INTRODUCTION: Ubiquitination is an important protein modification that regulates various essential cellular processes, including the functions of innate immune cells. Deubiquitinases are enzymes responsible for removing ubiquitin modification from substrates, and the regulation of deubiquitinases in macrophages during infection with Salmonella Typhimurium and Yersinia enterocolitica remains unknown. METHODS: To identify deubiquitinases regulated in human macrophages during bacterial infection, an activity-based proteomics screen was conducted. The effects of pharmacological inhibition of the identified deubiquitinase, USP8, were examined, including its impact on bacterial survival within macrophages and its role in autophagy regulation during Salmonella infection. RESULTS: Several deubiquiitnases were differentially regulated in infected macrophages. One of the deubiquitinases identified was USP8, which was downregulated upon Salmonella infection. Inhibition of USP8 was associated with a decrease in bacterial survival within macrophages, and it was found to play a distinct role in regulating autophagy during Salmonella infection. The inhibition of USP8 led to the downregulation of the p62 autophagy adaptor. DISCUSSION: The findings of this study suggest a novel role of USP8 in regulating autophagy flux, which restricts intracellular bacteria, particularly during Salmonella infection.
- MeSH
- autofagie MeSH
- deubikvitinasy metabolismus MeSH
- endopeptidasy genetika MeSH
- endozomální třídící komplexy pro transport genetika MeSH
- lidé MeSH
- Salmonella typhimurium metabolismus MeSH
- salmonelóza * MeSH
- thiolesterasa ubikvitinu genetika metabolismus MeSH
- ubikvitinace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.
Receptor tyrosine kinases (RTKs) form multiprotein complexes that initiate and propagate intracellular signals and determine the RTK-specific signalling patterns. Unravelling the full complexity of protein interactions within the RTK-associated complexes is essential for understanding of RTK functions, yet it remains an understudied area of cell biology. We describe a comprehensive approach to characterize RTK interactome. A single tag immunoprecipitation and phosphotyrosine protein isolation followed by mass-spectrometry was used to identify proteins interacting with fibroblast growth factor receptor 3 (FGFR3). A total of 32 experiments were carried out in two different cell types and identified 66 proteins out of which only 20 (30.3%) proteins were already known FGFR interactors. Using co-immunoprecipitations, we validated FGFR3 interaction with adapter protein STAM1, transcriptional regulator SHOX2, translation elongation factor eEF1A1, serine/threonine kinases ICK, MAK and CCRK, and inositol phosphatase SHIP2. We show that unappreciated signalling mediators exist for well-studied RTKs, such as FGFR3, and may be identified via proteomic approaches described here. These approaches are easily adaptable to other RTKs, enabling identification of novel signalling mediators for majority of the known human RTKs.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- buňky NIH 3T3 MeSH
- cyklin-dependentní kinasy genetika metabolismus MeSH
- elongační faktor 1 genetika metabolismus MeSH
- endozomální třídící komplexy pro transport genetika metabolismus MeSH
- fosfatidylinositol-3,4,5-trisfosfát-5-fosfatasy genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- fosforylace MeSH
- HEK293 buňky MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- lidé MeSH
- mapování interakce mezi proteiny MeSH
- myši MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- proteomika metody MeSH
- receptor fibroblastových růstových faktorů, typ 3 genetika metabolismus MeSH
- regulace genové exprese * MeSH
- signální transdukce genetika MeSH
- stanovení celkové genové exprese MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Endosomal sorting complexes required for transport (ESCRTs) are involved in the formation of multivesicular bodies and sorting of targeted proteins to the yeast vacuole. The deletion of seven genes encoding components of the ESCRT machinery render Saccharomyces cerevisiae cells sensitive to high extracellular CaCl2 concentrations as well as to low pH in media. In this work, we focused on intracellular pH (pHin) homeostasis of these mutants. None of the studied ESCRT mutants exhibited an altered pHin level compared to the wild type under standard growth conditions. Nevertheless, 60 min of CaCl2 treatment resulted in a more significant drop in pHin levels in these mutants than in the wild type, suggesting that pHin homeostasis is affected in ESCRT mutants upon the addition of calcium. Similarly, CaCl2 treatment caused a bigger pHin decrease in cells lacking the vacuolar Ca(2+)/H(+) antiporter Vcx1 which indicates a role for this protein in the maintenance of proper pHin homeostasis when cells need to cope with a high CaCl2 concentration in media. Importantly, ESCRT gene deletions in the vcx1Δ strain did not result in an increase in the CaCl2-invoked drop in the pHin levels of cells, which demonstrates a genetic interaction between VCX1 and studied ESCRT genes.
- MeSH
- antiportéry metabolismus MeSH
- delece genu MeSH
- endozomální třídící komplexy pro transport genetika metabolismus MeSH
- fyziologický stres * MeSH
- homeostáza * MeSH
- koncentrace vodíkových iontů MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae účinky léků genetika růst a vývoj fyziologie MeSH
- vápník metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.
- MeSH
- ADP-ribosylační faktory genetika metabolismus MeSH
- buněčné linie MeSH
- endozomální třídící komplexy pro transport genetika metabolismus MeSH
- erbB receptory metabolismus MeSH
- exozómy enzymologie genetika metabolismus MeSH
- fosfolipasa D genetika metabolismus MeSH
- HIV infekce genetika metabolismus virologie MeSH
- HIV-1 fyziologie MeSH
- lidé MeSH
- multivezikulární tělíska enzymologie genetika metabolismus MeSH
- proteiny buněčného cyklu genetika metabolismus MeSH
- proteiny vázající vápník genetika metabolismus MeSH
- synteniny genetika metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
