Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.
During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membranebound P2purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca 2+ ] i ) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y 4 agonist, MRS4062, caused a fast desensitizing [Ca 2+ ] i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca 2+ in the extracellular fluid. The biphasic [Ca 2+ ] i response to UTP was attenuated respectively by P2Y 4 blockers, like reactive blue-2 and suramin, and by the P2Y 11 antagonist, NF340. UTP and the P2Y 2 receptor agonist MRS2768 increased, whereas the selective P2Y 11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y 11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y 2 , P2Y 4 and P2Y 11 receptors. Data indicate that besides P2Y 4 and P2Y 2 receptors which are responsible for UTP-induced [Ca 2+ ] i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y 11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.
PEX13 and PEX14 are two core components of the so‐called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease‐protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a Nin‐Cout topology, and that PEX13 adopts a Nout‐Cin topology, thus exposing its carboxy‐terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery. Enzymes Trypsin, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/4.html; Proteinase K, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/21/64.html; Tobacco etch virus protease, http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/22/44.html.
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