bims-smemid 2024-07-14 papers

on Stress metabolism in mitochondrial dysfunction

Issue of 2024–07–14
six papers selected by
Deepti Mudartha, The International Institute of Molecular Mechanisms and Machines

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines.
The iAAA-mitochondrial protease YME1L1 regulates the degradation of the short-lived mitochondrial transporter SLC25A38.
A roadmap for ribosome assembly in human mitochondria.
The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.
An efficient method for immortalizing mouse embryonic fibroblasts.
Mouse vascularized adipose spheroids: an organotypic model for thermogenic adipocytes.

Mitochondrion. 2024 Jul 08. pii: S1567-7249(24)00091-6. [Epub ahead of print]78 101933

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines.

Harpreet Kaur, Omar Carrillo, Iraselia Garcia, Isaiah Ramos, Shaynah St Vallier, Patrick De La Torre, Alma Lopez, Megan Keniry, Daniel Bazan, Jorge Elizondo, K C Grishma, Lee Ann MacMillan-Crow, Robert Gilkerson.

  Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψm) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψm is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis. Previously, we demonstrated that L-OPA1 of H9c2 cardiomyoblasts is insensitive to loss of Δψm via challenge with the protonophore carbonyl cyanide chlorophenyl hydrazone (CCCP), but that CCCP-induced OPA1 processing is activated upon differentiation in media with low serum supplemented with all-trans retinoic acid (ATRA). Here, we show that this developmental induction of OPA1 processing in H9c2 cells is independent of ATRA; moreover, pretreatment of undifferentiated H9c2s with chloramphenicol (CAP), an inhibitor of mitochondrial protein synthesis, recapitulates the Δψm-sensitive OPA1 processing observed in differentiated H9c2s. L6.C11 and C2C12 myoblast lines display the same developmental and CAP-sensitive induction of OPA1 processing, demonstrating a general mechanism of OPA1 regulation in mammalian myoblast cell settings. Restoration of CCCP-induced OPA1 processing correlates with increased apoptotic sensitivity. Moreover, OPA1 knockdown indicates that intact OPA1 is necessary for effective myoblast differentiation. Taken together, our results indicate that a novel developmental mechanism acts to regulate OMA1-mediated OPA1 processing in myoblast cell lines, in which differentiation engages mitochondrial stress sensing.

Keywords:  Differentiation; Mitochondria; OMA1; OPA1; Transmembrane potential

DOI:  https://doi.org/10.1016/j.mito.2024.101933
bioRxiv. 2024 Jun 24. pii: 2024.05.12.593764. [Epub ahead of print]

The iAAA-mitochondrial protease YME1L1 regulates the degradation of the short-lived mitochondrial transporter SLC25A38.

Sijie Tan, Alisa Susan Dengler, Rami Zahi Darawsheh, Nora Kory.

Mitochondrial transporters facilitate the exchange of diverse metabolic intermediates across the inner mitochondrial membrane, ensuring an adequate supply of substrates and cofactors to support redox and biosynthetic reactions within the mitochondrial matrix. However, the regulatory mechanisms governing the abundance of these transporters, crucial for maintaining metabolic compartmentalization and mitochondrial functions, remain poorly defined. Through analysis of protein half-life data and mRNA-protein correlations, we identified SLC25A38, a mitochondrial glycine transporter, as a short- lived protein with a half-life of 4 hours under steady-state conditions. Pharmacological inhibition and genetic depletion of various cellular proteolytic systems revealed that SLC25A38 is rapidly degraded by the iAAA-mitochondrial protease YME1L1. Depolarization of the mitochondrial membrane potential induced by the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrozone prevented the degradation of SLC25A38. This dual regulation of SLC25A38 abundance by YME1L1 and mitochondrial membrane potential suggests a link between SLC25A38 turnover, the integrity of the inner mitochondrial membrane, and electron transport chain function. These findings open avenues for investigating whether mitochondrial glycine import coordinates with mitochondrial bioenergetics.

DOI: https://doi.org/10.1101/2024.05.12.593764
Nat Struct Mol Biol. 2024 Jul 11.

A roadmap for ribosome assembly in human mitochondria.

Elena Lavdovskaia, Elisa Hanitsch, Andreas Linden, Martin Pašen, Venkatapathi Challa, Yehor Horokhovskyi, Hanna P Roetschke, Franziska Nadler, Luisa Welp, Emely Steube, Marleen Heinrichs, Mandy Mong-Quyen Mai, Henning Urlaub, Juliane Liepe, Ricarda Richter-Dennerlein.

Mitochondria contain dedicated ribosomes (mitoribosomes), which synthesize the mitochondrial-encoded core components of the oxidative phosphorylation complexes. The RNA and protein components of mitoribosomes are encoded on two different genomes (mitochondrial and nuclear) and are assembled into functional complexes with the help of dedicated factors inside the organelle. Defects in mitoribosome biogenesis are associated with severe human diseases, yet the molecular pathway of mitoribosome assembly remains poorly understood. Here, we applied a multidisciplinary approach combining biochemical isolation and analysis of native mitoribosomal assembly complexes with quantitative mass spectrometry and mathematical modeling to reconstitute the entire assembly pathway of the human mitoribosome. We show that, in contrast to its bacterial and cytosolic counterparts, human mitoribosome biogenesis involves the formation of ribosomal protein-only modules, which then assemble on the appropriate ribosomal RNA moiety in a coordinated fashion. The presence of excess protein-only modules primed for assembly rationalizes how mitochondria cope with the challenge of forming a protein-rich ribonucleoprotein complex of dual genetic origin. This study provides a comprehensive roadmap of mitoribosome biogenesis, from very early to late maturation steps, and highlights the evolutionary divergence from its bacterial ancestor.

DOI: https://doi.org/10.1038/s41594-024-01356-w
EMBO Rep. 2024 Jul 09.

The IRE1α/XBP1 signaling axis drives myoblast fusion in adult skeletal muscle.

Aniket S Joshi, Meiricris Tomaz da Silva, Anirban Roy, Tatiana E Koike, Mingfu Wu, Micah B Castillo, Preethi H Gunaratne, Yu Liu, Takao Iwawaki, Ashok Kumar.

  Skeletal muscle regeneration involves a signaling network that regulates the proliferation, differentiation, and fusion of muscle precursor cells to injured myofibers. IRE1α, one of the arms of the unfolded protein response, regulates cellular proteostasis in response to ER stress. Here, we demonstrate that inducible deletion of IRE1α in satellite cells of mice impairs skeletal muscle regeneration through inhibiting myoblast fusion. Knockdown of IRE1α or its downstream target, X-box protein 1 (XBP1), also inhibits myoblast fusion during myogenesis. Transcriptome analysis revealed that knockdown of IRE1α or XBP1 dysregulates the gene expression of molecules involved in myoblast fusion. The IRE1α-XBP1 axis mediates the gene expression of multiple profusion molecules, including myomaker (Mymk). Spliced XBP1 (sXBP1) transcription factor binds to the promoter of Mymk gene during myogenesis. Overexpression of myomaker in IRE1α-knockdown cultures rescues fusion defects. Inducible deletion of IRE1α in satellite cells also inhibits myoblast fusion and myofiber hypertrophy in response to functional overload. Collectively, our study demonstrates that IRE1α promotes myoblast fusion through sXBP1-mediated up-regulation of the gene expression of multiple profusion molecules, including myomaker.

Keywords:  IRE1; Muscle Regeneration; Myoblast Fusion; XBP1; and Myomaker

DOI:  https://doi.org/10.1038/s44319-024-00197-4
bioRxiv. 2024 Jun 30. pii: 2024.06.27.601088. [Epub ahead of print]

An efficient method for immortalizing mouse embryonic fibroblasts.

Srisathya Srinivasan, Hsin-Yi Henry Ho.

Mouse embryonic fibroblasts (MEFs) derived from genetically modified mice are a valuable resource for studying gene function and regulation. The MEF system can also be combined with rescue studies to characterize the function of mutant genes/proteins, such as disease-causing variants. However, primary MEFs undergo senescence soon after isolation and passaging, making long-term genetic manipulations difficult. Previously described methods for MEF immortalization are often inefficient or alter the physiological properties of the cells. Here, we describe an optimized protocol for immortalizing MEFs via CRISPR-mediated deletion of the Tp53 gene. This method is highly efficient and consistently generates immortalized MEFs, or iMEFs, within 14 days. Importantly, iMEFs closely resemble the parent cell populations, and individual iMEFs can be cloned and expanded for subsequent genetic manipulation and characterization. We envision that this protocol can be adopted to immortalize other mouse primary cell types.
Key Features: CRISPR-based knockout of the Tp53 gene enables efficient immortalization of mouse embryonic fibroblasts (MEFs) in under 2 weeks. Immortalization requires a Neon electroporator or another comparable electroporation system to transfect cells with the Tp53 CRISPR constructs.

DOI: https://doi.org/10.1101/2024.06.27.601088
Front Endocrinol (Lausanne). 2024 ;15 1396965

Mouse vascularized adipose spheroids: an organotypic model for thermogenic adipocytes.

Laura Ingeborg Davidsen, Carolina E Hagberg, Victor Goitea, Stine Meinild Lundby, Steen Larsen, Morten Frendø Ebbesen, Natasha Stanic, Hande Topel, Jan-Wilhelm Kornfeld.

  Adipose tissues, particularly beige and brown adipose tissue, play crucial roles in energy metabolism. Brown adipose tissues' thermogenic capacity and the appearance of beige cells within white adipose tissue have spurred interest in their metabolic impact and therapeutic potential. Brown and beige fat cells, activated by environmental factors like cold exposure or by pharmacology, share metabolic mechanisms that drive non-shivering thermogenesis. Understanding these two cell types requires advanced, yet broadly applicable in vitro models that reflect the complex microenvironment and vasculature of adipose tissues. Here we present mouse vascularized adipose spheroids of the stromal vascular microenvironment from inguinal white adipose tissue, a tissue with 'beiging' capacity in mice and humans. We show that adding a scaffold improves vascular sprouting, enhances spheroid growth, and upregulates adipogenic markers, thus reflecting increased adipocyte maturity. Transcriptional profiling via RNA sequencing revealed distinct metabolic pathways upregulated in our vascularized adipose spheroids, with increased expression of genes involved in glucose metabolism, lipid metabolism, and thermogenesis. Functional assessment demonstrated increased oxygen consumption in vascularized adipose spheroids compared to classical 2D cultures, which was enhanced by β-adrenergic receptor stimulation correlating with elevated β-adrenergic receptor expression. Moreover, stimulation with the naturally occurring adipokine, FGF21, induced Ucp1 mRNA expression in the vascularized adipose spheroids. In conclusion, vascularized inguinal white adipose tissue spheroids provide a physiologically relevant platform to study how the stromal vascular microenvironment shapes adipocyte responses and influence activated thermogenesis in beige adipocytes.

Keywords:  3D spheroids; adipose tissue microenvironment; browning; crosstalk; metabolism; oxygraph; thermogenesis; vascularization

DOI:  https://doi.org/10.3389/fendo.2024.1396965