Biology subjects

Shoubridge, E. A.

Publications and source records attributed to Shoubridge, E. A..

8 recordsLinked to original sources

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↗

bioRxiv2022

Loss of mitochondrial Chchd10 or Chchd2 in zebrafish leads to an ALS-like phenotype and Complex I deficiency independent of the mt-ISR

Mutations in CHCHD10 and CHCHD2, coding for two paralogous mitochondrial proteins, have been identified in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), and Parkinsons disease (PD). Here we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish. We demonstrate that loss of either or both proteins leads to a motor impairment, reduced survival, and compromised neuromuscular junction (NMJ) integrity in larval zebrafish. Compensation by Chchd10 was observed in the chchd2-/- model, but not by Chchd2 in the chchd10 -/- model. The assembly of mitochondrial respiratory chain Complex I was impaired in chchd10 -/- and chchd2 -/- zebrafish larvae, but unexpectedly not in the double chchd10 -/- & chchd2 -/- model, suggesting that reduced mitochondrial Complex I cannot be solely responsible for the observed phenotypes, which are generally more severe in the double KO. Activation of the mitochondrial integrated stress response (mt-ISR) was only observed in the double KO model, possibly implicating this pathway in the recovery of the Complex I defect, and suggesting that Complex I assembly defect in our single KO is independent of the mt-ISR. Our results demonstrate that both proteins are required for normal vertebrate development, but their precise molecular function in the mitochondrial biology of motor neurons remains to be discovered.

developmental biology↗

bioRxiv2022

SPTLC1 variants associated with childhood onset amyotrophic lateral sclerosis produce distinct sphingolipid signatures through impaired interaction with ORMDL proteins

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Mutations in the SPTLC1 subunit of serine-palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased sphingolipid synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause the peripheral sensory neuropathy HSAN1 due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

biochemistry↗

bioRxiv2020

A CHCHD10 variant causing ALS elicits an unfolded protein response through the IRE1/XBP1 pathway

Mutations in CHCHD10, coding for a mitochondrial intermembrane space protein, are a rare cause of autosomal dominant amyotrophic lateral sclerosis (ALS). Mutation-specific toxic gain of function or haploinsuffuciency models have been proposed to explain pathogenicity. To decipher the metabolic dysfunction associated with the haploinsufficient p.R15L variant we integrated transcriptomic, metabolomic and proteomic data sets in patient cells subjected to nutrient stress. Patient cells had a complex I deficiency resulting in an increased NADH/NAD+ ratio, downregulation of the TCA cycle, and a reorganization of one carbon metabolism. This led to phosphorylation of AMPK, activation of an endoplasmic reticulum and mitochondrial unfolded protein response (UPR), and the production of GDF15 and FGF21, which are markers of mitochondrial disease. The endoplasmic reticulum UPR was mediated through the IRE1/XBP1 pathway, and was accompanied by reduced eIF2alpha phosphorylation, dephosphorylation of both JNK isoforms, and up regulation of several dual specific phosphatases. This study demonstrates that loss of CHCHD10 function elicits a striking energy deficit that activates cellular stress pathways, which may underlie the selective vulnerability of motor neurons.

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↗

bioRxiv2018

LONP1 is required for maturation of a subset of mitochondrial proteins and its loss elicits an integrated stress response

LONP1, a AAA+ mitochondrial protease, is implicated in protein quality control, but its substrates and precise role in this process remain poorly understood. Here we have investigated the role of human LONP1 in mitochondrial gene expression and proteostasis. Depletion of LONP1 resulted in partial loss of mtDNA, complete suppression of mitochondrial translation, a marked increase in the levels of a distinct subset of mitochondrial matrix proteins (SSBP1, MTERFD3, FASTKD2 and CLPX), and the accumulation of their unprocessed forms, with intact mitochondrial targeting sequences, in an insoluble protein fraction. Depletion of LONP1 produced massive matrix protein aggregates and activated the Integrated Stress Response (ISR) pathway. These results demonstrate that LONP1 is required for maturation of a subset of its client proteins and for maintenance of mitochondrial gene expression.

biochemistry↗