bims-resufa 2021-03-07 papers

Issue of 2021–03–07
two papers selected by
Vera Strogolova, Strong Microbials, Inc

Bio Protoc. 2019 Oct 05. 9(19): e3379

Isolation of Pure Mitochondria from Rat Kidneys and Western Blot of Mitochondrial Respiratory Chain Complexes.

Tamara Micakovic, Wiktoria Z Banczyk, Euan Clark, Bettina Kränzlin, Jörg Peters, Sigrid Christa Hoffmann.

Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.

Keywords: Angiotensin II type 2 receptor; Diabetic nephropathy; Mitochondria; Mitochondria isolation; Renin angiotensin system; Western blotting

DOI: https://doi.org/10.21769/BioProtoc.3379

Cells. 2021 Feb 20. pii: 452. [Epub ahead of print]10(2):

Upregulation of COX4-2 via HIF-1α in Mitochondrial COX4-1 Deficiency.

Liza Douiev, Chaya Miller, Shmuel Ruppo, Hadar Benyamini, Bassam Abu-Libdeh, Ann Saada.

Cytochrome-c-oxidase (COX) subunit 4 (COX4) plays important roles in the function, assembly and regulation of COX (mitochondrial respiratory complex 4), the terminal electron acceptor of the oxidative phosphorylation (OXPHOS) system. The principal COX4 isoform, COX4-1, is expressed in all tissues, whereas COX4-2 is mainly expressed in the lungs, or under hypoxia and other stress conditions. We have previously described a patient with a COX4-1 defect with a relatively mild presentation compared to other primary COX deficiencies, and hypothesized that this could be the result of a compensatory upregulation of COX4-2. To this end, COX4-1 was downregulated by shRNAs in human foreskin fibroblasts (HFF) and compared to the patient's cells. COX4-1, COX4-2 and HIF-1α were detected by immunocytochemistry. The mRNA transcripts of both COX4 isoforms and HIF-1 target genes were quantified by RT-qPCR. COX activity and OXPHOS function were measured by enzymatic and oxygen consumption assays, respectively. Pathways were analyzed by CEL-Seq2 and by RT-qPCR. We demonstrated elevated COX4-2 levels in the COX4-1-deficient cells, with a concomitant HIF-1α stabilization, nuclear localization and upregulation of the hypoxia and glycolysis pathways. We suggest that COX4-2 and HIF-1α are upregulated also in normoxia as a compensatory mechanism in COX4-1 deficiency.

Keywords: COX4-1; COX4-2; HIF-1α; cytochrome c oxidase; mitochondria

DOI: https://doi.org/10.3390/cells10020452