Giardia intestinalis is a globally important microbial pathogen with considerable public health, agricultural, and economic burden. Genome sequencing and comparative analyses have elucidated G. intestinalis to be a taxonomically diverse species consisting of at least eight different sub-types (assemblages A-H) that can infect a great variety of animal hosts, including humans. The best studied of these are assemblages A and B which have a broad host range and have zoonotic transmissibility towards humans where clinical Giardiasis can range from asymptomatic to diarrheal disease. Epidemiological surveys as well as previous molecular investigations have pointed towards critical genomic level differences within numerous molecular pathways and families of parasite virulence factors within assemblage A and B isolates. In this study, we explored the necessary machinery for the formation of vesicles and cargo transport in 89 Canadian isolates of assemblage A and B G. intestinalis. Considerable variability within the molecular complement of the endolysosomal ESCRT protein machinery, adaptor coat protein complexes, and ARF regulatory system have previously been reported. Here, we confirm inter-assemblage, but find no intra-assemblage variation within the trafficking systems examined. This variation includes losses of subunits belonging to the ESCRTIII as well as novel lineage specific duplications in components of the COPII machinery, ARF1, and ARFGEF families (BIG and CYTH). Since differences in disease manifestation between assemblages A and B have been controversially reported, our findings may well have clinical implications and even taxonomic, as the membrane trafficking system underpin parasite survival, pathogenesis, and propagation.
- MeSH
- Feces parasitology MeSH
- Genomics MeSH
- Genotype MeSH
- Giardia lamblia * MeSH
- Giardiasis * parasitology MeSH
- Humans MeSH
- Public Health MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Canada MeSH
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
- Autophagy MeSH
- Deubiquitinating Enzymes metabolism MeSH
- Endopeptidases genetics MeSH
- Endosomal Sorting Complexes Required for Transport genetics MeSH
- Humans MeSH
- Salmonella typhimurium metabolism MeSH
- Salmonella Infections * MeSH
- Ubiquitin Thiolesterase genetics metabolism MeSH
- Ubiquitination MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Exosomes are biological nanoscale spherical lipid bilayer vesicles, 40-160 nm in diameter, produced by most mammalian cells in both physiological and pathological conditions. Exosomes are formed via the endosomal sorting complex required for transport (ESCRT). The primary function of exosomes is mediating cell-to-cell communication. In terms of cancer, exosomes play important roles as mediators of intercellular communication, leading to tumor progression. Moreover, they can serve as biomarkers for cancer detection and progression. Therefore, their utilization in cancer therapies has been suggested, either as drug delivery carriers or as a diagnostic tool. However, exosomes were also reported to be involved in cancer drug resistance via transferring information of drug resistance to sensitive cells. It is important to consider the current knowledge regarding the role of exosomes in cancer, drug resistance, cancer therapies, and their clinical application in cancer therapies.
- MeSH
- Exosomes * physiology MeSH
- Carcinogenesis MeSH
- Drug Delivery Systems MeSH
- Humans MeSH
- Neoplasms * pathology MeSH
- Drug Carriers therapeutic use MeSH
- Mammals MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
[Figure: see text].
- MeSH
- Biological Evolution MeSH
- Cell Line MeSH
- Cytokinesis * MeSH
- Zebrafish MeSH
- Endosomal Sorting Complexes Required for Transport metabolism MeSH
- Phosphatidylinositol 3-Kinases genetics metabolism MeSH
- Phosphatidylinositol 4,5-Diphosphate metabolism MeSH
- Phosphatidylinositols metabolism MeSH
- Cataract metabolism pathology MeSH
- Humans MeSH
- Mutation MeSH
- Mice MeSH
- Lens, Crystalline cytology growth & development metabolism MeSH
- Aging, Premature MeSH
- Cell Cycle Proteins metabolism MeSH
- Zebrafish Proteins genetics metabolism MeSH
- Calcium-Binding Proteins metabolism MeSH
- Cellular Senescence * MeSH
- Tubulin metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural 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.
Exosomes are a heterogenous subpopulation of extracellular vesicles 30-150 nm in range and of endosome-derived origin. We explored the exosome formation through different systems, including the endosomal sorting complex required for transport (ESCRT) and ESCRT-independent system, looking at the mechanisms of release. Different isolation techniques and specificities of exosomes from different tissues and cells are also discussed. Despite more than 30 years of research that followed their definition and indicated their important role in cellular physiology, the exosome biology is still in its infancy with rapidly growing interest. The reasons for the rapid increase in interest with respect to exosome biology is because they provide means of intercellular communication and transmission of macromolecules between cells, with a potential role in the development of diseases. Moreover, they have been investigated as prognostic biomarkers, with a potential for further development as diagnostic tools for neurodegenerative diseases and cancer. The interest grows further with the fact that exosomes were reported as useful vectors for drugs.
- Publication type
- Journal Article MeSH
- Review MeSH
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
- Adaptor Proteins, Signal Transducing genetics metabolism MeSH
- NIH 3T3 Cells MeSH
- Cyclin-Dependent Kinases genetics metabolism MeSH
- Peptide Elongation Factor 1 genetics metabolism MeSH
- Endosomal Sorting Complexes Required for Transport genetics metabolism MeSH
- Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases genetics metabolism MeSH
- Phosphoproteins genetics metabolism MeSH
- Phosphorylation MeSH
- HEK293 Cells MeSH
- Homeodomain Proteins genetics metabolism MeSH
- Humans MeSH
- Protein Interaction Mapping MeSH
- Mice MeSH
- Protein Serine-Threonine Kinases genetics metabolism MeSH
- Proteomics methods MeSH
- Receptor, Fibroblast Growth Factor, Type 3 genetics metabolism MeSH
- Gene Expression Regulation * MeSH
- Signal Transduction genetics MeSH
- Gene Expression Profiling MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The integration of complementary molecular methods (including X-ray crystallography, NMR spectroscopy, small angle X-ray/neutron scattering, and computational techniques) is frequently required to obtain a comprehensive understanding of dynamic macromolecular complexes. In particular, these techniques are critical for studying intrinsically disordered protein regions (IDRs) or intrinsically disordered proteins (IDPs) that are part of large protein:protein complexes. Here, we explain how to prepare IDP samples suitable for study using NMR spectroscopy, and describe a novel SAXS modeling method (ensemble refinement of SAXS; EROS) that integrates the results from complementary methods, including crystal structures and NMR chemical shift perturbations, among others, to accurately model SAXS data and describe ensemble structures of dynamic macromolecular complexes.
- MeSH
- Endosomal Sorting Complexes Required for Transport chemistry metabolism MeSH
- Protein Conformation MeSH
- Crystallography, X-Ray methods MeSH
- Humans MeSH
- Magnetic Resonance Spectroscopy methods MeSH
- Mitogen-Activated Protein Kinases chemistry metabolism MeSH
- Models, Molecular MeSH
- Scattering, Radiation * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
- MeSH
- Potassium Channels, Voltage-Gated chemistry metabolism MeSH
- Endosomal Sorting Complexes Required for Transport metabolism MeSH
- Humans MeSH
- Microfilament Proteins chemistry metabolism MeSH
- Protein Multimerization * MeSH
- Proteasome Endopeptidase Complex chemistry metabolism MeSH
- Receptor, Fibroblast Growth Factor, Type 1 chemistry metabolism MeSH
- Saccharomyces cerevisiae Proteins chemistry metabolism MeSH
- Ubiquitination * MeSH
- Ubiquitin-Protein Ligases metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
... Receptors are Down-Regulated by Degradation in Lysosomes Early Endosomes Mature into Late Endosomes ESCRT ...
Sixth edition xxxiv, 1430 stran v různém stránkování : ilustrace (převážně barevné) ; 29 cm
- MeSH
- Cells * MeSH
- Molecular Biology MeSH
- Conspectus
- Biochemie. Molekulární biologie. Biofyzika
- NML Fields
- molekulární biologie, molekulární medicína
- NML Publication type
- učebnice vysokých škol
