Dystonin-deficient mice exhibit an intrinsic muscle weakness and an instability of skeletal muscle cytoarchitecture.
(17/2534)
Dystonia musculorum (dt) was originally described as a hereditary sensory neurodegeneration syndrome of the mouse. The gene defective in dt encodes a cytoskeletal linker protein, dystonin, that is essential for maintaining neuronal cytoskeletal integrity. In addition to the nervous system, dystonin is expressed in a variety of other tissues, including muscle. We now show that dystonin cross-links actin and desmin filaments and that its levels are increased during myogenesis, coinciding with the progressive reorganization of the intermediate filament network. A disorganization of cytoarchitecture in skeletal muscle from dt/dt mice was observed in ultrastructural studies. Myoblasts from dt/dt mice fused to form myotubes in culture; however, terminally differentiated myotubes contained incompletely assembled myofibrils. Another feature observed in dt/dt myotubes in culture and in skeletal muscle in situ was an accumulation and abnormal distribution of mitochondria. The diaphragm muscle from dt/dt mice was weak in isometric contractility measurements in vitro and was susceptible to contraction-induced sarcolemmal damage. Altogether, our data indicate that dystonin is a cross-linker of actin and desmin filaments in muscle and that it is essential for establishing and maintaining proper cytoarchitecture in mature muscle. (+info)
Structural and functional analysis of the rat metallothionein III genomic locus.
(18/2534)
Metallothionein III (MT III) has been reported to suppress neuronal growth in a rat in vitro model system. The protein and its specific mRNA are detected predominantly in the brain, differentiating MT III from the well-characterised archetypal metallothioneins. Isolation, sequencing and functional analysis of the rat MT III genomic locus indicated that, although the organisation of the gene was conserved between MT III and the more conventional metallothioneins, the 5' flanking region of the MT III gene was distinct. Within this region, a number of putative regulatory elements were identified, including the metal regulatory elements (MREs) characteristic of metallothionein promoters. However, despite their conservation in sequence with active elements, the MREs of MT III were unresponsive to zinc. A 'silencing element' was revealed within a 250 bp section of the MT III promoter which suppressed gene expression in two brain cell lines. The operation of this silencing region in conjunction with the inactive MREs may explain the distinct expression profile observed for MT III within the central nervous system and during neuronal development. (+info)
Functional roles of S100 proteins, calcium-binding proteins of the EF-hand type.
(19/2534)
A multigenic family of Ca2+-binding proteins of the EF-hand type known as S100 comprises 19 members that are differentially expressed in a large number of cell types. Members of this protein family have been implicated in the Ca2+-dependent (and, in some cases, Zn2+- or Cu2+-dependent) regulation of a variety of intracellular activities such as protein phosphorylation, enzyme activities, cell proliferation (including neoplastic transformation) and differentiation, the dynamics of cytoskeleton constituents, the structural organization of membranes, intracellular Ca2+ homeostasis, inflammation, and in protection from oxidative cell damage. Some S100 members are released or secreted into the extracellular space and exert trophic or toxic effects depending on their concentration, act as chemoattractants for leukocytes, modulate cell proliferation, or regulate macrophage activation. Structural data suggest that many S100 members exist within cells as dimers in which the two monomers are related by a two-fold axis of rotation and that Ca2+ binding induces in individual monomers the exposure of a binding surface with which S100 dimers are believed to interact with their target proteins. Thus, any S100 dimer is suggested to expose two binding surfaces on opposite sides, which renders homodimeric S100 proteins ideal for crossbridging two homologous or heterologous target proteins. Although in some cases different S100 proteins share their target proteins, in most cases a high degree of target specificity has been described, suggesting that individual S100 members might be implicated in the regulation of specific activities. On the other hand, the relatively large number of target proteins identified for a single S100 protein might depend on the specific role played by the individual regions that in an S100 molecule contribute to the formation of the binding surface. The pleiotropic roles played by S100 members, the identification of S100 target proteins, the analysis of functional correlates of S100-target protein interactions, and the elucidation of the three-dimensional structure of some S100 members have greatly increased the interest in S100 proteins and our knowledge of S100 protein biology in the last few years. S100 proteins probably are an example of calcium-modulated, regulatory proteins that intervene in the fine tuning of a relatively large number of specific intracellular and (in the case of some members) extracellular activities. Systems, including knock-out animal models, should be now used with the aim of defining the correspondence between the in vitro regulatory role(s) attributed to individual members of this protein family and the in vivo function(s) of each S100 protein. (+info)
New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.
(20/2534)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed. (+info)
Ethical aspects of neural tissue transplantation.
(21/2534)
The method of neural grafting is considered to be a very promising therapeutic strategy for the treatment of certain neurodegenerative disorders such as Parkinson's disease or Huntington's disease. During the last 15 years, clinical transplantation studies have been carried out worldwide in several hundreds of patients with Parkinson's disease. In these studies, primarily fetal mesencephalic tissue derived from aborted human fetuses has been used for implantation. Neural tissue transplantation gives rise to ethical issues in two different areas that need careful examination: the first, ethical problems linked to the use of tissue from aborted human fetuses; and the second, ethical issues concerning the graft recipients in clinical trials, i.e., his or her well-being, personality, and personal identity. (+info)
Biophysical characterization of lithostathine. Evidences for a polymeric structure at physiological pH and a proteolysis mechanism leading to the formation of fibrils.
(22/2534)
Lithostathine is a calcium carbonate crystal habit modifier. It is found precipitated under the form of fibrils in chronic calcifying pancreatitis or Alzheimer's disease. In order to gain better insight into the nature and the formation of fibrils, we have expressed and purified recombinant lithostathine. Analytical ultracentrifugation and quasi-elastic light scattering techniques were used to demonstrate that lithostathine remains essentially monomeric at acidic pH while it aggregates at physiological pH. Analysis of these aggregates by electron microscopy showed an apparently unorganized structure of numerous monomers which tend to precipitate forming regular unbranched fibrils. Aggregated forms seem to occur prior to the apparition of fibrils. In addition, we have demonstrated that these fibrils resulted from a proteolysis mechanism due to a specific cleavage of the Arg(11)-Ile(12) peptide bond. It is deduced that the NH(2)-terminal undecapeptide of lithostathine normally impedes fiber formation but not aggregation. A theoretical model explaining the formation of amyloid plaques in neurodegenerative diseases or stones in lithiasis starting from lithostathine is described. Therefore we propose that lithostathine, whose major function is unknown, defines a new class of molecules which is activated by proteolysis and is not involved in cytoskeleton nor intermediate filament functions. (+info)
Polyglutamine pathogenesis.
(23/2534)
An increasing number of neurodegenerative disorders have been found to be caused by expanding CAG triplet repeats that code for polyglutamine. Huntington's disease (HD) is the most common of these disorders and dentatorubral-pallidoluysian atrophy (DRPLA) is very similar to HD, but is caused by mutation in a different gene, making them good models to study. In this review, we will concentrate on the roles of protein aggregation, nuclear localization and proteolytic processing in disease pathogenesis. In cell model studies of HD, we have found that truncated N-terminal portions of huntingtin (the HD gene product) with expanded repeats form more aggregates than longer or full length huntingtin polypeptides. These shorter fragments are also more prone to aggregate in the nucleus and cause more cell toxicity. Further experiments with huntingtin constructs harbouring exogenous nuclear import and nuclear export signals have implicated the nucleus in direct cell toxicity. We have made mouse models of HD and DRPLA using an N-terminal truncation of huntingtin (N171) and full-length atrophin-1 (the DRPLA gene product), respectively. In both models, diffuse neuronal nuclear staining and nuclear inclusion bodies are observed in animals expressing the expanded glutamine repeat protein, further implicating the nucleus as a primary site of neuronal dysfunction. Neuritic pathology is also observed in the HD mice. In the DRPLA mouse model, we have found that truncated fragments of atrophin-1 containing the glutamine repeat accumulate in the nucleus, suggesting that proteolysis may be critical for disease progression. Taken together, these data lead towards a model whereby proteolytic processing, nuclear localization and protein aggregation all contribute to pathogenesis. (+info)
Properties of polyglutamine expansion in vitro and in a cellular model for Huntington's disease.
(24/2534)
Eight neurodegenerative diseases have been shown to be caused by the expansion of a polyglutamine stretch in specific target proteins that lead to a gain in toxic property. Most of these diseases have some features in common. A pathological threshold of 35-40 glutamine residues is observed in five of the diseases. The mutated proteins (or a polyglutamine-containing subfragment) form ubiquitinated aggregates in neurons of patients or mouse models, in most cases within the nucleus. We summarize the properties of a monoclonal antibody that recognizes specifically, in a Western blot, polyglutamine stretches longer than 35 glutamine residues with an affinity that increases with polyglutamine length. This indicates that the pathological threshold observed in five diseases corresponds to a conformational change creating a pathological epitope, most probably involved in the aggregation property of the carrier protein. We also show that a fragment of a normal protein carrying 38 glutamine residues is able to aggregate into regular fibrils in vitro. Finally, we present a cellular model in which the induced expression of a mutated full-length huntingtin protein leads to the formation of nuclear inclusions that share many characteristics with those observed in patients: those inclusions are ubiquitinated and contain only an N-terminal fragment of huntingtin. This model should thus be useful in studying a processing step that is likely to be important in the pathogenicity of mutated huntingtin. (+info)