bims-cytox1 2023-03-05 papers

Trends Cell Biol. 2023 Feb 28. pii: S0962-8924(23)00020-X. [Epub ahead of print]

The multifaceted mitochondrial OXA insertase.

Bettina Homberg, Peter Rehling, Luis Daniel Cruz-Zaragoza.

Most mitochondrial proteins are synthesized in the cytosol and transported into mitochondria by protein translocases. Yet, mitochondria contain their own genome and gene expression system, which generates proteins that are inserted in the inner membrane by the oxidase assembly (OXA) insertase. OXA contributes to targeting proteins from both genetic origins. Recent data provides insights into how OXA cooperates with the mitochondrial ribosome during synthesis of mitochondrial-encoded proteins. A picture of OXA emerges in which it coordinates insertion of OXPHOS core subunits and their assembly into protein complexes but also participates in the biogenesis of select imported proteins. These functions position the OXA as a multifunctional protein insertase that facilitates protein transport, assembly, and stability at the inner membrane.

Keywords: mitochondria; oxidase assembly; oxidative phosphorylation; protein translocation; ribosomes

DOI: https://doi.org/10.1016/j.tcb.2023.02.001

Proc Natl Acad Sci U S A. 2023 Mar 07. 120(10): e2216722120

FDX1-dependent and independent mechanisms of elesclomol-mediated intracellular copper delivery.

Mohammad Zulkifli, Amy N Spelbring, Yuteng Zhang, Shivatheja Soma, Si Chen, Luxi Li, Trung Le, Vinit Shanbhag, Michael J Petris, Tai-Yen Chen, Martina Ralle, David P Barondeau, Vishal M Gohil.

Recent studies have uncovered the therapeutic potential of elesclomol (ES), a copper-ionophore, for copper deficiency disorders. However, we currently do not understand the mechanism by which copper brought into cells as ES-Cu(II) is released and delivered to cuproenzymes present in different subcellular compartments. Here, we have utilized a combination of genetic, biochemical, and cell-biological approaches to demonstrate that intracellular release of copper from ES occurs inside and outside of mitochondria. The mitochondrial matrix reductase, FDX1, catalyzes the reduction of ES-Cu(II) to Cu(I), releasing it into mitochondria where it is bioavailable for the metalation of mitochondrial cuproenzyme- cytochrome c oxidase. Consistently, ES fails to rescue cytochrome c oxidase abundance and activity in copper-deficient cells lacking FDX1. In the absence of FDX1, the ES-dependent increase in cellular copper is attenuated but not abolished. Thus, ES-mediated copper delivery to nonmitochondrial cuproproteins continues even in the absence of FDX1, suggesting alternate mechanism(s) of copper release. Importantly, we demonstrate that this mechanism of copper transport by ES is distinct from other clinically used copper-transporting drugs. Our study uncovers a unique mode of intracellular copper delivery by ES and may further aid in repurposing this anticancer drug for copper deficiency disorders.

Keywords: FDX1; copper; cytochrome c oxidase; elesclomol; mitochondria

DOI: https://doi.org/10.1073/pnas.2216722120

Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2023 Feb;45(1): 9-15

[Excess Oxygen Supply for Different Time Periods Affect Energy Metabolism in Rat Alveolar Epithelial Type Ⅱ Cells].

Rong-Rong Huang, Shan-Shan Qu, Hong Guo, Su-Heng Chen, Chuan-Qi Yang, Jun-Mei Zhang, Yu-Lan Li.

Objective To observe the effect of excess oxygen supply for different time periods on the mitochondrial energy metabolism in alveolar epithelial type Ⅱ cells. Methods Rat RLE-6TN cells were assigned into a control group (21% O2 for 4 h) and excess oxygen supply groups (95% O2 for 1,2,3,and 4 h,res-pectively).The content of adenosine triphosphate (ATP),the activity of mitochondrial respiratory chain complex V,and the mitochondrial membrane potential were determined by luciferase assay,micro-assay,and fluorescent probe JC-1,respectively.Real-time fluorescence quantitative PCR was employed to determine the mRNA levels of NADH dehydrogenase subunit 1 (ND1),cytochrome b (Cytb),cytochrome C oxidase subunit I (COXI),and adenosine triphosphatase 6 (ATPase6) in the core subunits of mitochondrial respiratory chain complexes Ⅰ,Ⅲ,Ⅳ,and Ⅴ,respectively. Results Compared with the control group,excess oxygen supply for 1,2,3,and 4 h down-regulated the mRNA levels of ND1 (q=24.800,P<0.001;q=13.650,P<0.001;q=9.869,P<0.001;q=20.700,P<0.001),COXI (q=16.750,P<0.001;q=10.120,P<0.001;q=8.476,P<0.001;q=14.060,P<0.001),and ATPase6 (q=22.770,P<0.001;q=15.540,P<0.001;q=12.870,P<0.001;q=18.160,P<0.001).Moreover,excess oxygen supply for 1 h and 4 h decreased the ATPase activity (q=9.435,P<0.001;q=11.230,P<0.001) and ATP content (q=5.615,P=0.007;q=5.029,P=0.005).The excess oxygen supply for 2 h and 3 h did not cause significant changes in ATPase activity (q=0.156,P=0.914;q=3.197,P=0.116) and ATP content (q=0.859,P=0.557;q=1.273,P=0.652).There was no significant difference in mitochondrial membrane potential among the groups (F=0.303,P=0.869). Conclusion Short-term excess oxygen supply down-regulates the expression of the core subunits of mitochondrial respiratory chain complexes and reduces the activity of ATPase,leading to the energy metabolism disorder of alveolar epithelial type Ⅱ cells.

Keywords: alveolar epithelial type Ⅱ cell; energy metabolism; excess oxygen supply; mitochondria

DOI: https://doi.org/10.3881/j.issn.1000-503X.15096

Biol Open. 2023 Mar 15. pii: bio059844. [Epub ahead of print]12(3):

Defective COX1 expression in aging mice liver.

Steffen Witte, Angela Boshnakovska, Metin Özdemir, Arpita Chowdhury, Peter Rehling, Abhishek Aich.

Mitochondrial defects are associated with aging processes and age-related diseases, including cardiovascular diseases, neurodegenerative diseases and cancer. In addition, some recent studies suggest mild mitochondrial dysfunctions appear to be associated with longer lifespans. In this context, liver tissue is considered to be largely resilient to aging and mitochondrial dysfunction. Yet, in recent years studies report dysregulation of mitochondrial function and nutrient sensing pathways in ageing livers. Therefore, we analyzed the effects of the aging process on mitochondrial gene expression in liver using wildtype C57BL/6N mice. In our analyses, we observed alteration in mitochondrial energy metabolism with age. To assess if defects in mitochondrial gene expression are linked to this decline, we applied a Nanopore sequencing based approach for mitochondrial transcriptomics. Our analyses show that a decrease of the Cox1 transcript correlates with reduced respiratory complex IV activity in older mice livers.

Keywords: Ageing; Mitochondria; Nanopore; Transcriptomics

DOI: https://doi.org/10.1242/bio.059844

STAR Protoc. 2023 Feb 03. pii: S2666-1667(23)00046-1. [Epub ahead of print]4(1): 102088

Protocol for mitochondrial isolation and sub-cellular localization assay for mitochondrial proteins.

Danyi Zhou, Sheng Zhong, Xinyu Han, Dandan Liu, Hezhi Fang, Ya Wang.

Here, we provide a protocol to isolate mitochondria from cultured cells and extract differently located mitochondrial proteins. We detail steps to separate both integral and peripheral membrane proteins from soluble proteins using sonication. We describe the separation of integral membrane proteins from the peripheral membrane and soluble proteins using sodium carbonate extraction. Furthermore, we detail the use of proteinase K and Triton X-100 to distinguish outer membrane proteins from mitochondrial proteins.

Keywords: Cell Membrane; Cell culture; Cell separation/fractionation; Protein Biochemistry

DOI: https://doi.org/10.1016/j.xpro.2023.102088