Respiratory Chain Complexes and Supercomplexes Organization in Cells with Defective Complex III

Cytochrome b is the only subunit of complex III (CIII) encoded by the mitochondrial DNA. Constituting the central core of the enzyme, the protein is essential for both assembly and catalytic activity of the complex. CIII can associate with complex I (CI) and complex IV to form supercomplexes (SCs). MTCYB mutations can affect CIII only or both CIII and CI, as a consequence of the importance of CIII on the stability of CI. Here, we have investigated the effects of two pathogenic mutations affecting MTCYB: the p.278Y>C missense mutation, causing the substitution of conserved Tyr278 close to the QO site, and the ΔI300-P305 microdeletion, producing the loss of six aminoacids in the sixth transmembrane helix, but leaving the remaining of the MTCYB in frame. We have demonstrated that both MTCYB mutations severely impaired the activity of CIII: the missense mutation produced an oxidative damage of CIII due to increased superoxide production, whereas in cells bearing the ΔI300-P305 microdeletion, CIII was not detected, with consequent derangement also of CI. The detailed analysis of SCs organization revealed in both cases a strong perturbation of the CIII2+IV SC, together with an attempt to preserve the respirasome. These results favor the hypothesis that SCs not only preserve the structure and stability of respiratory complexes, but are essential for attenuating the mitochondrial dysfunction due to pathogenic mutations affecting the respiratory enzymes. Furthermore, the cells bearing ΔI300-P305 deletion showed a marked increase in complex II (CII) redox activity, associated with significant hydrogen peroxide production. It has been suggested that the enhanced CII activity is a compensatory mechanism due to the lacking of CI. Our results instead suggest that it might be a more general phenomenon for cell adaptation to respiratory chain dysfunction, being detected also in CIII-deficient cells where the hydrogen peroxide production is increased

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

Background: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently lim- ited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. Methods: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carry- ing different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamy- cin treatment. Results: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-muta- tion load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate- mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). Conclusions: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF

Colorectal Cancer Stage at Diagnosis Before vs During the COVID-19 Pandemic in Italy

IMPORTANCE Delays in screening programs and the reluctance of patients to seek medical attention because of the outbreak of SARS-CoV-2 could be associated with the risk of more advanced colorectal cancers at diagnosis. OBJECTIVE To evaluate whether the SARS-CoV-2 pandemic was associated with more advanced oncologic stage and change in clinical presentation for patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective, multicenter cohort study included all 17 938 adult patients who underwent surgery for colorectal cancer from March 1, 2020, to December 31, 2021 (pandemic period), and from January 1, 2018, to February 29, 2020 (prepandemic period), in 81 participating centers in Italy, including tertiary centers and community hospitals. Follow-up was 30 days from surgery. EXPOSURES Any type of surgical procedure for colorectal cancer, including explorative surgery, palliative procedures, and atypical or segmental resections. MAIN OUTCOMES AND MEASURES The primary outcome was advanced stage of colorectal cancer at diagnosis. Secondary outcomes were distant metastasis, T4 stage, aggressive biology (defined as cancer with at least 1 of the following characteristics: signet ring cells, mucinous tumor, budding, lymphovascular invasion, perineural invasion, and lymphangitis), stenotic lesion, emergency surgery, and palliative surgery. The independent association between the pandemic period and the outcomes was assessed using multivariate random-effects logistic regression, with hospital as the cluster variable. RESULTS A total of 17 938 patients (10 007 men [55.8%]; mean [SD] age, 70.6 [12.2] years) underwent surgery for colorectal cancer: 7796 (43.5%) during the pandemic period and 10 142 (56.5%) during the prepandemic period. Logistic regression indicated that the pandemic period was significantly associated with an increased rate of advanced-stage colorectal cancer (odds ratio [OR], 1.07; 95%CI, 1.01-1.13; P = .03), aggressive biology (OR, 1.32; 95%CI, 1.15-1.53; P < .001), and stenotic lesions (OR, 1.15; 95%CI, 1.01-1.31; P = .03). CONCLUSIONS AND RELEVANCE This cohort study suggests a significant association between the SARS-CoV-2 pandemic and the risk of a more advanced oncologic stage at diagnosis among patients undergoing surgery for colorectal cancer and might indicate a potential reduction of survival for these patients

Cellular and mitochondrial determination of low molecular mass organic acids by LC-MS/MS

A selective and sensitive method for the determination of low molecular mass organic acids (LMMOAs) in cell and mitochondrial extracts is presented. The analytical method consists in the separation by reversed phase liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS) of the LMMOAs like malic, succinic, formic and citric acids. These acids are among the cellular intermediates of the tricarboxylic acid cycle (TCA), thus their quantitation can provide essential information about the catabolic and anabolic processes occurring in cells under physiological and pathological conditions. The analytical method was fully validated in terms of linearity, detection and quantification limits, recovery and precision. Detection limits (LOD) for malic, succinic and fumaric acids were in the range of 1-10 nM, while 20 nM was obtained for citric acid. Analytical recovery in cell and mitochondrial extracts was found between 88 and 105% (CV% <7.1) and matrix effect was estimated to be less than 108%. The LC-MS/MS method applied to the quantification of TCA cycle metabolites revealed a different distribution of the four acids in cells and mitochondria, and it could be used to monitoring metabolic alterations associated with TCA cycle and oxidative phosphorylation dysfunctions

Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival

Mild phenotypes and proper supercomplex assembly in human cells carrying the homoplasmic m.15557G > A mutation in cytochrome b gene

Respiratory complex III (CIII) is the first enzymatic bottleneck of the mitochondrial respiratory chain both in its native dimeric form and in supercomplexes. The mammalian CIII comprises 11 subunits among which cytochrome b is central in the catalytic core, where oxidation of ubiquinol occurs at the Qo site. The Qo- or PEWY-motif of cytochrome b is the most conserved through species. Importantly, the highly conserved glutamate at position 271 (Glu271) has never been studied in higher eukaryotes so far and its role in the Q-cycle remains debated. Here we showed that the homoplasmic m.15557G\ua0>\ua0A/MT-CYB, which causes the p.Glu271Lys amino acid substitution predicted to dramatically affect CIII, induces a mild mitochondrial dysfunction in human transmitochondrial cybrids. Indeed, we found that the severity of such mutation is mitigated by the proper assembly of CIII into supercomplexes, which may favor an optimal substrate channeling and buffer superoxide production in vitro. This article is protected by copyright. All rights reserved

Unravelling the effects of the mutation m.3571insC/MT-ND1 on respiratory complexes structural organization

Mammalian respiratory complex I (CI) biogenesis requires both nuclear and mitochondria-encoded proteins and is mostly organized in respiratory supercomplexes. Among the CI proteins encoded by the mitochondrial DNA, NADH-ubiquinone oxidoreductase chain 1 (ND1) is a core subunit, evolutionary conserved from bacteria to mammals. Recently, ND1 has been recognized as a pivotal subunit in maintaining the structural and functional interaction among the hydrophilic and hydrophobic CI arms. A critical role of human ND1 both in CI biogenesis and in the dynamic organization of supercomplexes has been depicted, although the proof of concept is still missing and the critical amount of ND1 protein necessary for a proper assembly of both CI and supercomplexes is not defined. By exploiting a unique model in which human ND1 is allotopically re-expressed in cells lacking the endogenous protein, we demonstrated that the lack of this protein induces a stall in the multi-step process of CI biogenesis, as well as the alteration of supramolecular organization of respiratory complexes. We also defined a mutation threshold for the m.3571insC truncative mutation in mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1), below which CI and its supramolecular organization is recovered, strengthening the notion that a certain amount of human ND1 is required for CI and supercomplexes biogenesis