Localization and activity of metalloendopeptidase EC 3.4.24.15 (THOP1)
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Abstract
Thimet oligopeptidase 1, THOP1 (EC 3.4.24.15), is an 85kDa thiol-dependent
metalloendopeptidase implicated in a variety of neural processes and in the
neurodegeneration of Alzheimer's disease (AD). It has been postulated to play key roles in both neuropeptide production and degradation. Because of the potentially significant therapeutic applications of such a peptidase, a general characterization of the enzyme's localization and activity was undertaken. The distribution of THOP1 in rat and monkey brain, both regionally and at the cellular level was examined by immunohistochemical
techniques. THOP1 proteolytic activity was studied in rabbit, rat and monkey brain, as well as in cultured cell lines. Biochemical analyses indicate that the chromosomal localization of the THOP1 gene is at 19pl3.3, precluding strict genetic linkage to the AD risk factor, apolipoprotein E, on 19ql3.2. Various biochemical approaches showed that THOP1 is predominantly a cytosolic enzyme, which is also secreted. Contrary to reports in the literature, evidence is provided that the peptidase is not generally found in the
nuclear compartment. Full-length THOP1 was not found in nuclear extracts by western blot, although an immunoreactive band of 55kDa (p55) was observed. Nuclear extracts lack THOP1 activity, and p55 does not appear to be a degradation product of full-length THOP1. Stable and transient transfections with THOP1 cDNA do not lead to a change in
p55 levels in nuclei. Additionally, northern blot analysis failed to demonstrate an alternative start, stop, or splicing variant of THOP1 mRNA that would be consistent with the production of a 55kDa product. Therefore, we conclude that p55 is unrelated to THOP1 and that the binding of THOP1 antibodies is non-specific. The absence of THOP1 from the nucleus implies nuclear protein catabolism is significantly different from cytosolic protein catabolism. Our data show THOP1 to be a cytosolic and secreted
protein, encoded by a single mRNA species, transcribed from a region of chromosome 19pl3.3, giving rise to a single protein species. The characterization of THOP1 presented here may be important for future therapeutic intervention with the enzyme.
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