Biology subjects

Zaia, J.

Publications and source records attributed to Zaia, J..

12 recordsLinked to original sources

bioRxiv2026

ROCK inhibitor Ripasudil reduces Alzheimer pathological proteins and enhances cellular bioenergetics

Rho-associated kinase (ROCK) inhibitors have been explored as an Alzheimer Disease (AD) therapeutic agent. In this study, we aim to determine the efficacy of a potent ROCK inhibitor, ripasudil, in impacting AD pathological protein tau in mice. The time-course study showed that ripasudil rapidly penetrated mouse brains, reaching maximal concentration half an hour post-injection. To determine the efficacy of ripasudil, we injected AppNL-G-F knock-in mice with ripasudil at 30 mg/kg and sacrificed them after three hours. We used Tandem Mass Tag labels and compared the proteomic profiles of mouse brains exposed to ripasudil to those exposed to saline. We also treated AD-patient-derived Neural Stem Cells (NSCs) with ripasudil and performed a Seahorse Mito Stress Test to evaluate their impact on mitochondrial activity. Bioinformatics analysis of the proteomic profiles revealed differentially enriched components of chromosome. The Mito Stress Test of NSCs revealed significant increases in basal oxygen consumption, ATP-linked respiration, maximal respiration, and reserve capacity oxygen consumption compared to untreated NSCs. Importantly, we found a significant reduction of Tau and pTau 396 in brains of mice treated with ripasudil. Overall, our findings highlight the feasibility and promise of ROCK inhibitor ripasudil as an AD therapeutic agent.

neuroscience↗

bioRxiv2026

Proteomics of highly post-translationally modified proteins in kidney tubulointerstitium

Kidney disease affects over 850 million people and leads to degradation of the kidney cortical compartments and kidney failure. Interstitial fibrosis and tubular atrophy (IFTA) is a universal feature of chronic kidney disease, yet the sequence of tubulointerstitial matrix protein (matrisome) changes that underlie the development and progression of IFTA are not fully understood. In particular, post-translational modifications which may stabilize key matrisome components and result in irreversible and functionally detrimental interstitial fibrosis or tubular basement membrane thickening, have yet to be characterized. Post-translational modifications (e.g., acetylation) have been shown to contribute to kidney diseases, yet they have not been investigated in chronic kidney disease progression. Herein, we established feasibility of matrisome protein quantification in the tubulointerstitium, with concurrent matrisome protein post-translational modification quantification, in archived diagnostic human kidney biopsies, using laser capture microdissection, liquid chromatography tandem mass spectrometry (LC-MS/MS), and bioinformatics analysis. In this pilot study, we focused on two conditions associated with IFTA progression: diabetic nephropathy and kidney transplantation. We examined both healthy and chronically damaged tubulointerstitium in two human kidney biopsies, one representing each condition. We identified signature protein markers of tubulointerstitium (e.g., UMOD, SLC12A1) in abundance, confirming sample composition, and observed uniformity in protein abundance across replicates from the same biopsies. We found increases in certain collagen isoforms (COL1A1, COL6A3) and other ancillary proteins in areas of IFTA. We also successfully quantified post-translational modifications, confirming that hydroxylproline was the most abundant modification in healthy tubulointerstitium from the kidney transplant biopsy, and that hydroxyllysine was also highly abundant and could be confidently quantitated on two collagen isoforms (COL1A1, COL4A2). Lastly, we performed power analysis to determine the required sample size to achieve significant power and false discovery rate. Several proteins exhibited large Hedge effect sizes in the tubulointerstitium (ANPEP, PRODH2), indicating their potential usefulness as fibrosis signatures. Our study is a first step toward comprehensive characterization of tubulointerstitial matrisome changes, including post-translational modifications seen with IFTA progression. Subsequent studies will potentially reveal new molecular markers of key early events in chronic kidney disease progression, and new therapeutic targets for mitigating this process. Data are available via ProteomeXchange identifier PXD079025.

biochemistry↗

bioRxiv2026

The enteroviral protease target LSM14A operates outside of P-bodies to augment antiviral innate immunity

Antiviral innate immune networks in human cells comprise core components that serve as central signaling hubs and several context-dependent modulators whose role may be virus- or tissue-specific. One such modulator is LSM14A, which potentiates innate immune response but is not essential. We recently showed that enteroviruses deploy their protease activity to cleave LSM14A, thereby disabling its antiviral function. In this study, we probe the molecular mechanism by which LSM14A contributes to innate immunity. We show that although LSM14A predominantly localizes to processing bodies (P-bodies; PBs), this localization is not essential for its innate immune function. Likewise, association with peroxisomes does not contribute to its immune activity. Instead, an unbiased systems-level interactomic analysis reveals a distinct cohort of LSM14A-associated proteins that assemble outside canonical PBs and peroxisomes following infection with Sendai virus, a robust inducer of innate immunity. Functional interrogation of these interactors demonstrate that several are essential for LSM14A-dependent amplification of antiviral signaling. Together, these findings uncover a functional axis of LSM14A that operates independently of its canonical subcellular localizations and is mediated through a specialized interaction network, improving our understanding of how this protein reinforces the antiviral innate immune system.

microbiology↗

bioRxiv2024

Traffic Jam activates the Flamenco piRNA cluster locus and the Piwi pathway to ensure transposon silencing and Drosophila fertility.

Flamenco (Flam) is the most prominent piRNA cluster locus expressed in Drosophila ovarian follicle cells, and it is required for female fertility to silence gypsy/mdg4 transposons. To determine how Flam is regulated, we used promoter-bashing reporter assays in OSS cells to uncover novel enhancer sequences within the first exons of Flam. We confirmed the enhancer sequence relevance in vivo with new Drosophila Flam deletion mutants of these regions that compromised Flam piRNA expression and lowered female fertility from activated transposons. Our proteomic analysis of proteins associated with these enhancer sequences discovered the transcription factor Traffic Jam (TJ). Tj knockdowns in OSS cells caused a decrease in Flam transcripts, Flam piRNAs, and multiple Piwi pathway genes. A TJ ChIP-seq analysis from whole flies and OSS cells confirmed TJ binding exactly at the enhancer that was deleted in the new Flam mutant as well as at multiple Piwi pathway gene enhancers. Interestingly, TJ also bound the Long Terminal Repeats of transposons that had decreased expression after Tj knockdowns in OSS cells. Our study reveals the integral role TJ plays in the on-going arms race between selfish transposons and their suppression by the host Piwi pathway and the Flam piRNA cluster locus.

genetics↗

bioRxiv2023

Recurrent SARS-CoV-2 mutations at Spike D796 evade antibodies from pre-Omicron convalescent and vaccinated subjects

SARS-CoV-2 lineages of the Omicron variant rapidly became dominant in early 2022 and frequently cause human infections despite vaccination or prior infection with other variants. In addition to antibody-evading mutations in the Receptor Binding Domain, Omicron features amino acid mutations elsewhere in the Spike protein, however their effects generally remain ill-defined. The Spike D796Y substitution is present in all Omicron sub-variants and occurs at the same site as a mutation (D796H) selected during viral evolution in a chronically-infected patient. Here we map antibody reactivity to a linear epitope in the Spike protein overlapping position 796. We show that antibodies binding this region arise in pre-Omicron SARS-CoV-2 convalescent and vaccinated subjects, but that both D796Y and D796H abrogate their binding. These results suggest that D796Y contributes to the fitness of Omicron in hosts with pre-existing immunity to other variants of SARS-CoV-2 by evading antibodies targeting this site.

immunology↗

bioRxiv2023

RAMZIS: a bioinformatic toolkit for rigorous assessment of the alterations to glycoprotein structure that occur during biological processes

MotivationGlycosylation elaborates the structures and functions of glycoproteins; glycoproteins are common post-translationally modified proteins and are heterogeneous and non-deterministically synthesized as an evolutionarily driven mechanism that elaborates the functions of glycosylated gene products. While glycoproteins account for approximately half of all proteins, their macro- and micro-heterogeneity requires specialized proteomics data analysis methods as a given glycosite can be divided into several glycosylated forms, each of which must be quantified. Sampling of heterogeneous glycopeptides is limited by mass spectrometer speed and sensitivity, resulting in missing values. In conjunction with the low sample size inherent to glycoproteomics, this necessitated specialized statistical metrics to identify if observed changes in glycopeptide abundances are biologically significant or due to data quality limitations. ResultsWe developed an R package, Relative Assessment of m/z Identifications by Similarity (RAMZIS), that uses similarity metrics to guide biomedical researchers to a more rigorous interpretation of glycoproteomics data. RAMZIS uses contextual similarity to assess the quality of mass spectral data and generates graphical output that demonstrates the likelihood of finding biologically significant differences in glycosylation abundance dataset. Investigators can assess dataset quality, holistically differentiate glycosites, and identify which glycopeptides are responsible for glycosylation pattern expression change. Herein RAMZIS approach is validated by theoretical cases and by a proof-of-concept application. RAMZIS enables comparison between datasets too stochastic, small, or sparse for interpolation while acknowledging these issues in its assessment. Using our tool, researchers will be able to rigorously define the role of glycosylation and the changes that occur during biological processes. Availabilityhttps://github.com/WillHackett22/RAMZIS ContactJoseph Zaia, Boston University Medical Campus, 670 Albany St., rm 509, Boston, MA 02118 USA, (e) jzaia@bu.edu, (v) 1-617-358-2429 Supplementary informationSupplementary data are available

bioinformatics↗

bioRxiv2021

Community Evaluation of Glycoproteomics Informatics Solutions Reveals High-Performance Search Strategies of Glycopeptide Data

Glycoproteome profiling (glycoproteomics) is a powerful yet analytically challenging research tool. The complex tandem mass spectra generated from glycopeptide mixtures require sophisticated analysis pipelines for structural determination. Diverse software aiding the process have appeared, but their relative performance remains untested. Conducted through the HUPO Human Proteome Project - Human Glycoproteomics Initiative, this community study, comprising both developers and users of glycoproteomics software, evaluates the performance of informatics solutions for system-wide glycopeptide analysis. Mass spectrometry-based glycoproteomics datasets from human serum were shared with all teams. The relative team performance for N- and O-glycopeptide data analysis was comprehensively established and validated through orthogonal performance tests. Excitingly, several high-performance glycoproteomics informatics solutions were identified. While the study illustrated that significant informatics challenges remain, as indicated by a high discordance between annotated glycopeptides, lists of high-confidence (consensus) glycopeptides were compiled from the standardised team reports. Deep analysis of the performance data revealed key performance-associated search variables and led to recommendations for improved "high coverage" and "high accuracy" glycoproteomics search strategies. This study concludes that diverse software for comprehensive glycopeptide data analysis exist, points to several high-performance search strategies, and specifies key variables that may guide future software developments and assist informatics decision-making in glycoproteomics.

biochemistry↗

bioRxiv2020

Assignment of coronavirus spike protein site-specific glycosylation using GlycReSoft

Widely-available LC-MS instruments and methods allow users to acquire glycoproteomics data. Complex glycans, however, add a dimension of complexity to the data analysis workflow. In a sense, complex glycans are post-translationally modified post-translational modifications, reflecting a series of biosynthetic reactions in the secretory pathway that are spatially and temporally regulated. One problem is that complex glycan is micro-heterogeneous, multiplying the complexity of the proteome. Another is that glycopeptide glycans undergo dissociation during tandem MS that must be considered for tandem MS interpretation algorithms and quantitative tools. Fortunately, there are a number of algorithmic tools available for analysis of glycoproteomics LC-MS data. We summarize the principles for glycopeptide data analysis and show use of our GlycReSoft tool to analyze SARS-CoV-2 spike protein site-specific glycosylation.

bioinformatics↗

bioRxiv2020

Protocol for analysis of glycoproteomics LC-MS data using GlycReSoft

Summary/AbstractThe GlycReSoft software tool allows users to process glycoproteomics LC-MS data sets. The tool accepts proteomics database search results or a user-defined list of proteins in the sample. GlycReSoft processes LC-MS data to yield deconvoluted exact mass values. The user has the option to import a list of theoretical glycans from an external database, a curated glycan list, or a measured glycome. The tool assembles a list of theoretical glycopeptides from the lists of theoretical glycans and proteins, respectively. The program then scores the tandem mass spectra in the LC-MS data files and provides graphical views of the identified glycopeptides for each protein in the sample, and the set of glycoforms identified for each peptide sequence.

biochemistry↗

bioRxiv2020

Measuring site-specific glycosylation similarity between influenza A virus variants with statistical certainty

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

biochemistry↗

bioRxiv2019

Proteomic Profiling and Biological Characterization of Extracellular Vesicles Isolated from Human Alzheimer’s Disease Brain Tissues

IntroductionExtracellular vesicles (EVs) from human Alzheimers disease (AD) biospecimens contain amyloid-{beta} peptide (A{beta}) and tau. While AD EVs are known to affect brain disease pathobiology, their biochemical and molecular characterizations remain ill defined.\n\nMethodsEVs were isolated from the cortical grey matter of 20 AD and 18 control brains. Tau and A{beta} levels were measured by immunoassay. Differentially expressed EV proteins were assessed by quantitative proteomics and machine learning.\n\nResultsLevels of pS396 tau and A{beta} were significantly elevated in AD EVs. High levels of neuron- and glia- specific factors are detected in control and AD EVs, respectively. Machine learning identified ANXA5, VGF, GPM6A and ACTZ in AD EV compared to controls. They distinguished AD EVs from controls in the test sets with 88% accuracy.\n\nDiscussionIn addition to A{beta} and tau, ANXA5, VGF, GPM6A and ACTZ are new signature proteins in AD EVs.

biochemistry↗

bioRxiv2019

Complexity and ultrastructure of infectious extracellular vesicles from cells infected by nonenveloped virus

Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles secreted from host cells. Using biochemical and biophysical methods we identify multiple components in these secreted vesicles, including PV virions; positive and negative-sense viral RNA; essential viral replication proteins; ribosomal and regulatory cellular RNAs; and numerous host cell proteins, such as regulators of cellular metabolism and structural remodeling. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious extracellular vesicles containing both virions and a unique mat-like structure. Based on our biochemical data (western blot, RNA-Seq, and mass spectrometry), these mat-like structures are expected to be comprised of unencapsidated RNA and proteins. Our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral and host RNAs and proteins.\n\nImportanceThe family of picornaviridae is comprised of small positive-sense RNA viruses, many of which are significant human pathogens. Picornaviruses exploit secreted extracellular vesicles for cell-to-cell viral transmission without cell lysis, and poliovirus serves as a model system for picornaviruses that are not protected by a surrounding membrane (non-enveloped viruses). The structure and contents of these vesicles secreted by virus-infected cells are described here. In addition to mature virions, these vesicles carry negative-sense, template viral RNA and essential replication proteins, as well as cellular resources from the host. Their complex contents may comprise an enhanced virulence factor for propagation of infection, and understanding their structure and function is helping elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.

cell biology↗