Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32

Abstract

Bcl-2 homology domain (BH) 3-only proteins of the proapoptotic Bcl-2 subfamily play a key role as initiators of mitochondria-dependent apoptosis. To date, at least 10 mammalian BH3-only proteins have been identified, and it is now being realized that they have different roles and mechanisms of regulation in the transduction of apoptotic signals to mitochondria. Hrk/DP5 is one of the mammalian BH3-only proteins implicated in a variety of physiological and pathological apoptosis, yet the molecular mechanism involved in Hrk-mediated apoptosis remains poorly understood. In an attempt to identify cellular proteins participating in Hrkmediated apoptosis, we have conducted yeast two-hybrid screening for Hrk-interacting proteins and isolated p32, a mitochondrial protein that has been shown to form a channel consisting of its homotrimer. In vitro binding, co-immunoprecipitation, as well as immunocytochemical analyses verified specific interaction and colocalization of Hrk and p32, both of which depended on the presence of the highly conserved C-terminal region of p32. Importantly, Hrk-induced apoptosis was suppressed by the expression of p32 mutants lacking the N-terminal mitochondrial signal sequence (p32 (74-282)) and the conserved C-terminal region (p32 (1-221)), which are expected to inhibit binding of Hrk competitively to the endogenous p32 protein and to disrupt the channel function of p32, respectively. Furthermore, small interfering RNA-mediated knockdown of p32 conferred protection against Hrk-induced apoptosis. Altogether, these results suggest that p32 may be a key molecule that links Hrk to mitochondria and is critically involved in the regulation of Hrk-mediated apoptosis.

Inhibition of pathogen-induced apoptosis by a Coxiella burnetii type IV effector protein

“…Previous studies have shown that silencing of p32 reduced cisplatin-, harakiri-, and p14ARF-p16INK4a locusinduced apoptosis (21,22,25). Similarly, we found that p32 silencing conferred protection against UV-induced apoptosis.…”

“…Reducing the levels of p32 in mammalian cells has been shown to make them more resistant to apoptosis (21,22), suggesting that p32 has a proapoptotic activity. Consistent with these data, when p32 levels were reduced using siRNA ( Fig.…”

Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss

“…The dominant theory is that Hrk would inhibit the antiapoptotic function of other Bcl-2 family member by heterodimerizing with them. However, a homotetrameric protein, p32, was recently isolated in a two-hybrid screen by its ability to interact with Hrk (24). Hrk-mediated apoptosis requires tetrameric p32 to form a channel in the mitochondria membrane and destabilize the function of this organelle.…”

Arginase II Contributes to the Ca 2+ /CaMKII/eNOS Axis by Regulating Ca 2+ Concentration Between the Cytosol and Mitochondria in a p32‐Dependent Manner

“…Although p32 can be existed in multicellular compartments, it is predominantly targeted to mitochondria because of the mitochondrial targeting sequence contained in the 73N‐terminal amino acids 25. Functional studies have shown that p32 is required for the induction of mitochondria‐dependent cell death26, 27 and for the metabolic shift between oxidative phosphorylation and aerobic glycolysis 28. Moreover, the crystal structure of p32 has been determined and indicates that p32 can form a pore‐like homotrimer and may serve as a high‐capacity divalent cation storage protein 29.…”