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Murine Sirt3 protein isoforms have variable half-lives.

ABSTRACT: Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3 (aa 78-334). In this study, we stably expressed these variants in several cell lines. We found that Sirt3 M1 or M2 can be stably expressed with predominant mitochondrial localization. However, stable expression of Sirt3 M3 protein was consistently at very low levels. Fast proteasomal degradation contributes to the low expression of Sirt3 M3 protein, as proteasome inhibitor treatment increased Sirt3 M3 protein levels in these cells. Sirt3 M3 protein is ubiquitinated and the E3 ubiquitin ligase subunit Skp2 is involved in Sirt3 M3 protein degradation. Additionally, we found Sirt3 M3 protein can be produced from Sirt3 transcripts encoding longer M1 and M2 isoforms. To explore the mechanism underlying the instability of Sirt3 M3 protein, we found that Sirt3 M1 and M2 proteins, but not M3, specifically interact with HSP60. This suggests that heat shock proteins might play a role in the maintenance of Sirt3 protein stability.

SUBMITTER: Yang Y

PROVIDER: S-EPMC3185158 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Murine Sirt3 protein isoforms have variable half-lives.

Yang Yongjie Y Chen Ke Yun KY Tong Qiang Q

Gene 20110805 1-2

Sirt3 is a NAD+-dependent protein deacetylase mainly localized in mitochondria. Recent studies indicate that the murine Sirt3 gene expresses different transcript variants resulting in three possible Sirt3 protein isoforms with variable lengths at the N-terminus: M1 (aa 1-334), M2 (aa 15-334), and M3 (aa 78-334). In this study, we stably expressed these variants in several cell lines. We found that Sirt3 M1 or M2 can be stably expressed with predominant mitochondrial localization. However, stable ...[more]

PMID: 21840382

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Project description:Hypoxic areas are present in the majority of solid tumors, and hypoxia is associated with resistance to therapies and poor outcomes. A transmembrane protein that is upregulated by tumor cells that have adapted to hypoxic conditions is carbonic anhydrase IX (CAIX). Therefore, noninvasive imaging of CAIX could be of prognostic value, and it could steer treatment strategies. The aim of this study was to compare variants of CAIX-binding VHH B9, with and without a C-terminal albumin-binding domain with varying affinity (ABDlow and ABDhigh), for SPECT imaging of CAIX expression. The binding affinity and internalization of the various B9-variants were analyzed using SK-RC-52 cells. Biodistribution studies were performed in mice with subcutaneous SCCNij153 human head and neck cancer xenografts. Tracer uptake was determined by ex vivo radioactivity counting and visualized by SPECT/CT imaging. Furthermore, autoradiography images of tumor sections were spatially correlated with CAIX immunohistochemistry. B9-variants demonstrated a similar moderate affinity for CAIX in vitro. Maximal tumor uptake and acceptable tumor-to-blood ratios were found in the SCCNij153 model at 4 h post injection for [111In]In-DTPA-B9 (0.51 ± 0.08%ID/g and 8.1 ± 0.85, respectively), 24 h post injection for [111In]In-DTPA-B9-ABDlow (2.39 ± 0.44%ID/g and 3.66 ± 0.81, respectively) and at 72 h post injection for [111In]In-DTPA-B9-ABDhigh (8.7 ± 1.34%ID/g and 2.43 ± 0.15, respectively). An excess of unlabeled monoclonal anti-CAIX antibody efficiently inhibited tumor uptake of [111In]In-DTPA-B9, while only a partial reduction of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh uptake was found. Immunohistochemistry and autoradiography images showed colocalization of all B9-variants with CAIX expression; however, [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh also accumulated in non-CAIX expressing regions. Tumor uptake of [111In]In-DTPA-B9-ABDlow and [111In]In-DTPA-B9-ABDhigh, but not of [111In]In-DTPA-B9, could be visualized with SPECT/CT imaging. In conclusion, [111In]In-DTPA-B9 has a high affinity to CAIX and shows specific targeting to CAIX in head and neck cancer xenografts. The addition of ABD prolonged plasma half-life, increased tumor uptake, and enabled SPECT/CT imaging. This uptake was, however, partly CAIX- independent, precluding the ABD-tracers for use in hypoxia quantification in this tumor type.

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Murine Sirt3 protein isoforms have variable half-lives.

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Murine Sirt3 protein isoforms have variable half-lives.

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