bims-mitran 2022-07-03 papers

Issue of 2022‒07‒03
six papers selected by
Andreas Kohler

Methods Mol Biol. 2022 ;2497 243-254

In Vivo Analysis of Mitochondrial Protein Synthesis in Saccharomyces cerevisiae Mitochondrial tRNA Mutants.

I describe here a protocol for the analysis of mitochondrial protein synthesis as a useful tool to characterize the mitochondrial defects associated with mutations in mitochondrial tRNA genes. The yeast Saccharomyces cerevisiae mutants, bearing human equivalent pathogenic mutations, were used as a simple model for analysis. The mitochondrial proteins were labeled by L[35S]-methionine incorporation in growing cells, extracted from purified mitochondria, and fractionated by SDS-polyacrylamide gel electrophoresis followed by autoradiography. By this method, it is possible to distinguish different protein synthesis profiles in the analyzed mitochondrial tRNA mutants.

Keywords: Human equivalent mutations; In vivo L[35S]-methionine labeling; Mitochondria; Mitochondrial protein synthesis; Mitochondrial tRNA mutants; Saccharomyces cerevisiae

DOI: https://doi.org/10.1007/978-1-0716-2309-1_15

Nucleic Acids Res. 2022 Jun 29. pii: gkac548. [Epub ahead of print]

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine.

Egor A Syroegin, Elena V Aleksandrova, Yury S Polikanov.

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

DOI: https://doi.org/10.1093/nar/gkac548

Nature. 2022 Jun 29.

Mitochondrial RNA modifications shape metabolic plasticity in metastasis.

Sylvain Delaunay, Gloria Pascual, Bohai Feng, Kevin Klann, Mikaela Behm, Agnes Hotz-Wagenblatt, Karsten Richter, Karim Zaoui, Esther Herpel, Christian Münch, Sabine Dietmann, Jochen Hess, Salvador Aznar Benitah, Michaela Frye.

Aggressive and metastatic cancers show enhanced metabolic plasticity1, but the precise underlying mechanisms of this remain unclear. Here we show how two NOP2/Sun RNA methyltransferase 3 (NSUN3)-dependent RNA modifications-5-methylcytosine (m5C) and its derivative 5-formylcytosine (f5C) (refs.2-4)-drive the translation of mitochondrial mRNA to power metastasis. Translation of mitochondrially encoded subunits of the oxidative phosphorylation complex depends on the formation of m5C at position 34 in mitochondrial tRNAMet. m5C-deficient human oral cancer cells exhibit increased levels of glycolysis and changes in their mitochondrial function that do not affect cell viability or primary tumour growth in vivo; however, metabolic plasticity is severely impaired as mitochondrial m5C-deficient tumours do not metastasize efficiently. We discovered that CD36-dependent non-dividing, metastasis-initiating tumour cells require mitochondrial m5C to activate invasion and dissemination. Moreover, a mitochondria-driven gene signature in patients with head and neck cancer is predictive for metastasis and disease progression. Finally, we confirm that this metabolic switch that allows the metastasis of tumour cells can be pharmacologically targeted through the inhibition of mitochondrial mRNA translation in vivo. Together, our results reveal that site-specific mitochondrial RNA modifications could be therapeutic targets to combat metastasis.

DOI: https://doi.org/10.1038/s41586-022-04898-5

Trends Cell Biol. 2022 Jun 23. pii: S0962-8924(22)00138-6. [Epub ahead of print]

Arming a killer: mitochondrial regulation of CD8+ T cell cytotoxicity.

Miriam Lisci, Gillian M Griffiths.

While once regarded as ATP factories, mitochondria have taken the spotlight as important regulators of cellular homeostasis. The past two decades have witnessed an intensifying interest in the study of mitochondria in cells of the immune system, with many new and unexpected roles for mitochondria emerging. Immune cells offer intriguing insights as mitochondria appear to play different roles at different stages of T cell development, matching the changing functions of the cells. Here we briefly review the multifaceted roles of mitochondria during T cell differentiation, focusing on CD8+ cytotoxic T lymphocytes (CTLs) and we consider how mitochondrial dysfunction can contribute to CTL exhaustion. In addition, we highlight a newly appreciated role for mitochondria as homeostatic regulators of CTL-mediated killing and explore the emerging literature describing mechanisms linking cytosolic and mitochondrial protein synthesis.

Keywords: CD8; CTL; T cell; exhaustion; mitochondria; mitochondrial translation

DOI: https://doi.org/10.1016/j.tcb.2022.05.007

Methods Mol Biol. 2022 ;2497 221-242

Creation of Yeast Models for Evaluating the Pathogenicity of Mutations in the Human Mitochondrial Gene MT-ATP6 and Discovering Therapeutic Molecules.

Tribouillard-Tanvier Déborah, Dautant Alain, Godard François, Panja Chiranjit, di Rago Jean-Paul, Kucharczyk Roza.

Numerous diseases in humans have been associated with mutations of the mitochondrial genome (mtDNA). This genome encodes 13 protein subunits of complexes involved in oxidative phosphorylation (OXPHOS), a process that provides aerobic eukaryotes with the energy-rich adenosine triphosphate molecule (ATP). Mutations of the mtDNA may therefore have dramatic consequences especially in tissues and organs with high energy demand. Evaluating the pathogenicity of these mutations may be difficult because they often affect only a fraction of the numerous copies of the mitochondrial genome (up to several thousands in a single cell), which is referred to as heteroplasmy. Furthermore, due to its exposure to reactive oxygen species (ROS) produced in mitochondria, the mtDNA is prone to mutations, and some may be simply neutral polymorphisms with no detrimental consequences on human health. Another difficulty is the absence of methods for genetically transforming human mitochondria. Face to these complexities, the yeast Saccharomyces cerevisiae provides a convenient model for investigating the consequences of human mtDNA mutations in a defined genetic background. Owing to its good fermentation capacity, it can survive the loss of OXPHOS, its mitochondrial genome can be manipulated, and genetic heterogeneity in its mitochondria is unstable. Taking advantage of these unique attributes, we herein describe a method we have developed for creating yeast models of mitochondrial ATP6 gene mutations detected in patients, to determine how they impact OXPHOS. Additionally, we describe how these models can be used to discover molecules with therapeutic potential.

Keywords: ATP synthase; Drug screening; MT-ATP6 gene; Mitochondrial DNA mutations; Mitochondrial diseases; Mitochondrial transformation; Yeast

DOI: https://doi.org/10.1007/978-1-0716-2309-1_14

Front Genet. 2022 ;13 921610

Transcriptomic Analysis of Dysregulated Genes of the nDNA-mtDNA Axis in a Mouse Model of Dilated Cardiomyopathy.

Mark Ziemann, Wei Wu, Xiu-Ling Deng, Xiao-Jun Du.

Background: Mitochondrial dysfunction is implicated in the development of cardiomyopathy and heart failure. Transcription of mitochondrial DNA (mtDNA) encoded genes and subsequent protein synthesis are tightly regulated by nuclear DNA (nDNA) encoded proteins forming the nDNA-mtDNA axis. The scale of abnormalities in this axis in dilated cardiomyopathy (DCM) is unclear. We previously demonstrated, in a mouse DCM model with cardiac Mst1 overexpression, extensive downregulation of mitochondrial genes and mitochondrial dysfunction. Using the pre-acquired transcriptome sequencing database, we studied expression of gene sets of the nDNA-mtDNA axis. Methods: Using RNA-sequencing data from DCM hearts of mice at early and severe disease stages, transcriptome was performed for dysregulated nDNA-encoded gene sets that govern mtDNA transcription and in situ protein synthesis. To validate gene data, expression of a panel of proteins was determined by immunoblotting. Results: Relative to littermate controls, DCM hearts showed significant downregulation of all mtDNA encoded mRNAs, as well as mtDNA transcriptional activators. Downregulation was also evident for gene sets of mt-rRNA processing, aminoacyl-tRNA synthases, and mitoribosome subunits for in situ protein synthesis. Multiple downregulated genes belong to mitochondrial protein-importing machinery indicating compromised importing of proteins for mtDNA transcription and translation. Diverse changes were genes of mtRNA-binding proteins that govern maturation and stability of mtDNA-derived RNAs. Expression of mtDNA replicome genes was largely unchanged. These changes were similarly observed in mouse hearts at early and severe stages of DCM. Conclusion: Transcriptome revealed in our DCM model dysregulation of multiple gene sets of the nDNA-mtDNA axis, that is, expected to interfere with mtDNA transcription and in situ protein synthesis. Dysfunction of the nDNA-mtDNA axis might contribute to mitochondrial dysfunction and ultimately development of DCM.

Keywords: dilated cardiomyopathy; mitochondrial DNA; mitochondrial RNA; mitoribosome; nuclear DNA; transcriptome analysis

DOI: https://doi.org/10.3389/fgene.2022.921610