Biology subjects

Antonicka, H.

Publications and source records attributed to Antonicka, H..

8 recordsLinked to original sources

bioRxiv2026

Pathogenic variants of the mitochondrial copper chaperones SCO1 and SCO2 reshape their respective interactomes and disrupt cellular phospholipid metabolism

SCO1 and SCO2 are required for copper delivery to COX2, a copper-containing subunit of cytochrome c oxidase (COX), yet how mutations in these genes cause distinct, tissue-specific forms of human disease remains poorly understood. To gain further insight into the molecular underpinnings of this clinical heterogeneity, we used BioID to map the interactomes of four pathogenic SCO variants (SCO1 G132S, SCO1 P174L, SCO1 M294V and SCO2 E140K) and the wild-type proteins. While this approach identified many proteins common to both wild-type neighbourhoods, several potential interacting partners unique to each SCO protein were also observed that were consistent with their known roles in COX assembly. Follow-up analyses revealed that SCO1 interacts with COX16 and that this interaction is stabilized within the membrane by a coiled-coil helix-helix interface, with the soluble C-terminal region of COX16 physically bridging SCO1 and COX2 within a ternary complex to facilitate copper delivery. We further observed that COX16 abundance is relatively low in the brain and its association with SCO1 is most severely impaired by the M294V substitution associated with a fatal encephalopathy. Intriguingly, our BioID analyses also detected significant enrichment in each SCO neighbourhood for biosynthetic enzymes and lipases critical to phospholipid metabolism and found that the affinity for these candidate interactors was uniquely perturbed by various pathogenic variants of SCO1 and SCO2. Collectively, our data emphasize the potential of proximity labelling to further define the molecular roles of disease-causing variants that perturb mitochondrial function and suggest that SCO proteins impinge upon phospholipid metabolism.

molecular biology↗

bioRxiv2024

FASTKD5 processes mitochondrial pre-mRNAs at non-canonical cleavage sites

The regulation of mammalian mitochondrial gene expression is largely post-transcriptional and the first step in translating the 13 polypeptides encoded in mtDNA is endonucleolytic cleavage of the primary polycistronic transcripts. As the rRNAs and most of the mRNAs in mtDNA are flanked by tRNAs, the release of the mature RNAs occurs mostly by excision of the tRNAs. Processing the non-canonical mRNAs, not flanked by tRNAs, requires FASTKD5, but the molecular mechanism remains unknown. To investigate this, we created and characterized a knockout cell line to use as an assay system. The absence of FASTKD5 resulted in a severe combined OXPHOS assembly defect due to the inability to translate mRNAs with unprocessed 5-UTRs. Analysis of RNA processing of FASTKD5 variants allowed us to map amino acid residues essential for function. Remarkably, this map was RNA substrate-specific, arguing against a one size fits all model. A reconstituted in vitro system with purified FASTKD5 protein and synthetic RNA substrates showed that FASTKD5 on its own was able to cleave client substrates correctly, but not non-specific RNA sequences. These results establish FASTKD5 as the missing piece of the biochemical machinery required to completely process the primary mitochondrial transcript.

molecular biology↗

bioRxiv2024

SLC25A46 is in contact with lysosomes and plays a role in mitochondrial cholesterol homeostasis

Mitochondrial morphology reflects the dynamic equilibrium between fusion and fission events, controlled by cellular signaling. A cytoprotective response known as stress-induced mitochondrial hyperfusion (SIMH) is triggered by nutrient starvation and we show that the outer mitochondrial membrane protein SLC25A46 is required for this response. To unravel the cellular mechanisms involved, we conducted transcriptomic analysis on control human fibroblasts and SLC25A46 knock-out cells. Our analysis revealed a remarkable divergence in the transcriptional profile of proteins associated with lysosomal function and cholesterol binding and synthesis. Further investigations using live-cell imaging validated the presence of SLC25A46 at the majority of mitochondria-lysosome contact sites. Since mitochondria-lysosome contacts are linked to cholesterol transport, we investigated the involvement of SLC25A46 in cholesterol trafficking. The SLC25A46 knock-out cell line exhibited a decrease in mitochondrial cholesterol content and distinct alterations were observed in the pattern of cholesterol trafficking compared to control. Cholesterol supplementation in the SLC25A46 knock-out cell line rescued the mitochondrial fragmentation phenotype and restored the SIMH response, suggesting a role for SLC25A46 in maintaining mitochondrial cholesterol homeostasis. Summary blurbThe mitochondrial outer membrane protein SLC25A46 is required for SIMH triggered by nutrient starvation, localizes to lysosome contact sites and is involved in mitochondrial cholesterol homeostasis

cell biology↗

bioRxiv2023

BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from "multiple mitochondrial dysfunction" syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly.

biochemistry↗

bioRxiv2022

ESYT1 tethers the endoplasmic reticulum to mitochondria and is required for mitochondrial lipid and calcium homeostasis

Mitochondria interact with the endoplasmic reticulum (ER) at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). MERCs are crucial for a myriad of physiological functions including lipid synthesis and transport, and calcium signaling. Alterations in the structure, composition or regulation of MERCs contribute to the aetiology of many pathologies including neurodegenerative and metabolic diseases. The proteins mediating the formation of MERCs have been extensively studied in yeast, where the ER-mitochondria encounter structure (ERMES) complex mediates the transport of lipids between the ER and mitochondria via three lipid binding SMP-domain proteins. However, none of the SMP proteins of the ERMES complex have orthologues in mammals suggesting that alternate pathways have evolved in metazoans. Combining proximity labelling (BioID), confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain containing protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. The deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs, indicating that the ESYT1-SYN2JBP complex plays a role in tethering ER and mitochondria. Loss of this complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by re-expression of wild-type ESYT1 as well as an artificial mitochondria-ER tether. Together, these results reveal a novel function of ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs.

cell biology↗

bioRxiv2022

SLC25A46 localizes to sites of mitochondrial fission and fusion and loss of function variants alter the oligomerization states of MFN2 and OPA1

Mutations in SLC25A46, coding for an outer mitochondrial membrane protein, underlie a wide spectrum of neurodegenerative diseases associated with alterations in mitochondrial morphology, but the precise role of the protein remains unknown. We established an SLC25A46 knock-out cell line in human fibroblasts and studied the pathogenicity of three different variants (p.T142I, p.R257Q, p.E335D) introduced into the null background. Mitochondria were fragmented in the knock-out cell line and hyperfused in all pathogenic variants. The loss of SLC25A46 led to impaired cellular proliferation and striking abnormalities in mitochondrial cristae ultrastructure that were not rescued by expression of the pathogenic variants. SLC25A46 was present in discrete puncta at mitochondrial branch points and at tips of mitochondrial tubules, co-localizing with DRP1 and OPA1. Virtually all fission/fusion events were demarcated by the presence of an SLC25A46 focus. SLC25A46 co-immunoprecipitated with the fusion machinery, and loss of function altered the oligomerization state of OPA1 and MFN2. Proximity interaction mapping identified components of the ER membrane, lipid transfer proteins, and mitochondrial outer membrane proteins indicating that it is present at interorganellar contact sites important for lipid exchange. Consistent with this, SLC25A46 loss of function led to altered mitochondrial lipid composition, suggesting that it may facilitate interorganellar lipid flux or play a role in membrane remodeling associated with mitochondrial fusion and fission.

cell biology↗

bioRxiv2020

A high-density human mitochondrial proximity interaction network.

We used BioID, a proximity-dependent biotinylation assay, to interrogate 100 mitochondrial baits from all mitochondrial sub-compartments to create a high resolution human mitochondrial proximity interaction network. We identified 1465 proteins, producing 15626 unique high confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions, and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments, and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles. This proximity network will be a valuable resource for exploring the biology of uncharacterized mitochondrial proteins, the interactions of mitochondria with other cellular organelles, and will provide a framework to interpret alterations in sub-mitochondrial environments associated with mitochondrial disease. Bullet pointsO_LIWe created a high resolution human mitochondrial protein proximity map using BioID C_LIO_LIBait-bait analysis showed that the map has sub-compartment resolution and correlation analysis of preys identified functional clusters and assigned proteins to specific modules C_LIO_LIWe identified isoforms of matrix and IMS proteins with multiple cellular localizations and an endonuclease that localizes to both the matrix and the OMM C_LIO_LIOMM baits showed specific interactions with non-mitochondrial proteins reflecting organellar contact sites and protein dual localization C_LI

cell biology↗

bioRxiv2019

A proximity biotinylation map of a human cell

Compartmentalization is a defining characteristic of eukaryotic cells, partitioning cellular processes into discrete subcellular locations. High throughput microscopy1 and biochemical fractionation coupled with mass spectrometry2-6 have helped to define the proteomes of a variety of organelles and macromolecular structures. However, many other intracellular compartments have remained refractory to such approaches, due for example to difficulty in purifying non-membrane bound structures. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach for defining the composition of cellular compartments in living cells7-10. Here we present a BioID-based map of a human cell based on 192 markers from 32 different subcellular compartments, comprising 35,902 high confidence proximity interactions, and defining the intracellular locations of 4,145 unique proteins in HEK 293 cells. Our localization predictions meet or exceed previous-approaches, with higher specificity, and enabled the discovery of proteins at the mitochondrial outer membrane-endoplasmic reticulum (ER) interface that are critical for mitochondrial homeostasis. Based on this dataset, we have established humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to better understand BioID datasets.

molecular biology↗