Biology subjects

Ryan, M. T.

Publications and source records attributed to Ryan, M. T..

9 recordsLinked to original sources

bioRxiv2026

Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

Mitochondrial permeabilization by the apoptotic executioners BAK and BAX represents a critical stage of mitochondrial apoptosis and facilitates the release of pro-inflammatory mitochondrial DNA via herniation of the inner mitochondrial membrane. This study utilises TurboID proximity labelling to investigate the temporal changes of the BAK proximal proteome during mitochondrial herniation and apoptosis. In doing so, we detail a comprehensive BAK proximal proteome, both at steady state and during apoptosis and observe the loss of MICOS complex stability and proximity to the BAK pore as apoptosis proceeds. In addition, we identify the mitochondrial microprotein SLC35A4-MP proximal to the BAK pore and reveal a SLC35A4-MP dependent modulation of OPA1 processing. Furthermore, loss of SLC35A4-MP delays mitochondrial fragmentation in response to a variety of stressors, uncovering a previously unrecognised role for SLC35A4-MP in fine-tuning mitochondrial rearrangement during apoptotic stress.

cell biology↗

bioRxiv2026

Characterization of human Metaxin proteins reveals functional diversification of SAM37 homologs MTX1 and MTX3

The biogenesis of outer mitochondrial membrane {beta}-barrel proteins relies on the mitochondrial Sorting and Assembly Machinery (SAM) complex. In humans, the SAM complex contains SAM50 along with Metaxin (MTX) accessory subunits. MTX1 and MTX3 are homologous yet their functional similarities and differences have scarcely been investigated. Homozygous null mutations in the MTX2 gene are linked to a rare progeroid syndrome that causes severe depletion of MTX1. Here, we uncover unique phenotypes associated with the loss of MTX1 or MTX3 in human cells. Loss of MTX1 confers a deficiency in mitochondrial volume and causes network-wide mitochondrial morphology abnormalities. MTX3 loss resulted in negligible consequences for the biogenesis of {beta}-barrel proteins but resulted in increased mitochondrial mass. We also find that both MTX1 and MTX3 stability are dependent on the presence of MTX2, with MTX1 deficiency causing defective import and assembly. Collectively, our findings support the notion that MTX1 and MTX3 are functionally diverse homologs and are unlikely to be functionally redundant.

biochemistry↗

bioRxiv2022

Maternal and/or direct supplementation with a combination of a casein hydrolysate and yeast β-glucan on post-weaning performance and intestinal health in the pig

A 2 x 2 factorial experiment was conducted to investigate the effect of maternal supplementation from day 83 of gestation and/or direct supplementation from weaning of a bovine casein hydrolysate plus a yeast {beta}-glucan (CH-YBG) on pig performance and intestinal health on day ten post-weaning. Twenty cross bred gilts (Large White x Landrace) were randomly assigned to one of two dietary groups (n = 10 gilts/group): basal diet (basal sows) and basal diet supplemented with CH-YBG (supplemented sows) from day 83 of gestation until weaning (2g/sow/day). At weaning, 120 pigs (6 pigs/sow) were selected. The two dam groups were further divided, resulting in four experimental groups (10 replicates/group; 3 pigs/pen) as follows: 1) BB (basal sows + basal pigs); 2) BS (basal sows + supplemented pigs); 3) SB (supplemented sows + basal pigs); 4) SS (supplemented sows + supplemented pigs). Supplemented pigs were offered 0.5g CH-YBG/kg of feed for 10 days post-weaning. On day 10 post-weaning, 1 pig/pen was humanely sacrificed and samples were taken from the gastrointestinal tract for analysis. Pigs weaned from supplemented sows (SS, SB) had reduced faecal scores and incidence of diarrhoea (P<0.05) compared to pigs weaned from basal sows (BB, BS), with SS pigs not displaying the transient rise in faecal scores seen in the other three groups from day 3 to day 10 post-weaning (P<0.05). Pigs weaned from supplemented sows had reduced feed intake (P<0.05), improved feed efficiency (P<0.05), increased butyrate concentrations (P<0.05), increased abundance of Lactobacillus (P<0.05) and decreased abundance of Enterobacteriaceae and Campylobacteraceae (P<0.05) compared to pigs weaned from basal sows. In conclusion, a combination of both maternal and direct supplementation with CH-YBG is the desirable feeding strategy to ameliorate the post-weaning challenge in pig production systems.

developmental biology↗

bioRxiv2021

Caveolae, CAVIN1, and lipid peroxidation mediate the oxidative stress response

Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.

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↗

bioRxiv2019

Neuronal-specific function of hTim8a in Complex IV assembly provides insight into the molecular mechanism underlying Mohr-Tranebjaerg syndrome

Human Tim8a is a member of an intermembrane space chaperone network, known as the small TIM family, which transport hydrophobic membrane proteins through this compartment. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjaerg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells and consequently how lack of hTim8a leads to a neurodegenerative disease. We identified a novel cell-specific function of hTim8a in the assembly of Complex IV, which is mediated through a transient interaction with the copper chaperone COX17. Complex IV assembly defects in cells lacking hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c and Bax, which primes cells for cell death. Alleviation of oxidative stress using Vitamin E rescues cells from apoptotic vulnerability. We hypothesis that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

cell biology↗

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↗