Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore-forming protein p32
| Autor: | Kaori Sakurada, Fumio Tashiro, Dongchon Kang, Yukiko Gotoh, Yoshiyuki Kuchino, Jun Sunayama, Chifumi Kitanaka, Arata Tomiyama, N Itoh, Akinori Sugiyama, Y Ando |
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| Rok vydání: | 2004 |
| Předmět: |
Signal peptide
Recombinant Fusion Proteins Mutant Apoptosis Mitochondrion Biology Astrocytoma Pore forming protein Mitochondrial Proteins Transduction (genetics) Cell Line Tumor Proto-Oncogene Proteins Two-Hybrid System Techniques Chlorocebus aethiops Animals Humans Amino Acid Sequence RNA Small Interfering Molecular Biology Conserved Sequence Fluorescent Dyes Glutathione Transferase Sequence Deletion Inhibitor of apoptosis domain Binding Sites Rhodamines Neuropeptides Colocalization Nuclear Proteins Cell Biology Immunohistochemistry Precipitin Tests Peptide Fragments Cell biology body regions Microscopy Fluorescence COS Cells Apoptosis Regulatory Proteins Carrier Proteins Fluorescein-5-isothiocyanate Protein Binding |
| Zdroj: | Cell death and differentiation. 11(7) |
| ISSN: | 1350-9047 |
| Popis: | 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 Hrk-mediated 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. |
| Databáze: | OpenAIRE |
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