Mitochondrial E3 ubiquitin ligase MARCH5 controls mitochondrial fission and cell sensitivity to stress-induced apoptosis through regulation of MiD49 protein

Ubiquitin- and proteasome-dependent outer mitochondrial membrane (OMM)-associated degradation (OMMAD) is critical for mitochondrial and cellular homeostasis. However, the scope and molecular mechanisms of the OMMAD pathways are still not well understood. We report that the OMM-associated E3 ubiquitin ligase MARCH5 controls dynamin-related protein 1 (Drp1)-dependent mitochondrial fission and cell sensitivity to stress-induced apoptosis. MARCH5 knockout selectively inhibited ubiquitination and proteasomal degradation of MiD49, a mitochondrial receptor of Drp1, and consequently led to mitochondrial fragmentation. Mitochondrial fragmentation in MARCH5^−/− cells was not associated with inhibition of mitochondrial fusion or bioenergetic defects, supporting the possibility that MARCH5 is a negative regulator of mitochondrial fission. Both MARCH5 re-expression and MiD49 knockout in MARCH5^−/− cells reversed mitochondrial fragmentation and reduced sensitivity to stress-induced apoptosis. These findings and data showing MARCH5-dependent degradation of MiD49 upon stress support the possibility that MARCH5 regulation of MiD49 is a novel mechanism controlling mitochondrial fission and, consequently, the cellular response to stress.     

MCL1 meets its end during mitotic arrest

Evidence for the destruction of the anti-apoptotic protein MCL1 during prolonged mitotic arrest comes from three papers, one in The EMBO Journal and two in Nature, thus shedding light on the mechanism of apoptosis induction under these conditions.EMBO Rep (2011) advance online publication. doi:10.1038/nature09732EMBO Rep (2011) advance online publication. doi:10.1038/nature09779EMBO Rep (2011) advance online publication. doi:10.1038/emboj.2010.112During mitosis, eukaryotic cells have to properly align their chromosomes. Only after the kinetochore of each chromosome is attached to a polar microtubule can a cell satisfy the ‘spindle assembly checkpoint'', which prevents the mis-segregation of chromosomes. Failure to correctly segregate chromosomes before cell division might contribute to chromosome instability and tumorigenesis. To counteract chromosome aberrations, the cell initiates the apoptotic programme. It has become clear through the use of microtubule-poisoning, chemotherapeutic agents—such as paclitaxel and vincristine—that prolonged activation of the spindle checkpoint can induce mitotic arrest and, subsequently, programmed cell death. The molecular mechanisms responsible for initiating apoptosis during mitotic arrest have remained poorly defined. Two recent papers in Nature (Inuzuka et al, 2011; Wertz et al, 2011) and a report published by the Clarke group last year in The EMBO Journal (Harley et al, 2010) highlight the destruction of MCL1 during prolonged mitotic arrest and shed light on the mechanisms of apoptosis induction.Myeloid cell leukaemia 1 (MCL1) is an anti-apoptotic member of the B-cell lymphoma 2 (BCL2) family of proteins. MCL1, like BCL2 and BCLxL, prevents the downstream activation of BAX and BAK, which are responsible for mitochondrial outer-membrane permeabilization, initiation of the caspase cascade and induction of apoptosis (Youle & Strasser, 2008). Ubiquitination and proteolysis of MCL1 have been reported, but a mechanism for MCL1 degradation following spindle checkpoint activation remains unknown. Now, the studies referenced above suggest that degradation of MCL1 during prolonged mitotic arrest is essential for the induction of apoptosis. Given its prominent role in driving the cell cycle, as well as in safeguarding the fidelity of this process, it is not surprising that the ubiquitin-proteasome system (UPS) has a key role in dictating the activation of the intrinsic apoptotic pathway in cells arrested in mitosis. However, it is surprising that two E3 ubiquitin ligase complexes simultaneously facilitate this degradation event.Harley and colleagues describe the regulation of MCL1 by APC/C^Cdc20(anaphase-promoting complex/cyclosome and its activator Cdc20). This multi-subunit RING E3 ubiquitin ligase is active in mitosis, and ubiquitinates substrates such as securin and cyclin B, thereby allowing progression into anaphase. In their report, Harley and co-workers (2010) demonstrate a Cdk1/cyclin-B-mediated, site-specific phosphorylation (Thr 92 in humans) of MCL1 upon mitotic arrest, followed by its proteolytic destruction by APC/C^Cdc20. Thus, like the sand of an hourglass flipped at each entry into mitosis, the level of MCL1 steadily decreases. If time ‘runs out'' due to a prolonged mitotic arrest (that is, if MCL1 is completely destroyed), then apoptosis is initiated (Fig 1A). Both phosphorylation at Thr 92 and the presence of a conserved destruction or ‘D''-box motif (a characteristic of APC/C substrates) are required for MCL1 proteolysis, although the precise role of phosphorylation in promoting degradation remains unclear.Open in a separate windowFigure 1Two models for proteolytic destruction of MCL1 during mitotic arrest. (A) Schematic illustration of the effects of APC/C^Cdc20 and Cdk1/cyclin B on the degradation of MCL1 during prolonged arrest in mitosis. (B) Schematic illustration of the effects of SCFFbw7, JNK/p38/CKII and Cdk1/cyclin B on MCL1 degradation during mitotic arrest. PP2A is a protein phosphatase that is reported to associate with MCL1. Dashed lines represent inactive processes. Question marks denote unknown mechanisms. APC/C^Cdc20, anaphase-promoting complex/cyclosome and its activator Cdc20; SAC, spindle assembly checkpoint.Interestingly, the stability of MCL1 in asynchronous cells seems to be unaffected when the ability of APC/C^Cdc20 to target MCL1 is compromised by knockdown of Cdc20, or when phosphorylation at Thr 92 is ablated. Although the spindle assembly checkpoint is believed to inhibit APC/C^Cdc20 activity, the degradation of some targets, such as the CDK-inhibitor p21 and cyclin A, is not affected. Consequently, it is possible that MCL1 can be destroyed through Cdc20 during mitotic arrest.More recently, in two reports in Nature (Inuzuka et al, 2011; Wertz et al, 2011), it is shown that MCL1 interacts with another E3 ubiquitin ligase, SCF^Fbxw7. Similarly to the APC/C, the SCF (Skp1/Cul1/F-box protein) is a multi-subunit, RING E3 ubiquitin ligase. The F-box protein provides the specificity for target recognition, often by using specific interaction domains to bind to substrates. In the case of Fbxw7 (also known as Fbw7 and hCdc4), a series of WD40 domains form a pocket that dictates the binding of several substrates. For all known substrates, one or two phosphorylated degradation motifs (phospho-degrons) are recognized by Fbxw7 (Welcker & Clurman, 2008), and MCL1 seems to follow this trend. Briefly, two Fbxw7 degrons—Ser 121/Glu 125 and Ser 159/Thr 163—with different binding affinities were identified in MCL1. Inuzuka and colleagues report that these sites are phosphorylated in a GSK3-dependent manner, supporting a previous report that demonstrated a role for GSK3 in controlling MCL1 degradation (Maurer et al, 2006). They also demonstrate that Fbxw7 affects MCL1 stability during the DNA damage response. Wertz and co-workers provide evidence that, during mitotic arrest, the degrons in MCL1 are instead phosphorylated by JNK, p38 and CKII. Interestingly, when Wertz and colleagues investigated the degradation of MCL1 during mitotic arrest, they discovered a dependence on Fbxw7 similar to that reported for Cdc20 (Fig 1B). Furthermore, a functional Fbxw7–MCL1 interaction was required for the induction of apoptosis in ovarian cancer and T-ALL cell lines treated with microtubule-targeting chemotherapies. This observation presents a dilemma. Which ubiquitin ligase complex—APC/C^Cdc20 or SCF^Fbxw7—targets MCL1 for destruction during mitotic arrest? Do they compete or cooperate?There are several approaches that could be taken to investigate these questions. Perhaps the most promising direction is through understanding the role of various MCL1 phosphorylation events, particularly phosphorylation of Thr 92. The reports collectively demonstrate that Thr 92 and the Fbxw7 degrons are phosphorylated in mitotic cells. It is interesting that Thr 92 phosphorylation is specifically induced at mitosis, and Wertz and colleagues suggest that this event might drive the dissociation of a phosphatase to allow Fbxw7 degron phosphorylation (Fig 1B). However, the results so far are preliminary, and a more complete understanding of the mechanism by which Cdk1/cyclin B phosphorylation of MCL1 promotes proteolysis, and whether this is through Cdc20 and/or Fbxw7, is essential. Although MCL1 degradation after mitotic arrest is unlikely to be associated with the activity of GSK3, is there an induction of GSK3-dependent phosphorylation of MCL1 under other conditions? This important question has been studied previously, but it requires further investigation. Perhaps additional ‘priming'' kinases are involved, as is suspected to be the case for cyclin E and c-Myc, two other substrates of Fbxw7.The concept of a protein being targeted by two ubiquitin ligases is not new. For example, similarly to MCL1, p21 and MLL are targeted by both APC/C^Cdc20 and an SCF complex (SCF^Skp2). Several APC/C^Cdh1 substrates (for example, Cdc25A and claspin) are also degraded via SCFβ^TrCP (Frescas & Pagano, 2008). However, in these instances, APC/C and SCF target the substrates at different phases of the cell cycle. The case of MCL1 is less clear. Wertz and colleagues show that mitotic arrest specifically induces binding of MCL1 to Fbxw7. Conversely, Inuzuka and colleagues provide data suggesting that Fbxw7 loss affects the non-mitotic stability of MCL1. Additionally, in an earlier paper, the Fbxw7 degron was reported to be phosphorylated by GSK3 during cytokine withdrawal (Maurer et al, 2006). Thus, we are left with a picture in which Fbxw7 targets MCL1 during mitotic arrest, but it might also target MCL1 at other points during the cell cycle or in response to external stimuli. With regard to Cdc20-mediated degradation of MCL1, mutation of the D-box seems to stabilize MCL1 only during mitotic arrest, although Cdc20 remains bound to MCL1 in non-mitotic cells. Thus, there might be differences in the conditions for recognition by either Fbxw7 or Cdc20 that merit further investigation. It is also worth mentioning that deubiquitinating enzymes (DUBs) might counteract the activity of Fbxw7, Cdc20, or both. In fact, the DUB USP9X was found to associate with MCL1 (Schwickart et al, 2010).Assuming that both E3 ligases target the same pool of MCL1 at the same time during mitotic arrest, why are there two modes of regulation? It could be that the ligases cooperate to lower MCL1 levels. It is possible that Fbxw7 and Cdc20 together deplete MCL1 to a point at which apoptosis can be initiated; if either ligase is compromised, apoptotic induction is inefficient. Alternatively, there might be a particularly relevant growth condition or cell-type specificity that favours the activity of one complex over the other. For example, it is possible that in tissues that give rise to human cancers harbouring Fbxw7 mutations (for example, T-ALLs or ovarian carcinomas), SCF^Fbxw7 acts as the predominant ligase. Finally, there could be redundancy or competition between the different E3 ligases. Perhaps untransformed cells maintain both systems, to protect against apoptosis evasion in the face of spindle dysfunction. Alternatively, one or both of these systems might be compromised in the cell-culture models. Notably, the situation is further complicated by reports indicating that other ligases seem to affect MCL1 stability: Mule/Huwe1 (Zhong et al, 2005) and SCF^βTrCP (Ding et al, 2007). Silencing of Mule stabilizes MCL1, although Wertz and colleagues did not observe dramatic changes in MCL1 stability after Mule depletion during mitotic arrest. Instead, three groups did not observe stabilization of MCL1 after βTrCP silencing (Wertz et al, 2011; Inuzuka et al, 2011; Dehan et al, 2009). Moreover, the interaction between MCL1 and βTrCP seems to be mediated by BimEL (a βTrCP substrate), as indicated by increased binding under conditions when BimEL is degraded (rather than under conditions when MCL1 is degraded) and by the fact that some BimEL mutants lose their ability to bind to βTrCP, regardless of their binding to MCL1 (Dehan et al, 2009).Although the details of MCL1 regulation at mitotic arrest have only begun to unfold, it is clear that this pathway holds promise for furthering our understanding of the regulation of apoptosis. Microtubule-poisoning agents have historically been reliable chemotherapeutics, so, identifying cellular components that regulate MCL1 degradation during mitotic arrest is not only a way to stratify patients for a positive response to such drugs, but might also lead to the identification of novel targets for pharmacological intervention.     

Mitochondrial E3 ligase MARCH5 regulates FUNDC1 to fine‐tune hypoxic mitophagy

Mitophagy is an essential process for mitochondrial quality control and turnover. It is activated by two distinct pathways, one dependent on ubiquitin and the other dependent on receptors including FUNDC1. It is not clear whether these pathways coordinate to mediate mitophagy in response to stresses, or how mitophagy receptors sense stress signals to activate mitophagy. We find that the mitochondrial E3 ligase MARCH5, but not Parkin, plays a role in regulating hypoxia‐induced mitophagy by ubiquitylating and degrading FUNDC1. MARCH5 directly interacts with FUNDC1 to mediate its ubiquitylation at lysine 119 for subsequent degradation. Degradation of FUNDC1 by MARCH5 expression desensitizes mitochondria to hypoxia‐induced mitophagy, whereas knockdown of endogenous MARCH5 significantly inhibits FUNDC1 degradation and enhances mitochondrial sensitivity toward mitophagy‐inducing stresses. Our findings reveal a feedback regulatory mechanism to control the protein levels of a mitochondrial receptor to fine‐tune mitochondrial quality.     

MARCH5-mediated quality control on acetylated Mfn1 facilitates mitochondrial homeostasis and cell survival

Mitochondrial dynamics and quality control have a central role in the maintenance of cellular integrity. Mitochondrial ubiquitin ligase membrane-associated RING-CH (MARCH5) regulates mitochondrial dynamics. Here, we show that mitochondrial adaptation to stress is driven by MARCH5-dependent quality control on acetylated Mfn1. Under mitochondrial stress conditions, levels of Mfn1 were elevated twofold and depletion of Mfn1 sensitized these cells to apoptotic death. Interestingly, overexpression of Mfn1 also promoted cell death in these cells, indicating that a fine tuning of Mfn1 levels is necessary for cell survival. MARCH5 binds Mfn1 and the MARCH5-dependent Mfn1 ubiquitylation was significantly elevated under mitochondrial stress conditions along with an increase in acetylated Mfn1. The acetylation-deficient K491R mutant of Mfn1 showed weak interaction with MARCH5 as well as reduced ubiquitylation. Neither was observed in the acetylation mimetic K491Q mutant. In addition, MARCH5-knockout mouse embryonic fibroblast and MARCH5^H43W-expressing HeLa cells lacking ubiquitin ligase activity experienced rapid cell death upon mitochondrial stress. Taken together, a fine balance of Mfn1 levels is maintained by MARCH5-mediated quality control on acetylated Mfn1, which is crucial for cell survival under mitochondria stress conditions.     

The BH3-only protein NOXA serves as an independent predictor of breast cancer patient survival and defines susceptibility to microtubule targeting agents

Breast cancer (BC) treatment frequently involves microtubule-targeting agents (MTAs), such as paclitaxel, that arrest cells in mitosis. Sensitivity to MTAs is defined by a subset of pro- and anti-apoptotic BCL2 family proteins controlling mitochondrial apoptosis. Here, we aimed to determine their prognostic value in primary tumour samples from 92 BC patients. Our analysis identified high NOXA/PMAIP mRNA expression levels as an independent prognostic marker for improved relapse-free survival (RFS) and overall survival (OS) in multivariate analysis in BC patients, independent of their molecular subtype. Analysis of available TCGA datasets of 1060 BC patients confirmed our results and added a clear predictive value of NOXA mRNA levels for patients who received MTA-based therapy. In this TCGA cohort, 122 patients received MTA-treatment and high NOXA mRNA levels correlated with their progression-free interval (PFI) and OS. Our follow-up analyses in a panel of BC cell lines of different molecular subtypes identified NOXA protein expression as a key determinant of paclitaxel sensitivity in triple-negative breast cancer (TNBC) cells. Moreover, we noted highest additive effects between paclitaxel and chemical inhibition of BCLX, but not BCL2 or MCL1, documenting dependence of TNBC cells on BCLX for survival and paclitaxel sensitivity defined by NOXA expression levels.Subject terms: Cancer, Translational research     

The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division

We identify a mitochondrial E3 ubiquitin ligase, MARCH5, as a critical regulator of mitochondrial fission. MARCH5 RING mutants and MARCH5 RNA interference induce an abnormal elongation and interconnection of mitochondria indicative of an inhibition of mitochondrial division. The aberrant mitochondrial phenotypes in MARCH5 RING mutant-expressing cells are reversed by ectopic expression of Drp1, but not another mitochondrial fission protein Fis1. Moreover, as indicated by abnormal clustering and mitochondrial accumulation of Drp1, as well as decreased cellular mobility of YFP-Drp1 in cells expressing MARCH5 RING mutants, MARCH5 activity regulates the subcellular trafficking of Drp1, likely by impacting the correct assembly at scission sites or the disassembly step of fission complexes. Loss of this activity may account for the observed mitochondrial division defects. Finally, MARCH5 RING mutants and endogenous Drp1, but not wild-type MARCH5 or Fis1, co-assemble into abnormally enlarged clusters in a Drp1 GTPase-dependent manner, suggesting molecular interactions among these proteins. Collectively, our data suggest a model in which mitochondrial division is regulated by a MARCH5 ubiquitin-dependent switch.     

Mitofusin 1 is degraded at G<Subscript>2</Subscript>/M phase through ubiquitylation by MARCH5

Background

Mitochondria exhibit a dynamic morphology in cells and their biogenesis and function are integrated with the nuclear cell cycle. In mitotic cells, the filamentous network structure of mitochondria takes on a fragmented form. To date, however, whether mitochondrial fusion activity is regulated in mitosis has yet to be elucidated.

Findings

Here, we report that mitochondria were found to be fragmented in G₂ phase prior to mitotic entry. Mitofusin 1 (Mfn1), a mitochondrial fusion protein, interacted with cyclin B1, and their interactions became stronger in G₂/M phase. In addition, MARCH5, a mitochondrial E3 ubiquitin ligase, reduced Mfn1 levels and the MARCH5-mediated Mfn1 ubiquitylation were enhanced in G₂/M phase.

Conclusions

Mfn1 is degraded through the MARCH5-mediated ubiquitylation in G₂/M phase and the cell cycle-dependent degradation of Mfn1 could be facilitated by interaction with cyclin B1/Cdk1 complexes.

     

Genome‐wide siRNA screen reveals coupling between mitotic apoptosis and adaptation

The antimitotic anti‐cancer drugs, including taxol, perturb spindle dynamics, and induce prolonged, spindle checkpoint‐dependent mitotic arrest in cancer cells. These cells then either undergo apoptosis triggered by the intrinsic mitochondrial pathway or exit mitosis without proper cell division in an adaptation pathway. Using a genome‐wide small interfering RNA (siRNA) screen in taxol‐treated HeLa cells, we systematically identify components of the mitotic apoptosis and adaptation pathways. We show that the Mad2 inhibitor p31^comet actively promotes mitotic adaptation through cyclin B1 degradation and has a minor separate function in suppressing apoptosis. Conversely, the pro‐apoptotic Bcl2 family member, Noxa, is a critical initiator of mitotic cell death. Unexpectedly, the upstream components of the mitochondrial apoptosis pathway and the mitochondrial fission protein Drp1 contribute to mitotic adaption. Our results reveal crosstalk between the apoptosis and adaptation pathways during mitotic arrest.     

SUMOylation of MCL1 protein enhances its stability by regulating the ubiquitin-proteasome pathway

In cancers, apoptosis evasion through dysregulation of pro-apoptotic and anti-apoptotic intracellular signals is a recurring event. Accordingly, selective inhibition of specific proteins represents an exciting therapeutic opportunity. Myeloid cell leukemia 1 (MCL1) is an anti-apoptotic protein of the BCL-2 family, which is overexpressed in many cancers. Here, we demonstrate that MCL1 can be modified by the small ubiquitin-like modifier (SUMO) at K234 and K238 sites. The SUMOylation of MCL1 can improve its stability by inhibiting the MCL1 ubiquitin-proteasome pathway mediated by the Tripartite motif-containing 11 (TRIM11, a novel MCL1 ubiquitin E3 ligase that we identify in this study). Moreover, SUMOylation of MCL1 increases the proliferation of cancer cells by inhibiting apoptosis. These results suggest that the SUMOylation of MCL1 may play a significant role in the regulation of its function.     

Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model

Purpose

To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods

RGC5 cells transfected with expression constructs for MARCH5, MARCH5^H43W, Dpr1^K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results

Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5^H43W or inactive Drp^K38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5^H43W or Drp1^K38A did significantly delay cell death.

Conclusion

Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.     

Dual targeting of RIG-I and MAVS by MARCH5 mitochondria ubiquitin ligase in innate immunity

The mitochondrial antiviral signaling (MAVS) protein on the mitochondrial outer membrane acts as a central signaling molecule in the RIG-I-like receptor (RLR) signaling pathway by linking upstream viral RNA recognition to downstream signal activation. We previously reported that mitochondrial E3 ubiquitin ligase, MARCH5, degrades the MAVS protein aggregate and prevents persistent downstream signaling. Since the activated RIG-I oligomer interacts and nucleates the MAVS aggregate, MARCH5 might also target this oligomer. Here, we report that MARCH5 targets and degrades RIG-I, but not its inactive phosphomimetic form (RIG-I^S8E). The MARCH5-mediated reduction of RIG-I is restored in the presence of MG132, a proteasome inhibitor. Upon poly(I:C) stimulation, RIG-I forms an oligomer and co-expression of MARCH5 reduces the expression of this oligomer. The RING domain of MARCH5 is necessary for binding to the CARD domain of RIG-I. In an in vivo ubiquitination assay, MARCH5 transfers the Lys 48-linked polyubiquitin to Lys 193 and 203 residues of RIG-I. Thus, dual targeting of active RIG-I and MAVS protein oligomers by MARCH5 is an efficient way to switch-off RLR signaling. We propose that modulation of MARCH5 activity might be beneficial for the treatment of chronic immune diseases.     

NOXA, a sensor of proteasome integrity, is degraded by 26S proteasomes by an ubiquitin-independent pathway that is blocked by MCL-1

Ubiquitin (Ub)-mediated proteasome-dependent proteolysis is critical in regulating multiple biological processes including apoptosis. We show that the unstructured BH3-only protein, NOXA, is degraded by an Ub-independent mechanism requiring 19S regulatory particle (RP) subunits of the 26S proteasome, highlighting the possibility that other unstructured proteins reported to be degraded by 20S proteasomes in vitro may be bona fide 26S proteasome substrates in vivo. A lysine-less NOXA (NOXA-LL) mutant, which is not ubiquitinated, is degraded at a similar rate to wild-type NOXA. Myeloid cell leukemia 1, but not other anti-apoptotic BCL-2 family proteins, stabilizes NOXA by interaction with the NOXA BH3 domain. Depletion of 19S RP subunits, but not alternate proteasome activator REG subunits, increases NOXA half-life in vivo. A NOXA-LL mutant, which is not ubiquitinated, also requires an intact 26S proteasome for degradation. Depletion of the 19S non-ATPase subunit, PSMD1 induces NOXA-dependent apoptosis. Thus, disruption of 26S proteasome function by various mechanisms triggers the rapid accumulation of NOXA and subsequent cell death strongly implicating NOXA as a sensor of 26S proteasome integrity.     

The putative BH3 mimetic S1 sensitizes leukemia to ABT-737 by increasing reactive oxygen species, inducing endoplasmic reticulum stress, and upregulating the BH3-only protein NOXA

S1 is a putative BH3 mimetic proposed to inhibit BCL2 and MCL1 based on cell-free assays. However, we previously demonstrated that it failed to inhibit BCL2 or induce apoptosis in chronic lymphocytic leukemia (CLL) cells, which are dependent on BCL2 for survival. In contrast, we show here that S1 rapidly increases reactive oxygen species, initiates endoplasmic reticulum stress, and upregulates the BH3-only protein NOXA. The BCL2 inhibitors, ABT-737, ABT-263, and ABT-199, have demonstrated pro-apoptotic efficacy in cell lines, while ABT-263 and ABT-199 have demonstrated efficacy in early clinical trials. Resistance to these inhibitors arises from the upregulation of anti-apoptotic factors, such as MCL1, BFL1, and BCLXL. This resistance can be induced by co-culturing CLL cells on a stromal cell line that mimics the microenvironment found in patients. Since NOXA can inhibit MCL1, BFL1, and BCLXL, we hypothesized that S1 may overcome resistance to ABT-737. Here we demonstrate that S1 induces NOXA-dependent sensitization to ABT-737 in a human promyelocytic leukemia cell line (NB4). Furthermore, S1 sensitized CLL cells to ABT-737 ex vivo, and overcame resistance to ABT-737 induced by co-culturing CLL cells with stroma.     

MARCH5-FUNDC1 axis fine-tunes hypoxia-induced mitophagy

Mitophagy is responsible for removal of damaged mitochondria and is therefore a fundamental process in mitochondrial quality control. Both ubiquitin-dependent and receptor-dependent pathways are considered to mediate mitophagy. These distinct mechanisms may be activated in response to distinct mitochondrial stresses. An intriguing question is whether and how crosstalk occurs between the distinct pathways to coordinate mitophagy. We have uncovered a striking piece of evidence to demonstrate that the mitophagy receptor FUNDC1 is a substrate of MARCH5, a mitochondrially localized E3 ubiquitin ligase. In response to hypoxia, MARCH5 degrades redundant FUNDC1 to fine-tune hypoxia-induced mitophagy, whereas ablation of MARCH5 leads to accumulation of FUNDC1 and an exaggerated mitophagic phenotype. Mechanistic studies demonstrate that hypoxic insult enhances the interaction of FUNDC1 with MARCH5, which ubiquitinates FUNDC1 at lysine 119 for subsequent degradation. MARCH5-based ubiquitination and degradation of FUNDC1 circumvents injudicious removal of cellular mitochondria. However, severe hypoxic stress leads to dephosphorylation of FUNDC1, increasing mitophagic flux.