Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1

Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)–dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.     

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER–mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.     

Loss of OMA1 delays neurodegeneration by preventing stress-induced OPA1 processing in mitochondria

Proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 in mitochondria is emerging as a central regulatory hub that determines mitochondrial morphology under stress and in disease. Stress-induced OPA1 processing by OMA1 triggersmitochondrial fragmentation, which is associated with mitophagy and apoptosis in vitro. Here, we identify OMA1 as a critical regulator of neuronal survival in vivo and demonstrate that stress-induced OPA1 processing by OMA1 promotes neuronal death and neuroinflammatory responses. Using mice lacking prohibitin membrane scaffolds as a model of neurodegeneration, we demonstrate that additional ablation of Oma1 delays neuronal loss and prolongs lifespan. This is accompanied by the accumulation of fusion-active, long OPA1 forms, which stabilize the mitochondrial genome but do not preserve mitochondrial cristae or respiratory chain supercomplex assembly in prohibitin-depleted neurons. Thus, long OPA1 forms can promote neuronal survival independently of cristae shape, whereas stress-induced OMA1 activation and OPA1 cleavage limit mitochondrial fusion and promote neuronal death.     

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis.

Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses.

Results: When challenged with hydrogen peroxide (H₂O₂), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H₂O₂ challenge.

Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling.     

Regulation of mitochondrial morphology through proteolytic cleavage of OPA1

The dynamin-like GTPase OPA1, a causal gene product of human dominant optic atrophy, functions in mitochondrial fusion and inner membrane remodeling. It has several splice variants and even a single variant is found as several processed forms, although their functional significance is unknown. In yeast, mitochondrial rhomboid protease regulates mitochondrial function and morphology through proteolytic cleavage of Mgm1, the yeast homolog of OPA1. We demonstrate that OPA1 variants are synthesized with a bipartite-type mitochondrial targeting sequence. During import, the matrix-targeting signal is removed and processed forms (L-isoforms) are anchored to the inner membrane in type I topology. L-isoforms undergo further processing in the matrix to produce S-isoforms. Knockdown of OPA1 induced mitochondrial fragmentation, whose network morphology was recovered by expression of L-isoform but not S-isoform, indicating that only L-isoform is fusion-competent. Dissipation of membrane potential, expression of m-AAA protease paraplegin, or induction of apoptosis stimulated this processing along with the mitochondrial fragmentation. Thus, mammalian mitochondrial function and morphology is regulated through processing of OPA1 in a DeltaPsi-dependent manner.     

Membrane depolarization activates the mitochondrial protease OMA1 by stimulating self‐cleavage

Mitochondrial inner membrane fusion depends on the dynamin‐related GTPase OPA1 and the function of OPA1 is regulated by proteolytic cleavage. The mitochondrial proteases Yme1L and OMA1 cleave OPA1 at S2 and S1 sites, respectively. Here, we show that OMA1 is cleaved to a short form (S‐OMA1) by itself upon mitochondrial membrane depolarization; S‐OMA1 is degraded quickly but could be stabilized by CCCP treatment or Prohibitin knockdown in cells. In addition, OMA1 processing is positively correlated with OPA1 cleavage at the S1 site and the regulation of mitochondrial morphology. Thus, our results reveal the molecular mechanism for OMA1 activation toward OPA1 processing.     

Inducible vectors for expression in mammalian cells.

The most recent developments in mammalian cell inducible expression systems have involved the use of bacterial gene control elements and viral transactivator proteins. The combination of hybrid viral transactivator and bacterial repressor proteins, and simple chemical inducers can provide induction ratios of over 1000-fold. These developments will have applications in both cell-based research and the generation of transgenic animals.     

Afferent neurotransmission mediated by hemichannels in mammalian taste cells

In mammalian taste buds, ionotropic P2X receptors operate in gustatory nerve endings to mediate afferent inputs. Thus, ATP secretion represents a key aspect of taste transduction. Here, we characterized individual vallate taste cells electrophysiologically and assayed their secretion of ATP with a biosensor. Among electrophysiologically distinguishable taste cells, a population was found that released ATP in a manner that was Ca(2+) independent but voltage-dependent. Data from physiological and pharmacological experiments suggested that ATP was released from taste cells via specific channels, likely to be connexin or pannexin hemichannels. A small fraction of ATP-secreting taste cells responded to bitter compounds, indicating that they express taste receptors, their G-protein-coupled and downstream transduction elements. Single cell RT-PCR revealed that ATP-secreting taste cells expressed gustducin, TRPM5, PLCbeta2, multiple connexins and pannexin 1. Altogether, our data indicate that tastant-responsive taste cells release the neurotransmitter ATP via a non-exocytotic mechanism dependent upon the generation of an action potential.     

Inducible model for beta-six-mediated site-specific recombination in mammalian cells

The prokaryotic beta recombinase catalyzes site-specific recombination between two directly oriented minimal six sites in chromatin-integrated substrates. Here, we demonstrate that an enhanced green fluorescent protein (EGFP)-fused version of beta recombinase (beta-EGFP) is fully active, retaining most specific activity. It is used to develop a recombination-dependent activatable gene expression (RAGE) system based on the androgen receptor (AR) ligand-binding domain (LBD). Two hybrid molecules, a direct fusion of the LBD-AR to the C-terminus of beta recombinase (beta-AR) and a triple fusion of beta-EGFP to the same ligand-binding domain (beta-EGFP-AR), were engineered and their subcellular behavior, stability and catalytic activity were evaluated. Both chimeric beta recombinase proteins showed in vivo inducible recombinogenic activity dependent on addition of an androgen receptor agonist, although the beta-AR fusion protein demonstrated more accurate ligand-dependent translocation from cytoplasm to nucleus.     

Yeast KEX1 protease cleaves a prohormone processing intermediate in mammalian cells

A vaccinia virus vector was used to express the yeast KEX1 gene, which encodes a prohormone carboxypeptidase specific for the removal of basic amino acids from prohormone processing intermediates, in mammalian cells. When produced in BSC-40 cells, Kex1p was localized to the perinuclear region and conferred a large increase in enzymatic activity characteristic of this carboxypeptidase. Expression of the KEX1 gene together with the yeast KEX2 gene, which encodes a prohormone endopeptidase specific for cleavage at pairs of basic amino acids, and the mouse proopiomelanocortin (mPOMC) cDNA in BSC-40 cells resulted in the full conversion of mPOMC to mature peptides including gamma-lipotropin. This in vivo processing of mPOMC to mature peptides by the KEX2/KEX1 gene products demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.     

T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.     

Alkaline stress-induced autophagy is mediated by mTORC1 inactivation

The activation of autophagic pathway by alkaline stress was investigated. Various types of mammalian cells were subjected to alkaline stress by incubation in bicarbonate buffered media in humidified air containing atmospheric 0.04% CO(2) . The induction of autophagy following alkaline stress was evaluated by assessing the conversion of cytosolic LC3-I into lipidated LC3-II, the accumulation of autophagosomes, and the formation of autolysosomes. Colocalization of GFP-LC3 with endolysosomal marker in HeLa GFP-LC3 cells undergoing autophagic process by alkaline stress further demonstrates that autophagosomes triggered by alkaline stress matures into autolysosomes for the lysosome dependent degradation. We found that the inactivation of mTORC1 is important for the pathway leading to the induction of autophagy by alkaline stress since the expression of RhebQ64L, a constitutive activator of mTORC1, downregulates the induction of autophagy after alkaline stress in transfected human 293T cells. These results imply that activation of autophagic pathway following the inactivation of mTORC1 is important cellular events governing alkaline stress-induced cytotoxicity and clinical symptoms associated with alkalosis.     

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.