and increased cellular oxidative damage. Excessive mitochondrial fission was also observed, and we found that the activation of ROCK1 was responsible for this process. Inducible expression of muscle-specific constitutively active ROCK1 (mROCK1ca) exacerbated mitochondrial fragmentation and muscle wasting in CKD mice. Conversely, ROCK1 depletion (ROCK1-/-) alleviated these phenomena. Mechanistically, ROCK1 activation promoted the recruitment of Drp1 to mitochondria, thereby facilitating fragmentation. Notably, the pharmacological inhibition of ROCK1 mitigated muscle wasting by suppressing mitochondrial fission and oxidative stress. Our findings demonstrate that ROCK1 participates in CKD-induced muscle wasting by promoting mitochondrial fission and oxidative stress, and pharmacological suppression of ROCK1 could be a therapeutic strategy for combating muscle wasting in CKD conditions. Translational StatementProtein-energy wasting (PEW) is a prevalent issue among patients with chronic kidney disease (CKD) and is characterized by the loss of muscle mass. Our research uncovers a critical role that ROCK1 activation plays in muscle wasting induced by CKD. We found that ROCK1 is instrumental in causing mitochondrial fission, which leads to increased oxidative stress in muscle cells. By employing a pharmacological inhibitor, hydroxyfasudil, we were able to effectively curb ROCK1 activity, which in turn mitigated muscle wasting by reducing both mitochondrial fission and oxidative stress. These findings suggest that pharmacological inhibition of ROCK1 presents a promising therapeutic strategy for combating the muscle wasting associated with CKD.

cell biology↗

bioRxiv2023

High-throughput single-cell sorting by stimulated Raman-activated cell ejection

Single-cell sorting is essential to explore cellular heterogeneity in biology and medicine. Recently developed Raman-activated cell sorting (RACS) circumvents the limitations of fluorescence-activated cell sorting, such as the cytotoxicity of labels. However, the sorting throughputs of all forms of RACS are limited by the intrinsically small cross-section of spontaneous Raman scattering. Here, we report a stimulated Raman-activated cell ejection (S-RACE) platform that enables high-throughput single-cell sorting based on high-resolution multi-channel stimulated Raman chemical imaging, in situ image decomposition, and laser-induced cell ejection. The performance of this platform was illustrated by sorting a mixture of 1 m polymer beads, where 95% yield, 98% purity, and 14 events per second throughput were achieved. Notably, our platform allows live cell ejection, allowing for the growth of single colonies of bacteria and fungi after sorting. To further illustrate the chemical selectivity, lipid-rich Rhodotorula glutinis cells were successfully sorted from a mixture with Saccharomyces cerevisiae, confirmed by downstream quantitative PCR. Furthermore, by integrating a closed-loop feedback control circuit into the system, we realized real-time single-cell imaging and sorting, and applied this method to precisely eject regions of interest from a rat brain tissue section. The reported S-RACE platform opens exciting opportunities for a wide range of single-cell applications in biology and medicine. Significance statementImage-guided single-cell sorting is a potent tool in diverse biological applications. Current microfluidic cell sorting methods encounter challenges in handling smaller cells and are not applicable to tissue sections. To address these challenges, we have developed a stimulated Raman-activated cell ejection (S-RACE) platform, which is the first demonstration of single-cell ejection coupled with coherent Raman scattering. S-RACE allows label-free chemical imaging guided cell sorting through multispectral stimulated Raman scattering (SRS) imaging, on-the-fly image analysis, and laser-induced cell ejection. Versatile applications of S-RACE to a wide range of samples, such as polymer particles, single-live bacteria, single-live fungus, and tissue sections, are demonstrated.

bioengineering↗

bioRxiv2023

Circulating senescent myeloid cells drive blood brain barrier breakdown and neurodegeneration

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langerhans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors expressing MAPK activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some patients with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we show that LCH-ND is caused by myeloid cells that are clonal with peripheral LCH cells. We discovered that circulating BRAFV600E+ myeloid cells cause the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiate into senescent, inflammatory CD11a+ macrophages that accumulate in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced parenchymal infiltration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent novel and targetable mechanisms of ND.

cell biology↗

bioRxiv2023

TRIP13 localizes to synapsed chromosomes and functions as a dosage-sensitive regulator of meiosis

Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. The AAA+ ATPase TRIP13 and its orthologue Pch2 are instrumental in remodeling HORMA domain proteins. Meiosis-specific HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed chromosome homologues. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. Trip13-null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These findings confirm the previously reported phenotypes of the Trip13 hypomorph alleles. Trip13 heterozygous (Trip13+/-) mice also exhibit meiotic defects that are less severe than the Trip13-null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. The N- or C-terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres in knockin mice. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon chromosome synapsis in diverse organisms.

cell biology↗

bioRxiv2023

Structure-Activity Relationship Studies of Novel Gut-derived Lantibiotics Against Human Gut Commensals

Recent advances in sequencing techniques unveiled the vast potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) encoded in microbiomes. Class I lantibiotics such as nisin A, widely used as a food preservative, have been investigated for their efficacy in killing pathogens. However, the impact of nisin and nisin-like class I lantibiotics on commensal bacteria residing in the human gut remains unclear. Here, we report six gut-derived class I lantibiotics that are close homologs of nisin, four of which are novel. We applied an improved lantibiotic expression platform to produce and purify these lantibiotics for antimicrobial assays. We determined their minimal inhibitory concentration (MIC) against both Gram-positive human pathogens and gut commensals, and profiled the lantibiotic resistance genes in these pathogens and commensals. SAR studies with variants revealed key regions and residues that impact their antimicrobial properties. Our characterization and SAR studies of nisin-like lantibiotics against both pathogens and human gut commensals could shed light on the future development of lantibiotic-based therapeutics and food preservatives.

bioengineering↗

bioRxiv2023

Whole Genome Sequencing Based Analysis of Inflammation Biomarkers in the Trans-Omics for Precision Medicine (TOPMed) Consortium

Inflammation biomarkers can provide valuable insight into the role of inflammatory processes in many diseases and conditions. Sequencing based analyses of such biomarkers can also serve as an exemplar of the genetic architecture of quantitative traits. To evaluate the biological insight, which can be provided by a multi-ancestry, whole-genome based association study, we performed a comprehensive analysis of 21 inflammation biomarkers from up to 38,465 individuals with whole-genome sequencing from the Trans-Omics for Precision Medicine (TOPMed) program. We identified 22 distinct single-variant associations across 6 traits - E-selectin, intercellular adhesion molecule 1, interleukin-6, lipoprotein-associated phospholipase A2 activity and mass, and P-selectin - that remained significant after conditioning on previously identified associations for these inflammatory biomarkers. We further expanded upon known biomarker associations by pairing the single-variant analysis with a rare variant set-based analysis that further identified 19 significant rare variant set-based associations with 5 traits. These signals were distinct from both significant single variant association signals within TOPMed and genetic signals observed in prior studies, demonstrating the complementary value of performing both single and rare variant analyses when analyzing quantitative traits. We also confirm several previously reported signals from semi-quantitative proteomics platforms. Many of these signals demonstrate the extensive allelic heterogeneity and ancestry-differentiated variant-trait associations common for inflammation biomarkers, a characteristic we hypothesize will be increasingly observed with well-powered, large-scale analyses of complex traits.

genetics↗

bioRxiv2023

The 14-3-3/spastin pathway coordinates neurite regeneration and recovery of spinal cord injury by targeting microtubule dynamics

Axon regeneration is abortive in the central nervous system following injury. Orchestrating microtubule dynamics has emerged as a promising approach to improve axonal regeneration. The microtubule severing enzyme spastin is essential for axonal development and regeneration through remodeling of microtubule arrangement. To date, however, little is known regarding the mechanisms underlying spastin action in neural regeneration after spinal cord injury. Here, we use glutathione transferase pulldown and immunoprecipitation assays to demonstrate that 14-3-3 interacts with spastin, both in vivo and in vitro, via spastin Ser233 phosphorylation. Moreover, we show that 14-3-3 protects spastin from degradation by inhibiting the ubiquitination pathway and upregulates the spastin-dependent severing ability. Furthermore, the 14-3-3 agonist Fusicoccin (FC-A) promotes neurite outgrowth and regeneration in vitro which needs spastin activation. Western blot and immunofluorescence results revealed that 14-3-3 protein is upregulated in the neuronal compartment after spinal cord injury in vivo. In addition, administration of FC-A not only promotes locomotor recovery, but also nerve regeneration following spinal cord injury in both contusion and lateral hemisection models; however, application of spastin inhibitor spastazoline successfully reverses these phenomena. Taken together, these results indicate that 14-3-3 is a molecular switch that regulates spastin protein levels, and the small molecule 14-3-3 agonist FC-A effectively mediates the recovery of spinal cord injury in mice which requires spastin participation. HighlightsO_LIThe formation of the 14-3-3 and spastin protein complex requires phosphorylation of spastin at the S233 site. C_LIO_LI14-3-3 is involved in the phosphorylation-ubiquitination crosstalk of spastin, thus impacting the protein stability. C_LIO_LI14-3-3 agonists Fusicoccin-A can facilitate the repair of spinal cord injury in mice. C_LIO_LIActivation of spastin function is a prerequisite for nerve regeneration and recovery of spinal cord injury in mice. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/550600v3_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@ebabb6org.highwire.dtl.DTLVardef@5651caorg.highwire.dtl.DTLVardef@113df6borg.highwire.dtl.DTLVardef@1b0ea5a_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

A Conserved Electrostatic Membrane-Binding Surface in Synaptotagmin-Like Proteins Revealed Using Molecular Phylogenetic Analysis and Homology Modeling

Protein structure prediction has emerged as a core technology for understanding biomolecules and their interactions. Here, we combine homology-based structure prediction with molecular phylogenetic analysis to study the evolution of electrostatic membrane binding among vertebrate synaptotagmin-like proteins (Slps). Slp family proteins play key roles in the membrane trafficking of large dense-core secretory vesicles. Our previous experimental and computational study found that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to anionic phospholipids in the cytoplasmic leaflet of the plasma membrane through a large positively charged protein surface centered on a cluster of phosphoinositide-binding lysine residues. Because this surface contributes greatly to Slp-4 C2A domain membrane binding, we hypothesized that the net charge on the surface might be evolutionarily conserved. To test this hypothesis, the known C2A sequences of Slp-4 among vertebrates were organized by class (from mammalia to pisces) using molecular phylogenetic analysis. Consensus sequences for each class were then identified and used to generate homology structures, from which Poisson-Boltzmann electrostatic potentials were calculated. For comparison, homology structures and electrostatic potentials were also calculated for the five human Slp protein family members. The results demonstrate that the charge on the membrane-binding surface is highly conserved throughout the evolution of Slp-4, and more highly conserved than many individual residues among the human Slp family paralogs. Such molecular phylogenetic-driven computational analysis can help to describe the evolution of electrostatic interactions between proteins and membranes which are crucial for their function. Impact statementThe interior surface of eukaryotic plasma membranes is negatively charged, and many proteins that bind to it have correspondingly evolved a positively charged face. Here, we use techniques from evolutionary biology and computational biophysics to study the conservation of this positively charged surface in an important protein family. We find that the overall surface charge is highly conserved, more so than individual amino acids, consistent with its important role in electrostatic interaction with the membrane.

bioinformatics↗