Mitochondria-associated Degradation Research Articles

Phospholipase A2-activating protein induces mitophagy through anti-apoptotic MCL1-mediated NLRX1 oligomerization

Mitochondrial protein homeostasis is fine-tuned by diverse physiological processes such as mitochondria-associated degradation (MAD), which is regulated by valosin-containing protein (VCP) and its cofactors. As a cofactor of VCP, the mutation of phospholipase A-2-activating protein (PLAA) is the genetic cause of PLAA-associated neurodevelopmental disorder (PLAAND). However, the physiological and pathological roles of PLAA in mitochondria remain unclear. Here, we demonstrate that PLAA partially associates with mitochondria. Deficiency in PLAA increases mitochondrial reactive oxygen species (ROS) production, reduces mitochondrial membrane potential, inhibits mitochondrial respiratory activity and causes excessive mitophagy. Mechanically, PLAA interacts with myeloid cell leukemia-1 (MCL1) and facilitates its retro-translocation and proteasome-dependent degradation. The upregulation of MCL1 promotes the oligomerization of NLR family member X1 (NLRX1) and activation of mitophagy. Whereas downregulating NLRX1 abolishes MCL1 induced mitophagy. In summary, our data identify PLAA as a novel mediator of mitophagy by regulating MCL1-NLRX1 axis. We propose mitophagy as a target for therapeutic intervention in PLAAND.

Molecular Determinants of Mitochondrial Shape and Function and Their Role in Glaucoma.

Significance: Cells depend on well-functioning mitochondria for essential processes such as energy production, redox signaling, coordination of metabolic pathways, and cofactor biosynthesis. Mitochondrial dysfunction, metabolic decline, and protein stress have been implicated in the etiology of multiple late-onset diseases, including various ataxias, diabetes, sarcopenia, neuromuscular disorders, and neurodegenerative diseases such as parkinsonism, amyotrophic lateral sclerosis, and glaucoma. Recent Advances: New evidence supports that increased energy metabolism protects neuron function during aging. Key energy metabolic enzymes, however, are susceptible to oxidative damage making it imperative that the mitochondrial proteome is protected. More than 40 different enzymes have been identified as important factors for guarding mitochondrial health and maintaining a dynamic pool of mitochondria. Critical Issues: Understanding shared mechanisms of age-related disorders of neurodegenerative diseases such as glaucoma, Alzheimer's disease, and Parkinson's disease is important for developing new therapies. Functional mitochondrial shape and dynamics rely on complex interactions between mitochondrial proteases and membrane proteins. Identifying the sequence of molecular events that lead to mitochondrial dysfunction and metabolic stress is a major challenge. Future Directions: A critical need exists for new strategies that reduce mitochondrial protein stress and promote mitochondrial dynamics in age-related neurological disorders. Discovering how mitochondria-associated degradation is related to proteostatic mechanisms in mitochondrial compartments may reveal new opportunities for therapeutic interventions. Also, little is known about how protein and membrane contacts in the inner and outer mitochondrial membrane are regulated, even though they are pivotal for mitochondrial architecture. Future work will need to delineate the molecular details of these processes.

UBXD8 mediates mitochondria-associated degradation to restrain apoptosis and mitophagy.

The hexameric AAA-ATPase valosin-containing protein (VCP) is essential for mitochondrial protein quality control. How VCP is recruited to mammalian mitochondria remains obscure. Here we report that UBXD8, an ER- and lipid droplet-localized VCP adaptor, also localizes to mitochondria and locally recruits VCP. UBXD8 associates with mitochondrial and ER ubiquitin E3 ligases and targets their substrates for degradation. Remarkably, both mitochondria- and ER-localized UBXD8 can degrade mitochondrial and ER substrates in cis and in trans. UBXD8 also associates with the TOM complex but is dispensable for translocation-associated degradation. UBXD8 knockout impairs the degradation of the pro-survival protein Mcl1 but surprisingly sensitizes cells to apoptosis and mitochondrial stresses. UBXD8 knockout also hyperactivates mitophagy. We identify pro-apoptotic BH3-only proteins Noxa, Bik, and Bnip3 as novel UBXD8 substrates and determine that UBXD8 inhibits apoptosis via degrading Noxa and restrains mitophagy via degrading Bnip3. Collectively, our characterizations reveal UBXD8 as the major mitochondrial adaptor of VCP and unveil its role in apoptosis and mitophagy regulation.

ER-SURF: Riding the Endoplasmic Reticulum Surface to Mitochondria.

Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1) and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation (ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the current knowledge about the cooperation of the ER and mitochondria in the targeting and quality control of mitochondrial precursor proteins.

Quality control of the mitochondrion.

Mitochondria are essential organelles that execute and coordinate various metabolic processes in the cell. Mitochondrial dysfunction severely affects cell fitness and contributes to disease. Proper organellar function depends on the biogenesis and maintenance of mitochondria and its >1,000 proteins. As a result, the cell has evolved mechanisms to coordinate protein and organellar quality control, such as the turnover of proteins via mitochondria-associated degradation, the ubiquitin-proteasome system, and mitoproteases, as well as the elimination of mitochondria through mitophagy. Specific quality control mechanisms are engaged depending upon the nature and severity of mitochondrial dysfunction, which can also feed back to elicit transcriptional or proteomic remodeling by the cell. Here, we will discuss the current understanding of how these different quality control mechanisms are integrated and overlap to maintain protein and organellar quality and how they may be relevant for cellular and organismal health.

Mitochondrial redox-driven mitofusin 2 S-glutathionylation promotes neuronal necroptosis via disrupting ER-mitochondria crosstalk in cadmium-induced neurotoxicity

Reactive oxygen species (ROS)-mediated endoplasmic reticulum (ER) stress and mitochondrial dysfunction are known to affect the structural and functional damage in the neural system. Cadmium (Cd) is an environmental contaminant that is widely found in numerous environmental matrices and exhibits potential neurotoxic risk. However, it remains unclear how mitochondrial redox status induces, and whether Cd destabilizes, the ER-mitochondria crosstalk to have a toxic effect on the nervous system. Herein, in our present study, bioinformatics analysis revealed an important role of protein interaction and mitochondrial machinery in brain samples from Alzheimer’s disease (AD) patients. Furthermore, we established a neurotoxicity model in vivo and in vitro induced by cadmium chloride (CdCl2). We demonstrated that CdCl2 exposure disrupts the balance in mitochondrial redox represented by enhanced mitochondrial ROS (mitoROS) levels, which enhance mitofusin 2 (Mfn2) S-glutathionylation and interrupt the mitochondria-associated ER membranes (MAMs) for crosstalk between the ER and mitochondria to induce neuronal necroptosis. Mechanistically, it was shown that CdCl2 exposure significantly enhances the mitochondria-associated degradation (MAD) of Mfn2 via S-glutathionylation, which inhibits Mfn2 localization to the MAMs and subsequently leads to the formation of the RIPK1-RIPK3-p-MLKL complex (a key component of the necrosome) at MAMs, to promote neuronal necroptosis. Furthermore, the glutaredoxin 1 (Grx1) catalyzed and Mfn2 overexpression restored S-glu-Mfn2, MAMs perturbation, necrosome formation, and necroptosis in neurons induced by CdCl2 exposure in vitro. Moreover, the intervention with antioxidants to reduce mitochondrial redox, such as N-acetyl-l-cysteine (NAC) and mitochondria-targeted antioxidant Mito-TEMPO, reduced the S-glutathionylation of Mfn2 involved in the antagonism of CdCl2-induced necroptosis and neurotoxicity in vivo and in vitro. Taken together, our results are the first time to demonstrate that S-glutathionylation of Mfn2 promotes neuronal necroptosis via disruption of ER-mitochondria crosstalk in CdCl2-induced neurotoxicity, providing the novel mechanistic insight into how hazardous chemical-induced adverse effects in various organs and tissues could be interpreted by intraorganellar pathways under the control of MAMs components in neurons.

Mitochondria-Associated Degradation Pathway (MAD) Function beyond the Outer Membrane

SUMMARYThe mitochondria-associated degradation pathway (MAD) mediates ubiquitination and degradation of mitochondrial outer membrane (MOM) proteins by the proteasome. We find that the MAD, but not other quality-control pathways including macroautophagy, mitophagy, or mitochondrial chaperones and proteases, is critical for yeast cellular fitness under conditions of paraquat (PQ)-induced oxidative stress in mitochondria. Specifically, inhibition of the MAD increases PQ-induced defects in growth and mitochondrial quality and decreases chronological lifespan. We use mass spectrometry analysis to identify possible MAD substrates as mitochondrial proteins that exhibit increased ubiquitination in response to PQ treatment and inhibition of the MAD. We identify candidate substrates in the mitochondrial matrix and inner membrane and confirm that two matrix proteins are MAD substrates. Our studies reveal a broader function for the MAD in mitochondrial protein surveillance beyond the MOM and a major role for the MAD in cellular and mitochondrial fitness in response to chronic, low-level oxidative stress in mitochondria.

A protein quality control pathway at the mitochondrial outer membrane.

Maintaining the essential functions of mitochondria requires mechanisms to recognize and remove misfolded proteins. However, quality control (QC) pathways for misfolded mitochondrial proteins remain poorly defined. Here, we establish temperature-sensitive (ts-) peripheral mitochondrial outer membrane (MOM) proteins as novel model QC substrates in Saccharomyces cerevisiae. The ts- proteins sen2-1HAts and sam35-2HAts are degraded from the MOM by the ubiquitin-proteasome system. Ubiquitination of sen2-1HAts is mediated by the ubiquitin ligase (E3) Ubr1, while sam35-2HAts is ubiquitinated primarily by San1. Mitochondria-associated degradation (MAD) of both substrates requires the SSA family of Hsp70s and the Hsp40 Sis1, providing the first evidence for chaperone involvement in MAD. In addition to a role for the Cdc48-Npl4-Ufd1 AAA-ATPase complex, Doa1 and a mitochondrial pool of the transmembrane Cdc48 adaptor, Ubx2, are implicated in their degradation. This study reveals a unique QC pathway comprised of a combination of cytosolic and mitochondrial factors that distinguish it from other cellular QC pathways.

Mitochondria Associated Degradation Pathway (MAD) Function Beyond the Outer Membrane

The mitochondria-associated degradation (MAD) pathway mediates ubiquitination and removal of mitochondrial outer membrane (MOM) proteins, and targets them for degradation by the proteasome. We find that MAD, but not other mitochondrial control pathways including macroautophagy, mitophagy, or mitochondrial chaperones and proteases, is critical for yeast cellular fitness under conditions of paraquat (PQ) induced chronic, low-level oxidative stress in mitochondria. Specifically, inhibition of MAD results in increased PQ-induced defects in growth and mitochondrial quality, and reduced chronological lifespan. In light of this, we tested whether MAD function in mitochondrial proteostasis extends beyond the known substrates in MOM. Our mass spectrometry analysis revealed possible MAD substrates in different mitochondrial compartments. We confirmed that 2 matrix proteins, Kgd1p and Pim1p, are MAD substrates. Our studies reveal a broader function for MAD in mitochondrial protein surveillance beyond the MOM, and a major role in cellular and mitochondrial fitness in response to chronic, low level oxidative stress in mitochondria.

IRhom2 is essential for innate immunity to RNA virus by antagonizing ER- and mitochondria-associated degradation of VISA.

VISA (also known as MAVS, IPS-1 and Cardif) is an essential adaptor protein in innate immune response to RNA virus. The protein level of VISA is delicately regulated before and after viral infection to ensure the optimal activation and timely termination of innate antiviral response. It has been reported that several E3 ubiquitin ligases can mediate the degradation of VISA, but how the stability of VISA is maintained before and after viral infection remains enigmatic. In this study, we found that the ER-associated inactive rhomboid protein 2 (iRhom2) plays an essential role in mounting an efficient innate immune response to RNA virus by maintaining the stability of VISA through distinct mechanisms. In un-infected and early infected cells, iRhom2 mediates auto-ubiquitination and degradation of the E3 ubiquitin ligase RNF5 and impairs the assembly of VISA-RNF5-GP78 complexes, thereby antagonizes ER-associated degradation (ERAD) of VISA. In the late phase of viral infection, iRhom2 mediates proteasome-dependent degradation of the E3 ubiquitin ligase MARCH5 and impairs mitochondria-associated degradation (MAD) of VISA. Maintenance of VISA stability by iRhom2 ensures efficient innate antiviral response at the early phase of viral infection and ready for next round of response. Our findings suggest that iRhom2 acts as a checkpoint for the ERAD/MAD of VISA, which ensures proper innate immune response to RNA virus.

With the Help of MOM: Mitochondrial Contributions to Cellular Quality Control.

Mitochondria are essential organelles because they have key roles in cellular energy metabolism and many other metabolic pathways. Several quality control systems have evolved to ensure that dysfunctional mitochondria are either repaired or eliminated. The activities of these pathways are crucial for cellular health because they maintain functional mitochondria. In addition, the cytosolic ubiquitin-proteasome system (UPS) and the mitochondria-associated degradation pathway (MAD) share some of their core components, are functionally tightly interconnected, and mutually modulate their activities. Thus, the mitochondrial outer membrane (MOM) actively supports quality control systems in extramitochondrial compartments. Furthermore, mitochondrial quality surveillance systems also act on cytosolic or endoplasmic reticulum (ER) substrates and modulate immune responses. Therefore, mitochondria contribute to cellular quality control and homeostasis on several levels.

Doa1 targets ubiquitinated substrates for mitochondria-associated degradation.

Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion ofDOA1causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48(-Ufd1-Npl4)form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48(-Ufd1-Npl4)complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1-Cdc48(-Ufd1-Npl4)complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.

Doa1 is a MAD adaptor for Cdc48.

Dislocation of polypeptides from the mitochondrial outer membrane by the p97/Cdc48–Ufd1–Npl4 adenosine triphosphatase complex is essential for mitochondria-associated degradation and Parkin-mediated mitophagy. In this issue, Wu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510098) identify Doa1 as a pivotal adaptor that recruits substrates to Cdc48 for processing.

The small GTPase Arf1 modulates mitochondrial morphology and function.

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.

Mitochondrial quality control by the ubiquitin–proteasome system

Mitochondria perform multiple functions critical to the maintenance of cellular homoeostasis and their dysfunction leads to disease. Several lines of evidence suggest the presence of a MAD (mitochondria-associated degradation) pathway that regulates mitochondrial protein quality control. Internal mitochondrial proteins may be retrotranslocated to the OMM (outer mitochondrial membrane), multiple E3 ubiquitin ligases reside at the OMM and inhibition of the proteasome causes accumulation of ubiquitinated proteins at the OMM. Reminiscent of ERAD [ER (endoplasmic reticulum)-associated degradation], Cdc48 (cell division cycle 42)/p97 is recruited to stressed mitochondria, extracts ubiquitinated proteins from the OMM and presents ubiquitinated proteins to the proteasome for degradation. Recent research has provided mechanistic insights into the interaction of the UPS (ubiquitin-proteasome system) with the OMM. In yeast, Vms1 [VCP (valosin-containing protein) (p97)/Cdc48-associated mitochondrial-stress-responsive 1] protein recruits Cdc48/p97 to the OMM. In mammalian systems, the E3 ubiquitin ligase parkin regulates the recruitment of Cdc48/p97 to mitochondria, subsequent mitochondrial protein degradation and mitochondrial autophagy. Disruption of the Vms1 or parkin systems results in the hyper-accumulation of ubiquitinated proteins at mitochondria and subsequent mitochondrial dysfunction. The emerging MAD pathway is important for the maintenance of cellular and therefore organismal viability.