Browsing by Subject "PROTEOMIC ANALYSIS"

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  • Imle, Andrea; Abraham, Libin; Tsopoulidis, Nikolaos; Hoflack, Bernard; Saksela, Kalle; Fackler, Oliver T. (2015)
    Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Net usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Net to occur in infected human T lymphocytes, and to be conserved among lentiviral Net proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) Nef contributes to AIDS pathogenesis, but the underlying molecular mechanisms remain incompletely understood. An important aspect of Nef function is to facilitate virus replication by disrupting T lymphocyte actin dynamics in response to stimulation via its association with the host cell kinase PAK2. We report here that the molecular surface of Nef for PAK2 association also mediates interaction of Nef with EXOC and establish that PAK2 provides an essential adaptor function for the subsequent formation of Nef-EXOC complexes. PAK2 and EXOC specifically cooperate in the inhibition of actin dynamics and proximal signaling induced by T cell receptor engagement by Nef. These results establish EXOC as a functionally relevant Nef interaction partner, emphasize the suitability of the PAK2 interaction surface for future therapeutic interference with Nef function, and show that such strategies need to target activity-independent PAK2 functions.
  • Sulima, Anna; Bien, Justyna; Savijoki, Kirsi; Nareaho, Anu; Salamatin, Ruslan; Conn, David Bruce; Mlocicki, Daniel (2017)
    Background: A wide range of molecules are used by tapeworm metacestodes to establish successful infection in the hostile environment of the host. Reports indicating the proteins in the cestode-host interactions are limited predominantly to taeniids, with no previous data available for non-taeniid species. A non-taeniid, Hymenolepis diminuta, represents one of the most important model species in cestode biology and exhibits an exceptional developmental plasticity in its life-cycle, which involves two phylogenetically distant hosts, arthropod and vertebrate. Results: We identified H. diminuta cysticercoid proteins that were recognized by sera of H. diminuta-infected rats using two-dimensional gel electrophoresis (2DE), 2D-immunoblotting, and LC-MS/MS mass spectrometry. Proteomic analysis of 42 antigenic spots revealed 70 proteins. The largest number belonged to structural proteins and to the heat-shock protein (HSP) family. These results show a number of the antigenic proteins of the cysticercoid stage, which were present already in the insect host prior to contact with the mammal host. These are the first parasite antigens that the mammal host encounters after the infection, therefore they may represent some of the molecules important in host-parasite interactions at the early stage of infection. Conclusions: These results could help in understanding how H. diminuta and other cestodes adapt to their diverse and complex parasitic life-cycles and show universal molecules used among diverse groups of cestodes to escape the host response to infection.
  • Mlocicki, Daniel; Sulima, Anna; Bień, Justyna; Näreaho, Anu; Zawistowska-Deniziak, Anna; Basałaj, Katarzyna; Sałamatin, Rusłan; Conn, David Bruce; Savijoki, Kirsi (2018)
    In cestodiasis, mechanical and molecular contact between the parasite and the host activates the immune response of the host and may result in inflammatory processes, leading to ulceration and intestinal dysfunctions. The aim of the present study was to identify antigenic proteins of the adult cestode Hymenolepis diminuta by subjecting the total protein extracts from adult tapeworms to 2DE immunoblotting (two-dimensional electrophoresis combined with immunoblotting) using sera collected from experimentally infected rats. A total of 36 protein spots cross-reacting with the rat sera were identified using LC-MS/MS. As a result, 68 proteins, including certain structural muscle proteins (actin, myosin, and paramyosin) and moonlighters (heat shock proteins, kinases, phosphatases, and glycolytic enzymes) were identified; most of these were predicted to possess binding and/or catalytic activity required in various metabolic and cellular processes, and reported here as potential antigens of the adult cestode for the first time. As several of these antigens can also be found at the cell surface, the surface-associated proteins were extracted and subjected to in-solution digestion for LC-MS/MS identification (surfaceomics). As a result, a total of 76 proteins were identified, from which 31 proteins, based on 2DE immunoblotting, were predicted to be immunogenic. These included structural proteins actin, myosin and tubulin as well as certain moonlighting proteins (heat-shock chaperones) while enzymes with diverse catalytic activities were found as the most dominating group of proteins. In conclusion, the present study shed new light into the complexity of the enteric cestodiasis by showing that the H. diminuta somatic proteins exposed to the host possess immunomodulatory functions, and that the immune response of the host could be stimulated by diverse mechanisms, involving also those triggering protein export via yet unknown pathways.
  • Xu, Xiaomeng; Barreiro, Karina; Musante, Luca; Kretz, Oliver; Lin, Hanfei; Zou, Hequn; Huber, Tobias B.; Holthofer, Harry (2019)
    Purpose Urinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm-Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted "contaminant" in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently. Experimental design The uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS. Results Using HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap. Conclusions and clinical relevance The method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.
  • Palviainen, Mari; Saari, Heikki; Kärkkäinen, Olli; Pekkinen, Jenna; Auriola, Seppo; Yliperttula, Marjo; Puhka, Maija; Hanhineva, Kati; Siljander, Pia R-M (2019)
    One of the greatest bottlenecks in extracellular vesicle (EV) research is the production of sufficient material in a consistent and effective way using in vitro cell models. Although the production of EVs in bioreactors maximizes EV yield in comparison to conventional cell cultures, the impact of their cell growth conditions on EVs has not yet been established. In this study, we grew two prostate cancer cell lines, PC-3 and VCaP, in conventional cell culture dishes and in two-chamber bioreactors to elucidate how the growth environment affects the EV characteristics. Specifically, we wanted to investigate the growth condition-dependent differences by non-targeted metabolite profiling using liquid chromatography-mass spectrometry (LC-MS) analysis. EVs were also characterized by their morphology, size distribution, and EV protein marker expression, and the EV yields were quantified by NTA. The use of bioreactor increased the EV yield >100 times compared to the conventional cell culture system. Regarding morphology, size distribution and surface markers, only minor differences were observed between the bioreactor-derived EVs (BR-EVs) and the EVs obtained from cells grown in conventional cell cultures (C-EVs). In contrast, metabolomic analysis revealed statistically significant differences in both polar and non-polar metabolites when the BR-EVs were compared to the C-EVs. The results show that the growth conditions markedly affected the EV metabolite profiles and that metabolomics was a sensitive tool to study molecular differences of EVs. We conclude that the cell culture conditions of EV production should be standardized and carefully detailed in publications and care should be taken when EVs from different production platforms are compared with each other for systemic effects.
  • Suojalehto, Hille; Wolff, Henrik; Lindström, Irmeli; Puustinen, Anne (2018)
    Background: The mechanisms of work-related asthma (WRA) are incompletely delineated. Nasal cell samples may be informative about processes in the lower airways. Our aim was to determine the nasal protein expression profiles of WRA caused by different kind of exposures. Methods: We collected nasal brush samples from 82 nonsmoking participants, including healthy controls and WRA patients exposed to (i) protein allergens, (ii) isocyanates and (iii) welding fumes the day after relevant exposure. The proteome changes in samples were analysed by two-dimensional difference gel electrophoresis, and the differentially regulated proteins found were identified by mass spectrometry. Immunological comparison was carried out using Western blot. Results: We detected an average of 2500 spots per protein gel. Altogether, 228 protein spots were chosen for identification, yielding 77 different proteins. Compared to the controls, exposure to protein allergens had the largest effects on the proteome. Hierarchical clustering revealed that protein allergen- and isocyanate-related asthma had similar profiles, whereas asthma related to welding fumes differed. The highly overrepresented functional categories in the asthma groups were defence response, protease inhibitor activity, inflammatory and calcium signalling, complement activation and cellular response to oxidative stress. Immunological analysis confirmed the found abundance differences in galectin 10 and protein S100-A9 between the groups. Conclusions: Work-related asthma patients exposed to protein allergens and isocyanates elicit similar nasal proteome responses and the profiles of welders and healthy controls were alike. Revealed biological activities of the protein expression changes are associated with allergic inflammation and asthma.
  • Dumont, Vincent; Lehtonen, Sanna (2022)
    Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin-associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.
  • Fred, Senem Merve; Laukkanen, Liina; Brunello, Cecilia A.; Vesa, Liisa; Göös, Helka; Cardon, Iseline; Moliner, Rafael; Maritzen, Tanja; Varjosalo, Markku; Casarotto, Plinio C.; Castren, Eero (2019)
    Several antidepressant drugs activate tropomyosin-related kinase B (TRKB) receptor, but it remains unclear whether these compounds employ a common mechanism for TRKB activation. Here, using MS, we found that a single intraperitoneal injection of fluoxetine disrupts the interaction of several proteins with TRKB in the hippocampus of mice. These proteins included members of adaptor protein complex-2 (AP-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking ? subunit of the AP-2 complex (AP2M) was confirmed to be disrupted by both acute and repeated fluoxetine treatments. Of note, fluoxetine disrupted the coupling between full-length TRKB and AP2M, but not the interaction between AP2M and the TRKB C-terminal region, indicating that the fluoxetine-binding site in TRKB lies outside the TRKB:AP2M interface. ELISA experiments revealed that in addition to fluoxetine, other chemically diverse antidepressants, such as imipramine, rolipram, phenelzine, ketamine, and its metabolite 2R,6R-hydroxynorketamine, also decreased the interaction between TRKB and AP2M in vitro. Silencing the expression of AP2M in a TRKB-expressing mouse fibroblast cell line (MG87.TRKB) increased cell-surface expression of TRKB and facilitated its activation by brain-derived neurotrophic factor (BDNF), observed as levels of phosphorylated TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB, along with enhanced cell-surface expression of the receptor in cultured hippocampal neurons. Taken together, our results suggest that disruption of the TRKB:AP2M interaction is a common mechanism underlying TRKB activation by several chemically diverse antidepressants.
  • Geisslitz, Sabrina; Shewry, Peter; Brouns, Fred; America, Antoine H. P.; Caio, Giacomo Pietro Ismaele; Daly, Matthew; D'Amico, Stefano; De Giorgio, Roberto; Gilissen, Luud; Grausgruber, Heinrich; Huang, Xin; Jonkers, Daisy; Keszthelyi, Daniel; Larre, Colette; Masci, Stefania; Mills, Clare; Moller, Marie Sofie; Sorrells, Mark E.; Svensson, Birte; Zevallos, Victor F.; Weegels, Peter Louis (2021)
    Amylase/trypsin-inhibitors (ATIs) comprise about 2-4% of the total wheat grain proteins and may contribute to natural defense against pests and pathogens. However, they are currently among the most widely studied wheat components because of their proposed role in adverse reactions to wheat consumption in humans. ATIs have long been known to contribute to IgE-mediated allergy (notably Bakers' asthma), but interest has increased since 2012 when they were shown to be able to trigger the innate immune system, with attention focused on their role in coeliac disease which affects about 1% of the population and, more recently, in non-coeliac wheat sensitivity which may affect up to 10% of the population. This has led to studies of their structure, inhibitory properties, genetics, control of expression, behavior during processing, effects on human adverse reactions to wheat and, most recently, strategies to modify their expression in the plant using gene editing. We therefore present an integrated account of this range of research, identifying inconsistencies, and gaps in our knowledge and identifying future research needs. Note This paper is the outcome of an invited international ATI expert meeting held in Amsterdam, February 3-5 2020