Biochemistry and Pharmacology - Research Publications

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    Mtfp1 ablation enhances mitochondrial respiration and protects against hepatic steatosis
    Patitucci, C ; Hernandez-Camacho, JD ; Vimont, E ; Yde, S ; Cokelaer, T ; Chaze, T ; Gianetto, QG ; Matondo, M ; Gazi, A ; Nemazanyy, I ; Stroud, DA ; Hock, DH ; Donnarumma, E ; Wai, T (NATURE PORTFOLIO, 2023-12-20)
    Hepatic steatosis is the result of imbalanced nutrient delivery and metabolism in the liver and is the first hallmark of Metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD is the most common chronic liver disease and involves the accumulation of excess lipids in hepatocytes, inflammation, and cancer. Mitochondria play central roles in liver metabolism yet the specific mitochondrial functions causally linked to MASLD remain unclear. Here, we identify Mitochondrial Fission Process 1 protein (MTFP1) as a key regulator of mitochondrial and metabolic activity in the liver. Deletion of Mtfp1 in hepatocytes is physiologically benign in mice yet leads to the upregulation of oxidative phosphorylation (OXPHOS) activity and mitochondrial respiration, independently of mitochondrial biogenesis. Consequently, liver-specific knockout mice are protected against high fat diet-induced steatosis and metabolic dysregulation. Additionally, Mtfp1 deletion inhibits mitochondrial permeability transition pore opening in hepatocytes, conferring protection against apoptotic liver damage in vivo and ex vivo. Our work uncovers additional functions of MTFP1 in the liver, positioning this gene as an unexpected regulator of OXPHOS and a therapeutic candidate for MASLD.
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    Integrated multi-omics for rapid rare disease diagnosis on a national scale
    Lunke, S ; Bouffler, SEE ; Patel, CVV ; Sandaradura, SAA ; Wilson, M ; Pinner, J ; Hunter, MFF ; Barnett, CPP ; Wallis, M ; Kamien, B ; Tan, TYY ; Freckmann, M-L ; Chong, B ; Phelan, D ; Francis, D ; Kassahn, KSS ; Ha, T ; Gao, S ; Arts, P ; Jackson, MRSR ; Scott, HSS ; Eggers, S ; Rowley, S ; Boggs, K ; Rakonjac, A ; Brett, GRR ; de Silva, MGG ; Springer, A ; Ward, M ; Stallard, K ; Simons, C ; Conway, T ; Halman, A ; Van Bergen, NJJ ; Sikora, T ; Semcesen, LNN ; Stroud, DAA ; Compton, AGG ; Thorburn, DRR ; Bell, KMM ; Sadedin, S ; North, KNN ; Christodoulou, J ; Stark, Z (NATURE PORTFOLIO, 2023-07)
    Critically ill infants and children with rare diseases need equitable access to rapid and accurate diagnosis to direct clinical management. Over 2 years, the Acute Care Genomics program provided whole-genome sequencing to 290 families whose critically ill infants and children were admitted to hospitals throughout Australia with suspected genetic conditions. The average time to result was 2.9 d and diagnostic yield was 47%. We performed additional bioinformatic analyses and transcriptome sequencing in all patients who remained undiagnosed. Long-read sequencing and functional assays, ranging from clinically accredited enzyme analysis to bespoke quantitative proteomics, were deployed in selected cases. This resulted in an additional 19 diagnoses and an overall diagnostic yield of 54%. Diagnostic variants ranged from structural chromosomal abnormalities through to an intronic retrotransposon, disrupting splicing. Critical care management changed in 120 diagnosed patients (77%). This included major impacts, such as informing precision treatments, surgical and transplant decisions and palliation, in 94 patients (60%). Our results provide preliminary evidence of the clinical utility of integrating multi-omic approaches into mainstream diagnostic practice to fully realize the potential of rare disease genomic testing in a timely manner.
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    LINC00116-encoded microprotein mitoregulin regulates fatty acid metabolism at the mitochondrial outer membrane
    Zhang, S ; Guo, Y ; Fidelito, G ; Robinson, DRL ; Liang, C ; Lim, R ; Bichler, Z ; Guo, R ; Wu, G ; Xu, H ; Zhou, QD ; Singh, BK ; Yen, P ; Kappei, D ; Stroud, DA ; Ho, L (CELL PRESS, 2023-09-15)
    LINC00116 encodes a microprotein first identified as Mitoregulin (MTLN), where it was reported to localize to the inner membrane of mitochondria to regulate fatty acid oxidation and oxidative phosphorylation. These initial discoveries were followed by reports with differing findings about its molecular functions and submitochondrial localization. To clarify the apparent discrepancies, we constructed multiple orthogonal methods of determining the localization of MTLN, including split GFP-based reporters that enable efficient and reliable topology analyses for microproteins. These methods unequivocally demonstrate MTLN primarily localizes to the outer membrane of mitochondria, where it interacts with enzymes of fatty acid metabolism including CPT1B and CYB5B. Loss of MTLN causes the accumulation of very long-chain fatty acids (VLCFAs), especially docosahexaenoic acid (DHA). Intriguingly, loss of MTLN protects mice against western diet/fructose-induced insulin-resistance, suggests a protective effect of VLCFAs in this context. MTLN thus serves as an attractive target to control the catabolism of VLCFAs.
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    Loss of the large conductance calcium-activated potassium channel causes an increase in mitochondrial reactive oxygen species in glioblastoma cells
    Kulawiak, B ; Zochowska, M ; Bednarczyk, P ; Galuba, A ; Stroud, DA ; Szewczyk, A (SPRINGER HEIDELBERG, 2023-09)
    Mitochondrial potassium (mitoK) channels play an important role in cellular physiology. These channels are expressed in healthy tissues and cancer cells. Activation of mitoK channels can protect neurons and cardiac tissue against injury induced by ischemia-reperfusion. In cancer cells, inhibition of mitoK channels leads to an increase in mitochondrial reactive oxygen species, which leads to cell death. In glioma cell activity of the mitochondrial, large conductance calcium-activated potassium (mitoBKCa) channel is regulated by the mitochondrial respiratory chain. In our project, we used CRISPR/Cas9 technology in human glioblastoma U-87 MG cells to generate knockout cell lines lacking the α-subunit of the BKCa channel encoded by the KCNMA1 gene, which also encodes cardiac mitoBKCa. Mitochondrial patch-clamp experiments showed the absence of an active mitoBKCa channel in knockout cells. Additionally, the absence of this channel resulted in increased levels of mitochondrial reactive oxygen species. However, analysis of the mitochondrial respiration rate did not show significant changes in oxygen consumption in the cell lines lacking BKCa channels compared to the wild-type U-87 MG cell line. These observations were reflected in the expression levels of selected mitochondrial genes, organization of the respiratory chain, and mitochondrial morphology, which did not show significant differences between the analyzed cell lines. In conclusion, we show that in U-87 MG cells, the pore-forming subunit of the mitoBKCa channel is encoded by the KCNMA1 gene. Additionally, the presence of this channel is important for the regulation of reactive oxygen species levels in mitochondria.
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    Multi-omics identifies large mitoribosomal subunit instability caused by pathogenic MRPL39 variants as a cause of pediatric onset mitochondrial disease
    Amarasekera, SSC ; Hock, DH ; Lake, NJ ; Calvo, SE ; Gronborg, SW ; Krzesinski, E ; Amor, DJ ; Fahey, MC ; Simons, C ; Wibrand, F ; Mootha, VK ; Lek, M ; Lunke, S ; Stark, Z ; ostergaard, E ; Christodoulou, J ; Thorburn, DR ; Stroud, DA ; Compton, AG (OXFORD UNIV PRESS, 2023-07-20)
    MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (DNA). We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile-onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.
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    Mitochondrial E3 ubiquitin ligase MARCHF5 controls BAK apoptotic activity independently of BH3-only proteins
    Huang, AS ; San Chin, H ; Reljic, B ; Djajawi, TM ; Tan, IKL ; Gong, J-N ; Stroud, DA ; Huang, DCS ; van Delft, MF ; Dewson, G (SPRINGERNATURE, 2023-03)
    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 by restraining BAK activating conformation change 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.
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    Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins
    Healy, MD ; Sacharz, J ; McNally, KE ; McConville, C ; Tillu, VA ; Hall, RJ ; Chilton, M ; Cullen, PJ ; Mobli, M ; Ghai, R ; Stroud, DA ; Collins, BM (CELL PRESS, 2022-12-01)
    The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.
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    Caveolae sense oxidative stress through membrane lipid peroxidation and cytosolic release of CAVIN1 to regulate NRF2.
    Wu, Y ; Lim, Y-W ; Stroud, DA ; Martel, N ; Hall, TE ; Lo, HP ; Ferguson, C ; Ryan, MT ; McMahon, K-A ; Parton, RG (Elsevier BV, 2023-03-13)
    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 organelle in cellular homeostasis and wound response.
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    Two independent respiratory chains adapt OXPHOS performance to glycolytic switch
    Fernandez-Vizarra, E ; Lopez-Calcerrada, S ; Sierra-Magro, A ; Perez-Perez, R ; Formosa, LE ; Hock, DH ; Illescas, M ; Penas, A ; Brischigliaro, M ; Ding, S ; Fearnley, IM ; Tzoulis, C ; Pitceathly, RDS ; Arenas, J ; Martin, MA ; Stroud, DA ; Zeviani, M ; Ryan, MT ; Ugalde, C (CELL PRESS, 2022-11-01)
    The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.
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    Investigation of oxidative phosphorylation activity and complex composition in mitochondrial disease.
    Thompson, K ; Stroud, DA ; Thorburn, DR ; Taylor, RW (Elsevier, 2023)
    A multidisciplinary approach to the laboratory diagnosis of mitochondrial disease has long been applied, with crucial information provided by deep clinical phenotyping, blood investigations, and biomarker screening as well as histopathological and biochemical testing of biopsy material to support molecular genetic screening. In an era of second and third generation sequencing technologies, traditional diagnostic algorithms for mitochondrial disease have been replaced by gene agnostic, genomic strategies including whole-exome sequencing (WES) and whole-genome sequencing (WGS), increasingly supported by other 'omics technologies (Alston et al., 2021). Whether a primary testing strategy, or one used to validate and interpret candidate genetic variants, the availability of a range of tests aimed at determining mitochondrial function (i.e., the assessment of individual respiratory chain enzyme activities in a tissue biopsy or cellular respiration in a patient cell line) remains an important part of the diagnostic armory. In this chapter, we summarize several disciplines used in the laboratory investigation of suspected mitochondrial disease, including the histopathological and biochemical assessment of mitochondrial function, as well as protein-based techniques to assess the steady-state levels of oxidative phosphorylation (OXPHOS) subunits and assembly of OXPHOS complexes via traditional (immunoblotting) and cutting-edge (quantitative proteomic) approaches.