Abstract
Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.
Highlights
Numerous adult onset neurodegenerative diseases are caused by unstable, expanded CAG trinucleotide repeats within the coding region of an affected gene, which result in the synthesis of disease-specific proteins with expanded polyglutamine domains
Results from two independent experiments revealed that 93% of total tissue transglutaminase was found in the cytosol, 6% was extracted in the chromatin fraction, and 1% co-purified with the nuclear matrix
One pathogenic process that has been proposed to contribute to the neurodegeneration in Huntington’s disease, as well as other CAG trinucleotide repeat diseases, is the homodimerization or heterodimerization of the mutant polyglutamine-containing proteins with subsequent stabilization by transglutaminase resulting in the formation of poorly soluble protein aggregates [7, 15, 16]
Summary
Numerous adult onset neurodegenerative diseases are caused by unstable, expanded CAG trinucleotide repeats within the coding region of an affected gene, which result in the synthesis of disease-specific proteins with expanded polyglutamine domains. It had been hypothesized that tissue transglutaminase may be involved in the pathological process of the CAG repeat diseases [7] Tissue transglutaminase is both a signal transducing GTP-binding protein [8] and a transamidating enzyme [9]. Investigators have since demonstrated that peptides containing glutamine repeats are substrates for tissue transglutaminase [15] and that transglutaminase cross-links expanded polyglutamine domains with glyceraldehyde-3-phosphate dehydrogenase resulting in inactivation of the enzyme [16]. In an earlier investigation of GTP-binding proteins in rabbit liver nuclei, tissue transglutaminase was tentatively identified as a nuclear GTP-binding protein [17] In this previous study the in situ activity of the transglutaminase was not examined. Considering these and other findings, the purpose of this study was to determine the specific nuclear localization of tissue transglutaminase and the in situ activation of nuclear tissue transglutaminase
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