Random mutagenesis of PDZOmi domain and selection of mutants that specifically bind the Myc proto-oncogene and induce apoptosis

peer reviewedOmi is a mammalian serine protease that is localised in the mitochondria and released to the cytoplasm in response to apoptotic stimuli. Omi induces cell death in a caspase-dependent manner by interacting with the Xchromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity. Omi is synthesized as a precursor polypeptide and is processed to an active serene protease with a unique PDZ domain. PDZ domains recognise the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a b-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZOmi mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells. One such PDZm domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death. PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used. Our studies indicate that the PDZ domain of Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus

Regulation of HAX-1 anti-apoptotic protein by Omi/HtrA2 protease during cell death

Omi/HtrA2 is a nuclear-encoded mitochondrial serine protease that has a pro-apoptotic function in mammalian cells. Upon induction of apoptosis, Omi translocates to the cytoplasm and participates in caspase-dependent apoptosis by binding and degrading inhibitor of apoptosis proteins. Omi can also initiate caspase-independent apoptosis in a process that relies entirely on its ability to function as an active protease. To investigate the mechanism of Omi-induced apoptosis, we set out to isolate novel substrates that are cleaved by this protease. We identified HS1-associated protein X-1 (HAX-1), a mitochondrial anti-apoptotic protein, as a specific Omi interactor that is cleaved by Omi both in vitro and in vivo. HAX-1 degradation follows Omi activation in cells treated with various apoptotic stimuli. Using a specific inhibitor of Omi, HAX-1 degradation is prevented and cell death is reduced. Cleavage of HAX-1 was not observed in a cell line derived from motor neuron degeneration 2 mice that carry a mutated form of Omi that affects its proteolytic activity. Degradation of HAX-1 is an early event in the apoptotic process and occurs while Omi is still confined in the mitochondria. Our results suggest that Omi has a unique pro-apoptotic function in mitochondria that involves removal of the HAX-1 antiapoptotic protein. This function is distinct from its ability to activate caspase-dependent apoptosis in the cytoplasm by degrading inhibitor of apoptosis proteins

Characterization of a novel and specific inhibitor for the pro-apoptotic protease Omi/HtrA2

Omi/HtrA2 is a mammalian serine protease with high homology to bacterial HtrA chaperones. Omi/HtrA2 is localized in mitochondria and is released to the cytoplasm in response to apoptotic stimuli. Omi/HtrA2 induces cell death in a caspase-dependent manner by interacting with the inhibitor of apoptosis protein as well as in a caspase-independent manner that relies on its protease activity. We describe the identification and characterization of a novel compound as a specific inhibitor of the proteolytic activity of Omi/HtrA2. This compound (ucf-101) was isolated in a high throughput screening of a combinatorial library using bacterially made Omi-(134-458) protease and fluorescein-casein as a generic substrate. ucf-101 showed specific activity against Omi/HtrA2 and very little activity against various other serine proteases. This compound has a natural fluorescence that was used to monitor its ability to enter mammalian cells. ucf-101, when tested in caspase-9 (-/-) null fibroblasts, was found to inhibit Omi/HtrA2-induced cell death

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15

Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

ped/pea-15 is a ubiquitously expressed 15-kDa protein featuring a broad anti-apoptotic function. In a yeast two-hybrid screen, the pro-apoptotic Omi/HtrA2 mitochondrial serine protease was identified as a specific interactor of the ped/pea-15 death effector domain. Omi/HtrA2 also bound recombinant ped/pea-15 in vitro and co-precipitated with ped/pea-15 in 293 and HeLa cell extracts. In these cells, the binding of Omi/HtrA2 to ped/pea-15 was induced by UVC exposure and followed the mitochondrial release of Omi/HtrA2 into the cytoplasm. Upon UVC exposure, cellular ped/pea-15 protein expression levels decreased. This effect was prevented by the ucf-101 specific inhibitor of the Omi/HtrA2 proteolytic activity, in a dose-dependent fashion. In vitro incubation of ped/pea-15 with Omi/HtrA2 resulted in ped/pea-15 degradation. In intact cells, the inhibitory action of ped/pea-15 on UVC-induced apoptosis progressively declined at increasing Omi/HtrA2 expression. This further effect of Omi/HtrA2 was also inhibited by ucf-101. In addition, ped/pea-15 expression blocked Omi/HtrA2 co-precipitation with the caspase inhibitor protein XIAP and caspase 3 activation. Thus, in part, apoptosis following Omi/HtrA2 mitochondrial release is mediated by reduction in ped/pea-15 cellular levels. The ability of Omi/HtrA2 to relieve XIAP inhibition on caspases is modulated by the relative levels of Omi/HtrA2 and ped/pea-15

Regolazione dell'espressione del gene per l'adesione intercellulare ICAM-1 in cellule di melanoma

Dottorato di ricerca in medicina sperimentale. 7. ciclo. Coordinatore A. Gulino. Tutore S. MartinottiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Random Mutagenesis Of PdzOmi Domain And Selection Of Mutants That Specifically Bind The Myc Proto-Oncogene And Induce Apoptosis

Omi is a mammalian serine protease that is localized in the mitochondria and released to the cytoplasm in response to apoptotic stimuli. Omi induces cell death in a caspase-dependent manner by interacting with the X-chromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity. Omi is synthesized as a precursor polypeptide and is processed to an active serine protease with a unique PDZ domain. PDZ domains recognize the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a β-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZOmi mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells. One such PDZm domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death. PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used. Our studies indicate that the PDZ domain of Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus

Omi/Htra2 Protease Mediates Cisplatin-Induced Cell Death In Renal Cells

Omi/HtrA2 is a mitochondrial proapoptotic serine protease that is able to induce both caspase-dependent and caspase-independent cell death. After apoptotic stimuli, Omi is released to the cytoplasm where it binds and cleaves inhibitor of apoptosis proteins. In this report, we investigated the role of Omi in renal cell death following cisplatin treatment. Using primary mouse proximal tubule cells, as well as established renal cell lines, we show that the level of Omi protein is upregulated after treatment with cisplatin. This upregulation is followed by the release of Omi from mitochondria to the cytoplasm and degradation of XIAP. Reducing the endogenous level of Omi protein using RNA interference renders renal cells resistant to cisplatin-induced cell death. Furthermore, we show that the proteolytic activity of Omi is necessary and essential for cisplatin-induced cell death in this system. When renal cells are treated with Omi\u27s specific inhibitor, ucf-101, they become significantly resistant, to cisplatin-induced cell death. Ucf-101 was also able to minimize cisplatin-induced nephrotoxic injury in animals. Our results demonstrate that Omi is a major mediator of cisplatin-induced cell death in renal cells and suggest a way to limit renal injury by specifically inhibiting its proteolytic activity

Atf4 Interacts With Abro1/Kiaa0157 Scaffold Protein And Participates In A Cytoprotective Pathway

Abro1 (Abraxas brother 1), also known as KIAA0157, is a scaffold protein that recruits various polypeptides to assemble the BRISC (BRCC36 isopeptide) deubiquitinating enzyme (DUB) complex. The BRISC enzyme has a Lys63-linked deubiquitinating activity and is comprised of four known subunits: MERIT40 (mediator of Rap80 interactions and targeting 40. kDa), BRE (brain and reproductive organ-expressed), BRCC36 (BRCA1/BRCA2-containing complex, subunit 3) and Abro1. We have previously shown that Abro1 has a cytoprotective role that involves the BRISC DUB complex acting on specific Lys63-linked polyubiquitinated substrates. In this report we identify three members of the AP-1 (activating protein-1) family, the ATF4, ATF5 (activating transcription factor) and JunD proteins, as specific interactors of Abro1. The function of ATF4-Abro1 interaction was investigated under normal conditions as well as under cellular stress. Abro1 is predominantly cytoplasmic, but during cellular stress it enters the nucleus and co-localizes with ATF4. Furthermore, this interaction with ATF4 is necessary and essential for the cytoprotective function of Abro1 following oxidative stress. The ability of Abro1 to specifically interact with a number of transcription factors suggests a new mechanism of regulation of the BRISC DUB complex. This regulation involves the participation of at least three known members of the AP-1 family of transcription factors. © 2012 Elsevier B.V

Atf4 Interacts With Abro1/Kiaa0157 Scaffold Protein And Participates In A Cytoprotective Pathway

Abro1 (Abraxas brother 1), also known as KIAA0157, is a scaffold protein that recruits various polypeptides to assemble the BRISC (BRCC36 isopeptide) deubiquitinating enzyme (DUB) complex. The BRISC enzyme has a Lys63-linked deubiquitinating activity and is comprised of four known subunits: MERIT40 (mediator of Rap80 interactions and targeting 40. kDa), BRE (brain and reproductive organ-expressed), BRCC36 (BRCA1/BRCA2-containing complex, subunit 3) and Abro1. We have previously shown that Abro1 has a cytoprotective role that involves the BRISC DUB complex acting on specific Lys63-linked polyubiquitinated substrates. In this report we identify three members of the AP-1 (activating protein-1) family, the ATF4, ATF5 (activating transcription factor) and JunD proteins, as specific interactors of Abro1. The function of ATF4-Abro1 interaction was investigated under normal conditions as well as under cellular stress. Abro1 is predominantly cytoplasmic, but during cellular stress it enters the nucleus and co-localizes with ATF4. Furthermore, this interaction with ATF4 is necessary and essential for the cytoprotective function of Abro1 following oxidative stress. The ability of Abro1 to specifically interact with a number of transcription factors suggests a new mechanism of regulation of the BRISC DUB complex. This regulation involves the participation of at least three known members of the AP-1 family of transcription factors. © 2012 Elsevier B.V