To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.
Resveratrol is a naturally occurring phytoalexin with antioxidant and antiinflammatory properties. Recent studies suggest that resveratrol possesses anticancer effects, although its mechanism of action is not well understood. We now show that resveratrol inhibits Src tyrosine kinase activity and thereby blocks constitutive signal transducer and activator of transcription 3 (Stat3) protein activation in malignant cells. Analyses of resveratrol-treated malignant cells harboring constitutively-active Stat3 reveal irreversible cell cycle arrest of v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), human breast (MDA-MB-231), pancreatic (Panc-1), and prostate carcinoma (DU145) cell lines at the G 0 -G 1 phase or at the S phase of human breast cancer (MDA-MB-468) and pancreatic cancer (Colo-357) cells, and loss of viability due to apoptosis. By contrast, cells treated with resveratrol, but lacking aberrant Stat3 activity, show reversible growth arrest and minimal loss of viability. Moreover, in malignant cells harboring constitutively-active Stat3, including human prostate cancer DU145 cells and v-Src-transformed mouse fibroblasts (NIH3T3/v-Src), resveratrol treatment represses Stat3-regulated cyclin D1 as well as Bcl-x L and Mcl-1 genes, suggesting that the antitumor cell activity of resveratrol is in part due to the blockade of Stat3-mediated dysregulation of growth and survival pathways. Our study is among the first to identify Src-Stat3 signaling as a target of resveratrol, further defining the mechanism of antitumor cell activity of resveratrol and raising its potential application in tumors with an activated Stat3 profile.
Acute renal failure (ARF) is characterized by a very high mortality essentially unchanged over the past 40 years. Simple vertebrate models are needed to improve our understanding of ARF and facilitate the development of novel therapies for this clinical syndrome. Here, we demonstrate that gentamicin, a commonly used nephrotoxic antibiotic, causes larval zebrafish to develop ARF characterized by histological and functional changes that mirror aminoglycoside toxicity in higher vertebrates and inability of zebrafish to maintain fluid homeostasis. We developed a novel method to quantitate renal function in larval zebrafish and demonstrate a decline in glomerular filtration rate after gentamicin exposure. The antineoplastic drug cisplatin, whose use in humans is limited by kidney toxicity, also causes typical histological changes and a decline in renal function in larval zebrafish. A specific inhibitor of Omi/HtrA2, a serine protease implicated in cisplatin-induced apoptosis, prevented renal failure and increased survival. This protective effect was confirmed in a mouse model of cisplatin-induced nephrotoxicity. Therefore, zebrafish provides a unique model system, amenable to genetic manipulation and drug screening, to explore the pathophysiology of ARF and establish novel therapies with potential use in mammals. animal model; drug development; glomerular filtration; fluorescence ACUTE RENAL FAILURE (ARF) in the intensive care unit setting is associated with mortality rates of 50 to 70%, which have not changed for several decades (10, 32), and proven therapies remain elusive. Current mammalian models of ARF have been challenged as to their applicability to the human syndrome and are often difficult to interpret (4,14). The kidneys in the commonly used mouse or rat are inaccessible, and renal tubules and vessels cannot be visualized, with the exception of a very small subpopulation near the surface of the kidney (11). Cell culture systems, on the other hand, while more simple, are inadequate because of epithelial cell dedifferentiation and absence of the three-dimensional interaction of renal tubule, blood vessel, and immune cells, which plays a central role in the pathophysiology of ARF in humans (32). Furthermore, isolated cell systems are maintained in an environment containing growth factors and cytokines that do not reflect the local environment of epithelial cells in vivo in ARF. Early larval zebrafish kidneys are notable for their anatomic simplicity, consisting of a fused midline glomerulus with one nephron on each side, adjacent to the cardinal veins, while maintaining biological complexity inherent to the kidney of higher organisms. Zebrafish are translucent, facilitating microscopic observation along the entire length of the kidney. Microinjection of mRNA, DNA, and morpholinos (modified oligonucleotides with excellent antisense properties) is well tolerated. Morphological and functional changes can be correlated with protein expression after either knockdown (morpholinos) or overexpression (mRNA) (5). Their...
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.Omi/HtrA2 is a mitochondrial serine protease that is released to the cytoplasm upon induction of apoptosis (1-4). In the cytoplasm, Omi binds and cleaves IAPs 1 leading to activation of caspase-dependent apoptosis (5, 6). Omi can also induce caspase-independent apoptosis through an as yet unknown mechanism that requires its proteolytic activity (7,8). In addition to its pro-apoptotic function, Omi has another unique role in maintaining mitochondrial homeostasis, but the details of this mechanism are still unclear (9). The serine protease activity of Omi is necessary and essential for its normal function whether it acts as a pro-apoptotic protein in the cytoplasm or as a potential chaperone in the mitochondria (9). The proteolytic activity of Omi has been associated with autoprocessing to form the mature protein as well as cleavage of IAPs to activate caspase-dependent apoptosis (5,6,8). To understand the mechanism of Omi's function, it will be necessary to identify new substrates for this protease. These substrates might be mitochondrial or cytoplasmic proteins, and their degradation and removal by Omi could be part of the apoptotic process. In this report, we used the yeast two-hybrid system to isolate and characterize new Omi-interacting proteins. One of these interactors isolated from this screen was the HS1-associated protein X-1 (HAX-1) anti-apoptotic protein (10). HAX-1 interacted with Omi both in vitro and in vivo. Furthermore, HAX-1 was degraded and removed by Omi when cells were treated with various apoptotic stimuli. Using a specific inhibitor of the pro...
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.