Biology subjects

Xu, Z.

Publications and source records attributed to Xu, Z..

At least 217 records · Page 12Linked to original sources

bioRxiv2024

An orphan viral genome with controversial evolutionary status sheds light on a distinct lineage of flavi-like virus infecting plants

Advancements in high-throughput sequencing and associated bioinformatics methods have significantly expanded the RNA virus repertoire, including novel viruses with highly divergent genomes encoding orphan proteins that apparently lack homologous sequences. This absence of homologs in routine sequence similarity search complicates their taxonomic classification and raises a fundamental question: Do these orphan viral genomes represent bona fide viruses? In 2022, an orphan viral genome encoding a large polyprotein was identified in alfalfa (Medicago sativa) and named Snake River alfalfa virus (SRAV). Initially, SRAV was proposed to be within the flavi-like lineage of the family Flaviviridae. Subsequently, another research group showed its common occurrence in alfalfa but challenged its taxonomic position, suggesting it belongs to the family Endornaviridae rather than Flaviviridae. In this study, a large-scale analysis of 77 publicly available small RNA datasets indicated that SRAV could be detected across various tissues and cultivars of alfalfa, and has a broad geographical distribution. Moreover, profiles of the SRAV-derived small interfering RNAs (vsiRNAs) exhibited typical characteristics of virus in plant hosts. Through comprehensive evolutionary analysis, we demonstrated that SRAV should be a positive single-stranded RNA (ssRNA) flavi-like virus that infects alfalfa, rather than a member of the double-stranded RNA (dsRNA) of the family Endornaviridae. Our findings suggest that SRAV represents a unique class of plant-hosted flavi-like viruses with unusual genome organization and evolutionary status, differing from previously identified flavi-like viruses documented to infect plants. The latter shows a close evolutionary relationship to flavi-like viruses primarily found in plant-feeding invertebrates and lacks evidence of triggering host RNA interference (RNAi) responses so far. In summary, our study resolves the taxonomic controversy surrounding SRAV and suggests the potential existence of two distinct clades of plant-hosted flavi-like viruses with independent evolutionary origins. Furthermore, our research provides the first evidence of plant-hosted flavi-like viruses triggering the hosts RNAi antiviral response. The widespread occurrence of SRAV underscores its potential ecological significance in alfalfa, a crop of substantial economic importance.

evolutionary biology↗

bioRxiv2024

Detection of Nontoxic BoNT/A Levels in Post-Facial Botox Injection Breastmilk using a Multi-technique Approach

BackgroundThe use of cosmetic Botox (Botulinum Neurotoxin Type A, BoNT/A ) has become increasingly prevalent. Particularly after pregnancy, postpartum depression represents one major factor motivating women to use Botox even during the lactation and breastfeeding period. Currently, there is limited understanding of the impact of Botox on lactation and the potential of its active component passing into breastmilk and affecting the infant. MethodsBreastmilk samples were acquired from five women aged between 28 - 45 through a clinic in Suzhou, Jiangsu, P.R. China. Three sample sets ranged from 1 hour to 1 year after facial Botox treatments (64 U), whereas the remaining two sample sets were from women who never received Botox treatment. BoNT/A concentrations in samples were detected using standard Enzyme-Linked Immunosorbent Assay (ELISA), unreduced and reduced Western Blotting, confocal micro-Raman Spectroscopy, and Mass Spectrometry(LC-MS). FindingsFrom ELISA, breastmilk BoNT/A concentrations peaked at 33.4 pg/mL 4 days after Botox injection. BoNT/A concentrations were highest overall in the first week and around two months after injection. While non-reducing polyacrylamide gel electrophoresis (PAGE) showed a protein band of 150 kDa peaking at 48 hours, reduced SDS-PAGE detected a 100 kDa protein first peaking at 72 hours, then re-emerging after 7 days, respectively, and in line with previous observations by others. Interestingly, micro-Raman spectroscopy indicated additional Raman peaks at 6 hours and 48 hours that were not present in other breastmilk samples which were evaluated in this study. However, no clear indication of BoNT/A was detected in Mass Spectrometry (LC-MS). InterpretationThe amount of BoNT/A in breastmilk peaks around 48 hours, and at 2 months after facial injection. Even over a year after injection, BoNT/A can be detected. However, all quantities of BoNT/A detected in this study are highly likely to be safe for infants. Additionally, our study suggests that alternative methods, besides ELISA, may be utilized for the rapid detection of low concentrations of BoNT/A in body fluid samples. FundingDuke Kunshan University Start-Up funds, Duke Kunshan University Undergraduate Studies Signature Work Research Grant, Synear and Wang-Cai Biochemistry grants, and Kunshan Municipal Government research funding. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/595434v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@fcdd11org.highwire.dtl.DTLVardef@11591daorg.highwire.dtl.DTLVardef@11ab30eorg.highwire.dtl.DTLVardef@1e19311_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBoNT/A was detected in the breastmilk of 3 women after facial Botox injections of 64 U. C_LIO_LIToxin levels peaked in the first week and at around 2 months after injection. C_LIO_LIAll detected levels (up to 33.40 pg/mL) were significantly below the lethal dose for newborns. C_LI

molecular biology↗

bioRxiv2024

Large scale loss-of-function mutations during chicken evolution and domestication

Evolution and domestication are often driven by genetic innovations, yet the role of gene loss remains debated. Here, we present comparative genomic analyses of four indigenous chicken breeds from Yunnan Province, China and a reference red jungle fowl genome (GRCg6a). We identify extensive gene presence-absence variation and large numbers of pseudogenes, revealing highly dynamic gene repertoires among closely related chickens. By reconstructing ancestral gene content, we estimate that the common ancestor harbored at least 21,972 genes, of which 7,993 are dispensable. Each lineage has independently lost thousands of genes through both complete gene loss and pseudogenization. These loss-of-function events are non-random: pseudogenization mutations are biased toward gene termini, frequently fixed in populations, and enriched in specific biological pathways. Notably, patterns of gene loss recapitulate phylogenetic relationships, suggesting that loss-of-function mutations are shaped by selection rather than neutral drift. Analysis of four other chicken genomes assembled using PacBio HiFi reads draws the same conclusion. Thus, our results support a model in which large-scale loss-of-function mutations are a major driver of chicken evolution and domestication, consistent with the "less-is-more" hypothesis of adaptive evolution.

bioinformatics↗

bioRxiv2024

RNA/DNA Binding Protein TDP43 Regulates DNA Mismatch Repair Genes with Implications for Genome Stability

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions, including amyotrophic lateral sclerosis and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

biochemistry↗

bioRxiv2024

Development of a humanized anti-FABP4 monoclonal antibody for treatment of breast cancer

BackgroundBreast cancer, lung cancer, and colorectal cancer are the primary contributors to newly diagnosed cases among women, with breast cancer representing the second highest proportion of the total. The treatment protocols vary depends on different stages of breast cancer, and numerous clinical trials are ongoing based on the data derived from laboratory. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus offering a new target for breast cancer treatment. MethodsWe immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limit dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of S-V9 against FABP4 via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. ResultsHerein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. ConclusionsOur current evidence suggest that targeting FABP4 with humanized monoclonal antibodies represents a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.

cancer biology↗

bioRxiv2024

Unbiased metastatic niche-labeling identifies estrogen receptor-positive macrophages as a barrier of T cell infiltration during bone colonization

Microenvironment niches determine cellular fates of metastatic cancer cells. However, robust and unbiased approaches to identify niche components and their molecular profiles are lacking. We established Sortase A-Based Microenvironment Niche Tagging (SAMENT), which selectively labels cells encountered by cancer cells during metastatic colonization. SAMENT was applied to multiple cancer models colonizing the same organ and the same cancer to different organs. Common metastatic niche features include macrophage enrichment and T cell depletion. Macrophage niches are phenotypically diverse between different organs. In bone, macrophages express the estrogen receptor alpha (ER) and exhibit active ER signaling in male and female hosts. Conditional knockout of Esr1 in macrophages significantly retarded bone colonization by allowing T cell infiltration. ER expression was also discovered in human bone metastases of both genders. Collectively, we identified a unique population of ER+ macrophages in the metastatic niche and functionally tied ER signaling in macrophages to T cell exclusion during metastatic colonization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/593016v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@1cd65b6org.highwire.dtl.DTLVardef@1d099e2org.highwire.dtl.DTLVardef@26cd91org.highwire.dtl.DTLVardef@181f5f3_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LISAMENT is a robust metastatic niche-labeling approach amenable to single-cell omics. C_LIO_LIMetastatic niches are typically enriched with macrophages and depleted of T cells. C_LIO_LIDirect interaction with cancer cells induces ER expression in niche macrophages. C_LIO_LIKnockout of Esr1 in macrophages allows T cell infiltration and retards bone colonization. C_LI

cancer biology↗

bioRxiv2024

Single cell profiling of bone metastasis ecosystems from multiple cancer types reveals convergent and divergent mechanisms of bone colonization.

Bone represents congenial soil for metastatic seeds and is frequently affected by metastasis of multiple cancer types. The histological and molecular characteristics of bone metastases (BMs) are diverse but poorly understood. Herein, we performed single-cell RNA-seq on 34 BMs from 6 cancer types and identified 3 ecosystem archetypes characterized by enrichment of macrophages/osteoclasts (M{varphi}-OC), regulatory/exhausted T cells (Treg-Tex), and monocytes (Mono), respectively. Breast cancer BMs are mostly the M{varphi}-OC archetype driven by the osteolytic vicious cycle, whereas kidney cancers BMs mainly belong to the Treg-Tex archetype that lacks osteoclasts. Lung cancers BMs evenly distributed across all archetypes. Further analyses revealed parallel mechanisms of immunosuppression and bone remodeling. Elevated estrogen signaling distinguishes macrophages in the M{varphi}-OC subtype, which was investigated in a companion study. Together, we elucidated that divergent mechanisms toward bone colonization and that BMs of different origins can adopt the same mechanism through convergent evolution or adaptation. HIGHLIGHTSO_LIAnalyses of bone metastases from 6 cancer types revealed three immune archetypes. C_LIO_LIArchetypes diverge on immune trajectories, and features of tumor and stromal cells. C_LIO_LIDominant cell type in each archetype undergoes convergent evolution. C_LIO_LIRegulatory networks converge on osteoclasts and Tregs to drive archetype formation. C_LI Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/593027v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@82cef6org.highwire.dtl.DTLVardef@1e1b021org.highwire.dtl.DTLVardef@1f2881eorg.highwire.dtl.DTLVardef@1c6874a_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Multi-trait modeling and machine learning discover new markers associated with stem traits in alfalfa

Alfalfa biomass can be fractionated into leaf and stem components. Leaves comprise a protein-rich and highly digestible portion of biomass for ruminant animals, while stems constitute a high fiber and less digestible fraction, representing 50 to 70% of the biomass. However, little attention has focused on stem-related traits, which are a key aspect in improving the nutritional value and intake potential of alfalfa. This study aimed to identify molecular markers associated with four morphological traits in a panel of five populations of alfalfa generated over two cycles of divergent selection based on 16-h and 96-h in vitro neutral detergent fiber digestibility in stems. Phenotypic traits of stem color, presence of stem pith cells, winter standability, and winter injury were modeled using univariate and multivariate spatial mixed linear models (MLM), and the predicted values were used as response variables in genome-wide association studies (GWAS). The alfalfa panel was genotyped using a 3K DArTag SNP markers for the evaluation of the genetic structure and GWAS. Principal component and population structure analyses revealed differentiations between populations selected for high- and low-digestibility. Thirteen molecular markers were significantly associated with stem traits using either univariate or multivariate MLM. Additionally, support vector machine (SVM) and random forest (RF) algorithms were implemented to determine marker importance scores for stem traits and validate the GWAS results. The top-ranked markers from SVM and RF aligned with GWAS findings for solid stem pith, winter standability, and winter injury. Additionally, SVM identified additional markers with high variable importance for solid stem pith and winter injury. Most molecular markers were located in coding regions. These markers can facilitate marker-assisted selection to expedite breeding programs to increase winter hardiness or stem palatability. Author SummaryThe alfalfa stem constitutes a significant portion of forage yield, accounting for 50 to 70% of biomass yield, and influences various traits including plant height, standability, and digestibility. In our study, we identified thirteen molecular markers associated with stem color, presence of stem pith parenchyma, winter standability, and winter injury in a panel of five divergent stem digestibility populations. Multivariate trait modeling enhances the correlation among traits, thereby expanding the number of markers associated via GWAS. Similarly, machine learning algorithms increase the confidence of markers initially identified by GWAS and uncover new candidate regions that could serve as associated markers. Genes harboring markers associated to the four stem traits play roles in plant growth, response to plant injury, or tolerance to cold, underscoring their potential utility in enhancing traits such as cold tolerance and forage quality in alfalfa.

genomics↗

bioRxiv2024

Single-Cell Omics for Transcriptome CHaracterization (SCOTCH): isoform-level characterization of gene expression through long-read single-cell RNA sequencing

Recent advances in long-read single-cell transcriptome sequencing (lr-scRNA-Seq) enable full-length isoform profiling at single-cell resolution. We present SCOTCH (Single-Cell Omics for Transcriptome CHaracterization), an end-to-end, platform-independent pipeline for isoform characterization from lr-scRNA-Seq data, supporting Nanopore and PacBio sequencing as well as 10X Genomics and Parse Biosciences protocols. SCOTCH models isoforms as combinations of non-overlapping sub-exons and applies dynamic thresholding for robust isoform assignment while efficiently address ambiguous mapping issues. By refining sub-exon boundaries through integration of read coverage with existing annotations and applying an iterative clustering strategy to reconstruct novel transcripts, SCOTCH reliably recovers more true novel isoforms than existing splice-graph-based methods, with poly(A)-aware filtering further reducing false-positive structures. Extensive simulations demonstrate improved quantification of known isoforms and enhanced reconstruction of novel isoforms. Analyses of human blood and cerebral organoid datasets across multiple platforms further confirm SCOTCHs ability to resolve cell-type-specific transcriptome profiles and uncover experimentally supported novel isoforms.

bioinformatics↗

bioRxiv2024

A novel statistical framework for meta-analysis of total mediation effect with high dimensional omics mediators in large-scale genomic consortia

Meta-analysis is used to aggregate the effects of interest across multiple studies, while its methodology is largely underexplored in mediation analysis, particularly in estimating the total mediation effect of high-dimensional omics mediators. Large-scale genomic consortia, such as the Trans-Omics for Precision Medicine (TOPMed) program, comprise multiple cohorts with diverse technologies to elucidate the genetic architecture and biological mechanisms underlying complex human traits and diseases. Leveraging the recent established asymptotic standard error of the R-squared (R2)-based mediation effect estimation for high-dimensional omics mediators, we have developed a novel meta-analysis framework requiring only summary statistics and allowing inter-study heterogeneity. Whereas the proposed meta-analysis can uniquely evaluate and account for potential effect heterogeneity across studies due to, for example, varying genomic profiling platforms, our extensive simulations showed that the developed method was more computationally efficient and yielded satisfactory operating characteristics comparable to analysis of the pooled individual-level data when there was no inter-study heterogeneity. We applied the developed method to 8 TOPMed studies with over 5800 participants to estimate the mediation effects of gene expression on age-related variation in systolic blood pressure and sex-related variation in high-density lipoprotein (HDL) cholesterol. The proposed method is available in R package MetaR2M on GitHub.

bioinformatics↗

bioRxiv2024

Expanding the genome information on Bacillales for biosynthetic gene cluster discovery

This study showcases 121 new genomes of spore-forming Bacillales from strains collected globally from a variety of habitats, assembled using Oxford Nanopore long-read and MGI short-read sequences. Bacilli are renowned for their capacity to produce diverse secondary metabolites with use in agriculture, biotechnology, and medicine. These secondary metabolites are encoded within biosynthetic gene clusters (smBGCs). smBGCs have significant research interest due to their potential for the discovery of new bioactivate compounds. Our dataset includes 62 complete genomes, 2 at chromosome level, and 57 at contig level, covering a genomic size range from 3.50 Mb to 7.15 Mb. Phylotaxonomic analysis revealed that these genomes span 16 genera, with 69 of them belonging to Bacillus. A total of 1,176 predicted BGCs were identified by in silico genome mining. We anticipate that the open-access data presented here will expand the reported genomic information of spore-forming Bacillales and facilitate a deeper understanding of the genetic basis of Bacillales potential for secondary metabolite production.

microbiology↗

bioRxiv2024

Nanocluster-antibody-drug conjugates (NADC) as an intravesical precision theranostic agent for interstitial cystitis

Interstitial cystitis (IC) is a chronic inflammatory bladder disorder with recurring severe pain, lacking timely diagnostic and therapeutic options. Here, we propose a unitary theranostic nanocluster-antibody-drug conjugate (NADC) by covalently placing dihydroorotate dehydrogenase inhibitors (DHODHi) and ultrasmall gold quantum clusters (AuQCs) on a nerve growth factor (NGF) antagonistic antibody with simultaneous X-ray computed tomographic and near-infrared fluorescence imaging contrasts. Combining anti-inflammatory effects from all individual components, intravesical NADC specifically homed to mucosal lesions with NGF overexpression in voided bladder and capably alleviated inflammation in chronic, acute, and prophylactic IC models of rats, as revealed by behavioral and pathological evaluations. Transcriptomics unveiled cytokine modulation and concomitant inhibition of perturbed IL-17, NF-{kappa}B, TNF, and JAK-STAT signaling pathways. Notably, NADC indirectly remodeled the host bladder microbiota by differentially varying anti-inflammatory and pro-inflammatory bacteria diversities. Distinct from conventional nanoparticles conjugated with antibodies and drugs, NADC relies on the antibody framework, outperforms clinical standard-of-care agents, and represents an emerging category of precision theranostic agents with translational potential for IC theranostics in clinical practice.

microbiology↗

bioRxiv2024

Onco-Circuit Addiction and Onco-Nutrient mTORC1 Signaling Vulnerability in a Model of Aggressive T Cell Malignancy

How genetic lesions drive cell transformation and whether they can be circumvented without compromising function of non-transformed cells are enduring questions in oncology. Here we show that in mature T cells--in which physiologic clonal proliferation is a cardinal feature-- constitutive MYC transcription and Tsc1 loss in mice modeled aggressive human malignancy by reinforcing each others oncogenic programs. This cooperation was supported by MYC-induced large neutral amino acid transporter chaperone SLC3A2 and dietary leucine, which in synergy with Tsc1 deletion overstimulated mTORC1 to promote mitochondrial fitness and MYC protein overexpression in a positive feedback circuit. A low leucine diet was therapeutic even in late-stage disease but did not hinder T cell immunity to infectious challenge, nor impede T cell transformation driven by constitutive nutrient mTORC1 signaling via Depdc5 loss. Thus, mTORC1 signaling hypersensitivity to leucine as an onco-nutrient enables an onco-circuit, decoupling pathologic from physiologic utilization of nutrient acquisition pathways.

cancer biology↗

bioRxiv2024

HHIP protein interactions in lung cells provide insight into COPD pathogenesis

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The primary causes of COPD are environmental, including cigarette smoking; however, genetic susceptibility also contributes to COPD risk. Genome-Wide Association Studies (GWASes) have revealed more than 80 genetic loci associated with COPD, leading to the identification of multiple COPD GWAS genes. However, the biological relationships between the identified COPD susceptibility genes are largely unknown. Genes associated with a complex disease are often in close network proximity, i.e. their protein products often interact directly with each other and/or similar proteins. In this study, we use affinity purification mass spectrometry (AP-MS) to identify protein interactions with HHIP, a well-established COPD GWAS gene which is part of the sonic hedgehog pathway, in two disease-relevant lung cell lines (IMR90 and 16HBE). To better understand the network neighborhood of HHIP, its proximity to the protein products of other COPD GWAS genes, and its functional role in COPD pathogenesis, we create HUBRIS, a protein-protein interaction network compiled from 8 publicly available databases. We identified both common and cell type-specific protein-protein interactors of HHIP. We find that our newly identified interactions shorten the network distance between HHIP and the protein products of several COPD GWAS genes, including DSP, MFAP2, TET2, and FBLN5. These new shorter paths include proteins that are encoded by genes involved in extracellular matrix and tissue organization. We found and validated interactions to proteins that provide new insights into COPD pathobiology, including CAVIN1 (IMR90) and TP53 (16HBE). The newly discovered HHIP interactions with CAVIN1 and TP53 implicate HHIP in response to oxidative stress.

genetics↗

bioRxiv2024

Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAKs structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complexs quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

microbiology↗

bioRxiv2024

Cannabinoid CB2 receptors in primary sensory neurons are implicated in CB2 agonist-mediated suppression of paclitaxel-induced neuropathic nociception and sexually-dimorphic sparing of morphine tolerance

Cannabinoid CB2 agonists show therapeutic efficacy without the unwanted side effects commonly associated with direct activation of CB1 receptors. The G protein-biased CB2 receptor agonist LY2828360 attenuates the maintenance of chemotherapy-induced neuropathic nociception in male mice and blocks the development of morphine tolerance in this model. However, the specific cell types involved in this phenomenon have never been investigated and whether this therapeutic profile is observed in female mice remains poorly understood. We used conditional deletion of CB2 receptors from specific cell populations to determine the population(s) mediating the anti-allodynic and morphine-sparing effects of CB2 agonists. Anti-allodynic effects of structurally distinct CB2 agonists (LY2828360 and AM1710) were present in paclitaxel-treated CB2f/f mice of either sex. The anti-allodynic effect of the CB2 agonists were absent in conditional knockout (KO) mice lacking CB2 receptors in peripheral sensory neurons (AdvillinCRE/+; CB2f/f) but preserved in mice lacking CB2 receptors in CX3CR1 expressing microglia/macrophages (CX3CR1CRE/+; CB2f/f). The morphine-sparing effect of LY28282360 occurred in a sexually-dimorphic manner, being present in male mice but absent in female mice of any genotype. In mice with established paclitaxel-induced neuropathy, prior LY2828360 treatment (3 mg/kg per day i.p. x 12 days) blocked the subsequent development of morphine tolerance in male CB2f/f mice but was absent in male (or female) AdvillinCRE/+; CB2f/f mice. LY2828360-induced sparing of morphine tolerance was preserved in male CX3CR1CRE/+; CB2f/f mice, but this effect was not observed in female CX3CR1CRE/+; CB2f/f mice. Similarly, co-administration of morphine with a low dose of LY2828360 (0.1 mg/kg per day i.p. x 6 days) reversed tolerance to the anti-allodynic efficacy of morphine in paclitaxel-treated male CB2f/f mice, but this effect was absent in female CB2f/f mice and AdvillinCRE/+; CB2f/f mice of either sex. Additionally, LY2828360 (3 mg/kg per day i.p. x 8 days) delayed, but did not prevent, the development of paclitaxel-induced mechanical and cold allodynia in either CB2f/f or CX3CR1CRE/+; CB2f/f mice of either sex. Our studies reveal that CB2 receptors in primary sensory neurons are required for the anti-allodynic effects of CB2 agonists in a mouse model of paclitaxel-induced neuropathic nociception. We also find that CB2 agonists acting on primary sensory neurons produce a sexually-dimorphic sparing of morphine tolerance in males, but not female, paclitaxel-treated mice.

neuroscience↗

bioRxiv2024

Protein complex heterogeneity and topology revealed by electron capture charge reduction and surface induced dissociation

We illustrate the utility of native mass spectrometry (nMS) combined with a fast, tunable gas-phase charge reduction, electron capture charge reduction (ECCR), for the characterization of protein complex topology and glycoprotein heterogeneity. ECCR efficiently reduces the charge states of tetradecameric GroEL, illustrating Orbitrap m/z measurements to greater than 100,000 m/z. For pentameric C-reactive protein and tetradecameric GroEL, our novel device combining ECCR with surface induced dissociation (SID) reduces the charge states and yields more topologically informative fragmentation. This is the first demonstration that ECCR yields more native-like SID fragmentation. ECCR also significantly improved mass and glycan heterogeneity measurements of heavily glycosylated SARS-CoV-2 spike protein trimer and thyroglobulin dimer. Protein glycosylation is important for structural and functional properties and plays essential roles in many biological processes. The immense heterogeneity in glycosylation sites and glycan structure poses significant analytical challenges that hinder a mechanistic understanding of the biological role of glycosylation. Without ECCR, average mass determination of glycoprotein complexes is available only through charge detection mass spectrometry or mass photometry. With narrow m/z selection windows followed by ECCR, multiple glycoform m/z values are apparent, providing quick global glycoform profiling and providing a future path for glycan localization on individual intact glycoforms.

molecular biology↗

bioRxiv2024

Conformational landscape of soluble α-klotho revealed by cryogenic electron microscopy

-Klotho (KLA) is a type-1 membranous protein that can associate with fibroblast growth factor receptor (FGFR) to form co-receptor for FGF23. The ectodomain of unassociated KLA is shed as soluble KLA (sKLA) to exert FGFR/FGF23-independent pleiotropic functions. The previously determined X-ray crystal structure of the extracellular region of sKLA in complex with FGF23 and FGFR1c suggests that sKLA functions solely as an on-demand coreceptor for FGF23. To understand the FGFR/FGF23-independent pleiotropic functions of sKLA, we investigated biophysical properties and structure of apo-sKLA. Mass photometry revealed that sKLA can form a stable structure with FGFR and/or FGF23 as well as sKLA dimer in solution. Single particle cryogenic electron microscopy (cryo-EM) supported the dimeric structure of sKLA. Cryo-EM further revealed a 3.3[A] resolution structure of apo-sKLA that overlays well with its counterpart in the ternary complex with several distinct features. Compared to the ternary complex, the KL2 domain of apo-sKLA is more flexible. 3D variability analysis revealed that apo-sKLA adopts conformations with different KL1-KL2 interdomain bending and rotational angles. The potential multiple forms and shapes of sKLA support its role as FGFR-independent hormone with pleiotropic functions. A comprehensive understanding of the sKLA conformational landscape will provide the foundation for developing klotho-related therapies for diseases.

biophysics↗