The short variant of optic atrophy 1 (OPA1) improves cell survival under oxidative stress.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane-associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells

Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence.

Mitochondrial dynamics are tightly controlled by fusion and fission, and their dysregulation and excess reactive oxygen species (ROS) contribute to endothelial cell (EC) dysfunction. How redox signals regulate coupling between mitochondrial dynamics and endothelial (dys)function remains unknown. Here, we identify protein disulfide isomerase A1 (PDIA1) as a thiol reductase for the mitochondrial fission protein Drp1. A biotin-labeled Cys-OH trapping probe and rescue experiments reveal that PDIA1 depletion in ECs induces sulfenylation of Drp1 at Cys644, promoting mitochondrial fragmentation and ROS elevation without inducing ER stress, which drives EC senescence. Mechanistically, PDIA1 associates with Drp1 to reduce its redox status and activity. Defective wound healing and angiogenesis in diabetic or PDIA1+/- mice are restored by EC-targeted PDIA1 or the Cys oxidation-defective mutant Drp1. Thus, this study uncovers a molecular link between PDIA1 and Drp1 oxidoreduction, which maintains normal mitochondrial dynamics and limits endothelial senescence with potential translational implications for vascular diseases associated with diabetes or aging.This research was supported by NIH R01HL135584 (to M.U.-F.), NIH R21HL112293 (to M.U.-F.), NIH R01HL133613 (to T.F. and M.U.-F.), NIH R01HL116976 (to T.F. and M.U.-F.), NIH R01HL070187 (to T.F.), NIH R01HL112626 (to J.K.), Department of Veterans Affairs Merit Review Grant 2I01BX001232 (to T.F.), AHA 16GRNT31390032 (to M.U.-F.), AHA 15SDG25700406 (to S.V.), AHA 16POST27790038 (to A.D.), and NIH T32HL07829 (to R.C.). We thank Mr. Kyle Taylor at Keyence Corporation for assisting with taking images using the Keyence microscope; Dr. John O’Bryan at UIC for assisting with the BiFC assays; Dr. Leslie Poole at Wake Forest University for providing DCP-Bio1, as well as Dr. Jody Martin and the Center for Cardiovascular Research-supported Vector Core Facility at UIC for amplifying adenoviruses.S

Control of Mitochondrial Morphology Through Differential Interactions of Mitochondrial Fusion and Fission Proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria

Mitochondrial Membrane Dynamics—Functional Positioning of OPA1

The maintenance of mitochondrial energetics requires the proper regulation of mitochondrial morphology, and vice versa. Mitochondrial dynamins control mitochondrial morphology by mediating fission and fusion. One of them, optic atrophy 1 (OPA1), is the mitochondrial inner membrane remodeling protein. OPA1 has a dual role in maintaining mitochondrial morphology and energetics through mediating inner membrane fusion and maintaining the cristae structure. OPA1 is expressed in multiple variant forms through alternative splicing and post-translational proteolytic cleavage, but the functional differences between these variants have not been completely understood. Recent studies generated new information regarding the role of OPA1 cleavage. In this review, we will first provide a brief overview of mitochondrial membrane dynamics by describing fission and fusion that are mediated by mitochondrial dynamins. The second part describes OPA1-mediated fusion and energetic maintenance, the role of OPA1 cleavage, and a new development in OPA1 function, in which we will provide new insight for what OPA1 does and what proteolytic cleavage of OPA1 is for

Molecular interaction of Mitofusin 2 and its role in mitochondrial fusion

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2008.Mitochondria change their shape dynamically, mainly through fission and fusion. Dynamin-related GTPases have been shown to mediate remodeling of mitochondrial membranes during these processes. Mitochondrial fission in mammals is mediated by the dynamin-like protein DLP1/Drp1 that is recruited to the outer mitochondrial surface through the membrane-anchored protein hFis1. Another GTPase, Mitofusin (Mfn), is anchored at the outer mitochondrial membrane and mediates fusion of the outer membrane. Mammalian cells have two Mfn isoforms, Mfn1 and Mfn2, that share a conserved molecular structure. Either Mfn1 or Mfn2 can functionally replace each other in Mfn-null cells, suggesting their conserved role as fusion proteins as well. This thesis research centers on the mitochondrial fusion protein Mfn2. Specifically, it focuses on studying the effect of the Mfn2-induced mitochondrial shape change on mitochondrial function and the molecular mechanisms of mitochondrial fusion mediated by the Mfn2 protein. We found that overexpression of Mfn2 drastically changes mitochondrial morphology, forming mitochondrial clusters. High-resolution microscopic examination indicated that the mitochondrial cluster consisted of small fragmented mitochondria. Inhibiting mitochondrial fission prevented the cluster formation, supporting the notion that mitochondrial clusters are formed by fission-mediated mitochondrial fragmentation and subsequent aggregation. Mitochondrial clusters displayed a decrease in inner membrane potential and proton pumping activity, suggesting functional compromise of small fragmented mitochondria by Mfn2 overexpression; however, mitochondrial clusters still retained mitochondrial DNA. We found that cells containing clustered mitochondria lost cytochrome c from mitochondria and underwent caspase-mediated apoptosis. These results demonstrate that mitochondrial deformation impairs mitochondrial function, leading to apoptotic cell death and suggest the presence of an intricate form-function relationship of mitochondria. Because intra- and inter-molecular interactions play an important role in the membrane remodeling action of dynamin family proteins, we analyzed domain interactions of the Mfn2 molecule using genetic and biochemical approaches. We found that two hydrophobic heptad-repeat (HR) domains, HR1 and HR2, interact with each other, in addition to the already reported HR2 and HR2 interaction. Interestingly, we discovered that the region of Mfn2-HR1 interacting with HR2 also interacts with the C-terminal coiled-coil domain of the fission protein DLP1 (DLP1-CC). We identified mutations in the Mfn2-HR1 region that selectively disrupt the HR1/HR2 interaction and the Mfn2/DLP1 interaction. Morphological analyses indicated that the Mfn2/DLP1 interaction participates in mitochondrial fusion whereas the association of HR1 and HR2 of Mfn2 is inhibitory in the fusion process. These data suggest that DLP1 functions as a regulatory factor interacting differentially with Mfn2 and hFis1, which provides a novel mechanism for efficient execution of mitochondrial fusion and fission. We also explored the possible mechanisms by which the differential interactions of Mfn2-HR1 with DLP1 and Mfn2-HR2 can be regulated. We found that Mfn2-HR1 and DLP1-CC can be phosphorylated in vitro. In addition, mutations mimicking phosphorylated and dephosphorylated HR1 showed opposite effects on interaction with HR2 and DLP1, suggesting that HR1 phosphorylation plays a role in regulating the differential interactions of Mfn2-HR1. In conclusion, our results indicate that regulated interactions of fission/fusion machineries constitute the controlling mechanisms of mitochondrial shape change, which has a critical role in maintaining the proper function of mitochondria