bims-mibica 2024-04-07 papers

bims-mibica

Biomed News

on Mitochondrial bioenergetics in cancer

Issue of 2024‒04‒07
29 papers selected by
Kelsey Fisher-Wellman, East Carolina University

Targeting Metabolic Dependencies Fueling the TCA Cycle to Circumvent Therapy Resistance in Acute Myeloid Leukemia.
Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.
Human CCDC51 and yeast Mdm33 are functionally conserved mitochondrial inner membrane proteins that demarcate a subset of organelle fission events.
The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.
The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.
Long-range electron proton coupling in respiratory complex I - insights from molecular simulations of the quinone chamber and antiporter-like subunits.
Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.
Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.
Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.
A Human Brain Map of Mitochondrial Respiratory Capacity and Diversity.
Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway.
Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.
SCAF1 drives the compositional diversity of mammalian respirasomes.
PHGDH is Key to a Prognostic Multigene Signature and a Potential Therapeutic Target in Acute Myeloid Leukemia.
Hypoxic adaptation of mitochondria and its impact on tumor cell function.
Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice.
A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection.
An inner mitochondrial membrane microprotein from the SLC35A4 upstream ORF regulates cellular metabolism.
Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation, invasion, and migration of gastric cancer cells.
Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma.
Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.
idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans.
An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.
The energetics of cellular life transitions.
Mitochondrial energy metabolism-related gene signature as a prognostic indicator for pancreatic adenocarcinoma.
Peroxiredoxin 6 suppresses ferroptosis in lung endothelial cells.
Identification and structural characterization of small molecule inhibitors of PINK1.
Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.
mtDNA analysis using Mitopore.

Cancer Res. 2024 Apr 01. 84(7): 950-952

Targeting Metabolic Dependencies Fueling the TCA Cycle to Circumvent Therapy Resistance in Acute Myeloid Leukemia.

Emeline Boët, Jean-Emmanuel Sarry.

Acute myeloid leukemia (AML) is one of the most prevalent blood cancers, characterized by a dismal survival rate. This poor outcome is largely attributed to AML cells that persist despite treatment and eventually result in relapse. Relapse-initiating cells exhibit diverse resistance mechanisms, encompassing genetic factors and, more recently discovered, nongenetic factors such as metabolic adaptations. Leukemic stem cells (LSC) rely on mitochondrial metabolism for their survival, whereas hematopoietic stem cells primarily depend on glycolysis. Furthermore, following treatments such as cytarabine, a standard in AML treatment for over four decades, drug-persisting leukemic cells exhibit an enhanced reliance on mitochondrial metabolism. In this issue of Cancer Research, two studies investigated dependencies of AML cells on two respiratory substrates, α-ketoglutarate and lactate-derived pyruvate, that support mitochondrial oxidative phosphorylation (OXPHOS) following treatment with the imipridone ONC-213 and the BET inhibitor INCB054329, respectively. Targeting lactate utilization by interfering with monocarboxylate transporter 1 (MCT1 or SLC16A1) or lactate dehydrogenase effectively sensitized cells to BET inhibition in vitro and in vivo. In addition, ONC-213 affected αKGDH, a pivotal NADH-producing enzyme of the TCA cycle, to induce a mitochondrial stress response through ATF4 activation that diminished the expression of the antiapoptotic protein MCL1, consequently promoting apoptosis of AML cells. In summary, targeting these mitochondrial dependencies might be a promising strategy to kill therapy-naïve and treatment-resistant OXPHOS-reliant LSCs and to delay or prevent relapse. See related articles by Monteith et al., p. 1101 and Su et al., p. 1084.

DOI: https://doi.org/10.1158/0008-5472.CAN-24-0019
Nat Commun. 2024 Mar 30. 15(1): 2803

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies.

Saeed Daneshmandi, Jee Eun Choi, Qi Yan, Cameron R MacDonald, Manu Pandey, Mounika Goruganthu, Nathan Roberts, Prashant K Singh, Richard M Higashi, Andrew N Lane, Teresa W-M Fan, Jianmin Wang, Philip L McCarthy, Elizabeth A Repasky, Hemn Mohammadpour.

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via β2-adrenergic receptor (β2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using β2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

DOI: https://doi.org/10.1038/s41467-024-47096-9
bioRxiv. 2024 Mar 22. pii: 2024.03.21.586162. [Epub ahead of print]

Human CCDC51 and yeast Mdm33 are functionally conserved mitochondrial inner membrane proteins that demarcate a subset of organelle fission events.

Alia R Edington, Olivia M Connor, Madeleine Marlar-Pavey, Jonathan R Friedman.

Mitochondria are highly dynamic double membrane-bound organelles that exist in a semi- continuous network. Mitochondrial morphology arises from the complex interplay of numerous processes, including opposing fission and fusion dynamics and the formation of highly organized cristae invaginations of the inner membrane. While extensive work has examined the mechanisms of mitochondrial fission, it remains unclear how fission is coordinated across two membrane bilayers and how mitochondrial inner membrane organization is coupled with mitochondrial fission dynamics. Previously, the yeast protein Mdm33 was implicated in facilitating fission by coordinating with inner membrane homeostasis pathways. However, Mdm33 is not conserved outside fungal species and its precise mechanistic role remains unclear. Here, we use a bioinformatic approach to identify a putative structural ortholog of Mdm33 in humans, CCDC51 (also called MITOK). We find that the mitochondrial phenotypes associated with altered CCDC51 levels implicate the protein in mitochondrial fission dynamics. Further, using timelapse microscopy, we spatially and temporally resolve Mdm33 and CCDC51 to a subset of mitochondrial fission events. Finally, we show that CCDC51 can partially rescue yeast Δ mdm33 cells, indicating the proteins are functionally analogous. Our data reveal that Mdm33/CCDC51 are conserved mediators of mitochondrial morphology and suggest the proteins play a crucial role in maintaining normal mitochondrial dynamics and organelle homeostasis.

DOI: https://doi.org/10.1101/2024.03.21.586162
Acta Physiol (Oxf). 2024 Apr 05. e14143

The 2-oxoglutarate/malate carrier extends the family of mitochondrial carriers capable of fatty acid and 2,4-dinitrophenol-activated proton transport.

Kristina Žuna, Tatyana Tyschuk, Taraneh Beikbaghban, Felix Sternberg, Jürgen Kreiter, Elena E Pohl.

  AIMS: Metabolic reprogramming in cancer cells has been linked to mitochondrial dysfunction. The mitochondrial 2-oxoglutarate/malate carrier (OGC) has been suggested as a potential target for preventing cancer progression. Although OGC is involved in the malate/aspartate shuttle, its exact role in cancer metabolism remains unclear. We aimed to investigate whether OGC may contribute to the alteration of mitochondrial inner membrane potential by transporting protons.METHODS: The expression of OGC in mouse tissues and cancer cells was investigated by PCR and Western blot analysis. The proton transport function of recombinant murine OGC was evaluated by measuring the membrane conductance (Gm) of planar lipid bilayers. OGC-mediated substrate transport was measured in proteoliposomes using 14C-malate.
RESULTS: OGC increases proton Gm only in the presence of natural (long-chain fatty acids, FA) or chemical (2,4-dinitrophenol) protonophores. The increase in OGC activity directly correlates with the increase in the number of unsaturated bonds of the FA. OGC substrates and inhibitors compete with FA for the same protein binding site. Arginine 90 was identified as a critical amino acid for the binding of FA, ATP, 2-oxoglutarate, and malate, which is a first step towards understanding the OGC-mediated proton transport mechanism.
CONCLUSION: OGC extends the family of mitochondrial transporters with dual function: (i) metabolite transport and (ii) proton transport facilitated in the presence of protonophores. Elucidating the contribution of OGC to uncoupling may be essential for the design of targeted drugs for the treatment of cancer and other metabolic diseases.

Keywords:  SLC25A11; long‐chain fatty acids; mitochondrial transport; planar bilayer membranes; proton transport; total membrane conductance

DOI:  https://doi.org/10.1111/apha.14143
EMBO Rep. 2024 Apr 02.

The ER-SURF pathway uses ER-mitochondria contact sites for protein targeting to mitochondria.

Christian Koch, Svenja Lenhard, Markus Räschle, Cristina Prescianotto-Baschong, Anne Spang, Johannes M Herrmann.

  Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria in a post-translational reaction. Mitochondrial precursor proteins which use the ER-SURF pathway employ the surface of the endoplasmic reticulum (ER) as an important sorting platform. How they reach the mitochondrial import machinery from the ER is not known. Here we show that mitochondrial contact sites play a crucial role in the ER-to-mitochondria transfer of precursor proteins. The ER mitochondria encounter structure (ERMES) and Tom70, together with Djp1 and Lam6, are part of two parallel and partially redundant ER-to-mitochondria delivery routes. When ER-to-mitochondria transfer is prevented by loss of these two contact sites, many precursors of mitochondrial inner membrane proteins are left stranded on the ER membrane, resulting in mitochondrial dysfunction. Our observations support an active role of the ER in mitochondrial protein biogenesis.

Keywords:  Contact sites; ERMES; Endoplasmic reticulum; Mitochondria; Protein import

DOI:  https://doi.org/10.1038/s44319-024-00113-w
Biochem J. 2024 Apr 10. 481(7): 499-514

Long-range electron proton coupling in respiratory complex I - insights from molecular simulations of the quinone chamber and antiporter-like subunits.

Amina Djurabekova, Jonathan Lasham, Oleksii Zdorevskyi, Volker Zickermann, Vivek Sharma.

  Respiratory complex I is a redox-driven proton pump. Several high-resolution structures of complex I have been determined providing important information about the putative proton transfer paths and conformational transitions that may occur during catalysis. However, how redox energy is coupled to the pumping of protons remains unclear. In this article, we review biochemical, structural and molecular simulation data on complex I and discuss several coupling models, including the key unresolved mechanistic questions. Focusing both on the quinone-reductase domain as well as the proton-pumping membrane-bound domain of complex I, we discuss a molecular mechanism of proton pumping that satisfies most experimental and theoretical constraints. We suggest that protonation reactions play an important role not only in catalysis, but also in the physiologically-relevant active/deactive transition of complex I.

Keywords:  MD simulations; energy conversion; mitochondrial respiration; protein dynamics; proton pump

DOI:  https://doi.org/10.1042/BCJ20240009
Sci Adv. 2024 Apr 05. 10(14): eadl0389

Drp1 controls complex II assembly and skeletal muscle metabolism by Sdhaf2 action on mitochondria.

Zhenqi Zhou, Alice Ma, Timothy M Moore, Dane M Wolf, Nicole Yang, Peter Tran, Mayuko Segawa, Alexander R Strumwasser, Wenjuan Ren, Kai Fu, Jonathan Wanagat, Alexander M van der Bliek, Rachelle Crosbie-Watson, Marc Liesa, Linsey Stiles, Rebecca Acin-Perez, Sushil Mahata, Orian Shirihai, Mark O Goodarzi, Michal Handzlik, Christian M Metallo, David W Walker, Andrea L Hevener.

The dynamin-related guanosine triphosphatase, Drp1 (encoded by Dnm1l), plays a central role in mitochondrial fission and is requisite for numerous cellular processes; however, its role in muscle metabolism remains unclear. Here, we show that, among human tissues, the highest number of gene correlations with DNM1L is in skeletal muscle. Knockdown of Drp1 (Drp1-KD) promoted mitochondrial hyperfusion in the muscle of male mice. Reduced fatty acid oxidation and impaired insulin action along with increased muscle succinate was observed in Drp1-KD muscle. Muscle Drp1-KD reduced complex II assembly and activity as a consequence of diminished mitochondrial translocation of succinate dehydrogenase assembly factor 2 (Sdhaf2). Restoration of Sdhaf2 normalized complex II activity, lipid oxidation, and insulin action in Drp1-KD myocytes. Drp1 is critical in maintaining mitochondrial complex II assembly, lipid oxidation, and insulin sensitivity, suggesting a mechanistic link between mitochondrial morphology and skeletal muscle metabolism, which is clinically relevant in combatting metabolic-related diseases.

DOI: https://doi.org/10.1126/sciadv.adl0389
Sci Adv. 2024 Apr 05. 10(14): eadj7540

Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.

Enakshi D Sunassee, Riley J Deutsch, Victoria W D'Agostino, Pol Castellano-Escuder, Elizabeth A Siebeneck, Olga Ilkayeva, Brian T Crouch, Megan C Madonna, Jeffrey Everitt, James V Alvarez, Gregory M Palmer, Matthew D Hirschey, Nirmala Ramanujam.

Fewer than 20% of triple-negative breast cancer patients experience long-term responses to mainstay chemotherapy. Resistant tumor subpopulations use alternative metabolic pathways to escape therapy, survive, and eventually recur. Here, we show in vivo, longitudinal metabolic reprogramming in residual disease and recurrence of triple-negative breast cancer xenografts with varying sensitivities to the chemotherapeutic drug paclitaxel. Optical imaging coupled with metabolomics reported an increase in non-glucose-driven mitochondrial metabolism and an increase in intratumoral metabolic heterogeneity during regression and residual disease in resistant MDA-MB-231 tumors. Conversely, sensitive HCC-1806 tumors were primarily reliant on glucose uptake and minimal changes in metabolism or heterogeneity were observed over the tumors' therapeutic life cycles. Further, day-matched resistant HCC-1806 tumors revealed a higher reliance on mitochondrial metabolism and elevated metabolic heterogeneity compared to sensitive HCC-1806 tumors. Together, metabolic flexibility, increased reliance on mitochondrial metabolism, and increased metabolic heterogeneity are defining characteristics of persistent residual disease, features that will inform the appropriate type and timing of therapies.

DOI: https://doi.org/10.1126/sciadv.adj7540
bioRxiv. 2024 Mar 14. pii: 2024.03.13.583708. [Epub ahead of print]

Inhibition of Acyl-CoA Synthetase Long Chain Isozymes Decreases Multiple Myeloma Cell Proliferation and Causes Mitochondrial Dysfunction.

Connor S Murphy, Victoria E DeMambro, Samaa Fadel, Heather Fairfield, Carlos A Garter, Princess Rodriguez, Ya-Wei Qiang, Calvin P H Vary, Michaela R Reagan.

Multiple myeloma (MM) is an incurable cancer of plasma cells with a 5-year survival rate of 59%. Dysregulation of fatty acid (FA) metabolism is associated with MM development and progression; however, the underlying mechanisms remain unclear. Acyl-CoA synthetase long-chain family members (ACSLs) convert free long-chain fatty acids into fatty acyl-CoA esters and play key roles in catabolic and anabolic fatty acid metabolism. The Cancer Dependency Map data suggested that ACSL3 and ACSL4 were among the top 25% Hallmark Fatty Acid Metabolism genes that support MM fitness. Here, we show that inhibition of ACSLs in human myeloma cell lines using the pharmacological inhibitor Triascin C (TriC) causes apoptosis and decreases proliferation in a dose- and time-dependent manner. RNA-seq of MM.1S cells treated with TriC for 24 h showed a significant enrichment in apoptosis, ferroptosis, and ER stress. Proteomics of MM.1S cells treated with TriC for 48 h revealed that mitochondrial dysfunction and oxidative phosphorylation were significantly enriched pathways of interest, consistent with our observations of decreased mitochondrial membrane potential and increased mitochondrial superoxide levels. Interestingly, MM.1S cells treated with TriC for 24 h also showed decreased mitochondrial ATP production rates and overall lower cellular respiration.Implications: Overall, our data support the hypothesis that suppression of ACSL in human MM cells inhibit their growth and viability, indicating that ACSL proteins may be promising therapeutic targets in treating myeloma progression.

DOI: https://doi.org/10.1101/2024.03.13.583708
Res Sq. 2024 Mar 22. pii: rs.3.rs-4047706. [Epub ahead of print]

A Human Brain Map of Mitochondrial Respiratory Capacity and Diversity.

Martin Picard, Eugene Mosharov, Ayelet Rosenberg, Anna Monzel, Corey Osto, Linsey Stiles, Gorazd Rosoklija, Andrew Dwork, Snehal Bindra, Ya Zhang, Masashi Fujita, Madeline Mariani, Mihran Bakalian, David Sulzer, Philip De Jager, Vilas Menon, Orian Shirihai, John Mann, Mark Underwood, Maura Boldrini, Michel Thiebaut de Schotten.

Mitochondrial oxidative phosphorylation (OxPhos) powers brain activity 1,2 , and mitochondrial defects are linked to neurodegenerative and neuropsychiatric disorders 3,4 , underscoring the need to define the brain's molecular energetic landscape 5-10 . To bridge the cognitive neuroscience and cell biology scale gap, we developed a physical voxelization approach to partition a frozen human coronal hemisphere section into 703 voxels comparable to neuroimaging resolution (3x3x3 mm). In each cortical and subcortical brain voxel, we profiled mitochondrial phenotypes including OxPhos enzyme activities, mitochondrial DNA and volume density, and mitochondria-specific respiratory capacity. We show that the human brain contains a diversity of mitochondrial phenotypes driven by both topology and cell types. Compared to white matter, grey matter contains >50% more mitochondria. We show that the more abundant grey matter mitochondria also are biochemically optimized for energy transformation, particularly among recently evolved cortical brain regions. Scaling these data to the whole brain, we created a backward linear regression model integrating several neuroimaging modalities11, thereby generating a brain-wide map of mitochondrial distribution and specialization that predicts mitochondrial characteristics in an independent brain region of the same donor brain. This new approach and the resulting MitoBrainMap of mitochondrial phenotypes provide a foundation for exploring the molecular energetic landscape that enables normal brain functions, relating it to neuroimaging data, and defining the subcellular basis for regionalized brain processes relevant to neuropsychiatric and neurodegenerative disorders.

DOI: https://doi.org/10.21203/rs.3.rs-4047706/v1
Commun Biol. 2024 Mar 30. 7(1): 391

Cytosolic retention of HtrA2 during mitochondrial protein import stress triggers the DELE1-HRI pathway.

Paul Y Bi, Samuel A Killackey, Linus Schweizer, Damien Arnoult, Dana J Philpott, Stephen E Girardin.

Mitochondrial stress inducers such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and oligomycin trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged DELE1 showed that these stresses induced the cleavage of DELE1 to DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non-depolarizing MPIS. Surprisingly, while the mitochondrial protease OMA1 was crucial for DELE1 cleavage in HeLa cells, it was dispensable in HEK293T cells, suggesting that multiple proteases may be involved in DELE1 cleavage. In support, we identified a role for the mitochondrial protease, HtrA2, in mediating DELE1 cleavage into DELE1-VS, and showed that a Parkinson's disease (PD)-associated HtrA2 mutant displayed reduced DELE1 processing ability, suggesting a novel mechanism linking PD pathogenesis to mitochondrial stress. Our data further suggest that DELE1 is likely cleaved into DELE1-S in the cytosol, while the DELE1-VS form might be generated during halted translocation into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies a novel role for HtrA2 in DELE1 processing.

DOI: https://doi.org/10.1038/s42003-024-06107-7
J Physiol. 2024 Apr 02.

Impact of capillary and sarcolemmal proximity on mitochondrial structure and energetic function in skeletal muscle.

Hailey A Parry, T Bradley Willingham, Kevin A Giordano, Yuho Kim, Shureed Qazi, Jay R Knutson, Christian A Combs, Brian Glancy.

  Mitochondria within skeletal muscle cells are located either between the muscle contractile apparatus (interfibrillar mitochondria, IFM) or beneath the cell membrane (subsarcolemmal mitochondria, SSM), with several structural and functional differences reported between IFM and SSM. However, recent 3D imaging studies demonstrate that mitochondria are particularly concentrated in the proximity of capillaries embedded in sarcolemmal grooves rather than in proximity to the sarcolemma itself (paravascular mitochondria, PVM). To evaluate the impact of capillary vs. sarcolemmal proximity, we compared the structure and function of skeletal muscle mitochondria located either lateral to embedded capillaries (PVM), adjacent to the sarcolemma but not in PVM pools (SSM) or interspersed between sarcomeres (IFM). Mitochondrial morphology and interactions were assessed by 3D electron microscopy coupled with machine learning segmentation, whereas mitochondrial energy conversion was assessed by two-photon microscopy of mitochondrial membrane potential, content, calcium, NADH redox and flux in live, intact cells. Structurally, although PVM and SSM were similarly larger than IFM, PVM were larger, rounder and had more physical connections to neighbouring mitochondria compared to both IFM and SSM. Functionally, PVM had similar or greater basal NADH flux compared to SSM and IFM, respectively, despite a more oxidized NADH pool and a greater membrane potential, signifying a greater activation of the electron transport chain in PVM. Together, these data indicate that proximity to capillaries has a greater impact on resting mitochondrial energy conversion and distribution in skeletal muscle than the sarcolemma alone. KEY POINTS: Capillaries have a greater impact on mitochondrial energy conversion in skeletal muscle than the sarcolemma. Paravascular mitochondria are larger, and the outer mitochondrial membrane is more connected with neighbouring mitochondria. Interfibrillar mitochondria are longer and have greater contact sites with other organelles (i.e. sarcoplasmic reticulum and lipid droplets). Paravascular mitochondria have greater activation of oxidative phosphorylation than interfibrillar mitochondria at rest, although this is not regulated by calcium.

Keywords:  energy function; mitochondria; skeletal muscle

DOI:  https://doi.org/10.1113/JP286246
Nat Struct Mol Biol. 2024 Apr 04.

SCAF1 drives the compositional diversity of mammalian respirasomes.

Irene Vercellino, Leonid A Sazanov.

Supercomplexes of the respiratory chain are established constituents of the oxidative phosphorylation system, but their role in mammalian metabolism has been hotly debated. Although recent studies have shown that different tissues/organs are equipped with specific sets of supercomplexes, depending on their metabolic needs, the notion that supercomplexes have a role in the regulation of metabolism has been challenged. However, irrespective of the mechanistic conclusions, the composition of various high molecular weight supercomplexes remains uncertain. Here, using cryogenic electron microscopy, we demonstrate that mammalian (mouse) tissues contain three defined types of 'respirasome', supercomplexes made of CI, CIII2 and CIV. The stoichiometry and position of CIV differs in the three respirasomes, of which only one contains the supercomplex-associated factor SCAF1, whose involvement in respirasome formation has long been contended. Our structures confirm that the 'canonical' respirasome (the C-respirasome, CICIII2CIV) does not contain SCAF1, which is instead associated to a different respirasome (the CS-respirasome), containing a second copy of CIV. We also identify an alternative respirasome (A-respirasome), with CIV bound to the 'back' of CI, instead of the 'toe'. This structural characterization of mouse mitochondrial supercomplexes allows us to hypothesize a mechanistic basis for their specific role in different metabolic conditions.

DOI: https://doi.org/10.1038/s41594-024-01255-0
J Cancer. 2024 ;15(9): 2538-2548

PHGDH is Key to a Prognostic Multigene Signature and a Potential Therapeutic Target in Acute Myeloid Leukemia.

Jiagui Zhong, Kezhi Huang, Shaofan Xie, Ailian Tan, Jiaqin Peng, Danian Nie, Liping Ma, Yiqing Li.

  As a rate-limiting enzyme for the serine biosynthesis pathway (SSP) in the initial step, phosphoglycerate dehydrogenase (PHGDH) is overexpressed in many different tumors, and pharmacological or genetic inhibition of PHGDH promotes antitumor effects. In the present research, by analyzing several acute myeloid leukemia (AML) datasets in the Gene Expression Omnibus (GEO), we identified prognosis-related genes and constructed a multigene signature by univariate, multivariate Cox regression and LASSO regression. Subsequently, the multigene signature was confirmed through Cox, Kaplan-Meier, and ROC analyses in the validation cohort. Moreover, PHGDH acted as a risk factor and was correlated with inferior overall survival. We further analysed other datasets and found that PHGDH was overexpressed in AML. Importantly, the expression of PHGDH was higher in drug-resistant AML compared to drug-sensitive ones. In vitro experiments showed that inhibition of PHGDH induced apoptosis and reduced proliferation in AML cells, and these antitumor effects could be related to the Bcl-2/Bax signaling pathway by the noncanonical or nonmetabolic functions of PHGDH. In summary, we constructed a twenty-gene signature that could predicate prognosis of AML patients and found that PHGDH may be a potential target for AML treatment.

Keywords:  Acute myeloid leukemia; Gene signature; Overall survival; PHGDH; Therapeutic target

DOI:  https://doi.org/10.7150/jca.90822
Semin Cancer Biol. 2024 Mar 30. pii: S1044-579X(24)00022-1. [Epub ahead of print]100 28-38

Hypoxic adaptation of mitochondria and its impact on tumor cell function.

Martin Benej, Ioanna Papandreou, Nicholas C Denko.

  Mitochondria are the major sink for oxygen in the cell, consuming it during ATP production. Therefore, when environmental oxygen levels drop in the tumor, significant adaptation is required. Mitochondrial activity is also a major producer of biosynthetic precursors and a regulator of cellular oxidative and reductive balance. Because of the complex biochemistry, mitochondrial adaptation to hypoxia occurs through multiple mechanisms and has significant impact on other cellular processes such as macromolecule synthesis and gene regulation. In tumor hypoxia, mitochondria shift their location in the cell and accelerate the fission and quality control pathways. Hypoxic mitochondria also undergo significant changes to fundamental metabolic pathways of carbon metabolism and electron transport. These metabolic changes further impact the nuclear epigenome because mitochondrial metabolites are used as enzymatic substrates for modifying chromatin. This coordinated response delivers physiological flexibility and increased tumor cell robustness during the environmental stress of low oxygen.

Keywords:  Hypoxia; Mitochondria; Oxygen consumption; Reductive stress

DOI:  https://doi.org/10.1016/j.semcancer.2024.03.004
PNAS Nexus. 2024 Apr;3(4): pgae116

Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice.

Darren J Walsh, David J Bernard, Joanna L Fiddler, Faith Pangilinan, Madison Esposito, Denise Harold, Martha S Field, Anne Parle-McDermott, Lawrence C Brody.

  One-carbon metabolism is a complex network of metabolic reactions that are essential for cellular function including DNA synthesis. Vitamin B12 and folate are micronutrients that are utilized in this pathway and their deficiency can result in the perturbation of one-carbon metabolism and subsequent perturbations in DNA replication and repair. This effect has been well characterized in nuclear DNA but to date, mitochondrial DNA (mtDNA) has not been investigated extensively. Mitochondrial variants have been associated with several inherited and age-related disease states; therefore, the study of factors that impact heteroplasmy are important for advancing our understanding of the mitochondrial genome's impact on human health. Heteroplasmy studies require robust and efficient mitochondrial DNA enrichment to carry out in-depth mtDNA sequencing. Many of the current methods for mtDNA enrichment can introduce biases and false-positive results. Here, we use a method that overcomes these limitations and have applied it to assess mitochondrial heteroplasmy in mouse models of altered one-carbon metabolism. Vitamin B12 deficiency was found to cause increased levels of mitochondrial DNA heteroplasmy across all tissues that were investigated. Folic acid supplementation also contributed to elevated mitochondrial DNA heteroplasmy across all mouse tissues investigated. Heteroplasmy analysis of human data from the Framingham Heart Study suggested a potential sex-specific effect of folate and vitamin B12 status on mitochondrial heteroplasmy. This is a novel relationship that may have broader consequences for our understanding of one-carbon metabolism, mitochondrial-related disease and the influence of nutrients on DNA mutation rates.

Keywords:  cobalamin; folic acid; heteroplasmy; mitochondria; vitamin B12

DOI:  https://doi.org/10.1093/pnasnexus/pgae116
Biochem Biophys Res Commun. 2024 Mar 27. pii: S0006-291X(24)00372-3. [Epub ahead of print]709 149836

A genetically encoded fluorescent protein sensor for mitochondrial membrane damage detection.

Qian Liu, Dianbing Wang, Mengmeng Cui, Min Li, Xian-En Zhang.

  Mitochondria are essential cellular organelles; detecting mitochondrial damage is crucial in cellular biology and toxicology. Compared with existing chemical probe detection methods, genetically encoded fluorescent protein sensors can directly indicate cellular and molecular events without involving exogenous reagents. In this study, we introduced a molecular sensor system, MMD-Sensor, for monitoring mitochondrial membrane damage. The sensor consists of two molecular modules. Module I is a fusion structure of the mitochondrial localization sequence (MLS), AIF cleavage site sequence (CSS), nuclear localization sequence (NLS), N-terminus of mNeonGreen and mCherry. Module II is a fusion structure of the C-terminus of mNeonGreen, NLS sequence, and mtagBFP2. Under normal condition, Module I is constrained in the inner mitochondrial membrane anchored by MLS, while Module II is restricted to the nucleus by its NLS fusion component. If the mitochondrial membrane is damaged, CSS is cut from the inner membrane, causing Module I to shift into the nucleus guided by the NLS fusion component. After Module I enters the nucleus, the N- and C-terminus of mNeonGreen meet each other and rebuild its intact 3D structure through fragment complementation and thus generates green fluorescence in the nucleus. Dynamic migration of red fluorescence from mitochondria to the nucleus and generation of green fluorescence in the nucleus indicate mitochondrial membrane damage. Using the MMD-Sensor, mitochondrial membrane damage induced by various reagents, such as uncoupling agents, ATP synthase inhibitors, monovalent cationic carriers, and ROS, in HeLa and 293T cells are directly observed and evaluated.

Keywords:  Apoptosis-inducing factor; Bimolecular fluorescence complementation; Biosensor; Genetically encoded fluorescent protein sensor; Mitochondrial membrane damage

DOI:  https://doi.org/10.1016/j.bbrc.2024.149836
J Mol Biol. 2024 Apr 03. pii: S0022-2836(24)00154-2. [Epub ahead of print] 168559

An inner mitochondrial membrane microprotein from the SLC35A4 upstream ORF regulates cellular metabolism.

Andrea L Rocha, Victor Pai, Guy Perkins, Tina Chang, Jiao Ma, Qian Chu, Joan M Vaughan, Jolene K Diedrich, Mark H Ellisman, Alan Saghatelian.

  Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing translation under stress conditions. Computational and empirical methods have identified uORFs in the 5'-UTRs of approximately half of all mouse and human transcripts, making uORFs one the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs are under studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functionalmicroprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.

Keywords:  cellular metabolism; inner mitochondrial membrane; microprotein; mitochondria; upstream open reading frame (uORF)

DOI:  https://doi.org/10.1016/j.jmb.2024.168559
World J Gastrointest Oncol. 2024 Mar 15. 16(3): 991-1005

Mitochondrial carrier homolog 2 increases malignant phenotype of human gastric epithelial cells and promotes proliferation, invasion, and migration of gastric cancer cells.

Jing-Wen Zhang, Ling-Yan Huang, Ya-Ning Li, Ying Tian, Jia Yu, Xiao-Fei Wang.

  BACKGROUND: The precise role of mitochondrial carrier homolog 2 (MTCH2) in promoting malignancy in gastric mucosal cells and its involvement in gastric cancer cell metastasis have not been fully elucidated.AIM: To determine the role of MTCH2 in gastric cancer.
METHODS: We collected 65 samples of poorly differentiated gastric cancer tissue and adjacent tissues, constructed MTCH2-overexpressing and MTCH2-knockdown cell models, and evaluated the proliferation, migration, and invasion of human gastric epithelial cells (GES-1) and human gastric cancer cells (AGS) cells. The mitochondrial membrane potential (MMP), mitochondrial permeability transformation pore (mPTP) and ATP fluorescence probe were used to detect mitochondrial function. Mitochondrial function and ATP synthase protein levels were detected via Western blotting.
RESULTS: The expression of MTCH2 and ATP2A2 in gastric cancer tissues was significantly greater than that in adjacent tissues. Overexpression of MTCH2 promoted colony formation, invasion, migration, MMP expression and ATP production in GES-1 and AGS cells while upregulating ATP2A2 expression and inhibiting cell apoptosis; knockdown of MTCH2 had the opposite effect, promoting overactivation of the mPTP and promoting apoptosis.
CONCLUSION: MTCH2 can increase the malignant phenotype of GES-1 cells and promote the proliferation, invasion, and migration of gastric cancer cells by regulating mitochondrial function, providing a basis for targeted therapy for gastric cancer cells.

Keywords:  ATP synthase; ATP2A2; Gastric cancer; Mitochondrial carrier homolog 2; Mitochondrial permeability transformation pore

DOI:  https://doi.org/10.4251/wjgo.v16.i3.991
Cell Rep Med. 2024 Mar 23. pii: S2666-3791(24)00130-7. [Epub ahead of print] 101484

Targeting DNMT3A-mediated oxidative phosphorylation to overcome ibrutinib resistance in mantle cell lymphoma.

Nguyet-Minh Hoang, Yunxia Liu, Paul D Bates, Alexa R Heaton, Angelica F Lopez, Peng Liu, Fen Zhu, Ruoyu Chen, Apoorv Kondapelli, Xiyu Zhang, Paul E Selberg, Vu N Ngo, Melissa C Skala, Christian M Capitini, Lixin Rui.

  The use of Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib achieves a remarkable clinical response in mantle cell lymphoma (MCL). Acquired drug resistance, however, is significant and affects long-term survival of MCL patients. Here, we demonstrate that DNA methyltransferase 3A (DNMT3A) is involved in ibrutinib resistance. We find that DNMT3A expression is upregulated upon ibrutinib treatment in ibrutinib-resistant MCL cells. Genetic and pharmacological analyses reveal that DNMT3A mediates ibrutinib resistance independent of its DNA-methylation function. Mechanistically, DNMT3A induces the expression of MYC target genes through interaction with the transcription factors MEF2B and MYC, thus mediating metabolic reprogramming to oxidative phosphorylation (OXPHOS). Targeting DNMT3A with low-dose decitabine inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in a patient-derived xenograft mouse model. These findings suggest that targeting DNMT3A-mediated metabolic reprogramming to OXPHOS with decitabine provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory MCL.

Keywords:  DNMT3A; OXPHOS; gene regulation; ibrutinib resistance; mantle cell lymphoma

DOI:  https://doi.org/10.1016/j.xcrm.2024.101484
Genome Med. 2024 Apr 02. 16(1): 50

Leveraging new methods for comprehensive characterization of mitochondrial DNA in esophageal squamous cell carcinoma.

Xuehan Zhuang, Rui Ye, Yong Zhou, Matthew Yibo Cheng, Heyang Cui, Longlong Wang, Shuangping Zhang, Shubin Wang, Yongping Cui, Weimin Zhang.

  BACKGROUND: Mitochondria play essential roles in tumorigenesis; however, little is known about the contribution of mitochondrial DNA (mtDNA) to esophageal squamous cell carcinoma (ESCC). Whole-genome sequencing (WGS) is by far the most efficient technology to fully characterize the molecular features of mtDNA; however, due to the high redundancy and heterogeneity of mtDNA in regular WGS data, methods for mtDNA analysis are far from satisfactory.METHODS: Here, we developed a likelihood-based method dMTLV to identify low-heteroplasmic mtDNA variants. In addition, we described fNUMT, which can simultaneously detect non-reference nuclear sequences of mitochondrial origin (non-ref NUMTs) and their derived artifacts. Using these new methods, we explored the contribution of mtDNA to ESCC utilizing the multi-omics data of 663 paired tumor-normal samples.
RESULTS: dMTLV outperformed the existing methods in sensitivity without sacrificing specificity. The verification using Nanopore long-read sequencing data showed that fNUMT has superior specificity and more accurate breakpoint identification than the current methods. Leveraging the new method, we identified a significant association between the ESCC overall survival and the ratio of mtDNA copy number of paired tumor-normal samples, which could be potentially explained by the differential expression of genes enriched in pathways related to metabolism, DNA damage repair, and cell cycle checkpoint. Additionally, we observed that the expression of CBWD1 was downregulated by the non-ref NUMTs inserted into its intron region, which might provide precursor conditions for the tumor cells to adapt to a hypoxic environment. Moreover, we identified a strong positive relationship between the number of mtDNA truncating mutations and the contribution of signatures linked to tumorigenesis and treatment response.
CONCLUSIONS: Our new frameworks promote the characterization of mtDNA features, which enables the elucidation of the landscapes and roles of mtDNA in ESCC essential for extending the current understanding of ESCC etiology. dMTLV and fNUMT are freely available from https://github.com/sunnyzxh/dMTLV and https://github.com/sunnyzxh/fNUMT , respectively.

Keywords:  Esophageal squamous cell carcinoma; Mitochondrial DNA; Non-ref NUMTs; mtDNA copy number; mtDNA variants

DOI:  https://doi.org/10.1186/s13073-024-01319-2
bioRxiv. 2024 Mar 14. pii: 2024.03.13.584865. [Epub ahead of print]

idh-1 neomorphic mutation confers sensitivity to vitamin B12 via increased dependency on one-carbon metabolism in Caenorhabditis elegans.

Olga Ponomarova, Alyxandra N Starbard, Alexandra Belfi, Amanda V Anderson, Meera V Sundaram, Albertha J M Walhout.

The isocitrate dehydrogenase neomorphic mutation ( idh-1neo ) generates increased levels of cellular D-2-hydroxyglutarate (D-2HG), a proposed oncometabolite. However, the physiological effects of increased D-2HG and whether additional metabolic changes occur in the presence of an idh-1neo mutation are not well understood. We created a C. elegans model to study the effects of the idh-1neo mutation in a whole animal. Comparing the phenotypes exhibited by the idh-1neo to Δdhgd-1 (D-2HG dehydrogenase) mutant animals, which also accumulate D-2HG, we identified a specific vitamin B12 diet-dependent vulnerability in idh-1neo mutant animals that leads to increased embryonic lethality. Through a genetic screen we found that impairment of the glycine cleavage system, which generates one-carbon donor units, exacerbates this phenotype. Additionally, supplementation with an alternate source of one-carbon donors suppresses the lethal phenotype. Our results indicate that the idh-1neo mutation imposes a heightened dependency on the one-carbon pool and provides a further understanding how this oncogenic mutation rewires cellular metabolism.

DOI: https://doi.org/10.1101/2024.03.13.584865
J Cancer Res Clin Oncol. 2024 Apr 03. 150(4): 172

An algorithm-based technique for counting mitochondria in cells using immunohistochemical staining of formalin-fixed and paraffin-embedded sections.

Mai Sakashita, Noriko Motoi, Gaku Yamamoto, Emi Gambe, Masanori Suzuki, Yukihiro Yoshida, Shun-Ichi Watanabe, Yutaka Takazawa, Kazunori Aoki, Atsushi Ochiai, Shingo Sakashita.

  PURPOSE: Visualizing mitochondria in cancer cells from human pathological specimens may improve our understanding of cancer biology. However, using immunohistochemistry to evaluate mitochondria remains difficult because almost all cells contain mitochondria and the number of mitochondria per cell may have important effects on mitochondrial function. Herein, we established an objective system (Mito-score) for evaluating mitochondria using machine-based processing of hue, saturation, and value color spaces.METHODS: The Mito-score was defined as the number of COX4 (mitochondrial inner membrane) immunohistochemistry-positive pixels divided by the number of nuclei per cell. The system was validated using four lung cancer cell lines, normal tissues, and lung cancer tissues (199 cases).
RESULTS: The Mito-score correlated with MitoTracker, a fluorescent dye used to selectively label and visualize mitochondria within cells under a microscope (R2 = 0.68) and with the number of mitochondria counted using electron microscopy (R2 = 0.79). Histologically, the Mito-score of small cell carcinoma (57.25) was significantly lower than that of adenocarcinoma (147.5, p < 0.0001), squamous cell carcinoma (120.6, p = 0.0004), and large cell neuroendocrine carcinoma (111.8, p = 0.002).
CONCLUSION: The Mito-score method enables the analysis of the mitochondrial status of human formalin-fixed paraffin-embedded specimens and may provide insights into the metabolic status of cancer.

Keywords:  COX4; Cancer; MitoTracker; Mitochondria

DOI:  https://doi.org/10.1007/s00432-024-05653-1
Life Metab. 2024 Jun;pii: load051. [Epub ahead of print]3(3):

The energetics of cellular life transitions.

Anna S Monzel, Michael Levin, Martin Picard.

  Major life transitions are always difficult because change costs energy. Recent findings have demonstrated how mitochondrial oxidative phosphorylation (OxPhos) defects increase the energetic cost of living, and that excessive integrated stress response (ISR) signaling may prevent cellular identity transitions during development. In this perspective, we discuss general bioenergetic principles of life transitions and the costly molecular processes involved in reprograming the cellular hardware/software as cells shift identity. The energetic cost of cellular differentiation has not been directly quantified, representing a gap in knowledge. We propose that the ISR is an energetic checkpoint evolved to i) prevent OxPhos-deficient cells from engaging in excessively costly transitions, and ii) allow ISR-positive cells to recruit systemic energetic resources by signaling via the brain.

Keywords:  GDF15; development; energy; energy balance; mitochondria; signaling pathway

DOI:  https://doi.org/10.1093/lifemeta/load051
Front Pharmacol. 2024 ;15 1332042

Mitochondrial energy metabolism-related gene signature as a prognostic indicator for pancreatic adenocarcinoma.

Yu Ma, Ronghao Tang, Peilin Huang, Danhua Li, Meijian Liao, Shoucui Gao.

  Background: Pancreatic adenocarcinoma (PAAD) is a highly malignant gastrointestinal tumor and is associated with an unfavorable prognosis worldwide. Considering the effect of mitochondrial metabolism on the prognosis of pancreatic cancer has rarely been investigated, we aimed to establish prognostic gene markers associated with mitochondrial energy metabolism for the prediction of survival probability in patients with PAAD. Methods: Gene expression data were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases, and the mitochondrial energy metabolism-related genes were obtained from the GeneCards database. Based on mitochondrial energy metabolism score (MMs), differentially expressed MMRGs were established for MMs-high and MMs-low groups using ssGSEA. After the univariate Cox and least absolute and selection operator (LASSO) analyses, a prognostic MMRG signature was used in the multivariate Cox proportional regression model. Survival and immune cell infiltration analyses were performed. In addition, a nomogram based on the risk model was used to predict the survival probability of patients with PAAD. Finally, the expression of key genes was verified using quantitative polymerase chain reaction and immunohistochemical staining. Intro cell experiments were performed to evaluated the proliferation and invasion of pancreatic cancer cells. Results: A prognostic signature was constructed consisting of two mitochondrial energy metabolism-related genes (MMP11, COL10A1). Calibration and receiver operating characteristic (ROC) curves verified the good predictability performance of the risk model for the survival rate of patients with PAAD. Finally, immune-related analysis explained the differences in immune status between the two subgroups based on the risk model. The high-risk score group showed higher estimate, immune, and stromal scores, expression of eight checkpoint genes, and infiltration of M0 macrophages, which might indicate a beneficial response to immunotherapy. The qPCR results confirmed high expression of MMP11 in pancreatic cancer cell lines, and IHC also verified high expression of MMP11 in clinical pancreatic ductal adenocarcinoma tissues. In vitro cell experiments also demonstrated the role of MMP11 in cell proliferation and invasion. Conclusion: Our study provides a novel two-prognostic gene signature-based on MMRGs-that accurately predicted the survival of patients with PAAD and could be used for mitochondrial energy metabolism-related therapies in the future.

Keywords:  bioinformatics; immune; mitochondrial energy metabolism; pancreatic adenocarcinoma; prognostic signature

DOI:  https://doi.org/10.3389/fphar.2024.1332042
Free Radic Biol Med. 2024 Apr 03. pii: S0891-5849(24)00373-3. [Epub ahead of print]

Peroxiredoxin 6 suppresses ferroptosis in lung endothelial cells.

Julia María Torres-Velarde, Kaitlin N Allen, Andrea Salvador-Pascual, Roberto G Leija, Diamond Luong, Diana Daniela Moreno-Santillán, David C Ensminger, José Pablo Vázquez-Medina.

  Peroxiredoxin 6 (Prdx6) repairs peroxidized membranes by reducing oxidized phospholipids, and by replacing oxidized sn-2 fatty acyl groups through hydrolysis/reacylation by its phospholipase A2 (aiPLA2) and lysophosphatidylcholine acyltransferase activities. Prdx6 is highly expressed in the lung, and intact lungs and cells null for Prdx6 or with single-point mutations that inactivate either Prdx6-peroxidase or aiPLA2 activity alone exhibit decreased viability, increased lipid peroxidation, and incomplete repair when exposed to paraquat, hyperoxia, or organic peroxides. Ferroptosis is form of cell death driven by the accumulation of phospholipid hydroperoxides. We studied the role of Prdx6 as a ferroptosis suppressor in the lung. We first compared the expression Prdx6 and glutathione peroxidase 4 (GPx4) and visualized Prdx6 and GPx4 within the lung. Lung Prdx6 mRNA levels were five times higher than GPx4 levels. Both Prdx6 and GPx4 localized to epithelial and endothelial cells. Prdx6 knockout or knockdown sensitized lung endothelial cells to erastin-induced ferroptosis. Cells with genetic inactivation of either aiPLA2 or Prdx6-peroxidase were more sensitive to ferroptosis than WT cells, but less sensitive than KO cells. We then conducted RNA-seq analyses in Prdx6-depleted cells to further explore how the loss of Prdx6 sensitizes lung endothelial cells to ferroptosis. Prdx6 KD upregulated transcriptional signatures associated with selenoamino acid metabolism and mitochondrial function. Accordingly, Prdx6 deficiency blunted mitochondrial function and increased GPx4 abundance whereas GPx4 KD had the opposite effect on Prdx6. Moreover, we detected Prdx6 and GPx4 interactions in intact cells, suggesting that both enzymes cooperate to suppress lipid peroxidation. Notably, Prdx6-depleted cells remained sensitive to erastin-induced ferroptosis despite the compensatory increase in GPx4. These results show that Prdx6 suppresses ferroptosis in lung endothelial cells and that both aiPLA2 and Prdx6-peroxidase contribute to this effect. These results also show that Prdx6 supports mitochondrial function and modulates several coordinated cytoprotective pathways in the pulmonary endothelium.

Keywords:  14-3-3ε; Cell death; GPx4; Integrated stress response; Mitochondria; TP53; aiPLA(2)

DOI:  https://doi.org/10.1016/j.freeradbiomed.2024.04.208
Sci Rep. 2024 04 02. 14(1): 7739

Identification and structural characterization of small molecule inhibitors of PINK1.

Shafqat Rasool, Tara Shomali, Luc Truong, Nathalie Croteau, Simon Veyron, Bernardo A Bustillos, Wolfdieter Springer, Fabienne C Fiesel, Jean-François Trempe.

  Mutations in PINK1 and Parkin cause early-onset Parkinson's Disease (PD). PINK1 is a kinase which functions as a mitochondrial damage sensor and initiates mitochondrial quality control by accumulating on the damaged organelle. There, it phosphorylates ubiquitin, which in turn recruits and activates Parkin, an E3 ubiquitin ligase. Ubiquitylation of mitochondrial proteins leads to the autophagic degradation of the damaged organelle. Pharmacological modulation of PINK1 constitutes an appealing avenue to study its physiological function and develop therapeutics. In this study, we used a thermal shift assay with insect PINK1 to identify small molecules that inhibit ATP hydrolysis and ubiquitin phosphorylation. PRT062607, an SYK inhibitor, is the most potent inhibitor in our screen and inhibits both insect and human PINK1, with an IC50 in the 0.5-3 µM range in HeLa cells and dopaminergic neurons. The crystal structures of insect PINK1 bound to PRT062607 or CYC116 reveal how the compounds interact with the ATP-binding pocket. PRT062607 notably engages with the catalytic aspartate and causes a destabilization of insert-2 at the autophosphorylation dimer interface. While PRT062607 is not selective for PINK1, it provides a scaffold for the development of more selective and potent inhibitors of PINK1 that could be used as chemical probes.

Keywords:  Inhibitor; Kinase; Mitochondria; PTEN‐induced kinase 1 (PINK1); Parkinson disease; Ubiquitin

DOI:  https://doi.org/10.1038/s41598-024-58285-3
EMBO J. 2024 Apr 05.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

Joycelyn Tan, Sam Virtue, Dougall M Norris, Olivia J Conway, Ming Yang, Guillaume Bidault, Christopher Gribben, Fatima Lugtu, Ioannis Kamzolas, James R Krycer, Richard J Mills, Lu Liang, Conceição Pereira, Martin Dale, Amber S Shun-Shion, Harry Jm Baird, James A Horscroft, Alice P Sowton, Marcella Ma, Stefania Carobbio, Evangelia Petsalaki, Andrew J Murray, David C Gershlick, James A Nathan, James E Hudson, Ludovic Vallier, Kelsey H Fisher-Wellman, Christian Frezza, Antonio Vidal-Puig, Daniel J Fazakerley.

  The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Keywords:  Cell Culture; Hypoxia; Metabolism/Adipocytes; Oxygen Tension

DOI:  https://doi.org/10.1038/s44318-024-00084-7
Mol Ther Methods Clin Dev. 2024 Jun 13. 32(2): 101231

mtDNA analysis using Mitopore.

Jochen Dobner, Thach Nguyen, Mario Gustavo Pavez-Giani, Lukas Cyganek, Felix Distelmaier, Jean Krutmann, Alessandro Prigione, Andrea Rossi.

  Mitochondrial DNA (mtDNA) analysis is crucial for the diagnosis of mitochondrial disorders, forensic investigations, and basic research. Existing pipelines are complex, expensive, and require specialized personnel. In many cases, including the diagnosis of detrimental single nucleotide variants (SNVs), mtDNA analysis is still carried out using Sanger sequencing. Here, we developed a simple workflow and a publicly available webserver named Mitopore that allows the detection of mtDNA SNVs, indels, and haplogroups. To simplify mtDNA analysis, we tailored our workflow to process noisy long-read sequencing data for mtDNA analysis, focusing on sequence alignment and parameter optimization. We implemented Mitopore with eliBQ (eliminate bad quality reads), an innovative quality enhancement that permits the increase of per-base quality of over 20% for low-quality data. The whole Mitopore workflow and webserver were validated using patient-derived and induced pluripotent stem cells harboring mtDNA mutations. Mitopore streamlines mtDNA analysis as an easy-to-use fast, reliable, and cost-effective analysis method for both long- and short-read sequencing data. This significantly enhances the accessibility of mtDNA analysis and reduces the cost per sample, contributing to the progress of mtDNA-related research and diagnosis.

Keywords:  Mitopore; ONT; clinical mtDNA diagnosis; forensic mtDNA analysis; haplogroups; iPSCs quality control; long-read sequencing; mtDNA analysis; mtDNA heteroplasmy; webserver

DOI:  https://doi.org/10.1016/j.omtm.2024.101231