C-terminal RING Domain Research Articles

940. Neuroprotective Effects of XIAP Gene Therapy in Models of Neurodegenerative Diseases

The pathways and mechanisms leading to cell loss in neurodegerative diseases are still largely unknown. Neuronal cell death is thought to occur via apoptosis and the involvement of several caspases at the late stages of this process is well documented. X-linked inhibitor of apoptosis (XIAP) is a potent inhibitor of caspases 9, 3 and 7 and thus represents an attractive candidate as a potentially therapeutic neuroprotective factor. To test this hypothesis, we examined the effects of XIAP in several in vitro models of Parkinson's and Huntington's disease. Recent reports have documented the pivotal role of the proteasome pathway of protein turnover in the pathogenesis of neurodegenerative disorders and proteasome inhibitors have been employed to model neurodegeneration both in vitro and in vivo. In neuronal cell lines XIAP demonstrated significant protection against apoptosis induced by different proteasome inhibitors. Furthermore, XIAP almost completely prevented cells death induced by overexpression of alpha-synuclein and polyglatamine fusion proteins used to model familial Parkinson's and Huntington's diseases, respectively. To gain insight into the mechanisms of neuroprotective affects of XIAP we performed analysis of deletion and point mutants of this protein. Our experiments revealed that the N-terminal BIR1 domain as well as the C-terminal RING domain are dispensable for anti-apoptotic effects. Deletion or point mutation of the BIR2 domain or the juxtaposed linker region partially abrogated XIAP activity. In contrast, anti-apoptotic effect was lost when a deletion or a point mutation were introduced into the BIR3 domain. Caspases 3 and 7 are believed to be bound by the BIR2 and the linker regions while the caspase 9 is inactivated by the BIR3 domain. Thus, our data suggest that the protective affects of XIAP in these two models of Parkinson's and Huntington's diseases are mostly mediated by the anti-caspase 9 activity. To explore neuroprotective effects of XIAP in vivo we have generated a recombinant AAV (rAAV) vector expressing a stabilized truncated from of XIAP lacking the RING domain (dXIAP). Slowly progressing nigral degeneration triggered by proteasome inhibition is believed to be a close recapitulation of the events that mark sporadic Parkinson's disease in humans. Therefore, for this study we employed a novel PSI rat model of Parkinson's disease which is induced by inhibition of proteasome machinery. While treatment of the animals with PSI resulted in approximately 50% loss of dopaminergic neurons in the substantia nigra on the side injected with a control vector, all the cells were protected on the contralateral side injected with the dXIAP-overexpressing rAAV. Thus, dXIAP delivered by a rAAV provides a long-term protection of nigral neurons against death induced by proteasome inhibition. Furthermore, to our knowledge this is the first demonstration that neuronal loss can be prevented in this novel model of Parkinson's disease. Thus, our studies may provide a foundation for the use of dXIAP for neuroprotection, which can be further exploited for human gene therapy.

Inhibiting apoptosis in mammalian cell culture using the caspase inhibitor XIAP and deletion mutants.

Lower yields and poorer quality of biopharmaceutical products result from cell death in bioreactors. Such cell death may occur from necrosis but is more commonly associated with apoptosis. During the process of programmed cell death or apoptosis, caspases become activated and cause a cascade of events that eventually destroy the cell. XIAP is the most potent caspase inhibitor encoded in the mammalian genome. The effectiveness of XIAP and its deletion mutants was examined in two cell lines commonly utilized in commercial bioreactors: Chinese hamster ovary (CHO) and 293 human embryonic kidney (293 HEK) cells. CHO cells undergo apoptosis as a result of various insults, including Sindbis virus infection and serum deprivation. In this study, we demonstrate that 293 HEK cells undergo apoptosis during Sindbis virus infection and exposure to the toxins, etoposide and cisplatin. Two deletion mutants of XIAP were created; one containing three tandem baculovirus iap repeat (BIR) domains and the other containing only the C-terminal RING domain, lacking the BIRs. Viability studies were performed for cells expressing each mutant and the wild-type protein on transiently transfected cells, as stable pools, or as stable clonal cell populations after induction of apoptosis by serum deprivation, Sindbis virus infection, etoposide, and cisplatin treatment. Expression of the wild-type XIAP inhibited apoptosis significantly; however, the XIAP mutant containing the three BIRs provided equivalent or improved levels of apoptosis inhibition in all cases. Expression of the RING domain offered no protection and was pro-apoptotic in transient expression experiments. With the aid of an N-terminal YFP fusion to each protein, distribution within the cell was visualized, and the wild-type and mutants showed differing intracellular accumulation patterns. While the wild-type XIAP protein accumulated primarily in aggregates in the cytosol, the RING mutant was enriched in the nucleus. In contrast, the deletion mutant containing the three BIRs was distributed evenly throughout the cytosol. Thus, protein engineering of the XIAP protein can be used to alter the intracellular distribution pattern and improve the ability of this caspase inhibitor to protect against apoptosis for two mammalian cell lines.

Neuralized Encodes a Peripheral Membrane Protein Involved in Delta Signaling and Endocytosis

Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. We find that Dl endocytosis and N signaling are greatly stimulated by expression of neuralized ( neur). neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. We show that neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups.

C-IAP1 Is Cleaved by Caspases to Produce a Proapoptotic C-terminal Fragment

Although human c-IAP1 and c-IAP2 have been reported to possess antiapoptotic activity against a variety of stimuli in several mammalian cell types, we observed that full-length c-IAP1 and c-IAP2 failed to protect cells from apoptosis induced by Bax overexpression, tumor necrosis factor alpha treatment or Sindbis virus infection. However, deletion of the C-terminal RING domains of c-IAP1 and c-IAP2 restored antiapoptotic activity, indicating that this region negatively regulates the antiapoptotic function of the N-terminal BIR domain. This finding is consistent with the observation by others that the spacer region and RING domain of c-IAP1 functions as an E3 ligase, promoting autoubiquitination and degradation of c-IAP1. In addition, we found that c-IAP1 is cleaved during apoptosis to 52- and 35-kDa fragments. Both fragments contain the C-terminal end of c-IAP1 including the RING finger. In vitro cleavage of c-IAP1 with apoptotic cell extracts or with purified recombinant caspase-3 produced similar fragments. Furthermore, transfection of cells with the spacer-RING domain alone suppressed the antiapoptotic function of the N-terminal BIR domain of c-IAP1 and induced apoptosis. Optimal death-inducing activity of the spacer-RING required both the spacer region and the zinc-binding RING domain of c-IAP1 but did not require the caspase recruitment domain located within the spacer region. To the contrary, deletion of the caspase recruitment domain increased proapoptotic activity, apparently by stabilizing the C-terminal fragment.

Regulation of X Chromosome-Linked Inhibitor of Apoptosis Protein (XIAP) in Kainic Acid Induced Neuronal Cell Death in the Rat Hippocampus.

INTRODUCTION. Inhibitor of apoptosis protein (IAP) family consists of several antiapoptotic proteins conserved among species. The IAPs have a well-conserved motif of approximately 65 residues, called the baculovirus inhibitory repeat (BIR) (1). Baculovirus and drosophila IAPs have two, but most IAPs contain three BIR domains. Most of the IAPs also have a C-terminal RING domain which consists of conserved amino acids with zinc binding capacity. XIAP is one of the five known human IAPs and it binds directly and inhibits the activity of caspases (2). The BIR2 domain in XIAP is sufficient to mediate this inhibition (3). However little is known about the presence and function of XIAP in the nervous system. METHOD. In this study, we have investigated the expression and distribution of XIAP mRNA and protein in rat brain using RNase protection assay in addition to Western blotting and immunohistochemistry with a specific anti-XIAP antibody. Neuronal cell death upon kainic acid stimulation was investigated using TUNEL method and caspase-3 activation. RESULTS. Two transcripts of XIAP mRNA are present in rat brains, suggesting a heterogeneity of XIAP in the carboxyterminal region. Using a specific anti-XIAP antibody, XIAP protein was shown to be expressed in embryonic and adult brains with slightly higher levels in embryonic and neonatal brains compared to adults. There was a widespread distribution of XIAP immunoreactive cells in adult rat brains, among others in hippocampus, cortex and substantia nigra. Using double-labelling, it was shown that both neuronal and glia cells express XIAP. Kainic acid treatment is known to cause widespread neuronal cell death in hippocampus and amygdala. XIAP protein levels were increased in the hippocampus 12th and 24th after kainic acid. The intensity of XIAP staining was increased particularly in the hippocampal CA3 area. In the same area, many TUNEL positive and active caspase-3 positive cells were found. Caspase-3 is activated 24th after kainic acid in vivo and in vitro in cultured embryonic hippocampal neurons upon kainic acid stimulus. In hippocampus, XIAP does not colocalize with markers for cell death raising the possibility that XIAP is expressed in viable cells but not in dying cells. Using cultured embryonic hippocampal neurons, it was shown that XIAP becomes a target for active caspase-3 and that it is degradated into smaller pieces. DISCUSSION. XIAP mRNA is expressed in developing and adult rat brains, and the number of XIAP positive cells and XIAP protein levels were regulated by kainic acid. The largest change in XIAP immunoreactivity was observed in CA3 area, which was particularly vulnerable in this model. Labelling of cells with the TUNEL method and with antibody for

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control.

Members of the NF-kappaB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappaB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappaB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappaB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IkappaB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappaB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappaB via an IkappaB targeting mechanism. Functional coupling of NF-kappaB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.