Nejvíce citovaný článek - PubMed ID 21254760
The efforts to utilize microflow liquid chromatography hyphenated to tandem mass spectrometry (μLC-MS/MS) for deep-scale proteomic analysis are still growing. In this work, two-dimensional LC separation and peptide derivatization by a tandem mass tag (TMT) were used to assess the capability of μLC-MS/MS to reveal protein changes associated with the severe chronic anthracycline cardiotoxicity phenotype in comparison with nanoflow liquid chromatography (nLC-MS/MS). The analysis of the control and anthracycline-treated rabbit myocardium by μLC-MS/MS and nLC-MS/MS allowed quantification of 3956 and 4549 proteins, respectively, with 84% of these proteins shared in both data sets. Both nLC-MS/MS and μLC-MS/MS revealed marked global proteome dysregulation in severe anthracycline cardiotoxicity, with a significant change in approximately 55% of all detected proteins. The μLC-MS/MS analysis allowed less compressed and more precise determination of the TMT channel ratio and correspondingly broader fold-change protein distribution than nLC-MS/MS. The total number of significantly changed proteins was higher in nLC-MS/MS (2498 vs 2183, 1900 proteins shared), whereas the opposite was true for a number of significantly changed proteins with a fold-change cutoff ≥ 2 (535 vs 820). The profound changes concerned mainly proteins of cardiomyocyte sarcomeres, costameres, intercalated discs, mitochondria, and extracellular matrix. In addition, distinct alterations in immune and defense response were found with a remarkable involvement of type I interferon signaling that has been recently hypothesized to be essential for anthracycline cardiotoxicity pathogenesis. Hence, μLC-MS/MS was found to be a sound alternative to nLC-MS/MS that can be useful for comprehensive mapping of global myocardial proteome alterations such as those associated with severe anthracycline cardiotoxicity.
- Publikační typ
- časopisecké články MeSH
Micropterigidae is regarded as the sister group of all the other Lepidoptera, providing important insights into the evolution of Lepidoptera. However, the gene and protein profiles of silk from Micropterigidae have not yet been identified. In this study, we investigate the components of silk cocoons of the micropterigid species Neomicropteryx cornuta. Here we show that the protein fibroin heavy chain (FibH) is absent in the silk of N. cornuta and that the putative homolog of fibroin light chain (FibL) is also absent or severely altered. This is confirmed by transcriptome and genome analyses of the conserved regions in this species. The examination of the synteny around the fibH genes in several Lepidoptera and Trichoptera species shows that the genomic region containing this gene is absent in another micropterigid species, Micropterix aruncella. In contrast, we found putative orthologs of fibH and fibL in the representative transcripts of another distinct clade, Eriocraniidae. This study shows that the loss of FibH and the loss or severe divergence of FibL occurrs specifically in the family Micropterigidae and reveals dynamic evolutionary changes in silk composition during the early evolution of Lepidoptera. It also shows that silk proteins without FibH can form a solid cocoon.
- MeSH
- fibroiny * genetika chemie metabolismus MeSH
- fylogeneze MeSH
- hedvábí * genetika chemie metabolismus MeSH
- hmyzí proteiny * genetika metabolismus MeSH
- Lepidoptera * genetika metabolismus MeSH
- molekulární evoluce MeSH
- můry * genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- transkriptom MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fibroiny * MeSH
- hedvábí * MeSH
- hmyzí proteiny * MeSH
Hypertrophic cardiomyopathy (HCM) caused by autosomal-dominant mutations in genes coding for structural sarcomeric proteins, is the most common inherited heart disease. HCM is associated with myocardial hypertrophy, fibrosis and ventricular dysfunction. Hypoxia-inducible transcription factor-1α (Hif-1α) is the central master regulators of cellular hypoxia response and associated with HCM. Yet its exact role remains to be elucidated. Therefore, the effect of a cardiomyocyte-specific Hif-1a knockout (cHif1aKO) was studied in an established α-MHC719/+ HCM mouse model that exhibits the classical features of human HCM. The results show that Hif-1α protein and HIF targets were upregulated in left ventricular tissue of α-MHC719/+ mice. Cardiomyocyte-specific abolishment of Hif-1a blunted the disease phenotype, as evidenced by decreased left ventricular wall thickness, reduced myocardial fibrosis, disordered SRX/DRX state and ROS production. cHif1aKO induced normalization of pro-hypertrophic and pro-fibrotic left ventricular remodeling signaling evidenced on whole transcriptome and proteomics analysis in α-MHC719/+ mice. Proteomics of serum samples from patients with early onset HCM revealed significant modulation of HIF. These results demonstrate that HIF signaling is involved in mouse and human HCM pathogenesis. Cardiomyocyte-specific knockout of Hif-1a attenuates disease phenotype in the mouse model. Targeting Hif-1α might serve as a therapeutic option to mitigate HCM disease progression.
- Klíčová slova
- HIF1A, Hypertrophic cardiomyopathy, Hypertrophy, Hypoxia, Myocardial fibrosis,
- MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa * metabolismus genetika MeSH
- fibróza MeSH
- hypertrofická kardiomyopatie * metabolismus patologie genetika MeSH
- kardiomyocyty metabolismus patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši knockoutované MeSH
- myši MeSH
- sarkomery * metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- faktor 1 indukovatelný hypoxií - podjednotka alfa * MeSH
- Hif1a protein, mouse MeSH Prohlížeč
The RNA content is crucial for the formation of nuclear compartments, such as nuclear speckles and nucleoli. Phosphatidylinositol 4,5-bisphosphate (PIP2) is found in nuclear speckles, nucleoli, and nuclear lipid islets and is involved in RNA polymerase I/II transcription. Intriguingly, the nuclear localization of PIP2 was also shown to be RNA-dependent. We therefore investigated whether PIP2 and RNA cooperate in the establishment of nuclear architecture. In this study, we unveiled the RNA-dependent PIP2-associated (RDPA) nuclear proteome in human cells by mass spectrometry. We found that intrinsically disordered regions (IDRs) with polybasic PIP2-binding K/R motifs are prevalent features of RDPA proteins. Moreover, these IDRs of RDPA proteins exhibit enrichment for phosphorylation, acetylation, and ubiquitination sites. Our results show for the first time that the RDPA protein Bromodomain-containing protein 4 (BRD4) associates with PIP2 in the RNA-dependent manner via electrostatic interactions, and that altered PIP2 levels affect the number of nuclear foci of BRD4 protein. Thus, we propose that PIP2 spatiotemporally orchestrates nuclear processes through association with RNA and RDPA proteins and affects their ability to form foci presumably via phase separation. This suggests the pivotal role of PIP2 in the establishment of a functional nuclear architecture competent for gene expression.
- MeSH
- buněčné jádro * metabolismus genetika MeSH
- fosfatidylinositol-4,5-difosfát * metabolismus MeSH
- fosforylace MeSH
- jaderné proteiny * metabolismus genetika MeSH
- lidé MeSH
- proteiny buněčného cyklu metabolismus genetika MeSH
- proteiny obsahující bromodoménu MeSH
- RNA metabolismus genetika MeSH
- transkripční faktory * metabolismus genetika MeSH
- vazba proteinů MeSH
- vnitřně neuspořádané proteiny * metabolismus genetika chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- BRD4 protein, human MeSH Prohlížeč
- fosfatidylinositol-4,5-difosfát * MeSH
- jaderné proteiny * MeSH
- proteiny buněčného cyklu MeSH
- proteiny obsahující bromodoménu MeSH
- RNA MeSH
- transkripční faktory * MeSH
- vnitřně neuspořádané proteiny * MeSH
Cyclic electron transport around photosystem I (PSI) is essential for the protection of the photosynthetic apparatus in plants under diverse light conditions. This process is primarily mediated by Proton Gradient Regulation 5 protein/Proton Gradient Regulation 5-like photosynthetic phenotype 1 protein (PGR5/PGRL1) and NADH dehydrogenase-like complex (NDH). In angiosperms, NDH interacts with two PSI complexes through distinct monomeric antennae, LHCA5 and LHCA6, which is crucial for its higher stability under variable light conditions. This interaction represents an advanced evolutionary stage and offers limited insight into the origin of the PSI-NDH supercomplex in evolutionarily older organisms. In contrast, the moss Physcomitrium patens (Pp), which retains the lhca5 gene but lacks the lhca6, offers a glimpse into an earlier evolutionary stage of the PSI-NDH supercomplex. Here we present structural evidence of the Pp PSI-NDH supercomplex formation by single particle electron microscopy, demonstrating the unique ability of Pp to bind a single PSI in two different configurations. One configuration closely resembles the angiosperm model, whereas the other exhibits a novel PSI orientation, rotated clockwise. This structural flexibility in Pp is presumably enabled by the variable incorporation of LHCA5 within PSI and is indicative of an early evolutionary adaptation that allowed for greater diversity at the PSI-NDH interface. Our findings suggest that this variability was reduced as the structural complexity of the NDH complex increased in vascular plants, primarily angiosperms. This study not only clarifies the evolutionary development of PSI-NDH supercomplexes but also highlights the dynamic nature of the adaptive mechanisms of plant photosynthesis.
- Klíčová slova
- LHCA5, PSI‐NDH supercomplex, Physcomitrium patens, cyclic electron transport, single particle analysis, transmission electron microscopy,
- MeSH
- fotosyntéza MeSH
- fotosystém I (proteinový komplex) * metabolismus genetika MeSH
- mechy * genetika metabolismus MeSH
- NADH-dehydrogenasa metabolismus genetika MeSH
- rostlinné proteiny * metabolismus genetika MeSH
- světlosběrné proteinové komplexy metabolismus genetika chemie MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fotosystém I (proteinový komplex) * MeSH
- NADH-dehydrogenasa MeSH
- rostlinné proteiny * MeSH
- světlosběrné proteinové komplexy MeSH
Actomyosin contractility represents an ancient feature of eukaryotic cells participating in many developmental and homeostasis events, including tissue morphogenesis, muscle contraction and cell migration, with dysregulation implicated in various pathological conditions, such as cancer. At the molecular level, actomyosin comprises actin bundles and myosin motor proteins that are sensitive to posttranslational modifications like phosphorylation. While the molecular components of actomyosin are well understood, the coordination of contractility by extracellular and intracellular signals, particularly from cellular signalling pathways, remains incompletely elucidated. This study focuses on WNT/planar cell polarity (PCP) signalling, previously associated with actomyosin contractility during vertebrate neurulation. Our investigation reveals that the main cytoplasmic PCP proteins, Prickle and Dishevelled, interact with key actomyosin components such as myosin light chain 9 (MLC9), leading to its phosphorylation and localized activation. Using proteomics and microscopy approaches, we demonstrate that both PCP proteins actively control actomyosin contractility through Rap1 small GTPases in relevant in vitro and in vivo models. These findings unveil a novel mechanism of how PCP signalling regulates actomyosin contractility through MLC9 and Rap1 that is relevant to vertebrate neurulation.
- Klíčová slova
- MDCK cells, Xenopus embryos, actomyosin contractility, neurulation, planar cell polarity, vertebrates,
- MeSH
- aktomyosin * metabolismus MeSH
- fosforylace MeSH
- lehké řetězce myosinu metabolismus MeSH
- lidé MeSH
- myši MeSH
- neurulace * MeSH
- obratlovci metabolismus MeSH
- polarita buněk * MeSH
- protein dishevelled metabolismus genetika MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aktomyosin * MeSH
- lehké řetězce myosinu MeSH
- protein dishevelled MeSH
Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form (PCF) of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the 2 proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. Moreover, depletion of the mitochondrial proteasome results in increased levels of MaRF11. Thus, we have discovered a protein complex comprising TbPam18, TbPam16, and MaRF11, that controls maxicircle replication. We propose a working model in which the matrix protein MaRF11 functions downstream of the 2 integral inner membrane proteins TbPam18 and TbPam16. Moreover, we suggest that the levels of MaRF11 are controlled by the mitochondrial proteasome.
- MeSH
- mitochondriální DNA * genetika metabolismus MeSH
- mitochondriální proteiny metabolismus genetika MeSH
- mitochondrie metabolismus genetika MeSH
- molekulární evoluce MeSH
- protozoální proteiny * metabolismus genetika MeSH
- replikace DNA * MeSH
- Trypanosoma brucei brucei * metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- mitochondriální DNA * MeSH
- mitochondriální proteiny MeSH
- protozoální proteiny * MeSH
INTRODUCTION: Monoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 (PC1) maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report, we present clinical, genetic, histopathologic, and protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in 2 distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. METHODS: Whole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. RESULTS: We identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that cosegregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients' kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, in the endoplasmic reticulum (ER), but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease (CKD)-associated proteins. CONCLUSION: ALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.
Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 (RAB31) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.
- MeSH
- extracelulární vezikuly * metabolismus MeSH
- glykolýza * MeSH
- jaterní cirhóza * metabolismus patologie genetika MeSH
- jaterní hvězdicovité buňky * metabolismus patologie MeSH
- játra metabolismus patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- Rab proteiny vázající GTP metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Rab proteiny vázající GTP MeSH
High-grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients' ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of 'core ascitic EV-associated proteins' but also defined their subset unique to HGSC ascites. Using single-cell RNA sequencing data, we mapped the origin of HGSC-specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non-malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type-specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.
- Klíčová slova
- ascites, extracellular vesicles (EV), fallopian tube and peritoneum (HGSC), high-grade serous carcinoma of the ovary, macrophage, ovarian cancer (OC), tandem mass spectrometry (MS/MS), tumour microenvironment (TME),
- MeSH
- ascites metabolismus patologie MeSH
- extracelulární vezikuly * metabolismus MeSH
- lidé MeSH
- nádorové mikroprostředí MeSH
- nádory vaječníků * diagnóza MeSH
- proteomika MeSH
- serózní cystadenokarcinom * diagnóza genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
