Hip2 interacts with cyclin B1 and promotes its degradation through the ubiquitin proteasome pathway

Hip2, a ubiquitin conjugating enzyme, is involved in the suppression of cell death. The present study revealed that Hip2 regulates the stability of the apoptotic and cell cycle regulator cyclin B1. Hip2 was found to interact with cyclin B1 to promote its degradation through the ubiquitin proteasome pathway. As a result, Hip2 significantly blocked cell death induced by the cyclin B1 protein, suggesting that Hip2 is involved in the regulation of cyclin B1-mediated cell death.

Structured summary

MINT-8045218, MINT-8045231: Hip2 (uniprotkb:P61086) physically interacts (MI:0915) with cyclin-B1 (uniprotkb:P14635) by pull down (MI:0096)     

Identification of DR5 as a critical,NF-κB-regulated mediator of Smac-induced apoptosis

Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-α (TNFα) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNFα-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNFα/TNFR1, as the TNFα-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-κB (NF-κB) activation is critically required for apoptosis, as inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in IκBα-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-κB-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.     

Identification of a small-molecule inhibitor of the interaction between Survivin and Smac/DIABLO

The protein Survivin is selectively overexpressed in a variety of cancers, but not in normal tissues. It has been reported to be involved in cell survival and cell division. However, the molecular mechanisms involved in its function are not clear, although several binding partner proteins have been proposed to date. Here, we report the identification of a novel small molecule Survivin antagonist, which disrupts the Survivin-Smac/DIABLO interaction in cells. In order to identify Survivin-directed antagonists, we developed a high-throughput screening system based on AlphaScreen technology, which allows the identification of small molecules with the ability to inhibit the interaction of Survivin with Smac/DIABLO or INCENP in vitro. We screened chemical libraries, generated in-house, using this system and identified a 5-deazaflavin analog (compound 1) as a hit compound that selectively inhibited the interaction of Survivin with Smac/DIABLO but not INCENP. In cultured cells, compound 1 abrogated the formation of the complex between Survivin and Smac/DIABLO. In addition, this compound was able to sensitize cultured cells to doxorubicin-mediated DNA damage stress and synergistically enhance apoptotic cell death. Thus, the small-molecule inhibitor described here may serve as a proof-of-principle agent for discriminating between the multiple functions of Survivin.     

Changing the Apoptosis Pathway through Evolutionary Protein Design

One obstacle in de novo protein design is the vast sequence space that needs to be searched through to obtain functional proteins. We developed a new method using structural profiles created from evolutionarily related proteins to constrain the simulation search process, with functions specified by atomic-level ligand–protein binding interactions. The approach was applied to redesigning the BIR3 domain of the X-linked inhibitor of apoptosis protein (XIAP), whose primary function is to suppress the cell death by inhibiting caspase-9 activity; however, the function of the wild-type XIAP can be eliminated by the binding of Smac peptides. Isothermal calorimetry and luminescence assay reveal that the designed XIAP domains can bind strongly with the Smac peptides but do not significantly inhibit the caspase-9 proteolytic activity in vitro compared with the wild-type XIAP protein. Detailed mutation assay experiments suggest that the binding specificity in the designs is essentially determined by the interplay of structural profile and physical interactions, which demonstrates the potential to modify apoptosis pathways through computational design.     

Mycobacterium tuberculosis Hip1 Modulates Macrophage Responses through Proteolysis of GroEL2

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.     

Cellular inhibitor of apoptosis 1 and 2 are ubiquitin ligases for the apoptosis inducer Smac/DIABLO

Inhibitors of apoptosis (IAPs) are crucial regulators of programmed cell death. The mechanism by which IAPs prevent apoptosis has previously been attributed to the direct inhibition of caspases. The function of mammalian IAPs is counteracted by cell death inducer second mitochondria-derived activator of caspases (Smac)/DIABLO during apoptosis. Here we show that cIAP1 and cIAP2 are E3 ubiquitin-protein isopeptide ligases (ubiquitin ligases) for Smac. cIAPs stimulate Smac ubiquitination both in vivo and in vitro, leading to Smac degradation. cIAP1 and cIAP2 associate with overlapping but distinct subsets of E2 (ubiquitin carrier protein) ubiquitin-conjugating enzymes. The substrate-dependent E3 activity of cIAPs is mediated by their RING domains and is dependent on the specific interactions between cIAPs and Smac. Similarly, Drosophila IAP1 also possesses ubiquitin ligase activity that mediates the degradation of the Drosophila apoptosis inducers Grim and HID. These results suggest a novel and conserved mechanism by which IAPs block apoptosis through the degradation of death inducers.     

USP11-dependent selective cIAP2 deubiquitylation and stabilization determine sensitivity to Smac mimetics

Given their crucial role in apoptosis suppression, inhibitor of apoptosis proteins (IAPs) have recently become attractive targets for cancer therapy. Here, we report that cellular IAP2 (cIAP2) is specifically stabilized in several cancer cell lines, leading to resistance to Smac mimetics, such as BV6 and birinapant. In particular, our results showed that cIAP2 depletion, but not cIAP1 depletion, sensitized cancer cells to Smac mimetic-induced apoptosis. Ubiquitin-specific protease 11 (USP11) is a deubiquitylase that directly stabilizes cIAP2. USP11 overexpression is frequently found in colorectal cancer and melanoma and is correlated with poor survival. In our study, cancer cell lines expressing high levels of USP11 exhibited strong resistance to Smac mimetic-induced cIAP2 degradation. Furthermore, USP11 downregulation sensitized these cells to apoptosis induced by TRAIL and BV6 and suppressed tumor growth in a xenograft model. Finally, the TNFα/JNK pathway induced USP11 expression and maintained cIAP2 stability, suggesting an alternative TNFα-dependent cell survival pathway. Collectively, our data suggest that USP11-stabilized cIAP2 may serve as a barrier against IAP-targeted clinical approaches.Apoptosis is an inherent cell death program that is crucial for various physiological processes such as development, the immune response, and tumorigenesis.¹ This process is finely tuned by numerous cellular signaling pathways involving hundreds of pro-apoptotic and anti-apoptotic factors.^{2, 3} Inhibitor of apoptosis proteins (IAPs) are a conserved protein family containing the baculoviral IAP repeat (BIR) domain.⁴ There are eight human IAP proteins, including cellular IAP1 (cIAP1/BIRC2), cIAP2/BIRC3, X chromosome-linked IAP (XIAP/BIRC4), and melanoma IAP (ML-IAP/BIRC7).⁵ IAPs such as XIAP can exert their anti-apoptotic function through the BIR domain, which directly interacts with caspases.⁵ In addition, several IAPs contain a RING domain with E3 ubiquitin ligase activities, which are crucial for apoptosis suppression. In particular, the E3 ligase activities of cIAP1/2 are necessary to regulate tumor necrosis factor receptor (TNFR) signaling.⁶ Upon TNFR activation, cIAP1/2 is recruited to TNFR through TNFα receptor-associated factor 2 (TRAF2), leading to K63-linked polyubiquitylation of receptor interacting protein kinase 1 (RIPK1), which is essential for NF-κB-mediated cell survival.⁷ The lack of RIPK1 polyubiquitylation via cIAP1/2 depletion or the presence of CYLD deubiquitylase triggers TNFR complex IIa formation, thereby inducing caspase-8-dependent apoptosis.⁸ In addition, cIAP1/2 prevents the formation of the RIPK1-containing death complex ripoptosome in response to several stimuli including CD95, TNFα-related apoptosis-inducing ligand (TRAIL), genotoxic stress, and Toll-like receptor (TLR) activation.^{9, 10, 11, 12, 13}IAPs are frequently overexpressed in various human cancers, and their expression is associated with chemoresistance and poor clinical outcome.⁶ Therefore, inhibiting IAP function is an attractive strategy to treat cancer through the induction of apoptosis.^{5, 14} Upon apoptotic stimuli, IAPs are inhibited by the second mitochondria-derived activator of caspases (Smac),⁵ and this discovery led to the development of Smac mimetic peptides using the IAP binding motif containing four amino acids (Ala-Val-Pro-Ile). These peptides were shown to sensitize cells to apoptotic stimuli and efficiently suppress tumor growth in a xenograft model.^{15, 16} Subsequently, a number of small-molecule compounds mimicking the Smac mimetic peptide (Smac mimetics) were developed with improved pharmacological properties and IAP-binding affinity. Interestingly, Smac mimetics, such as BV6 and compound A, were found to induce autoubiquitylation and degradation of cIAP1/2.^{17, 18} Furthermore, cIAP1/2 depletion with Smac mimetics activates the non-canonical NF-κB pathway to induce autocrine TNFα production, which is essential for Smac mimetic-induced apoptotic cell death.^{18, 19}Because cIAP1 and cIAP2 show functional redundancy in TNFα-mediated survival, the depletion of both proteins is usually required for effective induction of cell death upon TNFα treatment.^{20, 21} However, there are several reports showing that cIAP2 expression, but not cIAP1 expression, renders cells resistant to Smac mimetic-induced cell death.^{20, 21} For example, cIAP2 upregulation via phosphoinositide 3-kinase (PI3K) upon compound 3 treatment in certain cell lines was shown to facilitate apoptosis evasion.²² In addition, treatment with compounds A and C led to cIAP1 dimerization, without cIAP2 dimerization, resulting in the autoubiquitylation and subsequent degradation of cIAP1. These findings may explain why cIAP1 is degraded more efficiently than cIAP2 upon treatment with Smac mimetics.²³ Alternatively, because cIAP2 degradation requires cIAP1, cIAP2 may become more stable when cIAP1 is depleted using Smac mimetics.²⁴ Direct cIAP deubiquitylation by OTUB1 or USP19 has been suggested to be responsible for cIAP stabilization;^{25, 26} however, these previous studies did not focus on the difference in stabilization between cIAP1 and cIAP2 and only provided general deubiquitylation-dependent mechanisms.^{25, 26}While several studies have supported hypotheses for how cIAP2 survives in the presence of Smac mimetics, numerous independent studies have also shown that cIAP2 can be efficiently degraded by Smac mimetics in various cell lines.^{27, 28, 29, 30, 31, 32} These observations suggest the existence of other factors that specifically regulate cIAP2 stability upon Smac mimetic treatment. In this study, we propose a new mechanism involving USP11-mediated cIAP2 regulation. We found that the differential destabilization of cIAP1 and cIAP2 is dependent on the presence of the cIAP2-specific deubiquitylase USP11. Mechanistically, USP11 can protect cIAP2 from Smac mimetic-mediated degradation, rendering cell lines with high USP11 expression unresponsive to Smac mimetic treatment. However, USP11 downregulation sensitized these cells to TNFα- or TRAIL-induced apoptosis in the presence of Smac mimetic and further suppressed tumor growth in a xenograft model. Corroborating these data, USP11 overexpression was observed in colon cancer and melanoma patients with poor clinical outcome. Finally, the TNFα/c-Jun N-terminal kinase (JNK) pathway induced USP11 expression, which was necessary for cIAP2 protein stabilization and its anti-apoptotic function. Thus, the identification of cIAP2-specific deubiquitylation indicates that more elaborate strategies should be developed for pharmaceutical therapies targeting cIAPs.     

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.     

Caspase activation and apoptosis in response to proteasome inhibitors

Several studies have indicated that proteasome inhibitors (PIs) are promising anticancer agents. We have discovered that PIs have the unique ability to activate effector caspases through a mitochondrial Bcl-2 inhibitable but caspase-9 independent pathway. Stabilization of released Smac induced by blockade of the proteasome could explain the apoptosome-independent cell death induced by PIs. In fact, Smac/DIABLO critically supports this PIs-dependent caspase activation. By using a new assay, we confirm that at a single cell level both Smac and PIs can activate caspases in the absence of the apoptosome. Moreover, we have observed two PIs-induced kinetics of caspase activation, with caspase-9 being still required for the rapid caspase activation in response to mitochondrial depolarization, but dispensable for the slow DEVDase activation. In summary, our data indicate that PIs can activate downstream caspases at least in part through Smac/DIABLO stabilization.     

Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase

Apoptotic cell death and survival is controlled by pro- and antiapoptotic proteins. Because these proteins act on each other, cell fate is dictated by the relative activity of pro- versus antiapoptotic proteins. Here we report that BRUCE, a conserved 528 kDa peripheral membrane protein of the trans-Golgi network, protects cells against apoptosis and functions as an inhibitor of apoptosis (IAP). By using wild-type and mutant forms we show that BRUCE inhibits caspase activity and apoptosis depending on its BIR domain. Upon apoptosis induction, BRUCE is antagonized by three mechanisms: first, through binding to Smac; second, by the protease HtrA2; and third, by caspase-mediated cleavage. In addition to its IAP activity BRUCE has the distinctive property of functioning as a chimeric E2/E3 ubiquitin ligase with Smac being a substrate. Our work suggests that, owing to its two activities and its localization, BRUCE may function as a specialized regulator of cell death pathways.     

A proteomic screen with Drosophila Opa1-like identifies Hsc70-5/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis

Mitochondrial morphology is regulated by conserved proteins involved in fusion and fission processes. The mammalian Optic atrophy 1 (OPA1) that functions in mitochondrial fusion is associated with Optic Atrophy and has been implicated in inner membrane cristae remodeling during cell death. Here, we show Drosophila Optic atrophy 1-like (Opa1-like) influences mitochondrial morphology through interaction with ‘mitochondria-shaping’ proteins like Mitochondrial assembly regulatory factor (Marf) and Drosophila Mitofilin (dMitofilin). To gain an insight into Opa1-like's network, we delineated bonafide interactors like dMitofilin, Marf, Serine protease High temperature requirement protein A2 (HTRA2), Rhomboid-7 (Rho-7) along with novel interactors such as Mortalin ortholog (Hsc70-5) from Drosophila mitochondrial extract. Interestingly, RNAi mediated down-regulation of hsc70-5 in Drosophila wing imaginal disc's peripodial cells resulted in fragmented mitochondria with reduced membrane potential leading to proteolysis of Opa1-like. Increased ecdysone activity induced dysfunctional fragmented mitochondria for clearance through lysosomes, an effect enhanced in hsc70-5 RNAi leading to increased cell death. Over-expression of Opa1-like rescues mitochondrial morphology and cell death in prepupal tissues expressing hsc70-5 RNAi. Taken together, we have identified a novel interaction between Hsc70-5/Mortalin and Opa1-like that influences cellular homeostasis through mitochondrial fusion.     

XIAP impairs Smac release from the mitochondria during apoptosis

X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases 3, 7 and 9, and mitochondrial Smac (second mitochondria-derived activator of caspase) release during apoptosis inhibits the activity of XIAP. In this study we show that cytosolic XIAP also feeds back to mitochondria to impair Smac release. We constructed a fluorescent XIAP-fusion protein by labelling NH₂- and COOH-termini with Cerulean fluorescent protein (C-XIAP-C). Immunoprecipitation confirmed that C-XIAP-C retained the ability to interact with Smac and impaired extrinsically and intrinsically activated apoptosis in response to tumour necrosis factor-related apoptosis-inducing ligand/cycloheximide and staurosporine. In C-XIAP-C-expressing cells, cytochrome c release from mitochondria proceeded normally, whereas Smac release was significantly prolonged and incomplete. In addition, physiological expression of native XIAP prolonged or limited Smac release in HCT-116 colon cancer cells and primary mouse cortical neurons. The Smac-binding capacity of XIAP, but not caspase inhibition, was central for mitochondrial Smac retention, as evidenced in experiments using XIAP mutants that cannot bind to Smac or effector caspases. Similarly, the release of a Smac mutant that cannot bind to XIAP was not impaired by C-XIAP-C expression. Full Smac release could however be provoked by rapid cytosolic C-XIAP-C depletion upon digitonin-induced plasma membrane permeabilization. Our findings suggest that although mitochondria may already contain pores sufficient for cytochrome c release, elevated amounts of XIAP can selectively impair and limit the release of Smac.     

Protection of pancreatic INS-1 ��-cells from glucose- and fructose-induced cell death by inhibiting mitochondrial permeability transition with cyclosporin A or metformin

Hyperglycemia is detrimental to β-cell viability, playing a major role in the progression of β-cell loss in diabetes mellitus. The permeability transition pore (PTP) is a mitochondrial channel involved in cell death. Recent evidence suggests that PTP inhibitors prevent hyperglycemia-induced cell death in human endothelial cells. In this work, we have examined the involvement of PTP opening in INS-1 cell death induced by high levels of glucose or fructose. PTP regulation was studied by measuring the calcium retention capacity in permeabilized INS-1 cells and by confocal microscopy in intact INS-1 cells. Cell death was analyzed by flow cytometry. We first reported that metformin and cyclosporin A (CsA) prevented Ca²⁺-induced PTP opening in permeabilized and intact INS-1 cells. We then showed that incubation of INS-1 cells in the presence of 30 mM glucose or 2.5 mM fructose induced PTP opening and led to cell death. As both metformin and CsA prevented glucose- and fructose- induced PTP opening, and hampered glucose- and fructose- induced cell death, we conclude that PTP opening is involved in high glucose- and high fructose- induced INS-1 cell death. We therefore suggest that preventing PTP opening might be a new approach to preserve β-cell viability.     

Down-modulation of nucleoporin RanBP2/Nup358 impaired chromosomal alignment and induced mitotic catastrophe

Chromosomal missegregation is a common feature of many human tumors. Recent studies have indicated a link between nucleoporin RanBP2/Nup358 and chromosomal segregation during mitosis; however, the molecular details have yet to be fully established. Observed through live cell imaging and flow cytometry, here we show that RNA interference-mediated knockdown of RanBP2 induced G2/M phase arrest, metaphase catastrophe and mitotic cell death. Furthermore, RanBP2 down-modulation disrupted importin/karyopherin β1 as well as the expression and localization of the Ran GTPase activating protein 1. We found that N-terminal of RanBP2 interacted with the N-terminal of importin β1. Moreover, at least a portion of RanBP2 partially localizes at the centrosome during mitosis. Notably, we also found that GTPase Ran is also involved in the regulation of RanBP2–importin β1 interaction. Overall, our results suggest that mitotic arrest and the following cell death were caused by depletion of RanBP2. Our findings point to a crucial role for RanBP2 in proper mitotic progression and faithful chromosomal segregation.     

A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its antitoxin and are found on the chromosomes of many bacteria including pathogens. Here we report how each of five such TA systems in E. coli affect bacterial cell death differently in liquid media and during biofilm formation. Of all these systems, only the TA system mazEF mediated cell death both in liquid media and during biofilm formation. At the other extreme, as our results have revealed here, the TA system dinJ-YafQ is unique in that it is involved only in the death process during biofilm formation. Cell death governed by mazEF and dinJ-YafQ seems to participate in biofilm formation through a novel mechanism.