The proteomic profile of hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. The goal of this study was to unravel new clues on the biological pathways leading to HIBM by proteomic comparison. Muscle cultures and biopsies were analyzed by two dimensional gel electrophoresis (2-DE) and the same biopsy extracts by isobaric tag for relative and absolute quantitation (iTRAQ). Proteins that were differentially expressed in all HIBM specimens versus all controls in each analysis were identified by mass spectrometry. The muscle cultures 2-DE analysis yielded 41 such proteins, while the biopsies 2-DE analysis showed 26 differentially expressed proteins. Out of the 400 proteins identified in biopsies by iTRAQ, 41 showed altered expression. In spite of the different nature of specimens (muscle primary cultures versus muscle biopsies) and of the different methods applied (2D gels versus iTRAQ) the differentially expressed proteins identified in each of the three analyses where related mainly to the same pathways, ubiquitination, stress response and mitochondrial processes, but the most robust cluster (30%) was assigned to cytoskeleton and sarcomere organization. Taken together, these findings indicate a possible novel function of GNE in the muscle filamentous apparatus that could be involved in the pathogenesis of HIBM.     

No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.     

Intravenous immune globulin in hereditary inclusion body myopathy: a pilot study

Background

Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive, adult onset, non-inflammatory neuromuscular disorder with no effective treatment. The causative gene, GNE, codes for UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, which catalyzes the first two reactions in the synthesis of sialic acid. Reduced sialylation of muscle glycoproteins, such as α-dystroglycan and neural cell adhesion molecule (NCAM), has been reported in HIBM.

Methods

We treated 4 HIBM patients with intravenous immune globulin (IVIG), in order to provide sialic acid, because IgG contains 8 μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive days followed by 3 doses of 400 mg/kg at weekly intervals.

Results

For all four patients, mean quadriceps strength improved from 19.0 kg at baseline to 23.2 kg (+22%) directly after IVIG loading to 25.6 kg (+35%) at the end of the study. Mean shoulder strength improved from 4.1 kg at baseline to 5.9 kg (+44%) directly after IVIG loading to 6.0 kg (+46%) at the end of the study. The composite improvement for 8 other muscle groups was 5% after the initial loading and 19% by the end of the study. Esophageal motility and lingual strength improved in the patients with abnormal barium swallows. Objective measures of functional improvement gave variable results, but the patients experienced improvements in daily activities that they considered clinically significant. Immunohistochemical staining and immunoblotting of muscle biopsies for α-dystroglycan and NCAM did not provide consistent evidence for increased sialylation after IVIG treatment. Side effects were limited to transient headaches and vomiting.

Conclusion

The mild benefits in muscle strength experienced by HIBM patients after IVIG treatment may be related to the provision of sialic acid supplied by IVIG. Other sources of sialic acid are being explored as treatment options for HIBM.     

Autophagy in a mouse model of distal myopathy with rimmed vacuoles or hereditary inclusion body myopathy

Distal myopathy with rimmed vacuoles (DMRV) or hereditary inclusion body myopathy (hIBM) is an autosomal recessive disorder clinically characterized by weakness that initially involves the distal muscles, although other muscles can be affected as well. Pathological hallmarks include the presence of rimmed vacuoles (RVs) and intracellular Congo red-positive depositions in vacuolated or nonvacuolated fibers. Mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which encodes the rate-limiting enzyme in sialic acid biosynthesis, are causative of DMRV/hIBM. Recently, we have generated a mouse model (Gne(-/-)hGNEV572L-Tg) for this disease, and have shown that these mice exhibit hyposialylation and intracellular amyloid deposition before the characteristic RVs are detected, indicating that autophagy is a downstream phenomenon to hyposialylation and amyloid deposition in DMRV/hIBM.     

Influence of UDP-GlcNAc 2-epimerase/ManNAc kinase mutant proteins on hereditary inclusion body myopathy

Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported to cause the disease. Ten of them, representing mutations in both functional domains of GNE, were recombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reduction in the two known GNE activities, thus revealing that mutations may also influence the function of the domain not harboring them. The extent of reduction strongly differs among the point mutants, ranging from only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activity in D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined by structural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis, respectively. We therefore generated models of the three-dimensional structures of the epimerase and the kinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-epimerase and glucokinase, respectively, and determined the localization of the HIBM mutations within these proteins. Whereas in the kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerase domain are mostly located at the protein surface. Otherwise, the different mutations result in different enzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role of the enzymatic function of GNE in the disease mechanism.     

Mitochondrial abnormalities and peripheral europathy in inflammatory myopathy, especially inclusion body myositis

Computer retrieval in a database, comprising 7,225 muscle cases, revealed that mitochondrial myopathies do not occur more frequently in inflammatory myopathies (3.74%) than in the whole series (3.69%). A more detailed study of inclusion body myositis (IBM), however, showed that severe mitochondrial alterations were apparent in about twice as many IBM cases as expected. This confirms recent studies of others although a causal relationship has thus far not been established. Identification of mitochondrial deletions by Southern blotting corresponded to the presence of severe structural abnormalities of mitochondria. Peripheral neuropathy of variable severity was noted in all cases of IBM and mitochondrial myopathy. By contrast, the association of severe mitochondrial abnormalities with polymyositis, systemic scleroderma, and vasculitis observed in some cases of the present series may be incidental or age dependent. (Mol Cell Biochem 174: 277–281, 1997)     

A novel autosomal dominant inclusion body myopathy linked to 7q22.1-31.1

We describe a novel autosomal dominant hereditary inclusion body myopathy (HIBM) that clinically mimics limb girdle muscular dystrophy in a Chinese family. We performed a detailed clinical assessment of 36 individuals spanning four generations. The age of onset ranged from the 30s to the 50s. Hip girdle, neck flexion and axial muscle weakness were involved at an early stage. This disease progressed slowly, and a shoulder girdle weakness appeared later in the disease course. Muscle biopsies showed necrotic, regenerating, and rimmed vacuolated fibers as well as congophilic inclusions in some of the fibers. Electron micrograph revealed cytoplasmic inclusions of 15-21 nm filaments. A genomewide scan and haplotype analyses were performed using an Illumina Linkage-12 DNA Analysis Kit (average spacing 0.58 cM), which traced the disease to a new locus on chromosome 7q22.1-31.1 with a maximum multi-point LOD score of 3.65. The critical locus for this unique disorder, which is currently referred to as hereditary inclusion body myopathy 4 (HIBM4), spans 8.78 Mb and contains 65 genes. This localization raises the possibility that one of the genes clustered within this region may be involved in this disorder.     

Colon cancer: genomics and apoptotic events

Colon cancer is the third most common cancer globally. The risk of developing colon cancer is influenced by a number of factors that include age and diet, but is primarily a genetic disease, resulting from oncogene over-expression and tumour suppressor gene inactivation. The induction and progression of the disease is briefly outlined, as are the cellular changes that occur in its progression. While colon cancer is uniformly amenable to surgery if detected at the early stages, advanced carcinomas are usually lethal, with metastases to the liver being the most common cause of death. Oncogenes and genetic mutations that occur in colon cancer are featured. The molecules and signals that act to eradicate or initiate the apoptosis cascade in cancer cells, are elucidated, and these include caspases, Fas, Bax, Bid, APC, antisense hTERT, PUMA, 15-LOX-1, ceramide, butyrate, tributyrin and PPARgamma, whereas the molecules which promote colon cancer cell survival are p53 mutants, Bcl-2, Neu3 and COX-2. Cancer therapies aimed at controlling colon cancer are reviewed briefly.     

The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy

In vivo growth of bacterial flagellar filaments by self-assembly of flagellin is promoted by a capping structure composed of a pentameric assembly of hook associated protein 2 (HAP2). Isolated native filaments with intact HAP2 cap exhibited higher melting temperature (deltaTm = 4 degrees C) and significantly increased resistance against heat-induced depolymerization than non-capped ones. Reconstituted filaments were also stabilized by HAP2 binding, but the obtained filament-HAP2 complexes were less stable than native assemblies. Their fast depolymerization at elevated temperatures and sensitivity to proteolysis indicated that native-like filament-HAP2 complexes are rarely obtained by in vitro reconstitution. A procedure was developed to isolate perfectly capped native filaments to facilitate high-resolution structural analysis.     

Global impairment of the ubiquitin-proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation

The highly conserved ubiquitin-proteasome system (UPS) controls the stability of most nuclear and cytoplasmic proteins and is therefore essential for virtually all aspects of cellular function. We have previously shown that the UPS is impaired in the presence of aggregated proteins that become deposited into cytoplasmic inclusion bodies (IBs). Here, we report that production of protein aggregates specifically targeted to either the nucleus or cytosol leads to global impairment of UPS function in both cellular compartments and is independent of sequestration of aggregates into IBs. The observation of severe UPS impairment in compartments lacking detectable aggregates or aggregation-prone protein, together with the lack of interference of protein aggregates on 26S proteasome function in vitro, suggests that UPS impairment is unlikely to be a consequence of direct choking of proteasomes by protein aggregates. These data suggest a common proteotoxic mechanism for nuclear and cytoplasmic protein aggregates in the pathogenesis of neurodegenerative disease.     

The economics of inclusion body processing

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.     

Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts.     

Properties of primary mouse myoblasts expanded in culture

We found that the convergently epidermal growth factor (EGF)-induced signal and the collagen-induced signal activate mitogen-activated protein kinase (MAPK), which induces migration. We examined the signaling mechanisms of EGF-induced cell migration on collagen using the A431 carcinoma cell. EGF (10 ng/ml) induced migration on collagen, but inhibited proliferation. Using a MAPK cascade inhibitor, PD98059, it was shown that EGF-induced migration on collagen was mediated by MAPK whereas EGF-induced migration on fibronectin and vitronectin was not. PD98059 also showed that activation of MAPK induced by EGF enhanced the adhesiveness of A431 cells to collagen. By Western blotting analysis, the kinetics of MAPK phosphorylation induced by EGF and collagen was examined separately, and convergently. First of all, EGF without collagen caused transient MAPK phosphorylation. Collagen without EGF caused MAPK to be immediately and transiently dephosphorylated, and rephosphorylated followed by sustained hyperphosphorylation. EGF together with collagen caused an immediate, and sustained, hyperphosphorylation. These facts suggest that the transient MAPK dephosphorylation induced by collagen is required for migration in order to maintain an appropriate level of sustained phosphorylation. Furthermore, we found that adhesion of A431 cells to collagen was blocked by the anti-beta1 integrin antibody or by the mixed antibodies composed of anti-alpha1, -alpha2, and -alpha3 antibodies, indicating that collagen-induced MAPK phosphorylation was mediated through alpha1beta1, alpha2beta1, and alpha3beta1 integrins.     

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。     

Linkage of hereditary distal myopathy with desmin accumulation to 2q

We are investigating the genetics of a large family with an autosomal dominant form of hereditary distal myopathy. This slowly progressive myopathy begins during early adulthood in the distal leg muscles, producing a gait disturbance. Cardiomyopathy is also present in most affected family members, manifesting itself as conduction block or congestive heart failure. Histologically, an accumulation of the protein, desmin, occurs in the subsarcolemmal spaces of myofibers. We have performed linkage analyses of this family, and have mapped the location of the gene causing the myopathy to human chromosome 2q33. The gene is within a 17-cM segment of chromosome 2q bounded by the DNA markers D2S2248 and D2S401. The best candidate gene for this myopathy is desmin.     

Biomechanical characterization of a desminopathy in primary human myoblasts

Heterozygous mutations of the human desmin gene on chromosome 2q35 cause hereditary and sporadic myopathies and cardiomyopathies. The expression of mutant desmin brings about partial disruption of the extra sarcomeric desmin cytoskeleton and abnormal protein aggregation in the sarcoplasm of striated muscle cells. The precise molecular pathways and sequential steps that lead from a desmin gene defect to progressive muscle damage are still unclear. We tested whether mutant desmin changes the biomechanical properties and the intrinsic mechanical stress response of primary cultured myoblasts derived from a patient carrying a heterozygous R350P desmin mutation. Compared to wildtype controls, undifferentiated mutant desmin myoblasts revealed increased cell death and substrate detachment in response to cyclic stretch on flexible membranes. Moreover, magnetic tweezer microrheometry of myoblasts using fibronectin-coated beads showed increased stiffness of diseased cells. Our findings provide the first evidence that altered mechanical properties may contribute to the progressive striated muscle pathology in desminopathies. We postulate that the expression of mutant desmin leads to increased mechanical stiffness, which results in excessive mechanical stress in response to strain and consecutively to increased mechanical vulnerability and damage of muscle cells.     

Genetic variability of foetal bovine myoblasts in primary culture

Selected strains of adult bovines and those which either have high muscle growth capacity or are double-muscled present particular characteristics of muscle fibres and collagen at slaughter that favour meat tenderness. For double-muscled bovines, it has been shown that these characteristics originated during foetal life. However, no studies have been done to determine the origin of muscle growth superiority in bovine with high muscle growth capacity compared to those with a low muscle growth capacity. Therefore, the objective of this study was to examine the proliferation and differentiation phases of myoblasts in primary culture taken from high and low muscle growth capacity foetuses at 110 days post-conception. These cultures were analysed on 1, 2, 3, 4, 6, 8, 10 days of culture. The proliferation phase was monitored by appropriate marker antibodies. The differentiation was studied by immunocytochemistry with specific antibodies for foetal, I, II (IIa and IIb), I and IIb, I and IIa myosin heavy chains (MHCs) and connectin respectively, and by immunoblotting with desmin antibody. A higher proliferation, a lower fusion and a delayed differentiation of the late markers namely MHCs fast (IIa and IIb) and connectin were shown in high muscle growth capacity foetuses compared to low capacity ones. The results indicate that the muscle growth superiority of high muscle growth capacity bovines seems, therefore, to have a similar foetal origin to that of double-muscled ones.