Biology subjects

Reljic, B.

Publications and source records attributed to Reljic, B..

8 recordsLinked to original sources

bioRxiv2022

E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.

cell biology↗

bioRxiv2021

Mitochondrial COA7 is a heme-binding protein involved in the early stages of complex IV assembly.

Cytochrome c oxidase assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked in patients to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here we show that the absence of COA7 leads to arrest of the complex IV assembly pathway at the initial step where the COX1 module is built, which requires incorporation of copper and heme cofactors. In solution, purified COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. Surprisingly, the crystal structure of COA7, determined to 2.4 [A] resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (/) repeats, tethered by disulfide bonds, with a structure entirely distinct from proteins with characterized heme binding activities. We therefore propose a role for COA7 in heme binding/chaperoning in the mitochondrial intermembrane space, this activity being crucial for and providing a missing link in complex IV biogenesis. Significance StatementAssembly factors play key roles in the biogenesis of many mitochondrial protein complexes regulating their stability, activity and incorporation of essential cofactors. COA7 is a metazoan-specific assembly factor, the absence or mutation of which in humans accompanies complex IV assembly defects and neurological conditions. Here we report the crystal structure of COA7 to 2.4 [A] resolution, revealing a banana-shaped molecule composed of five helix-turn-helix (/) repeats, tethered by disulfide bonds. Characterization of pathogenic variants reveals significantly lower stabilities, correlating with the associated disease outcomes. Fascinatingly, COA7 binds heme with micromolar affinity, despite the fact that the protein structure does not resemble previously characterized heme-binding proteins. This provides a possible missing link for heme handling in the mitochondrial intermembrane space.

biochemistry↗

bioRxiv2021

Multi-omics reveal intricate network of mitochondrial adaptations to training in human skeletal muscle

Mitochondrial defects are implicated in multiple diseases and aging. Exercise training is an accessible and inexpensive therapeutic intervention improving mitochondrial bioenergetics and quality of life. By combining a multi-omics approach with biochemical and in silico normalization, we removed the bias arising from the training-induced increase in human skeletal muscle mitochondrial content to unearth an intricate and previously undemonstrated network of differentially prioritized mitochondrial adaptations. We show that changes in hundreds of transcripts, proteins, and lipids are not stoichiometrically linked to the increase in mitochondrial content. We demonstrate that enhancing electron flow to oxidative phosphorylation (OXPHOS) is more important to improve ATP generation than increasing the abundance of the OXPHOS machinery, and that training-induced supercomplex formation does not confer enhancements in mitochondrial bioenergetics. Our study provides a new analytical approach allowing unbiased and in-depth investigations of training-induced mitochondrial adaptations, challenging our current understanding and calling for careful reinterpretation of previous findings.

cell biology↗

bioRxiv2020

Optic Atrophy-associated TMEM126A is an assembly factor for the ND4-module of Mitochondrial Complex I

Mitochondrial disease is a debilitating condition with a diverse genetic aetiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labelling interaction studies reveal that TMEM126A associates with the newly synthesised mtDNA-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. Importantly, we clarify that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I, helping to explain the differences in disease aetiology observed between these two genes.

biochemistry↗

bioRxiv2019

Dissecting the Roles of Mitochondrial Complex I Intermediate Assembly (MCIA) Complex Factors in the Biogenesis of Complex I

Mitochondrial Complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly factors. The Mitochondrial Complex I Intermediate Assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centred on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, while COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly.

biochemistry↗

bioRxiv2019

HIGD2A is required for assembly of the COX3 module of human mitochondrial complex IV

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. A number of assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. While in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

biochemistry↗

bioRxiv2018

BAX requires VDAC2 to mediate apoptosis and to limit tumor development

Intrinsic apoptosis is critical for normal physiology including the prevention of tumor formation. BAX and BAK are essential for mediating this process and for the cytotoxic action of many anticancer drugs. BAX and BAK are thought to act in a functionally redundant manner and are considered to be regulated similarly. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as essential for BAX, but not BAK, to function. The genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes that contain VDAC1, VDAC2 and VDAC3. By disrupting its localization to mitochondria, BAX is rendered completely ineffective. Moreover, we defined an interface unique to VDAC2 that is required to drive BAX activity. Consequently, interfering with this interaction or deleting VDAC2 phenocopied the loss of BAX, including impairing the killing of tumor cells by anti-cancer agents such as the BCL-2 inhibitor venetoclax. Furthermore, the ability of BAX to prevent tumor formation was attenuated in the absence of VDAC2. Taken together, our studies show for the first time that BAX-mediated apoptosis, but not BAK-mediated apoptosis, is critically dependent on VDAC2, hence revealing the differential regulation of BAX and BAK.

cell biology↗

bioRxiv2017

In the absence of apoptosis, myeloid cells arrest when deprived of growth factor, but remain viable without ATG5-dependent autophagy or uptake of glucose or glutamine.

Withdrawal of the growth factor interleukin 3 from IL3-dependent myeloid cells causes them to undergo Bax/Bak1-dependent apoptosis, whereas factor-deprived Bax-/-Bak1-/- cells remain viable, but arrest and shrink. It was reported that withdrawal of IL3 from Bax-/-Bak1-/- cells caused decreased expression of the glucose transporter Glut1, leading to reduced glucose uptake, so that arrested cells required Atg5-dependent autophagy for long-term survival. In other cell types, a decrease in Glut1 is mediated by the thioredoxin-interacting protein Txnip, which is induced in IL3-dependent myeloid cells when growth factor is removed. We mutated Atg5 and Txnip by CRISPR/Cas9 and found that Atg5-dependent autophagy was not necessary for the long-term viability of cycling or arrested Bax-/-Bak1-/- cells, and that Txnip was not required for the decrease in Glut1 expression in response to IL3 withdrawal. Surprisingly, Atg5-deficient Bax/Bak1 double mutant cells survived for several weeks in medium supplemented with 10% fetal bovine serum (FBS), without high concentrations of added glucose or glutamine. When serum was withdrawn, the provision of an equivalent amount of glucose present in 10% FBS (~0.5 mM) was sufficient to support cell survival for more than a week, in the presence or absence of IL3. Thus, Bax-/-Bak1-/- myeloid cells deprived of growth factor consume extracellular glucose to maintain long-term viability, without a requirement for Atg5-dependent autophagy.

cell biology↗