Biology subjects

Ori, A.

Publications and source records attributed to Ori, A..

32 records · Page 2Linked to original sources

bioRxiv2023

A comprehensive atlas of the aging vertebrate brain reveals signatures of progressive proteostasis dysfunction

Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brains biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules. One-Sentence SummaryTranslation pausing reshapes the aging brain proteome, revealing vulnerabilities in the biogenesis of nucleic-acid protein.

systems biology↗

bioRxiv2023

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development. One Sentence SummaryLoss of TDP-43 function results in the expression of de novo proteins from mis-spliced mRNA transcripts.

neuroscience↗

bioRxiv2022

ProteasomeID: quantitative mapping of proteasome interactomes and substrates for in vitro and in vivo studies

Proteasomes are essential molecular machines responsible for the degradation of proteins in eukaryotic cells. Altered proteasome activity has been linked to neurodegeneration, auto-immune disorders and cancer. Despite the relevance for human disease and drug development, no method currently exists to monitor proteasome composition and interactions in vivo in animal models. To fill this gap, we developed a strategy based on tagging of proteasomes with promiscuous biotin ligases and generated a new mouse model enabling the quantification of proteasome interactions by mass spectrometry. We show that biotin ligases can be incorporated in fully assembled proteasomes without negative impact on their activity. We demonstrate the utility of our method by identifying novel proteasome-interacting proteins, charting interactomes across mouse organs, and showing that proximity-labeling enables the identification of both endogenous and small molecule-induced proteasome substrates.

biochemistry↗

bioRxiv2022

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance leading to hippocampal network hypersynchrony

Specific autoantibodies against the NMDA-receptor (NMDAR) GluN1 subunit cause severe and debilitating NMDAR-encephalitis. Autoantibodies induce prototypic disease symptoms resembling schizophrenia, including psychosis and cognitive dysfunction. Using a mouse passive transfer model applying human monoclonal anti-GluN1-autoantibodies, we observed CA1 pyramidal neuron hypoexcitability, reduced AMPA-receptor (AMPAR) signaling, and faster synaptic inhibition resulting in disrupted excitatory-inhibitory balance. Functional alterations were supported by widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. At the network level, anti-GluN1-autoantibodies amplified gamma oscillations and disrupted theta-gamma coupling. A data-informed network model revealed that lower AMPAR strength and faster GABAA-receptor current kinetics chiefly account for these abnormal oscillations. As predicted by our model and evidenced experimentally, positive allosteric modulation of AMPARs alleviated aberrant gamma activity and thus reinforced the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-hypofunction-induced aberrant synaptic, cellular, and network dynamics provide new mechanistic insights into disease symptoms in NMDAR-encephalitis and schizophrenia.

neuroscience↗

bioRxiv2021

A systematic evaluation of 41 DNA methylation predictors across 101 data preprocessing and normalization strategies highlights considerable variation in algorithm performance

BackgroundDNA methylation (DNAm) based predictors hold great promise to serve as clinical tools for health interventions and disease management. While these algorithms often have high prediction accuracy and are associated with many disease-related phenotypes, the reliability of their performance remains to be determined. We therefore conducted a systematic evaluation across 101 different data processing strategies that preprocess and normalize DNAm data and assessed how each analytical strategy affects the reliability and prediction accuracy of 41 DNAm-based predictors. ResultsOur analyses were conducted in a large EPIC DNAm sample of the Jackson Heart Study (N=2,053) that included 146 pairs of technical replicate samples. By estimating the average absolute agreement between replicate pairs, we show that 32 out of 41 predictors (78%) demonstrate excellent test-retest reliability when appropriate data processing and normalization steps are implemented. Across all pairs of predictors, we find a moderate correlation in performance across analytical strategies (mean rho=0.40, SD=0.27), highlighting significant heterogeneity in performance across algorithms within a choice of an analytical pipeline. (Un)successful removal of technical variation furthermore significantly impacts downstream phenotypic association analysis, such as all-cause mortality risk associations. ConclusionsWe show that DNAm-based algorithms are sensitive to technical variation. The right choice of data processing and normalization pipeline is important to achieve reproducible estimates and improve prediction accuracy in downstream phenotypic association analyses. For each of the 41 DNAm predictors, we report its test-retest reliability and provide the best performing analytical strategy as a guideline for the research community. As DNAm-based predictors become more and more widely used, both for research purposes as well as for clinic applications, our work helps improve their performance and standardize their implementation.

genomics↗

bioRxiv2021

Novel biochemical, structural and systems insights into inflammatory signaling revealed by contextual interaction proteomics

Protein-protein interactions (PPI) represent the main mode of the proteome organization in the cell. In the last decade, several large-scale representations of PPI networks have captured generic aspects of the functional organization of network components, but mostly lack the context of cellular states. However, the generation of contextual representations of PPI networks is essential for structural and systems-level modeling of biological processes and remains an unsolved challenge. In this study we describe an integrated experimental/computational strategy to achieve a contextualized modeling of PPI. This strategy defines the composition, stoichiometry, spatio-temporal organization and cellular requirements for the formation of target assemblies. We used this approach to generate an integrated model of the formation principles and architecture of a large signalosome, the TNF-receptor signaling complex (TNF-RSC). Overall, we show that the integration of systems- and structure-level information provides a generic, largely unexplored link between the modular proteome and cellular function. Significance StatementIn this work, we propose a critical shift in the way we analyze, and think the study of, protein-protein interactions (PPI), and present an experimental and computational framework to model them in the cellular context. We applied this framework to the signalosome tumor necrosis factor receptor signaling complex (TNF-RSC), and generated an integrated model of its formation and architecture that provides new insights and resolved controversies regarding its organization and regulation. To achieve a contextual modelling of PPIs, we first optimized and developed, and then combined, approaches to map the composition of a target complex, its absolute stoichiometry, its spatial organization and assembly/disassembly dynamics, its temporal dependence on signaling, and its reliance on cellular resources.

systems biology↗

bioRxiv2021

Conserved exchange of paralog proteins during neuronal differentiation

Gene duplication enables the emergence of new functions by lowering the general evolutionary pressure. Previous studies have highlighted the role of specific paralog genes during cell differentiation, e.g., in chromatin remodeling complexes. It remains unexplored whether similar mechanisms extend to other biological functions and whether the regulation of paralog genes is conserved across species. Here, we analyze the expression of paralogs across human tissues, during development and neuronal differentiation in fish, rodents and humans. While ~80% of paralog genes are co-regulated, a subset of paralogs shows divergent expression profiles, contributing to variability of protein complexes. We identify 78 substitutions of paralog pairs that occur during neuronal differentiation and are conserved across species. Among these, we highlight a substitution between the paralogs SEC23A and SEC23B subunits of the COPII complex. Altering the ratio between these two proteins via RNAi-mediated knockdown is sufficient to influence neuron differentiation. We propose that remodeling of the vesicular transport system via paralog substitutions is an evolutionary conserved mechanism enabling neuronal differentiation.

systems biology↗

bioRxiv2021

Fetal-like reversion in the regenerating intestine is regulated by mesenchymal Asporin

Epithelial tissues undergo fetal-like cellular reprogramming to regenerate after damage1,2. Although the mesenchyme and the extracellular matrix (ECM) play critical roles in tissue homeostasis and regeneration2-5, their role in repurposing developmental programs in epithelium is unknown. To model epithelial regeneration, we culture intestinal epithelium on decellularized small intestinal scaffold (iECM), and identify Asporin (Aspn), an ECM bound proteoglycan, as a critical mediator of cellular reprogramming. Aspn is produced by the mesenchyme, and we show that its effect on epithelial Tgf{beta}-signalling via CD44 is critical for fetal-like conversion. Furthermore, we demonstrate that Aspn is transiently increased upon chemotherapy-induced damage and pivotal for a timely induction of the fetal-like state and tissue regeneration. In summary, we establish a platform for modelling epithelial injury responses ex vivo, and show that the mesenchymal Aspn-producing niche controls tissue repair by regulating epithelial fetal-like reprogramming.

developmental biology↗

bioRxiv2021

Computational and biochemical analysis of type IV Pilus dynamics and stability

Type IV pili (T4P) are distinctive dynamic filaments at the surface of many bacteria that can rapidly extend, retract and withstand strong forces. T4P are important virulence factors in many human pathogens, including Enterohemorrhagic Escherichia coli (EHEC). The structure of the EHEC T4P has been determined by integrating Nuclear Magnetic Resonance (NMR) and cryo-electron microscopy data. To better understand pilus assembly, stability and function, we performed a total of 108 s all-atom molecular dynamics simulations of wild-type and mutant T4P. Extensive characterization of the conformational landscape of T4P in different conditions of temperature, pH and ionic strength was complemented by targeted mutagenesis and biochemical analyses. Our simulations and NMR experiments revealed a conserved set of residues defining a novel calcium-binding site at the interface between three pilin subunits. Calcium binding enhanced T4P stability ex vivo and in vitro, supporting the role of this binding site as a potential pocket for drug design.

biochemistry↗

bioRxiv2020

Mapping sites of carboxymethyllysine modification on proteins reveals its consequences for proteostasis and cell proliferation

Posttranslational mechanisms play a key role in modifying the abundance and function of cellular proteins. Among these, modification by advanced glycation end products (AGEs) has been shown to accumulate during aging and age-associated diseases but specific protein targets and functional consequences remain largely unexplored. Here, we devised a proteomic strategy to identify specific sites of carboxymethyllysine (CML) modification, one of the most abundant AGEs. We identified over 1000 sites of CML modification in mouse and primary human cells treated with the glycating agent glyoxal. By using quantitative proteomics, we found that protein glycation triggers a proteotoxic response and directly affects the protein degradation machinery. We show that glyoxal induces cell cycle perturbation in primary endothelial cells and that CML modification reduces acetylation of tubulins and impairs microtubule dynamics. Our data demonstrate the relevance of AGE modification for cellular function and pinpoint specific protein networks that might become compromised during aging. HighlightsO_LIA peptide enrichment strategy allows mapping of CML modification in cells and tissues C_LIO_LICML modification competes with ubiquitination or acetylation of lysines C_LIO_LIGlyoxal treatment destabilizes the 26S proteasome C_LIO_LIGlyoxal arrests cell cycle and impairs microtubule dynamics via altering the tubulin code C_LI

biochemistry↗

bioRxiv2019

Late life metformin treatment limits cell survival and shortens lifespan by triggering an aging-associated failure of energy metabolism.

The diabetes drug metformin is to be clinically tested in aged humans to achieve health span extension, but little is known about responses of old non-diabetic individuals to this drug. By in vitro and in vivo tests we found that metformin shortens life span and limits cell survival when provided in late life, contrary to its positive early life effects. Mechanistically, metformin exacerbates aging-associated mitochondrial dysfunction towards respiratory failure, aggravated by the inability of old cells to upregulate glycolysis in response to metformin, leading to ATP exhaustion. The beneficial dietary restriction effect of metformin on lipid reserves is abrogated in old animals, contributing to metabolic failure, while ectopic stabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. The toxicity is also suspended in nematodes carrying diabetes-like insulin receptor insufficiency and showing prolonged resilience to metabolic stress induced by metformin. In sum, we uncovered an alarming metabolic decay triggered by metformin in late life which may limit its benefits for non-diabetic elderly patients. Novel regulators of life extension by metformin are also presented. HighlightsO_LILate life metformin treatment limits cell survival and shortens lifespan. C_LIO_LIMetformin exacerbates aging-associated mitochondrial dysfunction causing fatal ATP exhaustion. C_LIO_LIOld cells fail to upregulate glycolysis as a compensatory response to metformin. C_LIO_LIThe dietary restriction (DR) mimetic response to metformin is abrogated in old animals. C_LIO_LIPKA and not AMPK pathway instigates the early life DR response to metformin. C_LIO_LIStabilization of cellular ATP levels alleviates late life metformin toxicity in vitro and in vivo. C_LI

physiology↗

bioRxiv2019

Reduced proteasome activity in the aging brain results in ribosome stoichiometry loss and aggregation

A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short-lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post-transcriptional mechanisms that account for over 40% of the age-regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly / dis-assembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo. Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is the major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age-related neurodegeneration. HighlightsO_LIProgressive loss of stoichiometry affects multiple protein complexes C_LIO_LIRibosomes aggregate in old brains C_LIO_LIPartial reduction of proteasome activity is sufficient to induce loss of stoichiometry C_LIO_LIReduced proteasome levels are a major risk factor for early death in killifish C_LI

systems biology↗

bioRxiv2018

Rapid proteotyping reveals cancer biology and drug response determinants in the NCI-60 cells

We describe the rapid and reproducible acquisition of quantitative proteome maps for the NCI-60 cancer cell lines and their use to reveal cancer biology and drug response determinants. Proteome datasets for the 60 cell lines were acquired in duplicate within 30 working days using pressure cycling technology and SWATH mass spectrometry. We consistently quantified 3,171 proteotypic proteins annotated in the SwissProt database across all cell lines, generating a data matrix with 0.1% missing values, allowing analyses of protein complexes and pathway activities across all the cancer cells. Systematic and integrative analysis of the genetic variation, mRNA expression and proteomic data of the NCI-60 cancer cell lines uncovered complementarity between different types of molecular data in the prediction of the response to 240 drugs. We additionally identified novel proteomic drug response determinants for clinically relevant chemotherapeutic and targeted therapies. We anticipate that this study represents a significant advance toward the translational application of proteotypes, which reveal biological insights that are easily missed in the absence of proteomic data.

systems biology↗

bioRxiv2017

Species comparison of liver proteomes reveals enhanced lipid metabolism, reduced mitochondrial respiration and enhanced expression of detoxifying enzymes in the long-lived naked mole-rat.

Mammals display wide range of variation in their lifespan. Investigating the molecular networks that distinguish long-from short-lived species has proven useful to identify determinants of longevity. Here, we compared the liver of long-lived naked mole-rats (NMRs) and the phylogenetically closely related, shorter-lived, guinea pigs using an integrated omic approach. We found that NMRs livers display a unique expression pattern of mitochondrial proteins that result in distinct metabolic features of their mitochondria. For instance, we observed a generally reduced respiration rate associated with lower protein levels of respiratory chain components, particularly complex I, and increased capacity to utilize fatty acids. Interestingly, we show that the same molecular networks are affected during aging in both NMR and humans, supporting a direct link to the extraordinary longevity of both species. Finally, we identified a novel longevity pathway and validated it experimentally in the nematode C. elegans.

systems biology↗