bims-cytox1 2022-06-05 papers

Issue of 2022–06–05
three papers selected by
Gavin McStay, Staffordshire University

STAR Protoc. 2022 Jun 17. 3(2): 101359

Incorporation of reporter genes into mitochondrial DNA in budding yeast.

Magdalena Rzepka, Tamara Suhm, Martin Ott.

Many aspects of mitochondrial gene expression are still unknown, which can be attributed to limitations in molecular tools. Here, we present a protocol to introduce reporter genes into the mitochondrial genome of budding yeast, Saccharomyces cerevisiae. Mitochondrially encoded reporter constructs can be used to interrogate various aspects of mitochondrial gene expression. The power of this technique is exemplified by a mitochondrially encoded nanoluciferase, which allows to monitor levels of mitochondrial translation under a variety of growth conditions.

Keywords: Cell Biology; Genetics; Model Organisms; Molecular Biology

DOI: https://doi.org/10.1016/j.xpro.2022.101359

Front Neurol. 2022 ;13 873943

Compound Heterozygous COX20 Variants Impair the Function of Mitochondrial Complex IV to Cause a Syndrome Involving Ophthalmoplegia and Visual Failure.

Peizheng Li, Dandan Guo, Xiufang Zhang, Kunqian Ji, Hongbo Lv, Yanli Zhang, Zhichao Chen, Jun Ma, Yaofeng Fang, Yiming Liu.

The cytochrome c oxidase 20 (COX20) gene encodes a protein with a crucial role in the assembly of mitochondrial complex IV (CIV). Mutations in this gene can result in ataxia and muscle hypotonia. However, ophthalmoplegia and visual failure associated with COX20 mutation have not been examined previously. Moreover, the mechanism causing the phenotype of patients with COX20 variants to differ from that of patients with mutations in other genes impairing CIV assembly is unclear. In this investigation, the aim was to assess the relation between COX20 variants and CIV assembly. We performed detailed clinical, physical, and biochemical investigations of affected individuals. Western blotting, reverse transcription-polymerase chain reaction, and blue native-polyacrylamide gel electrophoresis were used to analyze the expression level of COX20 and oxidative phosphorylation. A Seahorse XF Cell Mito Stress Test and enzymatic activity analysis were performed to evaluate mitochondrial function. Whole-exome sequencing revealed the same compound heterozygous mutations (c.41A > G and c.222G > T, NM_198076) in COX20 in two siblings. This is the first description of ophthalmoplegia and visual failure associated with COX20 variants. In vitro analysis confirmed that the COX20 protein level was significantly decreased, impairing the assembly and activity of CIV in patients' fibroblast. Overexpression of COX20 using a transduced adenovirus partially restored the function of the patients' fibroblasts. Early-onset complex movement disorders may be closely related to COX20 variants. Our results broaden the clinical phenotypes of patients with COX20 variants showing ophthalmoplegia and visual failure. Additionally, dysfunction of COX20 protein can impair the assembly and activity of CIV.

Keywords: ataxia; cytochrome c oxidase 20 (COX20); mitochondrial dysfunction; neuropathy; whole-exome sequencings

DOI: https://doi.org/10.3389/fneur.2022.873943

Antimicrob Agents Chemother. 2022 Jun 01. e0015122

Deletion of cox7c Results in Pan-Azole Resistance in Aspergillus fumigatus.

Mingcong Chen, Guowei Zhong, Sha Wang, Peiying Chen, Lei Li.

In Aspergillus fumigatus, the most prevalent resistance to azoles results from mutational modifications of the azole target protein Cyp51A, but there are non-cyp51A mutants resistant to azoles, and the mechanisms underlying the resistance of these strains remain to be explored. Here, we identified a novel cytochrome c oxidase, cox7c (W56*), nonsense mutation in the laboratory and found that it caused reduced colony growth and resistance to multiantifungal agents. Meanwhile, we revealed that cold storage is responsible for increased tolerance of conidia to itraconazole (ITC) stress, which further advances azole-resistant mutations (cryopreservation→ITC tolerance→azole resistance). The deletion or mutation of cox7c results explicitly in resistance to antifungal-targeting enzymes, including triazoles, polyenes, and allylamines, required for ergosterol synthesis, or resistance to fungal ergosterol. A high-performance liquid chromatography (HPLC) assay showed that the cox7c knockout strain decreased intracellular itraconazole concentration. In addition, the lack of Cox7c resulted in the accumulation of intracellular heme B. We validated that an endogenous increase in, or the exogenous addition of, heme B was capable of eliciting azole resistance, which was in good accordance with the phenotypic resistance analysis of cox7c mutants. Furthermore, RNA sequencing verified the elevated transcriptional expression levels of multidrug transport genes. Additionally, lower itraconazole-induced reactive oxygen species generation in mycelia of a cox7c-deletion strain suggested that this reduction may, in part, contribute to drug resistance. These findings increase our understanding of how A. fumigatus's direct responses to azoles promote fungal survival in the environment and address genetic mutations that arise from patients or environments.

Keywords: Aspergillus fumigatus; antifungal resistance; cytochrome c oxidase; heme; reactive oxygen species

DOI: https://doi.org/10.1128/aac.00151-22