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Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

ABSTRACT: The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [1]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus [7-9]. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells [4, 10, 11] and genetics studies in C. elegans [3]. As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes.

SUBMITTER: Diener DR

PROVIDER: S-EPMC4318714 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

Diener Dennis R DR Lupetti Pietro P Rosenbaum Joel L JL

Current biology : CB 20150108 3

The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane [1]. Located near the base of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence [2-6]. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia- ...[more]

PMID: 25578910

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Project description:BackgroundCilia are critical for diverse functions, from motility to signal transduction, and ciliary dysfunction causes inherited diseases termed ciliopathies. Several ciliopathy proteins influence developmental signalling and aberrant signalling explains many ciliopathy phenotypes. Ciliary compartmentalisation is essential for function, and the transition zone (TZ), found at the proximal end of the cilium, has recently emerged as a key player in regulating this process. Ciliary compartmentalisation is linked to two protein complexes, the MKS and NPHP complexes, at the TZ that consist largely of ciliopathy proteins, leading to the hypothesis that ciliopathy proteins affect signalling by regulating ciliary content. However, there is no consensus on complex composition, formation, or the contribution of each component.ResultsUsing bioinformatics, we examined the evolutionary patterns of TZ complex proteins across the extant eukaryotic supergroups, in both ciliated and non-ciliated organisms. We show that TZ complex proteins are restricted to the proteomes of ciliated organisms and identify a core conserved group (TMEM67, CC2D2A, B9D1, B9D2, AHI1 and a single TCTN, plus perhaps MKS1) which are present in >50% of all ciliate/flagellate organisms analysed in each supergroup. The smaller NPHP complex apparently evolved later than the larger MKS complex; this result may explain why RPGRIP1L, which forms the linker between the two complexes, is not one of the core conserved proteins. We also uncovered a striking correlation between lack of TZ proteins in non-seed land plants and loss of TZ-specific ciliary Y-links that link microtubule doublets to the membrane, consistent with the interpretation that these proteins are structural components of Y-links, or regulators of their formation.ConclusionsThis bioinformatic analysis represents the first systematic analysis of the cohort of TZ complex proteins across eukaryotic evolution. Given the near-ubiquity of only 6 proteins across ciliated eukaryotes, we propose that the MKS complex represents a dynamic complex built around these 6 proteins and implicated in Y-link formation and ciliary permeability.

Project description:Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

Project description:BackgroundProteins are the most abundant component of kidney stone matrices and their presence may reflect the process of the stone's formation. Many studies have explored the proteomics of urinary stones and crystals. We sought to comprehensively identify the proteins found in kidney stones and to identify new, reliable biomolecules for use in nephrolithiasis research.MethodsWe conducted bioinformatics research in November 2020 on the proteomics of urinary stones and crystals. We used the ClusterProfiler R package to transform proteins into their corresponding genes and Ensembl IDs. In each study we located where proteomic results intersected to determine the 20 most frequently identified stone matrix proteins. We used the Human Protein Atlas to obtain the biological information of the 20 proteins and conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analysis to explore their biological functions. We also performed immunohistochemistry to detect the expression of the top five stone matrix proteins in renal tissue.ResultsWe included 19 relevant studies for analysis. We then identified 1,409 proteins in the stone matrix after the duplicates were removed. The 20 most-commonly identified stone matrix proteins were: S100A8, S100A9, uromodulin, albumin, osteopontin, lactotransferrin, vitamin K-dependent protein Z, prothrombin, hemoglobin subunit beta, myeloperoxidase, mannan-binding lectin serine protease 2, lysozyme C, complement C3, serum amyloid P-component, cathepsin G, vitronectin, apolipoprotein A-1, eosinophil cationic protein, fibrinogen alpha chain, and apolipoprotein D. GO and KEGG analysis revealed that these proteins were typically engaged in inflammation and immune response.Immunohistochemistry of the top five stone matrix proteins in renal tissue showed that the expression of S100A8, S100A9, and osteopontin increased, while uromodulin decreased in kidney stone patients. Albumin was rarely expressed in the kidney with no significant difference between healthy controls and kidney stone patients.ConclusionProteomic analysis revealed some common inflammation-related proteins in the kidney stone matrix. The role of these proteins in stone formation should be explored for their potential use as diagnostic biomarkers and therapeutic targets for urolithiasis.

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Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

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Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins.

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