No abstract

Lithium, a drug used to treat bipolar disorders, has a variety of neuroprotective mechanisms, including autophagy regulation, in various neuropsychiatric conditions. In neurodegenerative diseases, lithium enhances degradation of aggregate-prone proteins, including mutated huntingtin, phosphorylated tau, and α-synuclein, and causes damaged mitochondria to degrade, while in a mouse model of cerebral ischemia and Alzheimer's disease autophagy downregulation by lithium is observed. The signaling pathway of lithium as an autophagy enhancer might be associated with the mammalian target of rapamycin (mTOR)-independent pathway, which is involved in myoinositol-1,4,5-trisphosphate (IP 3 ) in Huntington's disease and Parkinson's disease. However, the mTOR-dependent pathway might be involved in inhibiting glycogen synthase kinase-3β (GSK3β) in other diseases. Lithium's autophagy-enhancing property may contribute to the therapeutic benefit of patients with neuropsychiatric disorders.

The present study demonstrates changes in the ET-receptor expression pattern in favor of the ETA receptor in human end-stage heart failure. Furthermore, activation of the cardiac ET system with increased tissue ET-1 concentrations in the failing myocardium is observed. This is more likely due to decreased clearance than to increased synthesis, because ppET-1 gene expression and ECE activity are unchanged.

Endothelin is a potent vasoconstrictive peptide that is produced by vascular endothelial cells; it is formed from its precursor, big endothelin, by endothelin-converting enzyme (ECE). In this work, ECE was studied using specific monoclonal antibodies. In immunoblotting, ECE was estimated to be a 300 kDa protein on SDS/PAGE under non-reducing conditions, and 130 kDa under reducing conditions. Cross-linking experiments revealed that ECE is composed of two disulphide-linked subunits. Localization of ECE was studied at the cellular and subcellular levels in various rat tissues and cells. High-level expression of ECE was observed in membrane fractions of simian virus 40-transformed rat endothelial cells by immunoblotting, but the immunoreactive band was absent form aortic smooth muscle cells and cytosolic fractions of endothelial cells. In immunohistochemical analysis, ECE was found to be localized in the endothelial cells of the aorta, lung, kidney, liver and heart. Confocal immunofluorescent microscopy showed that most of the ECE in endothelial cells and cells transfected with ECE cDNA was clustered along the plasma membrane. Intact COS or CHO cells transfected with ECE cDNA rapidly and efficiently cleaved big endothelin-1 added to the culture medium. Thus endothelial cells express ECE on the plasma membrane and the active site of the enzyme faces outside the cells, i.e. it is an ectoenzyme. Other than endothelial cells, ECE was also present in some secretory cells. The enzyme was abundant in the adrenal gland, and localized in chromaffin cells. ECE was also highly condensed in pancreatic islet beta cells. It is concluded that ECE and endothelin may be involved in the regulated secretion of hormones.

We investigated the role of periostin, an extracellular matrix protein, in the pathophysiology of osteoarthritis (OA). In OA, dysregulated gene expression and phenotypic changes in articular chondrocytes culminate in progressive loss of cartilage from the joint surface. The molecular mechanisms underlying this process are poorly understood. We examined periostin expression by immunohistochemical analysis of lesional and nonlesional cartilage from human and rodent OA knee cartilage. In addition, we used small interfering (si)RNA and adenovirus transduction of chondrocytes to knock down and upregulate periostin levels, respectively, and analyzed its effect on matrix metalloproteinase (MMP)-13, a disintegrin and MMP with thrombospondin motifs (ADAMTS)-4, and type II collagen expression. We found high periostin levels in human and rodent OA cartilage. Periostin increased MMP-13 expression dose [1-10 mg/ml (EC 50 0.5-1 mg/ml)] and time (24-72 h) dependently, significantly enhanced expression of ADAMTS4 mRNA, and promoted cartilage degeneration through collagen and proteoglycan degradation. Periostin induction of MMP-13 expression was inhibited by CCT031374 hydrobromide, an inhibitor of the canonical Wnt/b-catenin signaling pathway. In addition, siRNA-mediated knockdown of endogenous periostin blocked constitutive MMP-13 expression. These findings implicate periostin as a catabolic protein that promotes cartilage degeneration in OA by upregulating MMP-13 through canonical Wnt signaling.-Attur, M

. No significant difference was found in the specific activity and substrate specificity between the two isoforms. The expression level of ECE-I~ mRNA was higher than that of ECE-I~ in various rat cells and tissues, suggesting that the physiologically important isoform is ECE-la. The present findings verified the presence of two forms of ECE-1 over many species, which are created probably through alternative splicing.

BackgroundPerilipin A (PeriA) exclusively locates on adipocyte lipid droplets and is essential for lipid storage and lipolysis. Previously, we reported that adipocyte specific overexpression of PeriA caused resistance to diet-induced obesity and resulted in improved insulin sensitivity. In order to better understand the biological basis for this observed phenotype, we performed additional studies in this transgenic mouse model.Methodology and Principal FindingsWhen compared to control animals, whole body energy expenditure was increased in the transgenic mice. Subsequently, we performed DNA microarray analysis and real-time PCR on white adipose tissue. Consistent with the metabolic chamber data, we observed increased expression of genes associated with fatty acid β-oxidation and heat production, and a decrease in the genes associated with lipid synthesis. Gene expression of Pgc1a, a regulator of fatty acid oxidation and Ucp1, a brown adipocyte specific protein, was increased in the white adipose tissue of the transgenic mice. This observation was subsequently verified by both Western blotting and histological examination. Expression of RIP140, a regulator of white adipocyte differentiation, and the lipid droplet protein FSP27 was decreased in the transgenic mice. Importantly, FSP27 has been shown to control gene expression of these crucial metabolic regulators. Overexpression of PeriA in 3T3-L1 adipocytes also reduced FSP27 expression and diminished lipid droplet size.ConclusionsThese findings demonstrate that overexpression of PeriA in white adipocytes reduces lipid droplet size by decreasing FSP27 expression and thereby inducing a brown adipose tissue-like phenotype. Our data suggest that modulation of lipid droplet proteins in white adipocytes is a potential therapeutic strategy for the treatment of obesity and its related disorders.

Endothelin-converting enzyme-1 (ECE-1) is involved in the conversion of big endothelins (big ETs) into endothelins (ETs) and shows sequence similarity with neutral endopeptidase-24.11 (NEP). Unlike NEP, ECE-1 exists as a disulphide-linked dimer. Here we reveal that Cys412 is solely responsible for the dimerization of rat ECE-1. The C412S mutant enzyme, which existed as a monomer, showed no difference in glycosylation level, subcellular localization of clustering structure formation, but showed a higher K(m) and lower kcat for big ET-1 compared with the wild-type enzyme. These results indicate that dimerization of ECE-1 is preferential for effective conversion of big ETs into ETs. In addition, complete loss of activity in the mutants E592Q, E651Q and H716Q confirmed that these residues are responsible for catalytic activity, zinc binding and stabilization of the intermediate during the transition state respectively. In contrast, the catalytic properties of mutant enzymes containing a substitution at Arg129 or Glu752 were not markedly different from those of the wild-type enzyme, suggesting that these residues play only a minor role, if any, in substrate binding, in contrast with their role in NEP.