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Identification of 14-3-3? as a Mieap-interacting protein and its role in mitochondrial quality control.

ABSTRACT: Mieap, a p53-inducible protein, controls mitochondrial integrity by inducing the accumulation of lysosomal proteins within mitochondria. This phenomenon is designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria. To identify this novel Mieap-interacting protein(s), we performed a two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) on the proteins immunoprecipitated by an anti-Mieap antibody. We indentified 14-3-3? as one of the proteins that was included in the Mieap-binding protein complex when MALM was induced. The interaction between Mieap and 14-3-3? was confirmed on the exogenous and endogenous proteins. Interestingly, 14-3-3? was localized within mitochondria when MALM occurred. A 14-3-3? deficiency did not affect the accumulation of Mieap and lysosomal proteins within mitochondria, but dramatically inhibited the elimination of oxidized mitochondrial proteins. These results suggest that 14-3-3? plays a critical role in eliminating oxidized mitochondrial proteins during the MALM process by interacting with Mieap within mitochondria.

SUBMITTER: Miyamoto T

PROVIDER: S-EPMC3334856 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Identification of 14-3-3γ as a Mieap-interacting protein and its role in mitochondrial quality control.

Miyamoto Takafumi T Kitamura Noriaki N Ono Masaya M Nakamura Yasuyuki Y Yoshida Masaki M Kamino Hiroki H Murai Ryuya R Yamada Tesshi T Arakawa Hirofumi H

Scientific reports 20120424

Mieap, a p53-inducible protein, controls mitochondrial integrity by inducing the accumulation of lysosomal proteins within mitochondria. This phenomenon is designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria. To identify this novel Mieap-interacting protein(s), we performed a two-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL) on the proteins immunoprecipitated by an anti-Mieap antibody. We indentified 14-3- ...[more]

PMID: 22532927

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Project description:Doxorubicin (Dox) with cardiotoxicity and endotheliotoxicity limits its clinical application for cancer. The toxicitic mechanism involves excess ROS generation. 14-3-3s have the protective effects on various injured tissues and cells. Tetramethylpyrazine (TMP) is an alkaloid extracted from the rhizome of Ligusticum wallichii and has multiple bioactivities. We hypothesize that TMP has the protective effects on vascular endothelium by upregulating 14-3-3γ. To test the hypothesis, Dox-induced endotheliotoxicity was used to establish vascular endothelium injury models in mice and human umbilical vein endothelial cells. The effects of TMP were assessed by determining thoracic aortic strips' endothelium-dependent dilation (EDD), as well as LDH, CK, caspase-3, SOD, CAT, GSH-Px activities and MDA level in serum, apoptotic rate, and histopathological changes of vascular tissue (in vivo). Also, cell viability, LDH and caspase-3 activities, ROS generation, levels of NAD+/NADH and GSH/GSSG, MMP, mPTP opening, and apoptotic rate were evaluated (in vitro). The expression of 14-3-3γ and Bcl-2, as well as phosphorylation of Bad (S112), were determined by Western blot. Our results showed that Dox-induced injury to vascular endothelium was decreased by TMP via upregulating 14-3-3γ expression in total protein and Bcl-2 expression in mitochondria, activating Bad (S112) phosphorylation, maintaining EDD, reducing LDH, CK, and caspase-3 activities, thereby causing a reduction in apoptotic rate, and histopathological changes of vascular endothelium (in vivo). Furthermore, TMP increased cell viability and MMP levels, maintained NAD+/NADH, GSH/GSSG balance, decreased LDH and caspase-3 activities, ROS generation, mPTP opening, and apoptotic rate (in vitro). However, the protective effects to vascular endothelium of TMP were significantly canceled by pAD/14-3-3γ-shRNA, an adenovirus that caused knockdown 14-3-3γ expression, or ABT-737, a specific Bcl-2 inhibitor. In conclusion, this study is the first to demonstrate that TMP protects the vascular endothelium against Dox-induced injury via upregulating 14-3-3γ expression, promoting translocation of Bcl-2 to the mitochondria, closing mPTP, maintaining MMP, inhibiting RIRR mechanism, suppressing oxidative stress, improving mitochondrial function, and alleviating Dox-induced endotheliotoxicity.

Dataset Information

Identification of 14-3-3? as a Mieap-interacting protein and its role in mitochondrial quality control.

Publications

Identification of 14-3-3γ as a Mieap-interacting protein and its role in mitochondrial quality control.

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