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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 our direct detection of biotin-containing tags strategy increased protein identifications by enriching for BA-peptides instead of BA-proteins. We compared the performance of cleavable BA (DADPS) and uncleavable BA in the identification and TMT quantification of the NSP. DADPS identified and quantified more than double the number of peptides than uncleavable BA. Interrogation of the data revealed that multiple factors are responsible for the superior performance of DADPS.

Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.

The aim of this study was to identify, using proteomics, the molecular alterations caused by human serum exposure to Klebsiella pneumoniae ACH2. The analysis was performed under two different conditions, native serum from healthy donors and heat-inactivated serum (to inactivate the complement system), and at two different times, after 1 and 4 h of serum exposure. More than 1,000 bacterial proteins were identified at each time point. Enterobactin, a siderophore involved in iron uptake, and proteins involved in translation were upregulated at 1 h, while the chaperone ProQ and the glyoxylate cycle were identified after 4 h. Enzymes involved in the stress response were downregulated, and the SOD activity was validated using an enzymatic assay. In addition, an intricate metabolic adaptation was observed, with pyruvate and thiamine possibly involved in survival and virulence in the first hour of serum exposure. The addition of exogenous thiamine contributes to bacterial growth in human serum, corroborating this result. During 4 h of serum exposure, the glyoxylate cycle (GC) probably plays a central role, and the addition of exogenous succinate suppresses the GC, inducing a decrease in serum resistance. Therefore, serum exposure causes important changes in iron acquisition, the expression of virulence factors, and metabolic reprogramming, which could contribute to bacterial serum resistance.

Mutations in CCDC11 (cfap53) have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, explaining the multiciliation phenotype observed in Ccdc11-depleted cells. The small GTPase RhoA is critical for cytokinesis, and our Ccdc11 depletion phenotypes are reminiscent of RhoA loss of function. Here, we demonstrate that during cytokinesis CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA, and CCDC11 regulates total RhoA protein levels. Our results connect CCDC11 to cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis., (© 2024 The Author(s). Cytoskeleton published by Wiley Periodicals LLC.)

A personal narative of my time in the Hunt laboraotry and beyond is provided. The impact of the Hunt laboratory on the analysis of peptides and proteins by tandem mass spectrometry is described in the context of the time., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Single-cell proteomics has emerged as a powerful technology for unraveling the complexities of cellular heterogeneity, enabling insights into individual cell functions and pathologies. One of the primary challenges in single-cell proteomics is data generation, where low mass spectral signals often preclude the triggering of MS2 events. This challenge is addressed by Data Independent Acquisition (DIA), a data acquisition strategy that does not depend on peptide ion isotopic signatures to generate an MS2 event. In this study, we present data generated from the integration of DIA single-cell proteomics with a version of the DiagnoMass Proteomic Hub that was adapted to handle DIA data. DiagnoMass employs a hierarchical clustering methodology that enables the identification of tandem mass spectral clusters that are discriminative of biological conditions, thereby reducing the reliance on search engine biases for identifications. Nevertheless, a search engine (in this work, DIA-NN) can be integrated with DiagnoMass for spectral annotation. We used single-cell proteomic data from iPSC-derived neuroprogenitor cell cultures as a test study of this integrated approach. We were able to differentiate between control and Rett Syndrome patient cells to discern the proteomic variances potentially contributing to the disease's pathology. Our research confirms that the DiagnoMass-DIA synergy significantly enhances the identification of discriminative proteomic signatures, highlighting critical biological variations such as the presence of unique spectra that could be related to Rett Syndrome pathology.

Enterotoxigenic Escherichia coli (ETEC) cause hundreds of millions of cases of infectious diarrhea annually, predominantly in children from low-middle income regions. Notably, in children, as well as volunteers challenged with ETEC, diarrheal severity is significantly increased in blood group A (bgA) individuals. EtpA, is a secreted glycoprotein adhesin that functions as a blood group A lectin to promote critical interactions between ETEC and blood group A glycans on intestinal epithelia for effective bacterial adhesion and toxin delivery. EtpA is highly immunogenic resulting in robust antibody responses following natural infection and experimental challenge of volunteers with ETEC. To understand how EtpA directs ETEC-blood group A interactions and stimulates adaptive immunity, we mutated EtpA, mapped its glycosylation by mass-spectrometry (MS), isolated polyclonal (pAbs) and monoclonal antibodies (mAbs) from vaccinated mice and ETEC-infected volunteers, and determined structures of antibody-EtpA complexes by cryo-electron microscopy. Both bgA and mAbs that inhibited EtpA-bgA interactions and ETEC adhesion, bound to the C-terminal repeat domain highlighting this region as crucial for ETEC pathogen-host interaction. MS analysis uncovered extensive and heterogeneous N-linked glycosylation of EtpA and cryo-EM structures revealed that mAbs directly engage these unique glycan containing epitopes. Finally, electron microscopy-based polyclonal epitope mapping revealed antibodies targeting numerous distinct epitopes on N and C-terminal domains, suggesting that EtpA vaccination generates responses against neutralizing and decoy regions of the molecule. Collectively, we anticipate that these data will inform our general understanding of pathogen-host glycan interactions and adaptive immunity relevant to rational vaccine subunit design., Competing Interests: JMF is listed as the "Inventor" on U.S. patent 8323668 assigned to the University of Tennessee Research Foundation on December 4, 2012 that relates to use of EtpA related antigens in vaccine development. The other authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Standard single-cell (sc) proteomics of disease states inferred from multicellular organs or organoids cannot currently be related to single-cell physiology. Here, a scPatch-Clamp/Proteomics platform is developed on single neurons generated from hiPSCs bearing an Alzheimer's disease (AD) genetic mutation and compares them to isogenic wild-type controls. This approach provides both current and voltage electrophysiological data plus detailed proteomics information on single-cells. With this new method, the authors are able to observe hyperelectrical activity in the AD hiPSC-neurons, similar to that observed in the human AD brain, and correlate it to ≈1400 proteins detected at the single neuron level. Using linear regression and mediation analyses to explore the relationship between the abundance of individual proteins and the neuron's mutational and electrophysiological status, this approach yields new information on therapeutic targets in excitatory neurons not attainable by traditional methods. This combined patch-proteomics technique creates a new proteogenetic-therapeutic strategy to correlate genotypic alterations to physiology with protein expression in single-cells., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans., Competing Interests: Authors PL and YT were employed by company Acuitas Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guenaga, Alirezaei, Feng, Alameh, Lee, Baboo, Cluff, Wilson, Bale, Ozorowski, Lin, Tam, Diedrich, Yates, Paulson, Ward, Weissman and Wyatt.)

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.

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features., (© 2024. The Author(s).)

A protective HIV vaccine will likely need to induce broadly neutralizing antibodies (bnAbs). Vaccination with the germline-targeting immunogen eOD-GT8 60mer adjuvanted with AS01 B was found to induce VRC01-class bnAb precursors in 97% of vaccine recipients in the IAVI G001 phase 1 clinical trial; however, heterologous boost immunizations with antigens more similar to the native glycoprotein will be required to induce bnAbs. Therefore, we designed core-g28v2 60mer, a nanoparticle immunogen to be used as a first boost after eOD-GT8 60mer priming. We found, using a humanized mouse model approximating human conditions of VRC01-class precursor B cell diversity, affinity, and frequency, that both protein- and mRNA-based heterologous prime-boost regimens induced VRC01-class antibodies that gained key mutations and bound to near-native HIV envelope trimers lacking the N276 glycan. We further showed that VRC01-class antibodies induced by mRNA-based regimens could neutralize pseudoviruses lacking the N276 glycan. These results demonstrated that heterologous boosting can drive maturation toward VRC01-class bnAb development and supported the initiation of the IAVI G002 phase 1 trial testing mRNA-encoded nanoparticle prime-boost regimens.

Germline-targeting (GT) HIV vaccine strategies are predicated on deriving broadly neutralizing antibodies (bnAbs) through multiple boost immunogens. However, as the recruitment of memory B cells (MBCs) to germinal centers (GCs) is inefficient and may be derailed by serum antibody-induced epitope masking, driving further B cell receptor (BCR) modification in GC-experienced B cells after boosting poses a challenge. Using humanized immunoglobulin knockin mice, we found that GT protein trimer immunogen N332-GT5 could prime inferred-germline precursors to the V3-glycan-targeted bnAb BG18 and that B cells primed by N332-GT5 were effectively boosted by either of two novel protein immunogens designed to have minimum cross-reactivity with the off-target V1-binding responses. The delivery of the prime and boost immunogens as messenger RNA lipid nanoparticles (mRNA-LNPs) generated long-lasting GCs, somatic hypermutation, and affinity maturation and may be an effective tool in HIV vaccine development.

Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.

For decades, the expression of immediate early genes (IEGs) such as 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. 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 activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lyme disease (LD) caused by Borrelia burgdorferi is among the most important human vector borne diseases for which there is no effective prevention method. Identification of tick saliva transmission factors of the LD agent is needed before the highly advocated tick antigen-based vaccine could be developed. We previously reported the highly conserved Ixodes scapularis (Ixs) tick saliva serpin (S) 17 (IxsS17) was highly secreted by B. burgdorferi infected nymphs. Here, we show that IxsS17 promote tick feeding and enhances B. burgdorferi colonization of the host. We show that IxsS17 is not part of a redundant system, and its functional domain reactive center loop (RCL) is 100% conserved in all tick species. Yeast expressed recombinant (r) IxsS17 inhibits effector proteases of inflammation, blood clotting, and complement innate immune systems. Interestingly, differential precipitation analysis revealed novel functional insights that IxsS17 interacts with both effector proteases and regulatory protease inhibitors. For instance, rIxsS17 interacted with blood clotting proteases, fXII, fX, fXII, plasmin, and plasma kallikrein alongside blood clotting regulatory serpins (antithrombin III and heparin cofactor II). Similarly, rIxsS17 interacted with both complement system serine proteases, C1s, C2, and factor I and the regulatory serpin, plasma protease C1 inhibitor. Consistently, we validated that rIxsS17 dose dependently blocked deposition of the complement membrane attack complex via the lectin complement pathway and protected complement sensitive B. burgdorferi from complement-mediated killing. Likewise, co-inoculating C3H/HeN mice with rIxsS17 and B. burgdorferi significantly enhanced colonization of mouse heart and skin organs in a reverse dose dependent manner. Taken together, our data suggests an important role for IxsS17 in tick feeding and B. burgdorferi colonization of the host., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: When feeding on a vertebrate host, ticks secrete saliva, which is a complex mixture of proteins, lipids, and other molecules. Tick saliva assists the vector in modulating host hemostasis, immunity, and tissue repair mechanisms. While helping the vector to feed, its saliva modifies the site where pathogens are inoculated and often facilitates the infection process. The objective of this study is to uncover the variation in protein composition of Rhipicephalus microplus saliva during blood feeding., Methods: Ticks were fed on calves, and adult females were collected, weighed, and divided in nine weight groups, representing the slow and rapid feeding phases of blood feeding. Tick saliva was collected, and mass spectrometry analyses were used to identify differentially secreted proteins. Bioinformatic tools were employed to predict the structural and functional features of the salivary proteins. Reciprocal best hit analyses were used to identify conserved families of salivary proteins secreted by other tick species., Results: Changes in the protein secretion profiles of R. microplus adult female saliva during the blood feeding were observed, characterizing the phenomenon known as "sialome switching." This observation validates the idea that the switch in protein expression may serve as a mechanism for evading host responses against tick feeding. Cattle tick saliva is predominantly rich in heme-binding proteins, secreted conserved proteins, lipocalins, and protease inhibitors, many of which are conserved and present in the saliva of other tick species. Additionally, another remarkable observation was the identification of host-derived proteins as a component of tick saliva., Conclusions: Overall, this study brings new insights to understanding the dynamics of the proteomic profile of tick saliva, which is an important component of tick feeding biology. The results presented here, along with the disclosed sequences, contribute to our understanding of tick feeding biology and might aid in the identification of new targets for the development of novel anti-tick methods., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cryptococcus gattii is a primary pathogenic fungus that causes pneumonia. This species is also responsible for an outbreak in Vancouver, Canada, and spreading to the mainland and United States. The use of medical devices is often complicated by infections with biofilm-forming microbes with increased resistance to antimicrobial agents and host defense mechanisms. This study investigated the comparative proteome of C. gattii R265 (VGIIa) grown under planktonic and biofilm conditions. A brief comparison with C. neoformans H99 biofilm and the use of different culture medium and surface were also evaluated. Using Multidimensional Protein Identification Technology (MudPIT), 1819 proteins were identified for both conditions, where 150 (8.2%) were considered differentially regulated (up- or down-regulated and unique in biofilm cells). Overall, the proteomic approach suggests that C. gattii R265 biofilm cells are maintained by the induction of electron transport chain for reoxidation, and by alternative energy metabolites, such as succinate and acetate. SIGNIFICANCE: Since C. gattii is considered a primary pathogen and is one of the most virulent and less susceptible to antifungals, understanding how biofilms are maintained is fundamental to search for new targets to control this important mode of growth that is difficult to eradicate., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The β-coronavirus SARS-CoV-2 causes severe acute respiratory syndrome (COVID-19) in humans. It enters and infects epithelial airway cells upon binding of the receptor binding domain (RBD) of the virus entry protein spike to the host receptor protein Angiotensin Converting Enzyme 2 (ACE2). Here, we used coimmunoprecipitation coupled with bottom-up mass spectrometry to identify host proteins that engaged with the spike protein in human bronchial epithelial cells (16HBEo - ). We found that the spike protein bound to extracellular laminin and thrombospondin and endoplasmatic reticulum (ER)-resident DJB11 and FBX2 proteins. The ER-resident proteins UGGT1, CALX, HSP7A, and GRP78/BiP bound preferentially to the original Wuhan D614 over the mutated G614 spike protein in the more rapidly spreading Alpha SARS-CoV-2 strain. The increase in protein binding to the D614 spike might be explained by higher accessibility of cryptic sites in "RDB open" and "S2 only" D614 spike protein conformations and may enable SARS-CoV-2 to infect additional, ACE2-negative cell types. Moreover, a novel proteome-based cell type set enrichment analysis (pCtSEA) found that host factors like laminin might render additional cell types such as macrophages and epithelial cells in the nephron permissive to SARS-CoV-2 infection.

Differential precipitation of proteins (DiffPOP) is a simple technique for fractionating complex protein mixtures. Using stepwise addition of acidified methanol, ten distinct subsets of proteins can be selectively precipitated by centrifugation and identified by mass spectrometry-based proteomics. We have previously shown that the ability of a protein to resist precipitation can be altered by drug binding, which enabled us to identify a novel drug-target interaction. Here, we show that the addition of DiffPOP to a standard LC-MS proteomics workflow results in a three-dimensional separation of peptides that increases protein coverage and peptide identifications. Importantly, DiffPOP reveals solubility differences between proteoforms, potentially providing valuable insights that are typically lost in bottom-up proteomics.

The nuclear envelope (NE) is a subdomain of the ER with prominent roles in nuclear organization, which are largely mediated by its distinctive protein composition. We developed methods to reveal low-abundance transmembrane (TM) proteins concentrated at the NE relative to the peripheral ER. Using label-free proteomics that compared isolated NEs with 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., (© 2023 Cheng et al.)

Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here, we report that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo., (© 2023. The Author(s).)

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 β-cell biology, we used genome-editing to generate isogenic, clonal INS-1 insulinoma lines having 3.16 Mb deletions of the silent, maternal- (control) and active, paternal-allele (PWS). PWS β-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, differentially expressed genes identified by whole transcriptome studies included reductions in levels of mRNAs encoding these secreted peptides and the group of ER chaperones. 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 β-cells. Consistent with reduced ER chaperones levels, PWS INS-1 β-cells are more sensitive to ER stress, leading to earlier activation of all three arms of the unfolded protein response. Combined, the findings suggest that a chronic shortage of ER chaperones in PWS β-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 β-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 β-cell secretory pathway function., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Koppes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cells respond to perturbations such as inflammation by sensing changes in metabolite levels. Especially prominent is arginine, which has known connections to the inflammatory response. Aminoacyl-tRNA synthetases, enzymes that catalyse the first step of protein synthesis, can also mediate cell signalling. Here we show that depletion of arginine during inflammation decreased levels of nuclear-localized arginyl-tRNA synthetase (ArgRS). Surprisingly, we found that nuclear ArgRS interacts and co-localizes with serine/arginine repetitive matrix protein 2 (SRRM2), a spliceosomal and nuclear speckle protein, and that decreased levels of nuclear ArgRS correlated with changes in condensate-like nuclear trafficking of SRRM2 and splice-site usage in certain genes. These splice-site usage changes cumulated in the synthesis of different protein isoforms that altered cellular metabolism and peptide presentation to immune cells. Our findings uncover a mechanism whereby an aminoacyl-tRNA synthetase cognate to a key amino acid that is metabolically controlled during inflammation modulates the splicing machinery., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Prevention of infection and propagation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a high priority in the Coronavirus Disease 2019 (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, the 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 to pursue therapy., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines ., (© 2023. Springer Nature America, Inc.)

The Crotalus intermedius group is a clade of rattlesnakes consisting of several species adapted to a high elevation habitat, primarily in México. Crotalus tancitarensis was previously classified as C. intermedius , until individuals occurring on Cerro Tancítaro in Michoacán, México, were reevaluated and classified as a new species ( C. tancitarensis ) based on scale pattern and geographic location. This study aimed to characterize the venom of C. tancitarensis and compare the venom profile to those of other species within the Crotalus intermedius group using gel electrophoresis, biochemical assays, reverse-phase high performance liquid chromatography, mass spectrometry, and lethal toxicity (LD 50 ) assays. Results show that the venom profiles of species within the Crotalus intermedius group are similar, but with distinct differences in phospholipase A 2 (PLA 2 ), metalloproteinase PI (SVMP PI), and kallikrein-like serine proteinase (SVSP) activity and relative abundance. Proteomic analysis indicated that the highland forms produce venoms with 50-60 protein isoforms and a composition typical of type I rattlesnake venoms (abundant SVMPs, lack of presynaptic PLA 2 -based neurotoxins), as well as a diversity of typical Crotalus venom components such as serine proteinases, PLA 2 s, C-type lectins, and less abundant toxins (LAAOs, CRiSPs, etc.). The overall venom profile of C. tancitarensis appears most similar to C. transversus , which is consistent with a previous mitochondrial DNA analysis of the Crotalus intermedius group. These rattlesnakes of the Mexican highlands represent a radiation of high elevation specialists, and in spite of divergence of species in these Sky Island habitats, venom composition of species analyzed here has remained relatively conserved. The majority of protein family isoforms are conserved in all members of the clade, and as seen in other more broadly distributed rattlesnake species, differences in their venoms are largely due to relative concentrations of specific components.

The Planar cell polarity (PCP) pathway is known to mediate the function of the Wnt proteins in growth cone guidance. Here, we show that the PCP pathway may directly influence local protein synthesis within the growth cones. We found that Fragile X Messenger Ribonucleoprotein 1 (FMRP) interacts with Fzd3. This interaction is negatively regulated by Wnt5a, which induces FMRP phosphorylation. Knocking down FMRP via electroporating shRNAs into the dorsal spinal cord lead to a randomization of anterior-posterior turning of post-crossing commissural axons, which could be rescued by a FMRP rescue construct. Using RNAscope, we found that some of the FMRP target mRNAs encoding PCP components, PRICKLE2 and Celsr2, as well as regulators of cytoskeletal dynamics and components of cytoskeleton, APC, Cfl1, Map1b, Tubb3 and Actb, are present in the commissural neuron growth cones. Our results suggest that PCP signaling may regulate growth cone guidance, at least in part, by regulating local protein synthesis in the growth cones through via an interaction between Frizzled3 and FMRP., (Copyright © 2022. Published by Elsevier Ltd.)

Traditional mass spectrometry-based glycoproteomic approaches have been widely used for site-specific N-glycoform analysis, but a large amount of starting material is needed to obtain sampling that is representative of the vast diversity of N-glycans on glycoproteins. These methods also often include a complicated workflow and very challenging data analysis. These limitations have prevented glycoproteomics from being adapted to high-throughput platforms, and the sensitivity of the analysis is currently inadequate for elucidating N-glycan heterogeneity in clinical samples. Heavily glycosylated spike proteins of enveloped viruses, recombinantly expressed as potential vaccines, are prime targets for glycoproteomic analysis. Since the immunogenicity of spike proteins may be impacted by their glycosylation patterns, site-specific analysis of N-glycoforms provides critical information for vaccine design. Using recombinantly expressed soluble HIV Env trimer, we describe DeGlyPHER, a modification of our previously reported sequential deglycosylation strategy to yield a "single-pot" process. DeGlyPHER is an ultrasensitive, simple, rapid, robust, and efficient approach for site-specific analysis of protein N-glycoforms, that we developed for analysis of limited quantities of glycoproteins., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The fold and conformation of proteins are key to successful cellular function, but all techniques for protein structure determination are performed in an artificial environment with highly purified proteins. While protein conformations have been solved to atomic resolution and modern protein structure prediction tools rapidly generate near accurate models of proteins, there is an unmet need to uncover the conformations of proteins in living cells. Here, we describe Covalent Protein Painting (CPP), a simple and fast method to infer structural information on protein conformation in cells with a quantitative protein footprinting technology. CPP monitors the conformational landscape of the 3D proteome in cells with high sensitivity and throughput. A key advantage of CPP is its' ability to quantitatively compare the 3D proteomes between different experimental conditions and to discover significant changes in the protein conformations. We detail how to perform a successful CPP experiment, the factors to consider before performing the experiment, and how to interpret the results., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ixodes scapularis long-term blood feeding behavior is facilitated by a tick secreted bio adhesive (tick cement) that attaches tick mouthparts to skin tissue and prevents the host from dislodging the attached tick. Understanding tick cement formation is highly sought after as its disruption will prevent tick feeding. This study describes proteins that form the inner core layer of I. scapularis tick cement as disrupting these proteins will likely stop formation of the outer cortical layer. The inner core cement layer completes formation by 24 h of tick attachment. Thus, we used laser-capture microdissection to isolate cement from cryosections of 6 h and 24 h tick attachment sites and to distinguish between early and late inner core cement proteins. LC-MS/MS analysis identified 138 tick cement proteins (TCPs) of which 37 and 35 were unique in cement of 6 and 24 h attached ticks respectively. We grouped TCPs in 14 functional categories: cuticular protein (16%), tick specific proteins of unknown function, cytoskeletal proteins, and enzymes (13% each), enzymes (10%), antioxidant, glycine rich, scaffolding, heat shock, histone, histamine binding, proteases and protease inhibitors, and miscellaneous (3-6% each). Gene ontology analysis confirm that TCPs are enriched for bio adhesive properties. Our data offer insights into tick cement bonding patterns and set the foundation for understanding the molecular basis of I. scapularis tick cement formation., (© 2022. The Author(s).)

Broadly neutralizing antibodies (bnAbs) to the HIV envelope (Env) V2-apex region are important leads for HIV vaccine design. Most V2-apex bnAbs engage Env with an uncommonly long heavy-chain complementarity-determining region 3 (HCDR3), suggesting that the rarity of bnAb precursors poses a challenge for vaccine priming. We created precursor sequence definitions for V2-apex HCDR3-dependent bnAbs and searched for related precursors in human antibody heavy-chain ultradeep sequencing data from 14 HIV-unexposed donors. We found potential precursors in a majority of donors for only two long-HCDR3 V2-apex bnAbs, PCT64 and PG9, identifying these bnAbs as priority vaccine targets. We then engineered ApexGT Env trimers that bound inferred germlines for PCT64 and PG9 and had higher affinities for bnAbs, determined cryo-EM structures of ApexGT trimers complexed with inferred-germline and bnAb forms of PCT64 and PG9, and developed an mRNA-encoded cell-surface ApexGT trimer. These methods and immunogens have promise to assist HIV vaccine development., Competing Interests: Declaration of interests J.R.W., K.M.M., J.M.S., and W.R.S. are named inventors on patent applications filed by Scripps and IAVI regarding ApexGT immunogens in this manuscript., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transcription factors p63 and p73 have high similarity to the tumor suppressor protein p53. While the importance of p53 in DNA damage control is established, the functions of p63 or p73 remain elusive. Here, we analyzed nvp63, the cnidarian homologue of p63, that is expressed in the mesenteries of the starlet sea anemone Nematostella vectensis and that is activated in response to DNA damage. We used ultraviolet light (UV) to induce DNA damage and determined the chromatin-bound proteome with quantitative, bottom-up proteomics. We found that genotoxic stress or nvp63 knockdown recruited the protein nvPIWIL1, a homologue of the piRNA-binding PIWI protein family. Knockdown nvPIWIL1 increased protein expression from open reading frames (ORFs) that overlap with class I and II transposable element DNA sequences in the genome of N. vectensis . UV irradiation induced apoptosis, and apoptosis was reduced in the absence of nvp63 but increased with the loss of nvPIWIL1. Loss of nvp63 increased the presence of class I LTR and non-LTR retrotransposon but not of class II DNA transposon-associated protein products. These results suggest that an evolutionary early function of nvp63 might be to control genome stability in response to activation of transposable elements, which induce DNA damage during reintegration in the genome.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function. SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms., (Copyright © 2022 Schiapparelli et al.)

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 repress 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 mRNAs into mRNP granules and repress their translation. Additionally, this Rvb tethering to the mRNA drove further transcriptional upregulation of the target genes. Further, we found that depletion of Rvb2 caused decreased alternative glucose metabolism gene mRNA induction, but upregulation of protein synthesis during glucose starvation. 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., Competing Interests: YC, WH, ST, AH, VH, FX, JM, JY, BZ No competing interests declared, (© 2022, Chen et al.)

Emerin and lamin B receptor (LBR) are abundant transmembrane proteins of the nuclear envelope 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. Our analysis revealed 232 high confidence proximity partners 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, including two E3 ubiquitin ligases. Supporting these results, we found that 11/12 representative proximity relationships identified by TbID also were detected at the NE with the proximity ligation assay. 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.

Following health agencies warning, the use of animal origin supplements should be avoided in biological products proposed as therapy in humans. Platelet lysate and several other growth factors sources are alternatives to replace fetal calf serum, the current gold standard in clinical-grade cell culture. However, the platelet supplement's content lacks data due to different production methods. The principle behind these products relays on the lysis of platelets that release several proteins, some of which are contained in heterogeneous granules and coordinate biological functions. This study aims to analyze the composition and reproducibility of a platelet lysate produced with a standardized method, by describing several batches' protein and particle content using proteomics and dynamic light scattering. Proteomics data revealed a diversified protein content, with some related to essential cellular processes such as proliferation, morphogenesis, differentiation, biosynthesis, adhesion, and metabolism. It also detected proteins responsible for activation and binding of transforming growth factor beta, hepatocyte growth factor, and insulin-like growth factor. Total protein, biochemical, and growth factors quantitative data showed consistent and reproducible values across batches. Novel data on two major particle populations is presented, with high dispersion level at 231 ± 96 d.nm and at 30 ± 8 d.nm, possibly being an important way of protein trafficking through the cellular microenvironment. This experimental and descriptive analysis aims to support the content definition and quality criteria of a cell supplement for clinical applications., (© 2022 Wiley Periodicals LLC.)

While acetylated, RNA-binding-deficient TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) comprised of TDP-43-containing liquid outer shells and liquid centres of HSP70-family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we show that transient oxidative stress, proteasome inhibition or inhibition of the ATP-dependent chaperone activity of HSP70 provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independently of RNA binding or stress granules. Isotope labelling mass spectrometry was used to identify that phase-separated cytoplasmic TDP-43 is bound by the small heat-shock protein HSPB1. Binding is direct, mediated through TDP-43's 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. A decrease in HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion was identified in spinal motor neurons of patients with ALS containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Shotgun proteomics aims to identify and quantify the thousands of proteins in complex mixtures such as cell and tissue lysates and biological fluids. This approach uses liquid chromatography coupled with tandem mass spectrometry and typically generates hundreds of thousands of mass spectra that require specialized computational environments for data analysis. PatternLab for proteomics is a unified computational environment for analyzing shotgun proteomic data. PatternLab V (PLV) is the most comprehensive and crucial update so far, the result of intensive interaction with the proteomics community over several years. All PLV modules have been optimized and its graphical user interface has been completely updated for improved user experience. Major improvements were made to all aspects of the software, ranging from boosting the number of protein identifications to faster extraction of ion chromatograms. PLV provides modules for preparing sequence databases, protein identification, statistical filtering and in-depth result browsing for both labeled and label-free quantitation. The PepExplorer module can even pinpoint de novo sequenced peptides not already present in the database. PLV is of broad applicability and therefore suitable for challenging experimental setups, such as time-course experiments and data handling from unsequenced organisms. PLV interfaces with widely adopted software and community initiatives, e.g., Comet, Skyline, PEAKS and PRIDE. It is freely available at http://www.patternlabforproteomics.org ., (© 2022. Springer Nature Limited.)