Stress-induced non-vesicular release of prothymosin-α initiated by an interaction with S100A13, and its blockade by caspase-3 cleavage

Abstract

The nuclear protein prothymosin-a (ProTa), which lacks a signal peptide sequence, is released from neurons and astrocytes on ischemic stress and exerts a unique form of neuroprotection through an anti-necrotic mechanism. Ischemic stress-induced ProTa release is initiated by a nuclear release, followed by extracellular release in a non-vesicular manner, in C6 glioma cells. These processes are caused by ATP loss and elevated Ca 2 þ , respectively. S100A13, a Ca 2 þ -binding protein, was identified to be a major protein co-released with ProTa in an immunoprecipitation assay. The Ca 2 þ -dependent interaction between ProTa and S100A13 was found to require the C-terminal peptide sequences of both proteins. In C6 glioma cells expressing a D88-98 mutant of S100A13, serum deprivation caused the release of S100A13 mutant, but not of ProTa. When cells were administered apoptogenic compounds, ProTa was cleaved by caspase-3 to generate a C-terminal peptide-deficient fragment, which lacks the nuclear localization signal (NLS). However, there was no extracellular release of ProTa. All these results suggest that necrosisinducing stress induces an extacellular release of ProTa in a non-vesicular manner, whereas apoptosis-inducing stress does not, owing to the loss of its interaction with S100A13, a cargo molecule for extracellular release.

Regional Distribution and Cell Type-Specific Subcellular Localization of Prothymosin Alpha in Brain

“…The cytosol expression of ProTa in glial cells may be related to the fact that glial cells have more resistance to cell death stress than neurons. Recent in vitro study has postulated that ProTa is localized in the nuclei of primary culture of rat embryonic astrocytes (Matsunaga and Ueda 2010). This finding revealed a discrepancy from the present study, in which ProTa is found in both cytosol and nucleus of astrocytes.…”

“…This finding revealed a discrepancy from the present study, in which ProTa is found in both cytosol and nucleus of astrocytes. There are several reports hypothesizing that the fragmentation of ProTa was mediated by caspase-3 enzyme at C-terminal side located within the spacer region bipartite nuclear localization signal (NLS) in ProTa (Rubtsov et al 1997;Enkemann et al 2000;Matsunaga and Ueda 2010). It has recently been described that active caspase-3 is present in the nuclei of astrocytes in normal brain (Duran-Vilaregut et al 2010).…”

“…Our previous study reported that caspase-3 activation cleaved off the nuclear localization signal of ProTa in astrocytes and released it from the nucleus (Matsunaga and Ueda 2010). In addition, as adult astrocytes are known to have caspase-3 activity in the nucleus (Duran-Vilaregut et al 2010); we attempted to see whether this protease inhibitor might redistribute ProTa.…”

“…In addition, the administration of ProTa also inhibited the cerebral and retinal ischemia-induced necrosis as well as apoptosis through up-regulation of BDNF or erythropoietin/EPO (Ueda 2009;Fujita et al 2009;Ueda et al 2010). The most recent investigation demonstrate that ProTa is localized in the nuclei of cultured cortical neurons and astrocytes (Matsunaga and Ueda 2010). However, the pattern of ProTa expression in cells in the central nervous system is yet unknown.…”

Prothymosin-α Interacts with Mutant Huntingtin and Suppresses Its Cytotoxicity in Cell Culture

“…Protein extracts were resolved on 10 -15% SDS-PAGE gels and transferred onto nitrocellulose membranes (Whatman GmbH) either under standard transfer conditions or in 20 mM sodium acetate buffer, pH 5.0, followed by fixation with 0.5% glutaraldehyde (12,22). The membrane was blocked with 5% nonfat milk in 1ϫ Tris buffered saline supplemented with 0.5% Tween-20 and probed with rabbit anti-ProT␣ (1:500, Santa Cruz Biotechnology), mouse anti-FLAG (1:1,000, Sigma), mouse anti-V5 (Invitrogen, 1:2000), mouse anti-GFP (Syd Lab), goat anti-actin (1:500, Upstate), or mouse anti-GST (1:4,000, Abcam).…”