Membrane Repair Defects in Muscular Dystrophy Are Linked to Altered Interaction between MG53, Caveolin-3, and Dysferlin

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis and maintenance of sarcolemma integrity in striated muscle. During the life cycle of striated muscle, membrane repair is a fundamental process in maintaining cellular integrity, as shown by recent studies that link defective membrane repair to the progression of muscular dystrophy (1–3). Repair of the plasma membrane damage requires recruitment of intracellular vesicles to injury sites (4, 5). One protein that has been linked to membrane repair in skeletal muscle is dysferlin (6, 7), which is thought to fuse intracellular vesicles to patch the damaged membrane and restore sarcolemmal integrity following muscle injury. Like dysferlin, caveolin-3 (Cav3)3 is a muscle-specific protein, and many mutations in Cav3, including P104L, R26Q, and C71W, have been linked to muscular dystrophy (8–11). Despite extensive research efforts on Cav3 and dysferlin (12–14), the molecular function of these two proteins in membrane repair in muscle physiology and dystrophy have not been fully defined.Animal model studies reveal that either loss or gain of Cav3 function both result in dystrophic phenotypes in skeletal muscle (15, 16), suggesting that associated cellular components may be involved in the etiology of Cav3-related dystrophy. Although the discovery of dysferlin highlights the importance of membrane repair in the etiology of muscular dystrophy, dysferlin itself does not appear to participate in recruitment of intracellular vesicles because dysferlin^−/− muscle retains accumulation of vesicles near membrane damage sites (7). This indicates that proteins other than dysferlin are required for nucleation of intracellular vesicles at the sites of acute membrane damage. Recently, we discovered that MG53, a muscle-specific TRIM family protein (TRIM72), is an essential component of the acute membrane repair machinery. MG53 acts as a sensor of oxidation to nucleate recruitment of intracellular vesicles to the injury site for membrane patch formation (17). We also found that MG53 can regulate membrane budding and exocytosis in muscle cells, and this membrane-recycling function of MG53 can be modulated through a functional interaction with Cav3 (18).Here we present evidence that MG53 interacts with dysferlin to facilitate intracellular vesicle trafficking during repair of acute membrane damage. In addition, we show that transgenic overexpression of P104L-Cav3 in striated muscle produces defects in membrane repair that are linked to altered subcellular distribution of MG53 and dysferlin. Our results suggest that altered MG53 localization can be used as a marker for muscular dystrophy involving reduced sarcolemmal membrane repair capacity due to Cav3 mutation, and potentially, in other forms of dystrophy as well.     

UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells

Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.     

Synthesis of 2,4,6-trisubstituted pyrimidine and triazine heterocycles as antileishmanial agents

A series of 2,4,6 trisubstituted pyrimidines and triazines have been synthesized and screened for its in vitro antileishmanial activity profile in promastigote model. Nine compounds have shown > 94% inhibition against promastigotes at a concentration of 10 microg/mL.     

Proteomic surveillance of autoantigens in patients with Behcet's disease by a proteomic approach

To promote an understanding of autoimmunity in BD, we surveyed autoAgs in patients with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE‐WB, out of which eight spots were identified. They are enolase‐1, cofilin‐1, vimentin, Rho‐GDI β protein, tubulin‐like protein, and actin‐like proteins. The autoAbs to one of the identified proteins, cofilin‐1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin‐1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%) of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin‐1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs.     

Targeting of neutral cholesterol ester hydrolase to the endoplasmic reticulum via its N-terminal sequence

Neutral cholesterol ester hydrolase (NCEH) accounts for a large part of the nCEH activity in macrophage foam cells, a hallmark of atherosclerosis, but its subcellular localization and structure-function relationship are unknown. Here, we determined subcellular localization, glycosylation, and nCEH activity of a series of NCEH mutants expressed in macrophages. NCEH is a single-membrane-spanning type II membrane protein comprising three domains: N-terminal, catalytic, and lipid-binding domains. The N-terminal domain serves as a type II signal anchor sequence to recruit NCEH to the endoplasmic reticulum (ER) with its catalytic domain within the lumen. All of the putative N-linked glycosylation sites (Asn²⁷⁰, Asn³⁶⁷, and Asn³⁸⁹) of NCEH are glycosylated. Glycosylation at Asn²⁷⁰, which is located closest to the catalytic serine motif, is important for the enzymatic activity. Cholesterol loading by incubation with acetyl-LDL does not change the ER localization of NCEH. In conclusion, NCEH is targeted to the ER of macrophages, where it hydrolyzes CE to deliver cholesterol for efflux out of the cells.     

Biosynthesis of andrographolide in Andrographis paniculata

Andrographolide, a diterpene lactone, is isolated from Andrographis paniculata which is well known for its medicinal properties. The biosynthetic route to andrographolide was studied using [1-¹³C]acetate, [2-¹³C]acetate and [1,6-¹³C₂]glucose. The peak enrichment of eight carbon atoms in the ¹³C NMR spectra of andrographolide suggested that deoxyxylulose pathway (DXP) is the major biosynthetic pathway to this diterpene.The contribution of the mevalonic acid pathway (MVA) is indicated by the observed ¹³C-labeling pattern, and because the labeling patterns indicate a simultaneous contribution of both methyl erythritol phosphate (MEP) and MVA pathways it can be deduced that cross-talk occurs between plastids and cytoplasm.     

Structure-based drug design of tricyclic 8H-indeno[1,2-d][1,3]thiazoles as potent FBPase inhibitors

With the goal of improving metabolic stability and further enhancing FBPase inhibitory activity, a series of tricyclic 8H-indeno[1,2-d][1,3]thiazoles was designed and synthesized with the aid of structure-based drug design. Extensive SAR studies led to the discovery of 19a with an IC₅₀ value of 1 nM against human FBPase. X-ray crystallographic studies revealed that high affinity of 19a was due to the hydrophobic interaction arising from better shape complementarity and to the hydrogen bonding network involving the side chain on the tricyclic scaffold.     

Discovery of potent and orally active tricyclic-based FBPase inhibitors

With the aim of exploring the effect of tricyclic-based FBPase inhibitors in cells and in vivo, a series of prodrugs of tricyclic phosphonates was designed and synthesized. Introducing prodrug moieties into tricyclic-based phosphonates led to the discovery of prodrug 15c, which strongly inhibited glucose production in monkey hepatocytes. Furthermore, prodrug 15c lowered blood glucose levels in fasted cynomolgus monkeys.