Decreased Lysosomal Dipeptidyl Aminopeptidase I Activity in Cultured Human Skin Fibroblasts in Duchenne's Muscular Dystrophy

194

147

Section: Methodsmentioning

confidence: 99%

Mechanism of L-leucyl-L-leucine methyl ester-mediated killing of cytotoxic lymphocytes: dependence on a lysosomal thiol protease, dipeptidyl peptidase I, that is enriched in these cells.

Thiele

Lipsky

1990

194

147

“…The underlying cause of the increased Cl-permeability is unknown, but it may well be closely related to the primary etiology since it is detected in fibroblasts where no degenerative changes are apparent. Alterations in the lysosomal enzyme dipeptidyl aminopeptidase I, which we have previously described (6,7,9), are shown in Fig. 6 as a side effect rather than as a part of the direct pathway leading from gene to pathology.…”

mentioning

confidence: 90%

“…We have described three abnormalities in the lysosomes of DMD fibroblasts: (i) a 7-fold increase of cytoplasmic lamellar bodies (6,7); (ii) decreased activity of dipeptidyl aminopeptidase I, a lysosomal proteolytic enzyme; and (iii) decreased structure-linked latency of this enzyme. Decreased structurelinked latency of lysosome-bound dipeptidyl aminopeptidase I is correlated with a decrease in the apparent K1/2 (the concentration having half-maximal effect) for Cl1 of this chloride-requiring enzyme.…”

mentioning

confidence: 99%

Increased membrane permeability to chloride in Duchenne muscular dystrophy fibroblasts and its relationship to muscle function.

Pato¹,

Davis²,

Doughty³

et al. 1983

Previous studies have suggested an abnormality in Cl-metabolism in Duchenne muscular dystrophy (DMD) fibroblasts. In order to further characterize this abnormality, we have studied mCl-distribution and permeability in 11 DMD and 12 normal fibroblast lines. Under steady-state conditions Cl-efflux in fibroblasts is observed to be biphasic, revealing the presence of two major subcellular compartments. Each compartment contains approximately half of the cellular C1-. The faster of the two observed efflux components is significantly higher in DMD than in control fibroblasts (P < 0.001). To determine the results of a similar increase in Cl-permeability on skeletal muscle action potentials, we have simulated the effects of increased Cl-conductance on muscle by using a computer model. Effects on the simulated action potential include lower rates of membrane depolarization, lower overpotential, longer duration, and lower input resistance. These effects are similar to those actually observed in DMD muscle.Duchenne muscular dystrophy (DMD) is the most serious and rapidly progressive of the muscular dystrophies. Inherited as an X-linked recessive trait, the disease affects the skeletal muscle of boys in early childhood. Affected muscles show pseudohypertrophic changes secondary to the appearance of fat cells, macrophages, and fibroblasts between the necrotic muscle fibers (1). Ultrastructural changes in organelles within the fibers are suggestive of a compromised membrane. In the context of these pervasive cellular changes it has been difficult to identify the inherited biochemical abnormality. Recent investigations have, therefore, included studies of tissues such as erythrocytes, lymphocytes, and fibroblasts, which are not directly involved in the pathology of the disease (2-5).We have described three abnormalities in the lysosomes of DMD fibroblasts: (i) a 7-fold increase of cytoplasmic lamellar bodies (6, 7); (ii) decreased activity of dipeptidyl aminopeptidase I, a lysosomal proteolytic enzyme; and (iii) decreased structure-linked latency of this enzyme. Decreased structurelinked latency of lysosome-bound dipeptidyl aminopeptidase I is correlated with a decrease in the apparent K1/2 (the concentration having half-maximal effect) for Cl1 of this chloride-requiring enzyme. This difference in K1/2 is present only in intact lysosomes and disappears when the lysosomal membrane is disrupted (8). The requirement for an intact membrane and the correlation between latency and K112 for Cl-suggested that increased chloride permeability might be an important part of the etiology of DMD (9). In this study we 4732The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…DMD skin fibroblast populations in vitro express a variety of syndrome-specific cellbiological (6)(7)(8)(9)(10), and biochemical (11)(12)(13)(14)(15)(16)(17) abnormalities. Recent investigations into the protein turnover in DMD fibroblasts in vitro (18)(19)(20) (17) and from our laboratory (20) indicate a significant decrease in protein synthesis (17,20) and a significant increase in the degradation of short-and long-lived proteins in DMD fibroblast populations in vitro (20).…”

mentioning

confidence: 99%

Differential degradation of [35S]methionine polypeptides in Duchenne muscular dystrophy skin fibroblasts in vitro.

Rodemann¹,

Bayreuther²

1986

Rates of protein turnover have been measured in three normal and three Duchenne muscular dystrophy (DMD) skin fibroblast cell lines. Cell populations were analyzed at identical states with regard to cell number, state of topoinhibition, and cumulative population doublings (CPD). Net protein synthesis measured by the incorporation of [35SSmethionine in an 18-hr pulse was reduced by an average of 34%; degradation of total cellular protein measured after an 18-hr pulse with [35S]methionine and a 24-hr chase was enhanced by an average of 50% in DMD fibroblasts. Twodimensional gel electrophoresis analyses revealed that the breakdown of the majority of [35S]methionine polypeptides was markedly increased in DMD fibroblasts. Quantitative determinations of the differential degradation rates of 10 selected proteins in the tropomyosin region of two-dimensional gels were undertaken by scintillation counting and computer analyses. In three series of experiments, the degradation of the 10 proteins in DMD fibroblasts was enhanced by individual polypeptides between 12.0% and 151.2% as measured by scintillation counting or between 0.8% and 128% as determined by computer analyses.Duchenne muscular dystrophy (DMD) is an X-chromosomelinked recessive disorder, characterized by a progressive muscle degeneration in the primary organs of manifestation, the skeletal and cardiac muscles. The primary genetic defect of this disorder has not been defined; dystrophy of skeletal and cardiac muscles is considered to be the result of an abnormal protein metabolism (1-3). Analyses of the primary and/or secondary biochemical defects in degenerating muscle in DMD in vivo have been hampered by secondary changes-e.g., concomitant regeneration and degeneration of myoblasts, fibroblasts, and fat cells (1). The secondary changes make the selection of an appropriate reference base and of a representative control in vivo difficult.Numerous cellular and biochemical abnormalities have also been demonstrated in nerve, erythrocyte, and fibroblast cells in DMD patients (1, 4, 5). DMD skin fibroblast populations in vitro express a variety of syndrome-specific cellbiological (6-10), and biochemical (11-17) (20).In the present study, we analyzed net protein synthesis and degradation of total cellular protein and of individual proteins in DMD and normal skin fibroblasts by two-dimensional gel electrophoresis. We will provide evidence that in DMD skin fibroblasts the majority of proteins are affected by a decreased net synthesis and an enhanced degradation. Analyses ofthe degradation of individual [35S]methionine polypeptides in the tropomyosin region of two-dimensional gels revealed a significant polypeptide-specific increase in the rates of degradation in DMD fibroblasts. MATERIALS AND METHODSCell Cultures. Skin fibroblast populations of three male patients (6-15 yr old) with advanced to very advanced manifestations of the DMD syndrome and of three agematched normal male probands were established from skin biopsies as described elsewhere (14). Three DMD ...