Protéomique

Résumé

La protéomique désigne la science qui étudie les protéomes, c'est-à-dire l'ensemble des protéines d'une cellule, d'un organite, d'un tissu, d'un organe ou d'un organisme à un moment donné et sous des conditions données. Dans la pratique, la protéomique s'attache à identifier de manière globale les protéines extraites d'une culture cellulaire, d'un tissu ou d'un fluide biologique, leur localisation dans les compartiments cellulaires, leurs éventuelles modifications post-traductionnelles ainsi que leur quantité. Elle permet de quantifier les variations de leur taux d'expression en fonction du temps, de leur environnement, de leur état de développement, de leur état physiologique et pathologique, de l'espèce d'origine. Elle étudie aussi les interactions que les protéines ont avec d'autres protéines, avec l'ADN ou l'ARN, ou d'autres substances. La protéomique fonctionnelle étudie les fonctions de chaque protéine. La protéomique étudie enfin la structure primaire, secondaire et tertia

Source officielle

https://fr.wikipedia.org/wiki/Prot%C3%A9omique

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Background: Overexpression of the transsulfuration enzyme cystathionine-beta-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters. Methods: 8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS. Results: DS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. C-13 glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with the dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters. Conclusions: Increased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.

ELSEVIER2022

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Difficulties to replicate telomeres - the ends of our chromosomes - can cause telomere shortening andgenome instability. These difficulties are due to the repetitive DNA sequence and distinct structures at telomeresthat challenge the semi-conservative DNA replication machinery. Among the proteins that help toovercome the obstacles to telomere replication are factors known to be mutated in genetic diseases (forinstance WRN, BLM, RTEL1, CTC1), but we suspect that there are still factors and pathways unknown.DNA replication defects at telomeres manifest as smeary or multiple telomeric fluorescence in situ hybridization(FISH) signals on metaphase chromosomes - commonly called telomere fragility. To identify proteinsthat protect human telomeres from fragility, we depleted 71 proteins (including 8 controls) from HeLa cellsusing siRNA pools and analysed their metaphase chromosomes for fragile telomeres. The majority of theseproteins had been identified by QTIP-iPOND - a proteomic analysis of chromatin near telomere replicationforks. Among the 71 proteins that were depleted in the siRNA screen, we identified 29 novel proteins thatprevent telomere fragility and are therefore crucial to maintain genome stability. The telomere fragilityscreen validated QTIP-iPOND as a technique to identify telomere replication proteins. The hits identified inour telomere fragility screen may for example have functions in regulating telomeric chromatin compositionand compaction, regulating transcription of the telomeric long non-coding RNA TERRA and its associationwith telomeric chromatin, dealing with replication stress and stalled replication forks, modulating DNAdamage signalling and repair, modulating sister chromatid cohesion, and preventing oxidative stress.One of the 29 novel proteins identified in the telomere fragility screen is chromobox 8 (CBX8) whose functionat telomeres was investigated in further detail in this study. CBX8 is a component of the canonical polycombrepressive complex 1 (PRC1), an epigenetic regulator of gene expression. However, CBX8 also hasfunctions that are independent of the PRC1 complex. We show that human CBX8 prevents telomere fragilitycaused by RNA-DNA hybrids which could stall replication forks and induce homologous recombination.CBX8 depletion or knockout did however not affect telomeric RNA-DNA hybrid levels. We hypothesize thatCBX8 may be involved in repair activities at stalled replication forks that arise when the replisome encounterspersistent RNA-DNA hybrids. Moreover, we found that CBX8 localizes to telomeric repeat-binding factor2 (TRF2)-deficient telomeres in a manner that is dependent on RNA-DNA hybrids and ATM signalling.We demonstrate that H3K27me3 (trimethylated histone 3 lysine 27) marks, which have previously beensuggested to help recruitment of CBX proteins to chromatin via the CBX chromodomain, are dispensable forCBX8 recruitment to TRF2-deficient telomeres. Furthermore, we found that CBX8 promotes non-homologousend joining (NHEJ)-mediated telomere fusions of dysfunctional telomeres. We therefore hypothesizethat CBX8 is involved in repair activities that influence the DNA repair pathway choice at TRF2-deficient telomeres.

EPFL2022

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Assessing normalization methods in mass spectrometry-based proteome profiling of clinical samples

Loïc Dayon

Background: Large-scale proteomic studies have to deal with unwanted variability, especially when samples originate from different centers and multiple analytical batches are needed. Such variability is typically added throughout all the steps of a clinical research study, from human biological sample collection and storage, sample preparation, spectral data acquisition, to peptide and protein quantification. In order to remove such diverse and unwanted variability, normalization of the protein data is performed. There have been already several published reviews comparing normalization methods in the-omics field, but reports focusing on proteomic data generated with mass spectrometry (MS) are much fewer. Additionally, most of these reports have only dealt with small datasets. Results: As a case study, here we focused on the normalization of a large MS-based proteomic dataset obtained from an overweight and obese pan-European cohort, where different normalization methods were evaluated, namely: center standardize, quantile protein, quantile sample, global standardization, ComBat, median centering, mean centering, single standard and removal of unwanted variation (RUV); some of these are generic normalization methods while others have been specifically created to deal with genomic or metabolomic data. We checked how relationships between proteins and clinical variables (e.g., gender, levels of triglycerides or cholesterol) were improved after normalizing the data with the different methods. Conclusions: Some normalization methods were better adapted for this particular large-scale shotgun proteomic dataset of human plasma samples labeled with isobaric tags and analyzed with liquid chromatography-tandem MS. In particular, quantile sample normalization, RUV, mean and median centering showed very good perfor-mances, while quantile protein normalization provided worse results than those obtained with unnormalized data.

ELSEVIER SCI LTD2022

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Assessing normalization methods in mass spectrometry-based proteome profiling of clinical samples

Loïc Dayon

ELSEVIER SCI LTD2022

EPFL2022