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Proteolytic Pathway (proteolytic + pathway)
Selected AbstractsIncreased Bone Formation in Mice Lacking Plasminogen Activators,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2003E Daci Abstract Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. Introduction: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA,/,:uPA,/,). Materials and Methods: Bones of 2- to 7-day-old tPA,/,:uPA,/, and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. Results: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2×), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA,/,:uPA,/, mice. Conclusions: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix,as occurs during plasminogen activator deficiency,may in turn stimulate osteoblast function, resulting in increased bone formation. [source] The ubiquitin-proteasome system and its role in ethanol-induced disordersADDICTION BIOLOGY, Issue 1 2002Terrence M. Donohue Jr The levels of these proteins are controlled by their rates of degradation. Similarly, protein catabolism plays a crucial role in prolonging cellular life by destroying damaged proteins that are potentially cytotoxic. A major player in these catabolic reactions is the ubiquitin-proteasome system, a novel proteolytic system that has become the primary proteolytic pathway in eukaryotic cells. Ubiquitin-mediated proteolysis is now regarded as the major pathway by which most intracellular proteins are destroyed. Equally important, from a toxicological standpoint, is that the ubiquitin-proteasome system is also widely considered to be a cellular defense mechanism, since it is involved in the removal of damaged proteins generated by adduct formation and oxidative stress. This review describes the history and the components of the ubiquitin-proteasome system, its regulation and its role in pathological states, with the major emphasis on ethanol-induced organ injury. The available literature cited here deals mainly with the effects of ethanol consumption on the ubiquitin-proteasome pathway in the liver. However, since this proteolytic system is an essential pathway in all cells it is an attractive experimental model and therapeutic target in extrahepatic organs such as the brain and heart that are also affected by excessive alcohol consumption. [source] Differentiation-dependent sensitivity to cell death induced in the developing retina by inhibitors of the ubiquitin-proteasome proteolytic pathwayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2001D. D. C. Neves Abstract The effects of inhibitors of proteasome function were studied in the retina of developing rats. Explants from the retina of neonatal rats at postnatal day (P) 3 or P6 were incubated with various combinations of the proteasome inhibitor carbobenzoxyl-leucinyl-leucinyl-leucinal (MG132), the protein synthesis inhibitor anisomycin, or the adenylyl cyclase activator forskolin. MG132 induced cell death in a subset of cells within the neuroblastic (proliferative) layer of the retinal tissue. The cells sensitive to degeneration induced by either MG132 or anisomycin, were birthdated by bromodeoxyuridine injections. This showed that the MG132-sensitive population includes both proliferating cells most likely in their last round of cell division, and postmitotic undifferentiated cells, at a slightly earlier stage than the population, sensitive to anisomycin-induced cell death. The results show that sensitivity to cell death induced by proteasome inhibitors defines a window of development in the transition from the cell cycle to the differentiated state in retinal cells. [source] Calpain-dependent proteolysis of NF2 protein: Involvement in schwannomas and meningiomasNEUROPATHOLOGY, Issue 3 2000Yoriyoshi Kimura The neurofibromatosis type 2 (NF2) protein, known as merlin or schwannomin, is a tumor suppressor, and the NF2 gene has been found to be mutated in the majority of schwannomas and meningiomas, including both sporadically occurring and familial NF2 cases. Although the development of these tumors depends on the loss of merlin, the presence of tumors lacking detectable NF2 mutations suggests different mechanisms for inactivating merlin. Recent studies have demonstrated cleavage of merlin by calpain, a calcium-dependent neutral cysteine protease, and marked activation of the calpain system resulting in the degradation of merlin in these tumors. Increased turnover of merlin by calpain in some schwannomas and meningiomas exemplifies tumorigenesis linked to the calpain-mediated proteolytic pathway. [source] Ubiquitin C-terminal hydrolase-L1 (PGP9.5) expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines, neurotrophic factors or heat stressNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 2 2001J.-I. Satoh Dysfunction of the ubiquitin-dependent proteolytic pathway contributes to progressive accumulation of ubiquitinated protein inclusions in neurodegenerative disorders, such as Parkinson's disease (PD). Ubiquitin C-terminal hydrolase-L1 (UCH-L1), alternatively designated protein gene product 9.5 (PGP9.5), is a neural deubiquitinating enzyme which is identified as a principal constituent of Lewy bodies. To clarify the regulatory mechanism of UCH-L1 expression in human neural cells, we studied the constitutive, cytokine/neurotrophic factor-regulated, and heat stress-induced expression of UCH-L1 in cultured human neural cell lines by Western blot analysis. The constitutive expression of UCH-L1 was identified in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N). The levels of UCH-L1 expression were unaltered in these cell lines following treatment with TNF-,, IL-1,, BDNF, GDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate, and remained unchanged by exposure to heat stress. In contrast, its levels were elevated substantially in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation, accompanied with an increased expression of ,-synuclein and synaptophysin. These results indicate that UCH-L1 is expressed constitutively in human neual cell lines, where it is upregulated following induction of neuronal differentiation, but unaffected by exposure to heat stress, cytokines, or growth/differentiation factors which are supposed to be invloved in the nigral neuronal death and survival in PD. [source] Removal of the N-terminal hexapeptide from human ,2-microglobulin facilitates protein aggregation and fibril formationPROTEIN SCIENCE, Issue 5 2000G. Esposito Abstract The solution structure and stability of N-terminally truncated ,2-microglobulin (,N6,2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that ,N6,2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at ,M concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that ,N6,2-m is significantly less protected than its wild-type counterpart. Analysis of ,N6,2-m by NMR shows that this loss of protection occurs in , strands I, III, and part of II. At mM concentration gel filtration analysis shows that ,N6,2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of ,2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that ,N6,2-m could be a key intermediate of a proteolytic pathway of ,2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of ,2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between ,-strands far removed from this constrain is greatly perturbed. [source] A delayed leaf senescence mutant is defective in arginyl-tRNA:protein arginyltransferase, a component of the N-end rule pathway in ArabidopsisTHE PLANT JOURNAL, Issue 1 2002Satoko Yoshida Summary We have isolated a delayed-leaf-senescence mutant, designated dls1, from an Arabidopsis T-DNA line. Leaf senescence progresses more slowly in the dls1 mutant than in the wild-type plant in both age-dependent and dark-induced senescence. Genetic analysis revealed that the dls1 is a monogenic recessive mutation that cosegregated with the T-DNA insertion. Isolation of DNA flanking the T-DNA revealed that the T-DNA was inserted into the fourth intron of the AtATE1 gene, which encodes arginyl-tRNA:protein arginyltransferase (EC. 2.3.2.8, R-transferase), a component of the N-end rule proteolytic pathway in yeast and mammals that transfers arginine to the N-terminus of proteins with N-terminal glutamyl or aspartyl residues. AtATE1 transcripts were not detectable in the dls1 mutant by RT-PCR analysis. Introduction of a wild-type AtATE1 gene into the dls1 mutant complemented the dls1 phenotype. We also showed using a transient expression assay system, that the dls1 mutation results in a decreased degradation of proteins with Asp or Glu at their N-termini, and that the introduction of the wild-type AtATE1 gene reverses this deficiency. These results suggest that the normal progression of leaf senescence requires R-transferase activity, and that proteolysis by the N-end rule pathway has an important physiological function in the progress of leaf senescence in plants. [source] Protective effects of exercise preconditioning on hindlimb unloading-induced atrophy of rat soleus muscleACTA PHYSIOLOGICA, Issue 1 2009H. Fujino Abstract Aim:, A chronic decrease in the activation and loading levels of skeletal muscles as occurs with hindlimb unloading (HU) results in a number of detrimental changes. Several proteolytic pathways are involved with an increase in myofibrillar protein degradation associated with HU. Exercise can be used to counter this increase in proteolytic activity and, thus, may be able to protect against some of the detrimental changes associated with chronic decreased use. The purpose of the present study was to determine the potential of a single bout of preconditioning endurance exercise in attenuating the effects of 2 weeks of HU on the mass, phenotype and force-related properties of the soleus muscle in adult rats. Methods:, Male Wistar rats were subjected to HU for 2 weeks. One half of the rats performed a single bout of treadmill exercise for 25 min immediately prior to the 2 weeks of HU. Results:, Soleus mass, maximum tetanic tension, myofibrillar protein content, fatigue resistance and percentage of type I (slow) myosin heavy chain were decreased in HU rats. In addition, markers for the cathepsin, calpain, caspase and ATP-ubiquitin-proteasome proteolytic pathways were increased. The preconditioning endurance exercise bout attenuated all of the detrimental changes associated with HU, and increased HSP72 mRNA expression and protein levels. Conclusion:, These findings indicate that exercise preconditioning may be an effective countermeasure to the detrimental effects of chronic decreases in activation and loading levels on skeletal muscles and that an elevation in HSP72 may be one of the mechanisms associated with these responses. [source] Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009Tuomas Ryhänen Abstract The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells. [source] Programmed autologous cleavage of platelet receptorsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2007M. C. BERNDT Summary., Platelet adhesion receptors play a critical role in vascular pathophysiology, and control platelet adhesion, activation and aggregation in hemostasis, thrombotic disease and atherogenesis. One of the key emerging mechanisms for regulating platelet function is the programmed autologous cleavage of platelet receptors. Induced by ligand binding or platelet activation, proteolysis at extracellular (ectodomain shedding) or intracellular (cytoplasmic domain deactivation) sites down-regulates the adheso-signaling function of receptors, thereby controlling not only platelet responsiveness, but in the case of ectodomain shedding, liberating soluble ectodomain fragments into plasma where they constitute potential modulators or markers. This review discusses the underlying mechanisms for dual proteolytic pathways of receptor regulation, and the impact of these pathways on thrombus formation and stability in vivo. [source] | |||||||||||||