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In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification. [ABSTRACT FROM AUTHOR]

In order to understand the coordinated proteome changes associated with differentiation of a cultured cell pluripotency model, protein expression changes induced by treatment of NT2 embryonal carcinoma cells with retinoic acid were monitored by mass spectrometry. The relative levels of over 5000 proteins were mapped across distinct cell fractions. Analysis of the chromatin fraction revealed major abundance changes among chromatin proteins and epigenetic pathways between the pluripotent and differentiated states. Protein complexes associated with epigenetic regulation of gene expression, chromatin remodelling (e.g., SWI/SNF, NuRD) and histone-modifying enzymes (e.g., Polycomb, MLL) were found to be extensively regulated. We therefore investigated histone modifications before and after differentiation, observing changes in the global levels of lysine acetylation and methylation across the four canonical histone protein families, as well as among variant histones. We identified the set of proteins with affinity to peptides housing the histone marks H3K4me3 and H3K27me3, and found increased levels of chromatin-associated histone H3 tail trimming following differentiation that correlated with increased expression levels of cathepsin proteases. We further found that inhibition of cathepsins B and D reduces histone H3 clipping. Overall, the work reveals a global reorganization of the cell proteome congruent with differentiation, highlighting the key role of multiple epigenetic pathways, and demonstrating a direct link between cathepsin B and D activity and histone modification.

Ayodele Alaiya,1 Abeer Alshukairi,2 Zakia Shinwari,1 Mariam AL-Fares,3 Jawaher Alotaibi,4 Waleed AlOmaim,3 Ibtihaj Alsharif,5 Razan Bakheet,6 Layla Alharbi,5 Rabab Allam,1 Ayed Asiri,7 Ziad Memish,8 Khaldoun Alromaih,6 Maha Al-Mozaini5 1Proteomics Unit, Stem Cell and Tissue Re-Engineering Program, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia; 2Department of Medicine, King Faisal Specialist Hospital and Research Center, Jeddah, Kingdom of Saudi Arabia; 3Clinical Chemistry Laboratory, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 4Department of Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia; 5Immunocompromised Host Research Unit, Department of Infection and Immunity, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia; 6Centre for Genomic Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Kingdom of Saudi Arabia; 7Critical Care Services, Al Imam Abdulrahman Al Faisal Hospital, Riyadh, Kingdom of Saudi Arabia; 8Research and Innovation Center, King Saud Medical City, Ministry of Health, Riyadh, Kingdom of Saudi ArabiaCorrespondence: Ayodele AlaiyaProteomics Unit, Stem Cell and Tissue Re-Engineering Program, King Faisal Specialist Hospital and Research Centre, KFSH&RC, BOX 3354, Riyadh, 11211, Saudi ArabiaTel +966 114424178Email aalaiya@kfshrc.edu.saMaha Al-MozainiImmunocompromised Host Research Unit, Department of Infection and Immunity, KFSH&RC, Riyadh, Saudi ArabiaTel +966 114439632Email mmozaini@kfshrc.edu.saPurpose: This study aimed to understand the pathophysiology of host responses to infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/(COVID-19) and Middle East respiratory syndrome coronavirus (MERS-CoV) and to identify proteins for patient stratification with different grades of illness severity.Patients and Methods: Peripheral blood samples from 43 patients with different grades of COVID-19, 7 MERS-CoV patients admitted to the ICU, and 10 healthy subjects were analyzed using label-free quantitative liquid chromatography–mass spectrometry (LC–MS).Results: We identified 193 and 91 proteins that differed significantly between COVID-19 and MERS-CoV sample groups, respectively, and 49 overlapped between datasets. Only 10 proteins are diagnostic of asymptomatic cases, 12 are prognostic of recovery from severe illness, and 28 are prognostic of a fatal outcome of COVID-19. These proteins are implicated in virus-specific/related signaling networks. Notable among the top canonical pathways are humoral immunity, inflammation, acute-phase response signaling, liver X receptor/retinoid X receptor (LXR/RXR) activation, coagulation, and the complement system. Furthermore, we confirmed positive viral shedding in 11.76% of 51 additional peripheral blood samples, indicating that caution should be taken to avoid the possible risk of transfusion of infected blood products.Conclusion: We identified COVID-19 and MERS-CoV protein panels that have potential as biomarkers and might assist in the prognosis of SARS-CoV-2 infection. The identified markers further our understanding of COVID-19 disease pathophysiology and may have prognostic or therapeutic potential in predicting or managing host cell responses to human COVID-19 and MERS-CoV infections.Keywords: COVID-19, SARS-CoV, MERS-CoV, expression proteomics, biomarkers

Background: Human immunodeficiency virus-1 (HIV-1) exploits human host factors to complete its life cycle. Hence, discovery of HIV-regulated host proteins markers would better our understanding of the virus life-cycle and its contribution to pathogenesis and discovery of objective diagnostic and prognostic molecules. Methods: We conducted holistic total proteomics analysis of three closely related study populations including patients with HIV type-1 (HIV-1) and HIV type-2 (HIV-2) as well as HIV-1 elite controllers (HIV-1-EC). Peripheral blood plasma (PBP) samples were subjected to label-free quantitative liquid-chromatography tandem mass-spectrometry (LC–MS/MS). Results: Over 314 unique PBP protein species were identified of which 100 (approx. 32%) were significantly differentially expressed (≥2 to ∞ - fold-change; p

Human immunodeficiency virus-1 (HIV-1) exploits human host factors to complete its life cycle. Hence, discovery of HIV-regulated host proteins markers would better our understanding of the virus life-cycle and its contribution to pathogenesis and discovery of objective diagnostic and prognostic molecules. We conducted holistic total proteomics analysis of three closely related study populations including patients with HIV type-1 (HIV-1) and HIV type-2 (HIV-2) as well as HIV-1 elite controllers (HIV-1-EC). Peripheral blood plasma (PBP) samples were subjected to label-free quantitative liquid-chromatography tandem mass-spectrometry (LC–MS/MS). Over 314 unique PBP protein species were identified of which 100 (approx. 32%) were significantly differentially expressed (≥2 to ∞ - fold-change; p < 0.05) between the three sample cohorts. Of the 100 proteins, 91 were significantly changed between pairs of HIV-1 versus HIV-1-EC, while 83 of the 100 proteins differed significantly between HIV-2 and HIV-1-EC. Interestingly, 76 proteins (87.5%) overlap between the two data sets indicating that majority of these proteins share similar expression changes between HIV-1 and HIV-2 sample groups. Two of the identified proteins, XRCC5 and PSME1, were implicated in the early phase of the pathway network for HIV life cycle, while others were involved in infectious disease and disease of signal transduction. Among them were MAP2K1, RPL23A, RPS3, CALR, PRDX1, SOD2, LMNB1, PHB, and FGB. Despite the high degree of similarity in protein profiles of HIV-1 and HIV-2, six proteins differed significantly including ETFB, PHB2, S100A9, LMO2, PPP3R1 and Vif, a fragment of virion infectivity factor of HIV-1. Additionally, 15 proteins were uniquely expressed, and one of them (LSP1) is present only in HIV-1-EC but absent in HIV1 and HIV-2 and vice versa for the rest 14 proteins. Altogether, we have identified HIV-specific/related protein expression changes that might potentially be capable of early diagnosis and prognosis of HIV diseases and other related infectious diseases. [ABSTRACT FROM AUTHOR]

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease ( PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 ( PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. [ABSTRACT FROM AUTHOR]

The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.

Diagnostic classification accuracy is critical in expression proteomics to ensure that as many true differences as possible are identified with acceptable false-positive rates. We present a comparison of the diagnostic accuracy of iTRAQ with three label-free methods, peak area, spectral counting, and emPAI, for relative quantification using a spiked proteome standard. We provide the first validation of emPAI for intersample relative quantification and find clear differences among the four quantification approaches that could be considered when designing an experiment. Spectral counting was observed to perform surprisingly well in all regards. Peak area performed best for smaller fold differences and was shown to be capable of discerning a 1.1-fold difference with acceptable specificity and sensitivity. The performance of iTRAQ was dramatically worse than the label-free methods with low abundance proteins. Using the iTRAQ data set for validation, we also demonstrate a novel iTRAQ analysis regime that avoids the use of ratios in significance testing and outperforms a common commercial alternative.