Publications by authors named "John R Yates"

901 Publications

DeGlyPHER: An Ultrasensitive Method for the Analysis of Viral Spike -Glycoforms.

Anal Chem 2021 10 1;93(40):13651-13657. Epub 2021 Oct 1.

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California 92037, United States.

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single-pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each -glycosylation site. Though aimed at characterizing glycosylation of viral spike-proteins as potential vaccines, this method is applicable for the analysis of site-specific glycosylation of any glycoprotein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.1c03059DOI Listing
October 2021

Coupling of spliceosome complexity to intron diversity.

Curr Biol 2021 Sep 18. Epub 2021 Sep 18.

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address:

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cub.2021.09.004DOI Listing
September 2021

Clathrin packets move in slow axonal transport and deliver functional payloads to synapses.

Neuron 2021 09;109(18):2884-2901.e7

Department of Pathology, University of California, San Diego, La Jolla, CA, USA; Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA. Electronic address:

In non-neuronal cells, clathrin has established roles in endocytosis, with clathrin cages enclosing plasma membrane infoldings, followed by rapid disassembly and reuse of monomers. However, in neurons, clathrin is conveyed in slow axonal transport over days to weeks, and the underlying transport/targeting mechanisms, mobile cargo structures, and even its precise presynaptic localization and physiologic role are unclear. Combining live imaging, photobleaching/conversion, mass spectrometry, electron microscopy, and super-resolution imaging, we found that unlike in dendrites, where clathrin cages rapidly assemble and disassemble, in axons, clathrin and related proteins organize into stable "transport packets" that are unrelated to endocytosis and move intermittently on microtubules, generating an overall slow anterograde flow. At synapses, multiple clathrin packets abut synaptic vesicle (SV) clusters, and clathrin packets also exchange between synaptic boutons in a microtubule-dependent "superpool." Within synaptic boundaries, clathrin is surprisingly dynamic, continuously exchanging between local clathrin assemblies, and its depletion impairs SV recycling. Our data provide a conceptual framework for understanding clathrin trafficking and presynaptic targeting that has functional implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2021.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8457040PMC
September 2021

Where Are They Now...?

Authors:
John R Yates

J Proteome Res 2021 08;20(8):3775-3781

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.1c00582DOI Listing
August 2021

Phenotypic and functional variation in venom and venom resistance of two sympatric rattlesnakes and their prey.

J Evol Biol 2021 Sep 9;34(9):1447-1465. Epub 2021 Aug 9.

Department of Biology, San Diego State University, San Diego, CA, USA.

Predator-prey interactions often lead to the coevolution of adaptations associated with avoiding predation and, for predators, overcoming those defences. Antagonistic coevolutionary relationships are often not simple interactions between a single predator and prey but rather a complex web of interactions between multiple coexisting species. Coevolution between venomous rattlesnakes and small mammals has led to physiological venom resistance in several mammalian taxa. In general, viperid venoms contain large quantities of snake venom metalloproteinase toxins (SVMPs), which are inactivated by SVMP inhibitors expressed in resistant mammals. We explored variation in venom chemistry, SVMP expression, and SVMP resistance across four co-distributed species (California Ground Squirrels, Bryant's Woodrats, Southern Pacific Rattlesnakes, and Red Diamond Rattlesnakes) collected from four different populations in Southern California. Our aim was to understand phenotypic and functional variation in venom and venom resistance in order to compare coevolutionary dynamics of a system involving two sympatric predator-prey pairs to past studies that have focused on single pairs. Proteomic analysis of venoms indicated that these rattlesnakes express different phenotypes when in sympatry, with Red Diamonds expressing more typical viperid venom (with a diversity of SVMPs) and Southern Pacifics expressing a more atypical venom with a broader range of non-enzymatic toxins. We also found that although blood sera from both mammals were generally able to inhibit SVMPs from both rattlesnake species, inhibition depended strongly on the snake population, with snakes from one geographic site expressing SVMPs to which few mammals were resistant. Additionally, we found that Red Diamond venom, rather than woodrat resistance, was locally adapted. Our findings highlight the complexity of coevolutionary relationships between multiple predators and prey that exhibit similar offensive and defensive strategies in sympatry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jeb.13907DOI Listing
September 2021

Beyond factor H: The impact of genetic-risk variants for age-related macular degeneration on circulating factor-H-like 1 and factor-H-related protein concentrations.

Am J Hum Genet 2021 08 13;108(8):1385-1400. Epub 2021 Jul 13.

Stoller Biomarker Discovery Centre and Division of Cancer Sciences, School of Medical Sciences, Faculty of Biology, Medicine, and Health, The University of Manchester, Manchester, M13 9NQ, United Kingdom. Electronic address:

Age-related macular degeneration (AMD) is a leading cause of vision loss; there is strong genetic susceptibility at the complement factor H (CFH) locus. This locus encodes a series of complement regulators: factor H (FH), a splice variant factor-H-like 1 (FHL-1), and five factor-H-related proteins (FHR-1 to FHR-5), all involved in the regulation of complement factor C3b turnover. Little is known about how AMD-associated variants at this locus might influence FHL-1 and FHR protein concentrations. We have used a bespoke targeted mass-spectrometry assay to measure the circulating concentrations of all seven complement regulators and demonstrated elevated concentrations in 352 advanced AMD-affected individuals for all FHR proteins (FHR-1, p = 2.4 × 10; FHR-2, p = 6.0 × 10; FHR-3, p = 1.5 × 10; FHR-4, p = 1.3 × 10; FHR-5, p = 1.9 × 10) and FHL-1 (p = 4.9 × 10) when these individuals were compared to 252 controls, whereas no difference was seen for FH (p = 0.94). Genome-wide association analyses in controls revealed genome-wide-significant signals at the CFH locus for all five FHR proteins, and univariate Mendelian-randomization analyses strongly supported the association of FHR-1, FHR-2, FHR-4, and FHR-5 with AMD susceptibility. These findings provide a strong biochemical explanation for how genetically driven alterations in circulating FHR proteins could be major drivers of AMD and highlight the need for research into FHR protein modulation as a viable therapeutic avenue for AMD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajhg.2021.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387294PMC
August 2021

Aurora Kinase A proximity map reveals centriolar satellites as regulators of its ciliary function.

EMBO Rep 2021 Aug 25;22(8):e51902. Epub 2021 Jun 25.

Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey.

Aurora kinase A (AURKA) is a conserved kinase that plays crucial roles in numerous cellular processes. Although AURKA overexpression is frequent in human cancers, its pleiotropic functions and multifaceted regulation present challenges in its therapeutic targeting. Key to overcoming these challenges is to identify and characterize the full range of AURKA interactors, which are often weak and transient. Previous proteomic studies were limited in monitoring dynamic and non-mitotic AURKA interactions. Here, we generate the proximity interactome of AURKA in asynchronous cells, which consists of 440 proteins involving multiple biological processes and cellular compartments. Importantly, AURKA has extensive proximate and physical interactions to centriolar satellites, key regulators of the primary cilium. Loss-of-function experiments identify satellites as negative regulators of AURKA activity, abundance, and localization in quiescent cells. Notably, loss of satellites activates AURKA at the basal body, decreases centrosomal IFT88 levels, and causes ciliogenesis defects. Collectively, our results provide a resource for dissecting spatiotemporal regulation of AURKA and uncover its proteostatic regulation by satellites as a new mechanism for its ciliary functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embr.202051902DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339716PMC
August 2021

20 Years...2 Decades...10 Bienniums...5 Quadrenniums...4 Lustrums.

Authors:
John R Yates

J Proteome Res 2021 07 14;20(7):3401-3403. Epub 2021 Jun 14.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.1c00393DOI Listing
July 2021

Ciliary extracellular vesicles are distinct from the cytosolic extracellular vesicles.

J Extracell Vesicles 2021 04 27;10(6):e12086. Epub 2021 Apr 27.

Department of Biomedical & Pharmaceutical Sciences Chapman University Irvine California USA.

Extracellular vesicles (EVs) are cell-derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid-shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know the distinctive protein profiles of the differently sourced EVs (cilia vs cytosol). Here, we isolated EVs from ciliated wild-type (WT) and non-ciliated knockout (KO) mouse endothelial cells using fluid-shear flow followed by a conventional method of EV isolation. EVs isolated from WT and KO exhibited distinctive sizes. Differences in EV protein contents were studied using liquid chromatography with tandem mass spectrometry (LC-MS-MS) and proteomic comparative analysis, which allowed us to classify proteins between ciliary EVs and cytosolic EVs derived from WT and KO, respectively. A total of 79 proteins were exclusively expressed in WT EVs, 145 solely in KO EVs, and 524 in both EVs. Our bioinformatics analyses revealed 29% distinct protein classes and 75% distinct signalling pathways between WT and KO EVs. Based on our statistical analyses and in vitro studies, we identified NADPH-cytochrome P450 reductase (POR), and CD166 antigen (CD166) as potential biomarkers for ciliary and cytosolic EVs, respectively. Our protein-protein interaction network analysis revealed that POR, but not CD166, interacted with either established or strong ciliopathy gene candidates. This report shows the unique differences between EVs secreted from cilia and the cytosol. These results will be important in advancing our understanding of human genetic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jev2.12086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8077156PMC
April 2021

Penicillium oxalicum secretomic analysis identify plant cell wall degrading enzymes important for fruit juice extraction.

J Food Sci Technol 2021 May 6;58(5):1764-1775. Epub 2020 Aug 6.

Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS Brazil.

Pectinases and other carbohydrate-active enzymes are important for the food industry, mainly for juice processing. In addition, the use of peels to produce enzymes can aggregate value to these agro-industrial residues and at the end of the process enhance qualitatively and quantitatively the juice production. In this work, three different extracts produced by LS09 using agro-industrial residues were optimized and analyzed by mass spectrometry. It was observed an increased production of pectinases in the medium containing orange peel and optimized for production of pectin lyase and pectinesterase (PE). Interestingly, not only pectinases, but also different plant cell wall degrading enzymes (i.e. glucanases, xylanases, arabinases), with a higher ratio (42/73) was identified in the medium optimized for PE. The crude extracts produced by also reveal the potential for application in the fruit juice industry, showing an increased yield and qualitative characteristics of extracted juices. The presence of other cell wall-degrading enzymes identified by proteomics, reinforce the combination for obtaining clarified and depectinized juice in a single step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s13197-020-04688-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021675PMC
May 2021

Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy.

Mol Psychiatry 2021 Apr 22. Epub 2021 Apr 22.

Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41380-021-01104-2DOI Listing
April 2021

Exposure to a sublethal menadione concentration modifies the mycelial secretome and conidial enzyme activities of Metarhizium anisopliae sensu lato and increases its virulence against Rhipicephalus microplus.

Microbiol Res 2021 Jul 7;248:126753. Epub 2021 Apr 7.

Graduate Program in Veterinary Science, Department of Animal Parasitology, Federal Rural University of Rio de Janeiro, Seropédica, RJ 23790-000, Brazil. Electronic address:

Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC and LT, particularly at 10 conidia mL. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micres.2021.126753DOI Listing
July 2021

Protein Footprinting via Covalent Protein Painting Reveals Structural Changes of the Proteome in Alzheimer's Disease.

J Proteome Res 2021 05 19;20(5):2762-2771. Epub 2021 Apr 19.

Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.

Misfolding and aggregation of amyloid-β peptide and hyperphosphorylated tau are molecular markers of Alzheimer's disease (AD), and although the 3D structures of these aberrantly folded proteins have been visualized in exquisite detail, no method has been able to survey protein folding across the proteome in AD. Here, we present covalent protein painting (CPP), a mass spectrometry-based protein footprinting approach to quantify the accessibility of lysine ε-amines for covalent modification at the surface of natively folded proteins. We used CPP to survey the reactivity of 2645 lysine residues and therewith the structural proteome of HEK293T cells and found that reactivity increased upon mild heat shock. CPP revealed that the accessibility of lysine residues for covalent modification in tubulin-β (TUBB), in succinate dehydrogenase (SHDB), and in amyloid-β peptide (Aβ) is altered in human postmortem brain samples of patients with neurodegenerative diseases. The structural alterations of TUBB and SHDB in patients with AD, dementia with Lewy bodies (DLB), or both point to broader perturbations of the 3D proteome beyond Aβ and hyperphosphorylated tau.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c00912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8477671PMC
May 2021

Site-Specific O-Glycosylation Analysis of SARS-CoV-2 Spike Protein Produced in Insect and Human Cells.

Viruses 2021 03 25;13(4). Epub 2021 Mar 25.

Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence is difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S-insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v13040551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8064498PMC
March 2021

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.

Mol Psychiatry 2021 Jul 16;26(7):3586-3613. Epub 2021 Mar 16.

Department of Psychiatry, University of California San Diego, La Jolla, CA, USA.

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41380-021-01052-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443683PMC
July 2021

S-nitrosylated TDP-43 triggers aggregation, cell-to-cell spread, and neurotoxicity in hiPSCs and in vivo models of ALS/FTD.

Proc Natl Acad Sci U S A 2021 03;118(11)

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037;

Rare genetic mutations result in aggregation and spreading of cognate proteins in neurodegenerative disorders; however, in the absence of mutation (i.e., in the vast majority of "sporadic" cases), mechanisms for protein misfolding/aggregation remain largely unknown. Here, we show environmentally induced nitrosative stress triggers protein aggregation and cell-to-cell spread. In patient brains with amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), aggregation of the RNA-binding protein TDP-43 constitutes a major component of aberrant cytoplasmic inclusions. We identify a pathological signaling cascade whereby reactive nitrogen species cause S-nitrosylation of TDP-43 (forming SNO-TDP-43) to facilitate disulfide linkage and consequent TDP-43 aggregation. Similar pathological SNO-TDP-43 levels occur in postmortem human FTD/ALS brains and in cell-based models, including human-induced pluripotent stem cell (hiPSC)-derived neurons. Aggregated TDP-43 triggers additional nitrosative stress, representing positive feed forward leading to further SNO-TDP-43 formation and disulfide-linked oligomerization/aggregation. Critically, we show that these redox reactions facilitate cell spreading in vivo and interfere with the TDP-43 RNA-binding activity, affecting SNMT1 and phospho-(p)CREB levels, thus contributing to neuronal damage in ALS/FTD disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2021368118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980404PMC
March 2021

Borrelia burgdorferi infection modifies protein content in saliva of Ixodes scapularis nymphs.

BMC Genomics 2021 Mar 4;22(1):152. Epub 2021 Mar 4.

Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America.

Background: Lyme disease (LD) caused by Borrelia burgdorferi is the most prevalent tick-borne disease. There is evidence that vaccines based on tick proteins that promote tick transmission of B. burgdorferi could prevent LD. As Ixodes scapularis nymph tick bites are responsible for most LD cases, this study sought to identify nymph tick saliva proteins associated with B. burgdorferi transmission using LC-MS/MS. Tick saliva was collected using a non-invasive method of stimulating ticks (uninfected and infected: unfed, and every 12 h during feeding through 72 h, and fully-fed) to salivate into 2% pilocarpine-PBS for protein identification using LC-MS/MS.

Results: We identified a combined 747 tick saliva proteins of uninfected and B. burgdorferi infected ticks that were classified into 25 functional categories: housekeeping-like (48%), unknown function (18%), protease inhibitors (9%), immune-related (6%), proteases (8%), extracellular matrix (7%), and small categories that account for <5% each. Notably, B. burgdorferi infected ticks secreted high number of saliva proteins (n=645) than uninfected ticks (n=376). Counter-intuitively, antimicrobial peptides, which function to block bacterial infection at tick feeding site were suppressed 23-85 folds in B. burgdorferi infected ticks. Similar to glycolysis enzymes being enhanced in mammalian cells exposed to B. burgdorferi : eight of the 10-glycolysis pathway enzymes were secreted at high abundance by B. burgdorferi infected ticks. Of significance, rabbits exposed to B. burgdorferi infected ticks acquired potent immunity that caused 40-60% mortality of B. burgdorferi infected ticks during the second infestation compared to 15-28% for the uninfected. This might be explained by ELISA data that show that high expression levels of immunogenic proteins in B. burgdorferi infected ticks.

Conclusion: Data here suggest that B. burgdorferi infection modified protein content in tick saliva to promote its survival at the tick feeding site. For instance, enzymes; copper/zinc superoxide dismutase that led to production of HO that is toxic to B. burgdorferi were suppressed, while, catalase and thioredoxin that neutralize HO, and pyruvate kinase which yields pyruvate that protects Bb from HO killing were enhanced. We conclude data here is an important resource for discovery of effective antigens for a vaccine to prevent LD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12864-021-07429-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930271PMC
March 2021

SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in .

Elife 2021 Feb 15;10. Epub 2021 Feb 15.

Department of Biology, Johns Hopkins University, Baltimore, United States.

Piwi-interacting RNAs (piRNAs) play essential roles in silencing repetitive elements to promote fertility in metazoans. Studies in worms, flies, and mammals reveal that piRNAs are expressed in a sex-specific manner. However, the mechanisms underlying this sex-specific regulation are unknown. Here we identify SNPC-1.3, a male germline-enriched variant of a conserved subunit of the small nuclear RNA-activating protein complex, as a male-specific piRNA transcription factor in . SNPC-1.3 colocalizes with the core piRNA transcription factor, SNPC-4, in nuclear foci of the male germline. Binding of SNPC-1.3 at male piRNA loci drives spermatogenic piRNA transcription and requires SNPC-4. Loss of leads to depletion of male piRNAs and defects in male-dependent fertility. Furthermore, TRA-1, a master regulator of sex determination, binds to the promoter and represses its expression during oogenesis. Loss of TRA-1 targeting causes ectopic expression of and male piRNAs during oogenesis. Thus, sexually dimorphic regulation of expression coordinates male and female piRNA expression during germline development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.60681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884074PMC
February 2021

Site-specific O-glycosylation analysis of SARS-CoV-2 spike protein produced in insect and human cells.

bioRxiv 2021 Feb 10. Epub 2021 Feb 10.

Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen, Denmark.

Enveloped viruses hijack not only the host translation processes, but also its glycosylation machinery, and to a variable extent cover viral surface proteins with tolerogenic host-like structures. SARS-CoV-2 surface protein S presents as a trimer on the viral surface and is covered by a dense shield of N-linked glycans, and a few O-glycosites have been reported. The location of O-glycans is controlled by a large family of initiating enzymes with variable expression in cells and tissues and hence difficult to predict. Here, we used our well-established O-glycoproteomic workflows to map the precise positions of O-linked glycosylation sites on three different entities of protein S - insect cell or human cell-produced ectodomains, or insect cell derived receptor binding domain (RBD). In total 25 O-glycosites were identified, with similar patterns in the two ectodomains of different cell origin, and a distinct pattern of the monomeric RBD. Strikingly, 16 out of 25 O-glycosites were located within three amino acids from known N-glycosites. However, O-glycosylation was primarily found on peptides that were unoccupied by N-glycans, and otherwise had low overall occupancy. This suggests possible complementary functions of O-glycans in immune shielding and negligible effects of O-glycosylation on subunit vaccine design for SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2021.02.03.429627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872350PMC
February 2021

Identification of sulfenylation patterns in trophozoite stage Plasmodium falciparum using a non-dimedone based probe.

Mol Biochem Parasitol 2021 03 26;242:111362. Epub 2021 Jan 26.

Department of Biochemistry and Molecular Biology, Interdisciplinary Research Center, Justus Liebig University Giessen, Giessen, Germany. Electronic address:

Plasmodium falciparum causes the deadliest form of malaria. Adequate redox control is crucial for this protozoan parasite to overcome oxidative and nitrosative challenges, thus enabling its survival. Sulfenylation is an oxidative post-translational modification, which acts as a molecular on/off switch, regulating protein activity. To obtain a better understanding of which proteins are redox regulated in malaria parasites, we established an optimized affinity capture protocol coupled with mass spectrometry analysis for identification of in vivo sulfenylated proteins. The non-dimedone based probe BCN-Bio1 shows reaction rates over 100-times that of commonly used dimedone-based probes, allowing for a rapid trapping of sulfenylated proteins. Mass spectrometry analysis of BCN-Bio1 labeled proteins revealed the first insight into the Plasmodium falciparum trophozoite sulfenylome, identifying 102 proteins containing 152 sulfenylation sites. Comparison with Plasmodium proteins modified by S-glutathionylation and S-nitrosation showed a high overlap, suggesting a common core of proteins undergoing redox regulation by multiple mechanisms. Furthermore, parasite proteins which were identified as targets for sulfenylation were also identified as being sulfenylated in other organisms, especially proteins of the glycolytic cycle. This study suggests that a number of Plasmodium proteins are subject to redox regulation and it provides a basis for further investigations into the exact structural and biochemical basis of regulation, and a deeper understanding of cross-talk between post-translational modifications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molbiopara.2021.111362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218313PMC
March 2021

Emergency preparation and mitigation for COVID-19 response in an integrated pharmacy practice model.

Am J Health Syst Pharm 2021 03;78(8):705-711

Department of Pharmacy, University of Tennessee Medical Center, Knoxville, TN, USA.

Purpose: The purpose of this descriptive report is to share experiences in crisis response planning and risk mitigation at a university health system department of pharmacy with an integrated clinical practice model in the early months of the coronarvirus disease 2019 (COVID-19) pandemic.

Summary: The department of pharmacy's COVID-19 pandemic response included successful planning and implementation of measures to maintain pharmacy operations and minimize COVID-19 exposure of patients and staff. These measures included ensuring adequate personnel staffing using flexible staffing solutions, ongoing assessment of supply chain integrity, and continuation of integrated clinical pharmacy services 24/7 throughout the initial phase of the COVID-19 pandemic. Information technology (IT) and educational program modifications are also discussed.

Conclusion: This report describes successful crisis planning and risk mitigation in the setting of COVID-19, which was facilitated by the department of pharmacy's integrated clinical practice model. This model enabled uninterrupted personnel scheduling, supply chain integrity, continued provision of 24/7 integrated clinical services, adaptive use of IT tools, and continuation of educational programs. The experiences described may be instructive to other pharmacy departments in evaluating their response to the COVID-19 pandemic and in planning for similar pandemic or other emergency scenarios.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/ajhp/zxab015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7929428PMC
March 2021

A Hetero-Multimeric Chitinase-Containing and Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion.

Front Cell Infect Microbiol 2020 8;10:615343. Epub 2021 Jan 8.

Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, United States.

Malaria parasites are transmitted by  mosquitoes. During its life cycle in the mosquito vector the ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, , have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that PgCHT2 and its ortholog, PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in and ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric parasites expressing to replace 1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric ookinetes express monomeric PfCHT1, but a HMW complex containing is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2020.615343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821095PMC
June 2021

The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.

Nat Commun 2021 01 21;12(1):502. Epub 2021 Jan 21.

Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, 92093, USA.

The multifunctional nucleocapsid (N) protein in SARS-CoV-2 binds the ~30 kb viral RNA genome to aid its packaging into the 80-90 nm membrane-enveloped virion. The N protein is composed of N-terminal RNA-binding and C-terminal dimerization domains that are flanked by three intrinsically disordered regions. Here we demonstrate that the N protein's central disordered domain drives phase separation with RNA, and that phosphorylation of an adjacent serine/arginine rich region modulates the physical properties of the resulting condensates. In cells, N forms condensates that recruit the stress granule protein G3BP1, highlighting a potential role for N in G3BP1 sequestration and stress granule inhibition. The SARS-CoV-2 membrane (M) protein independently induces N protein phase separation, and three-component mixtures of N + M + RNA form condensates with mutually exclusive compartments containing N + M or N + RNA, including annular structures in which the M protein coats the outside of an N + RNA condensate. These findings support a model in which phase separation of the SARS-CoV-2 N protein contributes both to suppression of the G3BP1-dependent host immune response and to packaging genomic RNA during virion assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20768-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820290PMC
January 2021

Rising Stars in Proteomics and Metabolomics.

Authors:
John R Yates

J Proteome Res 2021 02 14;20(2):1105-1106. Epub 2021 Jan 14.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jproteome.0c01026DOI Listing
February 2021

The INO80 Complex Regulates Epigenetic Inheritance of Heterochromatin.

Cell Rep 2020 12;33(13):108561

Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Electronic address:

One key aspect of epigenetic inheritance is that chromatin structures can be stably inherited through generations after the removal of the signals that establish such structures. In fission yeast, the RNA interference (RNAi) pathway is critical for the targeting of histone methyltransferase Clr4 to pericentric repeats to establish heterochromatin. However, pericentric heterochromatin cannot be properly inherited in the absence of RNAi, suggesting the existence of mechanisms that counteract chromatin structure inheritance. Here, we show that mutations of components of the INO80 chromatin-remodeling complex allow pericentric heterochromatin inheritance in RNAi mutants. The ability of INO80 to counter heterochromatin inheritance is attributed to one subunit, Iec5, which promotes histone turnover at heterochromatin but has little effects on nucleosome positioning at heterochromatin, gene expression, or the DNA damage response. These analyses demonstrate the importance of the INO80 chromatin-remodeling complex in controlling heterochromatin inheritance and maintaining the proper heterochromatin landscape of the genome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.108561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896557PMC
December 2020

The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans.

Mol Cell 2021 02 29;81(3):546-557.e5. Epub 2020 Dec 29.

Department of Molecular, Cell and Systems Biology, University of California, Riverside, Riverside, CA, USA. Electronic address:

Eukaryotic cells regulate 5'-triphosphorylated RNAs (ppp-RNAs) to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR-1 (phosphatase that interacts with RNA and ribonucleoprotein particle 1) family of RNA polyphosphatases remove both the β and γ phosphates from ppp-RNAs. Here, we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RNA-dependent RNA polymerases (RdRPs) and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and Dicer-independent Argonaute pathways and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.12.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867642PMC
February 2021

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex.

Nucleic Acids Res 2021 04;49(7):3603-3616

Department of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa1183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8053116PMC
April 2021

HSP70 chaperones RNA-free TDP-43 into anisotropic intranuclear liquid spherical shells.

Science 2021 02 17;371(6529). Epub 2020 Dec 17.

Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, CA, USA.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding-deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named "anisosomes", have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)-dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43-containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.abb4309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8286096PMC
February 2021
-->