Dissection of DIAP1 Functional Domains via a Mutant Replacement Strategy

Yokokura, Takakazu; Dresnek, Doug; Huseinovic, Neda; Lisi, Simonetta; Abdelwahid, Eltyeb; Bangs, Peter Lawrence; White, Kristin

doi:10.1074/jbc.m409691200

Cited by 42 publications

(64 citation statements)

References 52 publications

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“…Ditzel concludes that DIAP1 can be degraded via two distinct ubiquitin-mediated mechanisms: (i) RING-finger-mediated autoubiquitinating pathway 15 and (ii) caspase-mediated processing followed by rapid degradation of the released fragment via the N-end rule pathway. Yokokura et al 26 have also shown that apoptosis induces caspase-mediated processing of DIAP1, in the same site. However, in contrast to Ditzel et al, 19 they found that the cleavage of DIAP1 promotes its further degradation via autoubiquitination, that requires the RING-finger domain of the protein.…”

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confidence: 92%

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

et al. 2007

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Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates. Recent studies indicate that ubiquitination plays an important role in regulating apoptosis. This is partially mediated by the inhibitors of apoptosis proteins (IAPs), a centrally important group of cell death regulators, many of them are ubiquitin ligases. IAPs suppress cell death by inactivating different proapoptotic factors, some by targeting them for ubiquitination and subsequent proteasomal degradation. At least eight IAPs have been identified in mammals. In Drosophila melanogaster, expression of DIAP1 and DIAP2 1 can suppress apoptosis that occurs both during normal development as well as following overexpression of proapoptotic factors such as Reaper (Rpr). 2 Loss of DIAP1 function results in early embryonic death as a consequence of massive apoptosis. [3][4][5] The IAP family is characterized structurally by one-three Nterminal copies of Baculovirus IAP Repeat (BIR) domains. DIAP1 contains two copies of BIR that bind effector and initiator caspases. In some cases, this binding leads to ubiquitination and subsequent degradation of the caspase. 2 IAP-mediated inactivation of caspases is effectively inhibited by a family of proapoptotic proteins that share an IAP-binding tetra-peptide motif at their N-termini. Among those proteins are Smac/Diablo in mammals, and Rpr, Hid and Grim (RHG) in Drosophila. It was shown that RHG proteins abrogate effectively DIAP1-mediated inactivation of Dronc and DrICE. 6 DIAP1 contains also a C-terminal RING-finger motif that serves to recruit the E2 component of the ubiquitin conjugation machinery. For RING-finger E3s, their best-characterized activity is self-ubiquitination thought to regulate their cellular level, but it is assumed th...

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confidence: 92%

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

et al. 2007

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“…Specifically, the N terminus of DIAP1 is an intramolecular inhibitor of DIAP1, keeping DIAP1 in an auto-inhibited conformation, unable to bind and inhibit DrICE. 51,[57][58][59] After caspase cleavage at D20 (likely by DrICE), the inhibitory N terminus is removed and now activated DIAP1 can bind to DrICE and inhibit it. Because DIAP1 is a substrate of DrICE before it becomes an inhibitor, the wt/class C heterotetramer would produce less-active DIAP1 and thus would be less-efficiently inhibited.…”

Section: Discussionmentioning

confidence: 99%

Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila

Lindblad

Garnett

et al. 2015

Cell Death Differ

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Caspases are the executioners of apoptosis. Although much is known about their physiological roles and structures, detailed analyses of missense mutations of caspases are lacking. As mutations within caspases are identified in various human diseases, the study of caspase mutants will help to elucidate how caspases interact with other components of the apoptosis pathway and how they may contribute to disease. DrICE is the major effector caspase in Drosophila required for developmental and stressinduced cell death. Here, we report the isolation and characterization of six de novo drICE mutants, all of which carry point mutations affecting amino acids conserved among caspases in various species. These six mutants behave as recessive loss-offunction mutants in a homozygous condition. Surprisingly, however, two of the newly isolated drICE alleles are gain-of-function mutants in a heterozygous condition, although they are loss-of-function mutants homozygously. Interestingly, they only behave as gain-of-function mutants in the presence of an apoptotic signal. These two alleles carry missense mutations affecting conserved amino acids in close proximity to the catalytic cysteine residue. This is the first time that viable gain-of-function alleles of caspases are described in any intact organism and provides a significant exception to the expectation that mutations of conserved amino acids always abolish the pro-apoptotic activity of caspases. We discuss models about how these mutations cause the gain-offunction character of these alleles. Cell Death and Differentiation (2016) 23, 723-732; doi:10.1038/cdd.2015.144; published online 6 November 2015Apoptosis is a major form of programmed cell death. 1 The core apoptotic machinery is evolutionary conserved with caspases as the fundamental components. [1][2][3] Caspases are specific cysteine proteases that are produced as inactive zymogens composed of an N-terminal pro-domain, a large subunit region with the catalytic cysteine residue, and a small subunit region at the carboxyl end. 2,4 Depending on their structures and functions, caspases are grouped into initiator and effector caspases. 2,3 Initiator caspases possess long pro-domains, which facilitate the recruitment of initiator caspases into cell death signaling complexes for activation. 2,3,5 Effector caspases are activated by initiator caspase complexes through proteolytic processing, cleaving off the pro-domain and separating the large and small subunits. The active effector caspase is a tetramer composed of two large and two small subunits and contains two catalytic sites. 2,3 Activated effector caspases cleave many protein targets to trigger the physiological and morphological changes characteristic of apoptosis. In mammals, apoptotic initiator caspases are Caspase-8, -9, -2 and -10, and effector caspases involved in apoptosis are 3 Of the seven Drosophila caspases, only the initiator caspase Dronc (Caspase-9-like) and the effector caspases DrICE and Dcp-1 (Caspase-3-like) have been implicated in apoptosis in imagina...

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“…Although DIAP1 has been shown as a substrate for N-end rule-mediated degradation and could interact with isolated UBR domains of multiple UBR family members, no single UBR protein has ever been identified in the regulation of DIAP1. 20,21 We for the first time characterized Ubr3, a highly conserved UBR protein, as an antiapoptotic factor, which controls the function rather than the stability of DIAP1. Interestingly, the role of Ubr3 in apoptosis pathway cannot be replaced by other UBR proteins such as Ubr1.…”

Section: Discussionmentioning

confidence: 99%

“…N-DIAP1 (-V5) is a truncated DIAP1, from which the first 20 N-terminal amino acids are removed. In M-DIAP1 (-V5), the N 21 was replaced by M 21 .…”

Section: Methodsmentioning

confidence: 99%

“…Ditzel et al 20 first discovered that DIAP1 can be cleaved by effector caspases at its NH2 terminus, exposing a binding motif for UBR-box-containing E3 ligases and subsequently be degraded by the N-end rule-mediated degradation. Later, Yokokura et al 21 reported that the N-end rule pathway does not have a major role in the turnover of N-terminally truncated DIAP1. More recently, Ditzel et al 22 reported that the UBRbinding motif of DIAP1 is essential for its anti-apoptotic function, indicating UBR family proteins regulate apoptosis via ways other than destroying DIAP1.…”

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Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

et al. 2014

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Apoptosis has essential roles in a variety of cellular and developmental processes. Although the pathway is well studied, how the activities of individual components in the pathway are regulated is less understood. In Drosophila, a key component in apoptosis is Drosophila inhibitor of apoptosis protein 1 (DIAP1), which is required to prevent caspase activation. Here, we demonstrate that Drosophila CG42593 (ubr3), encoding the homolog of mammalian UBR3, has an essential role in regulating the apoptosis pathway. We show that loss of ubr3 activity causes caspase-dependent apoptosis in Drosophila eye and wing discs. Our genetic epistasis analyses show that the apoptosis induced by loss of ubr3 can be suppressed by loss of initiator caspase Drosophila Nedd2-like caspase (Dronc), or by ectopic expression of the apoptosis inhibitor p35, but cannot be rescued by overexpression of DIAP1. Importantly, we show that the activity of Ubr3 in the apoptosis pathway is not dependent on its Ring-domain, which is required for its E3 ligase activity. Furthermore, we find that through the UBR-box domain, Ubr3 physically interacts with the neoepitope of DIAP1 that is exposed after caspase-mediated cleavage. This interaction promotes the recruitment and ubiquitination of substrate caspases by DIAP1. Together, our data indicate that Ubr3 interacts with DIAP1 and positively regulates DIAP1 activity, possibly by maintaining its active conformation in the apoptosis pathway. Cell Death and Differentiation (2014) 21, 1961-1970 doi:10.1038/cdd.2014 published online 22 August 2014 Morphogenesis in multicellular organisms is a process with a balanced control of cell proliferation and cell death. To maintain this homeostasis, superfluous or unwanted cells are usually removed promptly via programmed cell death or apoptosis. 1,2 Compelling evidence has shown that dysregulation of apoptosis results in a variety of diseases, such as cancer, neurodegenerative disorders and autoimmune diseases. [3][4][5][6] The apoptotic machinery is conserved from invertebrates to vertebrates. Drosophila has been used as an excellent model to study apoptosis because of its advantages in genetic manipulation. A crucial step in apoptosis is the cascade activation of initiator and effector caspases that eventually causes cell death. Under normal circumstances, the activities of caspases are kept in check by a conserved family of anti-apoptotic proteins termed inhibitor of apoptosis proteins (IAPs). The Drosophila genome encodes four IAPs, including Drosophila inhibitor of apoptosis protein 1 (DIAP1), DIAP2, DBruce and Deterin. 7-10 Among these four proteins, DIAP1 is stringently required to prevent caspase activation. 11,12 Although the requirement of DIAP1 in the apoptosis pathway is well documented, it is unclear how the activity of DIAP1 is regulated during development.The covalent attachment of ubiquitin to proteins is a crucial regulatory mechanism in many developmental and physiological processes. 13 Ubiquitination is a catalytic cascade involving ubiquitin-ac...

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Dissection of DIAP1 Functional Domains via a Mutant Replacement Strategy

Cited by 42 publications

References 52 publications

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

Genetic characterization of two gain-of-function alleles of the effector caspase DrICE in Drosophila

Ubr3 E3 ligase regulates apoptosis by controlling the activity of DIAP1 in Drosophila

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