Adaptor fehérjék vizsgálata a vaszkuláris endotéliumban

Vascular endothelial cell monolayer acts as a semiselective barrier between blood and the interstitium. Phosphorylation level of many cytoskeleton and cytoskeleton-associated proteins playing crucial role in the EC barrier function is critical to tissue and organ function. ERM-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We found unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A. Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, our results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells. TIMAP, TGF-β inhibited membrane-associated protein, is most abundant in endothelial cells with a regulatory effect on the endothelial barrier function, yet little is known about its interacting partners. RACK1, receptor for activated protein kinase C, serves as an anchor in multiple signaling pathways. We found that RACK1 binds to the TIMAP-PP1c complex in endothelial cells. WD1-4 repeats of RACK1 were identified as critical regions of the interaction both with TIMAP and farnesyl transferase. Phosphorylation of TIMAP by activation of the cAMP/PKA pathway reduced the amount of TIMAP-RACK1 complex and enhanced translocation of TIMAP to the cell membrane in vascular endothelial cells. However, both membrane localization of TIMAP and transendothelial resistance were attenuated after RACK1 depletion. Farnesyl transferase, the enzyme responsible for prenylation and consequent membrane localization of TIMAP, is present in the RACK1-TIMAP complex in control cells, but it does not co-immunoprecipitate with TIMAP after RACK1 depletion. Our results suggest that transient parallel linkage of TIMAP and farnesyl transferase to RACK1 could ensure prenylation and transport of TIMAP to the plasma membrane where it may attend in maintaining the endothelial barrier as a phosphatase regulator. A vaszkuláris endotél sejtek szemiszelektív barrierként választják el a keringő vért a környező szövetektől. A jól működő endotél barrier funció fenntartásában számos citoszkeletális és citoszkeleton asszociált fehérje foszforilációja fontos szerepet játszik. Az EBP50 (ezrin-radixin-moesin-binding phosphoprotein 50), egy olyan foszforilálható adaptor fehérje, mely epitél sejtekben magas expressziós szinttel rendelkezik azonban endotél sejtekben még nem vizsgálták. Immunfluoreszcens festéssel, valamint sejtfrakcionálással az EBP50 fehérjét az interfázisban lévő endotél sejtek magi régiójában detektáltuk. A mitózis profázisában foszforiláció-függő módon a sejtek citoplazmájába transzlokálódott és kolokalizáció volt megfigyelhető a protein foszfatáz 2A enzimmel. Az EBP50 foszforilációt utánzó mutánsát expresszáló sejtek gyorsabb in vitro sebgyógyálást mutattak, ami az EBP50 sejtosztódásban betöltött szabályozó szerepére utal. Immunprecipitációs és pull down kísérletekkel sikerült igazolnuk az EBP50 és a PP2A (A, C, és Bα) alegységei közötti sejtciklus függő kölcsönhatást. Eredményeink arra utalnak, hogy a PP2A enzim szerepet játszik az EBP50 fehérje defoszforilálásában normál sejtekben a sejtciklus során. A TIMAP (TGFβ inhibited membrane-associated protein) endotél sejtekben rendelkezik a legmagasabb expressziós szinttel, szerepéről és kölcsönható partnereiről azonban csak keveset tudunk. A RACK1 (Receptor for Activated C Kinase 1) adaptor fehérje, számos jelátviteli útvonalban játszik szerepet. Eredményeink alapján endotél sejtekben a RACK1 a TIMAP-PP1c komplexhez kötődik. A kölcsönhatásban a RACK1 WD1-4 doménjei vesznek részt, melyhez kötődik továbbá a farnezil-transzferáz enzim is. A cAMP/PKA útvonal aktiválásával elért TIMAP foszforiláció csökkentette a RACK1-TIMAP komplex mennyiségét, valamint a TIMAP membránban való feldúsulásához vezetett. A RACK1 depléciójával a TIMAP membránlokalizációja valamint a farnezil-transzferáz enzimmel való kölcsönhatása is megszűnt. Eredményeink alapján a RACK1 adaptor fehérje biztosítja a TIMAP prenilációját az ehhez szükséges farnezil-transzferáz enzimmel való kölcsönhatással

Regulation of merlin by protein phosphatase 1-TIMAP and EBP50 in endothelial cells

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Cell Cycle Dependent Association of EBP50 with Protein Phosphatase 2A in Endothelial Cells

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells

TIMAP-protein phosphatase 1-complex controls endothelin-1 production via ECE-1 dephosphorylation

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Role of Human Corneal Stroma-Derived Mesenchymal-Like Stem Cells in Corneal Immunity and Wound Healing

Corneal tissue regeneration is of crucial importance for maintaining normal vision. We aimed to isolate and cultivate human corneal stroma-derived mesenchymal stem-like cells (CSMSCs) from the central part of cadaver corneas and study their phenotype, multipotency, role in immunity and wound healing. The isolated cells grew as monolayers in vitro, expressed mesenchymal- and stemness-related surface markers (CD73, CD90, CD105, CD140b), and were negative for hematopoietic markers as determined by flow cytometry. CSMSCs were able to differentiate in vitro into fat, bone and cartilage. Their gene expression profile was closer to bone marrow-derived MSCs (BMMSCs) than to limbal epithelial stem cells (LESC) as determined by high-throughput screening. The immunosuppressive properties of CSMSCs were confirmed by a mixed lymphocyte reaction (MLR), while they could inhibit proliferation of activated immune cells. Treatment of CSMSCs by pro-inflammatory cytokines and toll-like receptor ligands significantly increased the secreted interleukin-6 (IL-6), interleukin-8 (IL-8) and C-X-C motif chemokine 10 (CXCL-10) levels, as well as the cell surface adhesion molecules. CSMSCs were capable of closing a wound in vitro under different stimuli. These cells thus contribute to corneal tissue homeostasis and play an immunomodulatory and regenerative role with possible implications in future cell therapies for treating sight-threatening corneal diseases

The Proteasome Activators Blm10/PA200 Enhance the Proteasomal Degradation of N-Terminal Huntingtin.

The Blm10/PA200 family of proteasome activators modulates the peptidase activity of the core particle (20S CP). They participate in opening the 20S CP gate, thus facilitating the degradation of unstructured proteins such as tau and Dnm1 in a ubiquitin- and ATP-independent manner. Furthermore, PA200 also participates in the degradation of acetylated histones. In our study, we use a combination of yeast and human cell systems to investigate the role of Blm10/PA200 in the degradation of N-terminal Huntingtin fragments (N-Htt). We demonstrate that the human PA200 binds to N-Htt. The loss of Blm10 in yeast or PA200 in human cells results in increased mutant N-Htt aggregate formation and elevated cellular toxicity. Furthermore, Blm10 in vitro accelerates the proteasomal degradation of soluble N-Htt. Collectively, our data suggest N-Htt as a new substrate for Blm10/PA200-proteasomes and point to new approaches in Huntington\u27s disease (HD) research

Indolepropionic Acid, a Metabolite of the Microbiome, Has Cytostatic Properties in Breast Cancer by Activating AHR and PXR Receptors and Inducing Oxidative Stress

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Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o.q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer