Biology subjects

Yates, J. R.

Publications and source records attributed to Yates, J. R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Structural and Functional Insights into the Evolution of SARS-CoV-2 KP.3.1.1 Spike Protein

The JN.1-sublineage KP.3.1.1 recently emerged as the globally prevalent SARS-CoV-2 variant, demonstrating increased infectivity and antibody escape. We investigated how mutations and a deletion in the KP.3.1.1 spike protein (S) affect ACE2 binding and antibody escape. Mass spectrometry revealed a new glycan site at residue N30 and altered glycoforms at neighboring N61. Cryo-EM structures showed that the N30 glycan and rearrangement of adjacent residues did not significantly change the overall spike structure, up-down ratio of the receptor-binding domains (RBDs), or ACE2 binding. Furthermore, a KP.3.1.1 S structure with hACE2 further confirmed an epistatic effect between F456L and Q493E on ACE2 binding. Our analysis shows SARS-CoV-2 variants that emerged after late 2023 are now incorporating reversions to residues found in other sarbecoviruses, including the N30 glycan, Q493E, and others. Overall, these results inform on the structural and functional consequences of the KP.3.1.1 mutations, the current SARS-CoV-2 evolutionary trajectory, and immune evasion.

immunology↗

bioRxiv2024

Acetylation of lysine 82 initiates TDP-43 nuclear loss of function by disrupting its nuclear import

The hallmark of a spectrum of age-dependent neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS), is a TDP-43 proteinopathy that includes nuclear loss of function and cytoplasmic aggregation. Here, reduced proteasome activity, as naturally occurs during aging, is shown to inhibit nuclear import of TDP-43. Quantitative mass spectrometry is used to determine that TDP-43 is the protein whose nuclear localization is most perturbed upon reduction in proteasome activity, culminating in elevated cytoplasmic TDP-43. Interaction of importin-1 with the bipartite classical nuclear localization sequence (cNLS) of TDP-43 is shown to be disrupted by partial proteasome inhibition but maintained by replacement with a PY-NLS that is recognized by importin-{beta}2. Mechanistically, this nuclear depletion of TDP-43 is shown to be driven by ubiquitination or acetylation of lysines 79, 82, and 84 within the cNLS when proteasome activity is reduced in human neurons. Specifically, acetylation at lysine 82 is sufficient to abolish TDP-43 binding to importin-1 and subsequent nuclear import of TDP-43. Moreover, using acetylation-specific TDP-43 antibodies, we detected acetylation of lysine 82 in the motor cortex of sporadic ALS patients but not control subjects. Our findings demonstrate that post-translational acetylation at lysine 82 of TDP-43 drives disruption of its importin-1-mediated nuclear import and is sufficient to initiate TDP-43 nuclear loss of function and cytoplasmic accumulation, evidence supporting acetylation as a plausible initiator of TDP-43 proteinopathies.

neuroscience↗

bioRxiv2024

Acid cleavable biotin-alkyne improves sensitivity for direct detection of biotin labeled peptides in BONCAT analysis.

BONCAT (Biorthogonal noncanonical amino acid tagging) is a labeling strategy that covalently adds a biotin-alkyne (BA) to methionine analogs via a click reaction. When methionine analogs are incorporated into a proteome, enrichment of the BA-labeled proteins allows the detection of newly synthesized proteins (NSP) by mass spectrometry. We previously reported that using our Direct Detection of Biotin-containing Tags (DidBIT) strategy, protein identifications and confidence are increased by enriching for BA-peptides instead of BA-proteins. We compared cleavable BA (DADPS) and uncleavable BA in the identification and TMT quantification of NSP. More than fifty percent more proteins were identified and quantified using DADPS than with uncleavable BA. Interrogation of the data revealed that multiple factors are responsible for the superior performance of DADPS.

biochemistry↗

bioRxiv2024

DiDBiT-TMT: A novel method to quantify changes in the proteomic landscape induced by neural plasticity

Withdrawal StatementThe authors have withdrawn this manuscript because during the peer review process in the journal, it was discovered that one of the processed proteomic data groups (the chemical LTP group) was mistakenly processed from the raw data for the norepinephrine group, resulting in an apparent complete overlap between the two conditions. For a correct analysis, please refer to the currently published peer-reviewed publication through the following DOI: https://doi.org/10.1021/acs.jproteome.4c00180 Therefore, the authors do not wish this preprint to be cited as reference for the project. If you have any questions, please contact the corresponding author.

neuroscience↗

bioRxiv2024

Diverse competitor B cell responses to a germline-targeting priming immunogen in human Ig loci transgenic mice

Broadly neutralizing antibodies (bnAbs) have promise to protect against HIV infection, but induction of bnAbs by immunization is an unsolved vaccine design challenge. Germline-targeting priming immunogens aim to initiate the induction of bnAbs by specifically activating rare bnAb-precursor B cells that can subsequently be matured using suitable heterologous boosting and shepherding immunogens. Several pre-clinical studies, and the IAVI G001 human clinical trial, have demonstrated the ability of a germline-targeting priming immunogen, eOD-GT8 60mer, to induce precursors of the VRC01 class of bnAbs. However, much less is known about B cells induced against other epitopes of the immunogen. Here, we performed unbiased analysis of B cells induced by eOD-GT8 60mers in Intelliselect Transgenic mice (Kymice) that are transgenic for the human Ig loci and produce human-like BCRs. B cells isolated with intact eOD-GT8 60mer nanoparticles showed a large diversity of non-VRC01-class B cells, with 38% unique clonotypes and only 5% of BCRs belonging to public lineages shared among all animals. We found that many competitors recognize epitopes in close proximity to or overlapping with the VRC01 epitope. These results indicate that optimal boosting of VRC01-class bnAb-precursor B cells primed by eOD-GT8 60mer might require a first-boost immunogen that minimizes recognition of competitor B cells, and such competitors isolated from Kymice could serve as valuable reagents for boost development.

immunology↗

bioRxiv2023

In vivo mapping of protein-protein interactions of schizophrenia risk factors generates an interconnected disease network.

Genetic analyses of Schizophrenia (SCZ) patients have identified thousands of risk factors. In silico protein-protein interaction (PPI) network analysis has provided strong evidence that disrupted PPI networks underlie SCZ pathogenesis. In this study, we performed in vivo PPI analysis of several SCZ risk factors in the rodent brain. Using endogenous antibody immunoprecipitations coupled to mass spectrometry (MS) analysis, we constructed a SCZ network comprising 1612 unique PPI with a 5% FDR. Over 90% of the PPI were novel, reflecting the lack of previous PPI MS studies in brain tissue. Our SCZ PPI network was enriched with known SCZ risk factors, which supports the hypothesis that an accumulation of disturbances in selected PPI networks underlies SCZ. We used Stable Isotope Labeling in Mammals (SILAM) to quantitate phencyclidine (PCP) perturbations in the SCZ network and found that PCP weakened most PPI but also led to some enhanced or new PPI. These findings demonstrate that quantitating PPI in perturbed biological states can reveal alterations to network biology.

neuroscience↗

bioRxiv2023

Wiskott-Aldrich Syndrome Protein Regulates Nucleolar Organization and Function in Innate Immune Response

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by the dysfunction of the WAS protein (WASP). Using an isogenic macrophage model derived from genome edited induced pluripotent stem cells we demonstrated that WASP functions in the nucleolus, which plays important roles in immune regulation. The absence of WASP resulted in smaller and misshapen nucleoli, decreased fibrillar center territory, and impaired ribosomal RNA (rRNA) transcription. The nucleolar and rRNA phenotypes were confirmed in WAS patient samples. Furthermore, WASP interacts with nucleolar proteins, including nucleophosmin 1 (NPM1) and fibrillarin (FBL). NPM1 deficiency is known to cause elevated cytokine expression following lipopolysaccharide (LPS) stimulation. Consistently, WASP deficient cells displayed lower levels of NPM1 and a heightened inflammatory cytokine response to LPS, which was rescued by overexpressing NPM1. Together, our research provides novel insights into the critical role of WASP in nucleolar function and the modulation of inflammatory cytokine production.

cell biology↗

bioRxiv2023

In vivo Protein Footprinting Reveals the Dynamic Conformational Changes of Proteome of Multiple Tissues in Progressing Alzheimer's Disease

Numerous studies have investigated changes in protein expression at the system level using proteomic mass spectrometry, but only recently have studies explored the structure of proteins at the proteome level. We developed covalent protein painting (CPP), a protein footprinting method that quantitatively labels exposed lysine, and have now extended the method to whole intact animals to measure surface accessibility as a surrogate of in vivo protein conformations. We investigated how protein structure and protein expression change as Alzheimers disease (AD) progresses by conducting in vivo whole animal labeling of AD mice. This allowed us to analyze broadly protein accessibility in various organs over the course of AD. We observed that structural changes of proteins related to energy generation, carbon metabolism, and metal ion homeostasis preceded expression changes in the brain. We found that proteins in certain pathways undergoing structural changes were significantly co-regulated in the brain, kidney, muscle, and spleen.

systems biology↗

bioRxiv2023

Evolving spike-protein N-glycosylation in SARS-CoV-2 variants

Since >3 years, SARS-CoV-2 has plunged humans into a colossal pandemic. Henceforth, multiple waves of infection have swept through the human population, led by variants that were able to partially evade acquired immunity. The co-evolution of SARS-CoV-2 variants with human immunity provides an excellent opportunity to study the interaction between viral pathogens and their human hosts. The heavily N-glycosylated spike-protein of SARS-CoV-2 plays a pivotal role in initiating infection and is the target for host immune-response, both of which are impacted by host-installed N-glycans. Using highly-sensitive DeGlyPHER approach, we compared the N-glycan landscape on spikes of the SARS-CoV-2 Wuhan-Hu-1 strain to seven WHO-defined variants of concern/interest, using recombinantly expressed, soluble spike-protein trimers, sharing same stabilizing-mutations. We found that N-glycan processing is conserved at most sites. However, in multiple variants, processing of N-glycans from high mannose- to complex-type is reduced at sites N165, N343 and N616, implicated in spike-protein function.

biochemistry↗

bioRxiv2023

Do perineuronal nets stabilize the engram of a synaptic circuit?

Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. SignificanceIn this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.

neuroscience↗

bioRxiv2023

Phosphorylation of pyruvate dehydrogenase marks the inhibition of in vivo neuronal activity

For decades, the expression of immediate early genes (IEGs) such as c-fos has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity (i.e., inhibition). Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected neuronal inhibition across the brain induced by a wide range of factors including general anesthesia, sensory experiences, and natural behaviors. Thus, as an in vivo marker for neuronal inhibition, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

neuroscience↗

bioRxiv2023

Comparative membrane proteomics reveals diverse cell regulators concentrated at the nuclear envelope

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, largely mediated by its distinctive protein composition. We developed methods to reveal novel, low abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs to cytoplasmic membranes, we first identified proteins with apparent NE enrichment. In subsequent authentication, ectopically expressed candidates were analyzed by immunofluorescence microscopy to quantify their targeting to the NE in cultured cells. Ten proteins from a validation set were found to associate preferentially with the NE, including oxidoreductases, enzymes for lipid biosynthesis and regulators of cell growth and survival. We determined that one of the validated candidates, the palmitoyltransferase Zdhhc6, modifies the NE oxidoreductase Tmx4 and thereby modulates its NE levels. This provides a functional rationale for the NE concentration of Zdhhc6. Overall, our methodology has revealed a group of previously unrecognized proteins concentrated at the NE and additional candidates. Future analysis of these can potentially unveil new mechanistic pathways associated with the NE.

cell biology↗

bioRxiv2022

Shared and distinctive neighborhoods of emerin and LBR revealed by proximity labeling and quantitative proteomics

Emerin and LBR are abundant transmembrane proteins of the nuclear envelope (NE) that are concentrated at the inner nuclear membrane (INM). Although both proteins interact with chromatin and nuclear lamins, they have distinctive biochemical and functional properties. Here we have deployed proximity labeling using the engineered biotin ligase TurboID (TbID) and quantitative proteomics to compare the neighborhoods of emerin and LBR in cultured mouse embryonic fibroblasts (MEFs). Our analysis revealed 232 high confidence proximity partners (HCPP) that interact selectively with emerin and/or LBR, 49 of which are shared by both. These included previously characterized NE-concentrated proteins, as well as a host of additional proteins not previously linked to emerin or LBR functions. Many of these are TM proteins of the ER and include two E3 ubiquitin ligases. Using the proximity ligation assay as an orthogonal approach, we validated the interactions described by proximity labeling for 11/12 proteins analyzed, supporting the robustness of our analysis. Overall, this work presents methodology that may be used for large-scale mapping of the landscape of the INM and reveals a group of new proteins with potential functional connections to emerin and LBR.

cell biology↗

bioRxiv2022

Targeted protein S-nitrosylation of ACE2 as potential treatment to prevent spread of SARS-CoV-2 infection

Prevention of infection and propagation of SARS-CoV-2 is of high priority in the COVID-19 pandemic. Here, we describe S-nitrosylation of multiple proteins involved in SARS-CoV-2 infection, including angiotensin converting enzyme 2 (ACE2), the receptor for viral entry. This reaction prevents binding of ACE2 to the SARS-CoV-2 Spike protein, thereby inhibiting viral entry, infectivity, and cytotoxicity. Aminoadamantane compounds also inhibit coronavirus ion channels formed by envelope (E) protein. Accordingly, we developed dual-mechanism aminoadamantane nitrate compounds that inhibit viral entry and thus spread of infection by S-nitrosylating ACE2 via targeted delivery of the drug after E-protein channel blockade. These non-toxic compounds are active in vitro and in vivo in the Syrian hamster COVID-19 model, and thus provide a novel avenue for therapy.

microbiology↗

bioRxiv2021

A genome-edited β-cell model of Prader-Willi syndrome reveals chronic deficits in endoplasmic reticulum chaperones and insulin secretion

Prader-Willi syndrome (PWS) is a multisystem disorder with neurobehavioral, metabolic, and hormonal phenotypes, caused by loss of expression of a paternally-expressed imprinted gene cluster. Prior evidence from a PWS mouse model identified abnormal pancreatic islet development with retention of aged insulin and deficient insulin secretion. To determine the collective roles of PWS genes in {beta}-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal (control) or active, paternal PWS-alleles. PWS {beta}-cells demonstrated a significant cell-autonomous reduction in basal and glucose-stimulated insulin secretion. Further, proteomic analyses revealed reduced levels of cellular and secreted hormones, including all insulin peptides and amylin, concomitant with reduction of at least ten endoplasmic reticulum (ER) chaperones, including GRP78 and GRP94. Critically, transcriptomic studies demonstrated that the broad reduction of ER chaperones originated from transcriptional downregulation without corresponding changes for Ins1 and Ins2. In contrast to the dosage compensation previously seen for ER chaperones in Grp78 or Grp94 gene knockouts or knockdown, compensation is precluded by the stress-independent deficiency of ER chaperones in PWS {beta}-cells. Consistent with reduced ER chaperones levels, PWS INS-1 {beta}-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. These findings suggest that a chronic shortage of ER chaperones in PWS {beta}-cells leads to a deficiency of protein folding and/or delay in ER transit of insulin and other cargo. In summary, our results illuminate the pathophysiological basis of pancreatic {beta}-cell hormone deficits in PWS, with evolutionary implications for the multigenic PWS-domain, and indicate that PWS-imprinted genes coordinate concerted regulation of ER chaperone biosynthesis and {beta}-cell secretory pathway function.

genetics↗

bioRxiv2021

Rvb1/Rvb2 proteins couple transcription and translation during glucose starvation

During times of unpredictable stress, organisms must adapt their gene expression to maximize survival. Along with changes in transcription, one conserved means of gene regulation during conditions that quickly represses translation is the formation of cytoplasmic phase-separated mRNP granules such as P-bodies and stress granules. Previously, we identified that distinct steps in gene expression can be coupled during glucose starvation as promoter sequences in the nucleus are able to direct the subcellular localization and translatability of mRNAs in the cytosol. Here, we report that Rvb1 and Rvb2, conserved ATPase proteins implicated as protein assembly chaperones and chromatin remodelers, were enriched at the promoters and mRNAs of genes involved in alternative glucose metabolism pathways that we previously found to be transcriptionally upregulated but translationally downregulated during glucose starvation in yeast. Engineered Rvb1/Rvb2-binding on mRNAs was sufficient to sequester the mRNAs into phase-separated granules and repress their translation. Additionally, this Rvb-tethering to the mRNA drove further transcriptional upregulation of the target genes. Overall, our results point to Rvb1/Rvb2 coupling transcription, mRNA granular localization, and translatability of mRNAs during glucose starvation. This Rvb-mediated rapid gene regulation could potentially serve as an efficient recovery plan for cells after stress removal.

molecular biology↗

bioRxiv2021

Heat shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

While the RNA binding protein TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) with TDP-43-containing liquid outer shells and liquid centers of HSP70 family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including ALS. Here we show that transient oxidative stress, proteasome inhibition, or inhibition of HSP70s ATP-dependent chaperone activity provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independent of RNA binding or stress granules. Isotope labeling mass spectrometry is used to identify that phase separated cytoplasmic TDP-43 is primarily bound by the small heat shock protein HSPB1. Binding is direct, mediated through TDP-43s RNA binding and low complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced, TDP-43 droplets. Decrease of HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion is identified within ALS-patient spinal motor neurons containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

cell biology↗

bioRxiv2021

Arg-tRNA synthetase links inflammatory metabolism to RNA splicing via nuclear condensates

Cells respond to perturbations like inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Here, we found that depletion of arginine during inflammation decreased levels of a nuclear form of arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal protein and nuclear speckle component and that arginine depletion impacted both condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a novel mechanism whereby a tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery.

molecular biology↗