bims-cytox1 2022-03-13 papers

Issue of 2022‒03‒13
four papers selected by
Gavin McStay
Staffordshire University

RNA. 2022 Mar 07. pii: rna.079097.122. [Epub ahead of print]

Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation.

Yusuke Kimura, Hironori Saito, Tatsuya Osaki, Yasuhiro Ikegami, Taisei Wakigawa, Yoshiho Ikeuchi, Shintaro Iwasaki.

Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.

Keywords: FACS; FUNCAT; HPG; Mitochondria; Translation

DOI: https://doi.org/10.1261/rna.079097.122

Anal Biochem. 2022 Mar 05. pii: S0003-2697(22)00102-6. [Epub ahead of print] 114646

How many molecules of mitochondrial complex I are in a cell?

Fariha Ansari, Belem Yoval, Zoya Niatsetskaya, Vadim Ten, Ilka Wittig, Alexander Galkin.

Mitochondrial complex I is the only enzyme responsible for oxidation of matrix NADH and regeneration of NAD+ for catabolism. Nuclear and mtDNA mutations, assembly impairments, and enzyme damage are implicated in inherited diseases, ischemia-reperfusion injury, neurodegeneration, and tumorogenesis. Here we introduce a novel method to measure the absolute content of complex I. The method is based on flavin fluorescence scanning of a polyacrylamide gel after separation of complexes by Clear Native electrophoresis. Using mouse primary astrocytes as an example, we calculated an average value of 2.2 × 105 complex I molecules/cell. Our method can be used for accurate quantification of complex I content.

Keywords: Astrocytes; Flavin mononucleotide; Fluorescence; Mitochondrial complex I; Respiratory chain

DOI: https://doi.org/10.1016/j.ab.2022.114646

Cells. 2022 Mar 06. pii: 908. [Epub ahead of print]11(5):

Direct Interaction of Mitochondrial Cytochrome c Oxidase with Thyroid Hormones: Evidence for Two Binding Sites.

Ilya P Oleynikov, Roman V Sudakov, Natalia V Azarkina, Tatiana V Vygodina.

Thyroid hormones regulate tissue metabolism to establish an energy balance in the cell, in particular, by affecting oxidative phosphorylation. Their long-term impact is mainly associated with changes in gene expression, while the short-term effects may differ in their mechanisms. Our work was devoted to studying the short-term effects of hormones T2, T3 and T4 on mitochondrial cytochrome c oxidase (CcO) mediated by direct contact with the enzyme. The data obtained indicate the existence of two separate sites of CcO interaction with thyroid hormones, differing in their location, affinity and specificity to hormone binding. First, we show that T3 and T4 but not T2 inhibit the oxidase activity of CcO in solution and on membrane preparations with Ki ≈ 100-200 μM. In solution, T3 and T4 compete in a 1:1 ratio with the detergent dodecyl-maltoside to bind to the enzyme. The peroxidase and catalase partial activities of CcO are not sensitive to hormones, but electron transfer from heme a to the oxidized binuclear center is affected. We believe that T3 and T4 could be ligands of the bile acid-binding site found in the 3D structure of CcO by Ferguson-Miller's group, and hormone-induced inhibition is associated with dysfunction of the K-proton channel. A possible role of this interaction in the physiological regulation of the enzyme is discussed. Second, we find that T2, T3, and T4 inhibit superoxide generation by oxidized CcO in the presence of excess H2O2. Inhibition is characterized by Ki values of 0.3-5 μM and apparently affects the formation of O2●- at the protein surface. The second binding site for thyroid hormones presumably coincides with the point of tight T2 binding on the Va subunit described in the literature.

Keywords: bile acid-binding site; cytochrome oxidase; regulation; steroid hormones; superoxide generation; thyroid hormones

DOI: https://doi.org/10.3390/cells11050908

J Biol Chem. 2022 Mar 04. pii: S0021-9258(22)00239-3. [Epub ahead of print] 101799

Temperature-dependent structural transition following X-ray-induced metal center reduction in oxidized cytochrome c oxidase.

Izumi Ishigami, Silvia Russi, Aina Cohen, Syun-Ru Yeh, Denis L Rousseau.

Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in the inner membrane of mitochondria. It contains four metal redox centers, two of which, CuB and heme a3, form the binuclear center (BNC), where dioxygen is reduced to water. Crystal structures of CcO in various forms have been reported, from which ligand binding states of the BNC and conformations of the protein matrix surrounding it have been deduced to elucidate the mechanism by which the oxygen reduction chemistry is coupled to proton translocation. However, metal centers in proteins can be susceptible to X-ray-induced radiation damage, raising questions about the reliability of conclusions drawn from these studies. Here, we used microspectroscopy-coupled X-ray crystallography to interrogate how the structural integrity of bovine CcO in the fully oxidized state (O) is modulated by synchrotron radiation. Spectroscopic data showed that, upon X-ray exposure, O was converted to a hybrid O* state where all the four metal centers were reduced, but the protein matrix was trapped in the genuine O conformation and the ligands in the BNC remained intact. Annealing the O* crystal above the glass transition temperature induced relaxation of the O* structure to a new R* structure, wherein the protein matrix converted to the fully reduced R conformation with the exception of Helix X, which partly remained in the O conformation due to incomplete dissociation of the ligands from the BNC. We conclude from these data that reevaluation of reported CcO structures obtained with synchrotron light sources is merited.

Keywords: Crystallography; bioenergetics; metastable states; optical absorption; synchrotron radiation

DOI: https://doi.org/10.1016/j.jbc.2022.101799