Mitochondrial Respiratory Complex Assembly Research Articles

A FRET-based respirasome assembly screen identifies spleen tyrosine kinase as a target to improve muscle mitochondrial respiration and exercise performance in mice

Aerobic muscle activities predominantly depend on fuel energy supply by mitochondrial respiration, thus, mitochondrial activity enhancement may become a therapeutic intervention for muscle disturbances. The assembly of mitochondrial respiratory complexes into higher-order “supercomplex” structures has been proposed to be an efficient biological process for energy synthesis, although there is controversy in its physiological relevance. We here established Förster resonance energy transfer (FRET) phenomenon-based live imaging of mitochondrial respiratory complexes I and IV interactions using murine myoblastic cells, whose signals represent in vivo supercomplex assembly of complexes I, III, and IV, or respirasomes. The live FRET signals were well correlated with supercomplex assembly observed by blue native polyacrylamide gel electrophoresis (BN-PAGE) and oxygen consumption rates. FRET-based live cell screen defined that the inhibition of spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase that belongs to the SYK/ zeta-chain-associated protein kinase 70 (ZAP-70) family, leads to an increase in supercomplex assembly in murine myoblastic cells. In parallel, SYK inhibition enhanced mitochondrial respiration in the cells. Notably, SYK inhibitor administration enhances exercise performance in mice. Overall, this study proves the feasibility of FRET-based respirasome assembly assay, which recapitulates in vivo mitochondrial respiration activities.

Beyond a platform protein for the degradosome assembly: The Apoptosis-Inducing Factor as an efficient nuclease involved in chromatinolysis.

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA-degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

Ccn6 Is Required for Mitochondrial Integrity and Skeletal Muscle Function in Zebrafish.

Mutations in the CCN6 (WISP3) gene are linked with a debilitating musculoskeletal disorder, termed progressive pseudorheumatoid dysplasia (PPRD). Yet, the functional significance of CCN6 in the musculoskeletal system remains unclear. Using zebrafish as a model organism, we demonstrated that zebrafish Ccn6 is present partly as a component of mitochondrial respiratory complexes in the skeletal muscle of zebrafish. Morpholino-mediated depletion of Ccn6 in the skeletal muscle leads to a significant reduction in mitochondrial respiratory complex assembly and activity, which correlates with loss of muscle mitochondrial abundance. These mitochondrial deficiencies are associated with notable architectural and functional anomalies in the zebrafish muscle. Taken together, our results indicate that Ccn6-mediated regulation of mitochondrial respiratory complex assembly/activity and mitochondrial integrity is important for the maintenance of skeletal muscle structure and function in zebrafish. Furthermore, this study suggests that defects related to mitochondrial respiratory complex assembly/activity and integrity could be an underlying cause of muscle weakness and a failed musculoskeletal system in PPRD.

W196 and the β-Hairpin Motif Modulate the Redox Switch of Conformation and the Biomolecular Interaction Network of the Apoptosis-Inducing Factor.

The human apoptosis-inducing factor (hAIF) is a moonlight flavoprotein involved in mitochondrial respiratory complex assembly and caspase-independent programmed cell death. These functions might be modulated by its redox-linked structural transition that enables hAIF to act as a NAD(H/+) redox sensor. Upon reduction with NADH, hAIF undergoes a conformational reorganization in two specific insertions—the flexible regulatory C-loop and the 190-202 β-harpin—promoting protein dimerization and the stabilization of a long-life charge transfer complex (CTC) that modulates its monomer-dimer equilibrium and its protein interaction network in healthy mitochondria. In this regard, here, we investigated the precise function of the β-hairpin in the AIF conformation landscape related to its redox mechanism, by analyzing the role played by W196, a key residue in the interaction of this motif with the regulatory C-loop. Mutations at W196 decrease the compactness and stability of the oxidized hAIF, indicating that the β-hairpin and C-loop coupling contribute to protein stability. Kinetic studies complemented with computational simulations reveal that W196 and the β-hairpin conformation modulate the low efficiency of hAIF as NADH oxidoreductase, contributing to configure its active site in a noncompetent geometry for hydride transfer and to stabilize the CTC state by enhancing the affinity for NAD+. Finally, the β-hairpin motif contributes to define the conformation of AIF's interaction surfaces with its physiological partners. These findings improve our understanding on the molecular basis of hAIF's cellular activities, a crucial aspect for clarifying its associated pathological mechanisms and developing new molecular therapies.

Mitochondrial Supercomplexes: Physiological Organization and Dysregulation in Age-Related Neurodegenerative Disorders.

Several studies suggest that the assembly of mitochondrial respiratory complexes into structures known as supercomplexes (SCs) may increase the efficiency of the electron transport chain, reducing the rate of production of reactive oxygen species. Therefore, the study of the (dis)assembly of SCs may be relevant for the understanding of mitochondrial dysfunction reported in brain aging and major neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Here we briefly reviewed the biogenesis and structural properties of SCs, the impact of mtDNA mutations and mitochondrial dynamics on SCs assembly, the role of lipids on stabilization of SCs and the methodological limitations for the study of SCs. More specifically, we summarized what is known about mitochondrial dysfunction and SCs organization and activity in aging, AD and PD. We focused on the critical variables to take into account when postmortem tissues are used to study the (dis)assembly of SCs. Since few works have been performed to study SCs in AD and PD, the impact of SCs dysfunction on the alteration of brain energetics in these diseases remains poorly understood. The convergence of future progress in the study of SCs structure at high resolution and the refinement of animal models of AD and PD, as well as the use of iPSC-based and somatic cell-derived neurons, will be critical in understanding the biological relevance of the structural remodeling of SCs.

CCN6 regulates mitochondrial respiratory complex assembly and activity.

Cellular communication network factor 6 (CCN6) mutations are linked with Progressive Pseudo Rheumatoid Dysplasia (PPRD) a debilitating musculoskeletal disorder. The function of CCN6 and the mechanism of PPRD pathogenesis remain unclear. Accordingly, we focused on the functional characterization of CCN6 and CCN6 mutants. Using size exclusion chromatography and native polyacrylamide gel electrophoresis we demonstrated that CCN6 is present as a component of the mitochondrial respiratory complex in human chondrocyte lines. By means of siRNA-mediated transfection and electron microscopy we showed that moderate reduction in CCN6 expression decreases the RER- mitochondria inter-membrane distance. Parallel native PAGE, immunoblotting and Complex I activity assays furthermore revealed increase in both mitochondrial distribution of CCN6 and mitochondrial respiratory complex assembly/activity in CCN6 depleted cells. CCN6 mutants resembling those linked with PPRD, which were generated by CRISPR-Cas9 technology displayed low level of expression of mutant CCN6 protein and inhibited respiratory complex assembly/activity. Electron microscopy and MTT assay of the mutants revealed abnormal mitochondria and poor cell viability. Taken together, our results indicate that CCN6 regulates mitochondrial respiratory complex assembly/activity as part of the mitochondrial respiratory complex by controlling the proximity of RER with the mitochondria, and CCN6 mutations disrupt mitochondrial respiratory complex assembly/activity resulting in mitochondrial defects and poor cell viability.

CCN6 REGULATES MITOCHONDRIAL RESPIRATORY COMPLEX ASSEMBLY AND ACTIVITY

BackgroundCCN6 is a multidomain protein of mesenchymal origin. Mutations in CCN6 lead to PPRD (Progressive Pseudo Rheumatoid Dysplasia), which is associated with cartilage loss, muscle wasting and restricted skeletal development. Mechanism of PPRD pathogenesis and its connection with CCN6 is unclear. Previously we documented that CCN6 localizes to mitochondria and regulates ATP production. Here we have investigated the molecular mechanism of this regulation in context of PPRD.Objective To check whether CCN6 regulates mitochondrial respiratory complex assembly and activity. To check if engineering PPRD causing CCN6 mutations alters the function of the mitochondrial respiratory complex.MethodsCCN6 expression was depleted by siRNA transfection in C28/I2 chondrocyte cell line. CCN6 mutants were generated by CRISPR‐Cas9 technology. CCN6 association with respiratory complex was assessed by 2D BN/SDS PAGE & size‐exclusion chromatography. Mitochondrial respiratory complex activity/assembly was assessed spectrophotometrically and by BN‐PAGE in‐gel assay. Transmission electron microscopy (TEM) was used to study mitochondrial morphology.ResultsSize‐exclusion chromatography and 2D BN/SDS PAGE of mitochondria reveals that CCN6 is associated with Complex I/Super complexes of mitochondrial electron transport chain. Partial depletion of CCN6 by siRNA shows close association of RER with mitochondria and alteration of its mitochondrial distribution relative to cytosol, thereby resulting in increased Complex I activity and assembly. CCN6 mutation (truncation in exon 5, similar to PPRD linked mutation), however, results in disrupted complex I activity and assembly. CCN6 mutation also correlates with distorted mitochondria and mitophagy.ConclusionCCN6 is required for the regulation of mitochondrial respiratory complex activity/assembly in chondrocytes, which involves controlled juxtaposition of RER‐mitochondria. Mutations in CCN6 disrupt respiratory complex activity/assembly and cause mitophagy.Support or Funding InformationThis work is supported by a grant from the Department of Science and Technology, Grant No‐SB/SO/BB‐016/2014 and Department of Biotechnology, Grant No‐BT/PR24244/MED/12/764/2017, Ministry of Science and Technology, Government of India. D.K.P. is supported by the Council of Scientific and Industrial Research (CSIR) fellowship, A.S is supported by DBT‐RA programme and A.G is supported by University Grant Commission (UGC), Government of India.CCN6 migrates as a high molecular weight complex with mitochondrial respiratory complex proteins.CCN6 remains as high molecular weight complex in mitochondria along with subunits of mitochondrial electron transport chain including complex I subunit NDUFB8 as shown by 2D BN‐PAGE/SDS‐PAGE (A) and size exclusion chromatography (B) of mitochondrial lysate from C28/I2 cells (chondrocyte line).Figure 1CCN6 regulates mitochondrial respiratory complex assembly and activity.(A) Increased Complex I assembly and activity was documented by BN‐PAGE western blot with NDUFB8 (Complex I subunit) antibody and in‐gel Complex I activity assay in CCN6 siRNA transfected (CCN6 partially depleted) C‐28/I2 cells (CCN6 si) as compared to control cells (Cont.si). (B) Transmission Electron Microscopy (TEM) micrographs showing decreased RER‐Mitochondria distance in CCN6 siRNA transfected cells. (C) BN‐PAGE western blot with NDUFB8 antibody and in‐gel Complex I activity of CRISPR Cas9 mediated PPRD causing CCN6 mutants shows reduced Complex I/Super complex assembly and activity. (D) TEM micrographs shows presence of mitophagy in CCN6 mutant cells.. RER: Rough endoplasmic reticulum. M: MitochondriaFigure 2

Hydroxytyrosol influences exercise-induced mitochondrial respiratory complex assembly into supercomplexes in rats

Hydroxytyrosol (HT) has been demonstrated to improve mitochondrial function, both in sedentary and in exercised animals. Herein, we assessed the effects of two different doses of HT on exercise-induced mitochondrial respiratory complex (C) assembly into supercomplexes (SCs) and the relation of the potential results to OPA1 levels and oxidative stress. Wistar rats were allocated into six groups: sedentary (SED), sedentary consuming 20 mg/kg/d of HT (SED-20), sedentary consuming 300 mg/kg/d of HT (SED-300); exercised (EXE), exercised consuming 20 mg/kg/d of HT (EXE-20) and exercised consuming 300 mg/kg/d of HT (EXE-300). Animals were exercised and/or supplemented for 10 weeks, and assembly of SCs, mitochondrial oxidative status and expression of OPA1 were quantified in the gastrocnemius muscle. Both EXE and EXE-20 animals exhibited increased assembly of CI into SCs, but this effect was absent in EXE-300 animals. Levels of CIII2 assembled into SCs were only increased in EXE-20 animals. Notably EXE-300 animals showed a decreased relative expression of s-OPA1 isoforms. Therefore, HT exerted dose-dependent effects on SC assembly in exercised animals. Although the mechanisms leading to SCs assembly in response to exercise and HT are unclear, it seems that a high HT dose can prevent SCs assembly during exercise by decreasing the expression of the s-OPA1 isoforms.

Mitochondrial stress management: a dynamic journey

Mitochondria undergo continuous challenges in the course of their life, from their generation to their degradation. These challenges include the management of reactive oxygen species, the proper assembly of mitochondrial respiratory complexes and the need to balance potential mutations in the mitochondrial DNA. The detection of damage and the ability to keep it under control is critical to fine-tune mitochondrial function to the organismal energy needs. In this review, we will analyze the multiple mechanisms that safeguard mitochondrial function in light of in crescendo damage. This sequence of events will include initial defense against excessive reactive oxygen species production, compensation mechanisms by the unfolded protein response (UPRmt), mitochondrial dynamics and elimination by mitophagy.

Partial suppression of l-galactono-1,4-lactone dehydrogenase causes significant reduction in leaf water loss through decreasing stomatal aperture size in Arabidopsis

In higher plants, l-galactono-1,4-lactone dehydrogenase (GLDH) participates in the biosynthesis of ascorbic acid (AsA) and the assembly of mitochondrial respiratory complex I. In this work, homozygous Arabidopsis thaliana mutant (gldhRNAi3-11) plants with approximately 40 % of the GLDH activity of wild type (WT) controls were developed by RNA interference, and were found to be viable under standard laboratory conditions. Compared with WT controls, gldhRNAi3-11 plants showed about 20 % decrease in the contents of reduced AsA and total AsA. Like previously recorded for several AsA-deficient mutants (vtc1-1, vtc2-1, vtc3-1 and vtc4-1) grown under long day (LD) conditions, gldhRNAi3-11 plants exhibited an early flowering phenotype in LD environment. Interestingly, relative to WT control, vtc1-1 and vtc4-1, gldhRNAi3-11 had significantly lower leaf water loss rate. Further analysis indicated that reduced stomatal aperture size was likely responsible for the lower water loss displayed by gldhRNAi3-11 leaves. Thus, our work demonstrates that partial suppression of GLDH activity confers significant reduction in leaf water loss through decreasing stomatal aperture size in Arabidopsis. The novel phenotypes displayed by gldhRNAi3-11 plants, the new insights obtained by this work, and their implications on further study of GLDH function in higher plants are discussed.

The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease.

Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phblp and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phblp and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately.