Dentatorubral-pallidoluysian Atrophy Protein Research Articles

Tissue Transglutaminase-Catalyzed Formation of High-Molecular-Weight Aggregatesin VitroIs Favored with Long Polyglutamine Domains: A Possible Mechanism Contributing to CAG-Triplet Diseases

To investigate possible biochemical mechanisms underlying the “toxic gain of function” associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-Mraggregates—a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral–pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptidesin vitro,resulting in the formation of high-Mraggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-Mrprotein aggregates, thereby contributing to cell death.

Suppression of aggregate formation and apoptosis by transglutaminase inhibitors in cells expressing truncated DRPLA protein with an expanded polyglutamine stretch.

To elucidate the molecular mechanisms whereby expanded polyglutamine stretches elicit a gain of toxic function, we expressed full-length and truncated DRPLA (dentatorubral-pallidoluysian atrophy) cDNAs with or without expanded CAG repeats in COS-7 cells. We found that truncated DRPLA proteins containing an expanded polyglutamine stretch form filamentous peri- and intranuclear aggregates and undergo apoptosis. The apoptotic cell death was partially suppressed by the transglutaminase inhibitors cystamine and monodansyl cadaverine (but not putrescine), suggesting involvement of a transglutaminase reaction and providing a potential basis for the development of therapeutic measures for CAG-repeat expansion diseases.

Dentatorubral Pallidoluysian Atrophy (DRPLA) Protein Is Cleaved by Caspase-3 during Apoptosis

Dentatorubral pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder. It is associated with an abnormal CAG repeat expansion resulting in formation of a protein with an elongated polyglutamine stretch. However, neither the physiological roles of the DRPLA gene product nor molecular mechanisms of its pathogenesis have yet been elucidated. Here we report that the DRPLA protein is cleaved at a site near the N terminus during apoptosis induced by VP-16, staurosporine, or glucocorticoid. Moreover, the in vitro translated DRPLA protein is cleaved by recombinant caspase-3, a member of the cysteine protease family, which is thought to be a main executioner of apoptosis. Using mutant DRPLA proteins, the cleavage site was identified as 106DSLDG110. The cleavage, however, was not modulated by the length of the polyglutamine stretch. These findings suggest that the DRPLA protein is one of the physiological substrates of caspase-3, and its cleavage may result in structural and biochemical alterations associated with apoptosis.

Expression of dentatorubral-pallidoluysian atrophy (DRPLA) proteins in patients

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is caused by expansion of a CAG trinucleotide repeat. The mutant gene is translated into protein whose electrophoretic mobility correlates to the number of expanded CAG trinucleotide repeats, indicating that the protein carries an expanded glutamine repeat. Using two polyclonal antibodies raised against the DRPLA gene product in immunoblotting, we determined the untruncated DRPLA proteins, and showed that the amounts of mutant and wild-type DRPLA proteins were similar in DRPLA brain tissues and lymphoblastoid cells, suggesting that regulation of the level of translation of the DRPLA gene is not central to the development of the disease.

Characterization of dentatorubral-pallidoluysian atrophy proteins using two-dimensional electrophoretic analysis

The genetic defect dentatorubral-pallidoluysian atrophy (DRPLA) is known to be an expansion of a CAG trinucleotide repeat. The mutant gene has been demonstrated to be translated into protein, and this protein contains an expanded polyglutamine region. In the present study, we have demonstrated using two-dimensional gel electrophoresis and immunoblotting that the DRPLA gene products consist of a number of isoelectric variants in brain tissue and lymphoblastoid cells. The identification of isoelectric variants indicates that there may be multiple stages of post-translational modification. DRPLA proteins from brain tissue possess fewer basic isoelectric variants than those from lymphoblastoid cells, suggesting that there is different post-translational modification steps in brain tissue and lymphoblastoid cell.

Dentatorubral-pallidoluysian atrophy proteins in lymphoblastoid cells.

The genetic defect responsible for dentatorubral-pallidoluysian atrophy (DRPLA) is an expansion of a CAG trinucleotide repeat. The DRPLA gene is translated into protein in the brain. In this study, we demonstrate that the wild-type and mutant genes are also translated into proteins, p190 and p205, in lymphoblastoid cells. The correlation between the age of onset and the expansion of polyglutamine stretch shown by slower electrophoretic mobility suggests that the polyglutamine stretch is directly involved in the acceleration of the disease process. Moreover, analysis of the protein in lymphoblastoid cells can be used as a diagnostic procedure for DRPLA.

Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH.

At least five adult-onset neurodegenerative diseases, including Huntingtin disease (HD), and dentatorubral-pallidoluysian atrophy (DRPLA) are produced by genes containing a variably increased CAG repeat within the coding region. The size range of the repeats is similar in all diseases; unaffected individuals have fewer than 30 CAG repeats, whereas affected patients usually have more than 40 repeats. The size of the inherited CAG repeat correlates with the severity and age of disease onset. The CAG triplet repeat produces a polyglutamine domain in the expressed proteins. All of these diseases are inherited in a dominant fashion, and a pathologic gain of function in gene carriers has been proposed. We sought to identify proteins in the brain that selectively interact with polyglutamine-domain proteins, hypothesizing that the polyglutamine domain may determine protein-protein interactions.