bims-micpro 2025-09-07 papers

Sci Adv. 2025 Aug 29. 11(35): eads7381

Abnormal mitochondrial structure and function in brown adipose tissue of SLC35A4-MP knockout mice.

Andréa L Rocha, Christian Schmedt, Guy Perkins, Antonio Pinto, Jolene K Diedrich, Huanqi Shan, Kaja Plucińska, Eduardo Vieira de Souza, Joan M Vaughan, Mark Foster, Srinath C Sampath, Srihari C Sampath, Paul Cohen, Mark H Ellisman, Alan Saghatelian.

Uncovering the role of upstream open reading frames (uORFs) challenges conventional views of one protein per messenger RNA and reveals the capacity of some uORFs to encode microproteins that contribute to cellular biology and physiology. This study explores the functional role of a recently identified mitochondrial microprotein, SLC35A4-MP, in the brown adipose tissue of mice. Our findings reveal dynamic regulation of SLC35A4-MP expression during primary brown adipocyte differentiation in vitro and during cold exposure or high-fat diet (HFD)-induced obesity in mice. Using a knockout mouse model, we show that loss of SLC35A4-MP disrupts mitochondrial lipid composition, decreasing cardiolipins and phosphatidylethanolamine in brown adipose tissue from HFD-fed mice. SLC35A4-MP deficiency also impairs mitochondrial activity, alters mitochondrial number and morphology, and promotes inflammation. Knockout mice accumulate acylcarnitines during cold exposure, indicating defective fatty acid oxidation. These findings reveal SLC35A4-MP as a previously unrecognized microprotein in regulating mitochondrial function and tissue lipid metabolism, adding to the growing list of functional endogenous microproteins.

DOI: https://doi.org/10.1126/sciadv.ads7381

Circ Heart Fail. 2025 Sep 04. e013381

Mitochondrial sORF-Encoded Peptide MODICA Protects the Heart From Doxorubicin-Induced Cardiac Injury by Suppressing VDAC Oligomerization.

Jialing Wu, Kang Li, Youchen Yan, Xingfeng Xu, Ting Xu, He Xu, Huimin Zhou, Tailai Du, Yan Li, Chen Liu, Xinxue Liao, Yugang Dong, Jing-Song Ou, Yili Chen, Zhan-Peng Huang.

BACKGROUND: Doxorubicin (DOX) cardiotoxicity increases cardiovascular risk in cancer patients, mainly through mitochondrial damage. However, the underlying mechanisms remain unclear, and whether mitochondrial short open reading frame-encoded peptides can mitigate DOX-induced cardiotoxicity is unknown.
METHODS: Five adeno-associated viruses expressing mitochondrial short open reading frame-encoded peptides under the cardiac troponin T promoter, including MODICA (mito-SEP protector against DOX-induced cardiac injury), were screened in a DOX-induced cardiotoxicity mouse model (n=3-5 per group). Male and female mice were randomized to adeno-associated virus-CTRL or adeno-associated virus-MODICA, respectively, combined with saline or DOX treatment. Sample sizes were: males-saline-CTRL (n=4), saline-MODICA (n=4), DOX-CTRL (n=11), DOX-MODICA (n=10); females-saline-CTRL (n=8), saline-MODICA (n=10), DOX-CTRL (n=10), DOX-MODICA (n=13). MODICA-heterozygous mice generated by CRISPR/Cas9 were also included: saline-WT (n=7), saline-heterozygous (n=4), DOX-WT (n=11), DOX-heterozygous (n=8). Echocardiography was performed at baseline and after 2 weeks of DOX treatment; myocardial tissue and serum samples were collected for molecular and histological analyses.
RESULTS: The mitochondrial short open reading frame-encoded peptide MODICA was identified through biochemical analysis and functional screening in a DOX-induced cardiac injury model. MODICA localizes to the outer mitochondrial membrane and is significantly downregulated by DOX (1.00±0.08 versus 0.42±0.09; P<0.001). Cardiac-specific overexpression of MODICA via adeno-associated viruses significantly attenuated DOX-induced cardiac injury in both males and females (fractional shortening: males 38.86% versus 51.54%, P<0.001; females 39.81% versus 51.39%, P<0.001, DOX-CTRL versus DOX-MODICA) and was supported by bulk RNA-seq analysis. Conversely, MODICA deficiency exacerbated DOX-induced injury, resulting in reduced fractional shortening (40.37% versus 31.85%, P<0.001; DOX-WT versus DOX-heterozygous) and increased cardiac fibrosis (P=0.009). Proteomic analyses revealed that MODICA interacts with apoptosis-related voltage-dependent anion channel proteins, inhibiting their DOX-induced oligomerization (P<0.001) on the outer mitochondrial membrane, thereby reducing mitochondrial permeability, decreasing cardiomyocyte apoptosis and improving calcium handling.
CONCLUSIONS: Our study shows that the mitochondrial short open reading frame-encoded peptide MODICA alleviates DOX-induced cardiac dysfunction and may represent a therapeutic target against DOX cardiotoxicity.

Keywords: animals; heart diseases; peoptides; risk factors; sample size

DOI: https://doi.org/10.1161/CIRCHEARTFAILURE.125.013381

Diabetes. 2025 Sep 02. pii: db240527. [Epub ahead of print]

Proteogenomic Discovery of Novel Open Reading Frames With HLA Immune Presentation on Human β-Cells.

Kathryn Walters, Roberto Castro-Gutierrez, Soumyadeep Sarkar, Amber Baldwin, Alexandra S Baker, Ali H Shilleh, Amanda M Anderson, Maki Nakayama, Tim Fugman, Ernesto S Nakayasu, Neelanjan Mukherjee, Holger A Russ.

ARTICLE HIGHLIGHTS: We developed a cell type-specific proteogenomic approach to reveal novel or unannotated open reading frames (nuORFs) using transcriptomics, ribosomal profiling, and proteomic analysis of human pancreatic β-cells using stem cell-derived β-cells and/or cadaveric islets. Our analysis revealed translational regulation of β-cell-specific pathways during differentiation and identified 965 nuORFs, with a majority exhibiting protein support and substantial β-cell specificity. A primate-specific ORF located in the 5' untranslated region of the type 1 diabetes risk gene TYK2 may act as a translational activator. We provide HLA class I immunopeptidomic data from cytokine-stimulated human β-cells and demonstrate their utility in coculture assays with autoreactive T-cell transductants. Taken together, our results define the human β-cell translatome, an important resource to the research field.

DOI: https://doi.org/10.2337/db24-0527

Biochemistry. 2025 Sep 05.

Structural Elements of Dwarf Open Reading Frame Required for Activation of the Sarco-Endoplasmic Reticulum Calcium Pump.

M'Lynn E Fisher, Justin R Gregory, Stan T J Aanhane, M Joanne Lemieux, Howard S Young.

The sarco-endoplasmic reticulum calcium pump (SERCA) is a P-type ATPase that plays a critical role in intracellular calcium signaling. SERCA maintains the calcium gradient between the cytosol and the sarco-endoplasmic reticulum, which is essential for a variety of physiological events including the muscle contraction-relaxation cycle. In cardiac muscle, SERCA is regulated by transmembrane peptides phospholamban (PLN) and dwarf open reading frame (DWORF). These peptides encode the opposing functions of SERCA inhibition by PLN and SERCA activation by DWORF, though the underlying mechanisms remain unclear. Herein, we investigated structural elements of DWORF expected to play a role in SERCA activation. We first measured SERCA activity in the absence and presence of DWORF variants targeting Leu12 and Pro15. These residues were selected based on sequence alignment with PLN. Leu12 and Pro15 of DWORF align with the essential residues Leu31 and Asn34 of PLN, which are required for SERCA inhibition. We found that both residues are required for SERCA activation by DWORF and that substitution of Pro15 (to Ala, Asn, or Leu) resulted in potent inhibition of SERCA. We next investigated the roles of Gly21, Ile23, and Gly25 in SERCA activation and DWORF oligomerization. These residues are part of a common helix interaction motif, GxxxG (Gly21-Trp-Ile-Val-Gly25) found in DWORF, which is unique among the regulins. The data suggest that the GxxxG motif does not play a role in DWORF oligomerization. Instead, this motif appears to interact with SERCA and provides a smooth interface that promotes activation and avoids inhibitory interactions with SERCA.

DOI: https://doi.org/10.1021/acs.biochem.5c00305

Circ Res. 2025 Sep 05.

DWORF Gene Therapy Improves Cardiac Calcium Handling and Mitochondrial Function.

Omar Brito-Estrada, Yasuhide Kuwabara, Aaron M Gibson, Keira R Hassel, Michael L Kamradt, Joseph P Verry, N Scott Blair, Michael J Bround, Jiuzhou Huo, Jeffery D Molkentin, Catherine A Makarewich.

BACKGROUND: Calcium (Ca2+) dysregulation is a hallmark of heart failure, impairing excitation-contraction coupling and contributing to pathological remodeling. The SERCA2a (sarco/endoplasmic reticulum Ca2+ ATPase isoform 2a) mediates Ca2+ reuptake into the sarcoplasmic reticulum (SR) during diastole, but its activity declines in failing hearts. DWORF (dwarf open reading frame), a newly identified cardiac microprotein, enhances SERCA2a activity and improves cardiomyocyte Ca2+ cycling and contractility. SR Ca2+ release also influences mitochondrial metabolism and ATP production. Here, we investigated whether DWORF overexpression improves SR Ca2+ handling, augments mitochondrial Ca2+ signaling, and protects against heart failure progression.
METHODS: Transgenic and adeno-associated virus approaches were used to overexpress DWORF in the heart. Mice underwent transverse aortic constriction to model pressure overload-induced heart failure. Cardiac function, mitochondrial metabolism, SR Ca2+ uptake, and remodeling were assessed.
RESULTS: Mitochondria from DWORF transgenic hearts displayed increased basal respiration, maximal respiration, and spare respiratory capacity, correlating with enhanced mitochondrial Ca2+ uptake kinetics. Western blot analysis showed elevated levels of active PDH (pyruvate dehydrogenase) and mitochondrial Ca2+ uniporter expression in DWORF transgenic hearts, supporting a role for DWORF in Ca2+-driven metabolic regulation. Similarly, MyoAAV-mediated DWORF overexpression enhanced mitochondrial respiration and increased levels of active PDH in adult mice. Following TAC, MyoAAV-DWORF-treated mice maintained higher left ventricular function and were protected from further deterioration compared with controls. This benefit was observed when DWORF gene therapy was delivered preventively at the time of pressure overload or after heart failure was already established. DWORF gene therapy also attenuated remodeling, with lower heart weight and lung weight-to-tibia length ratios. Seahorse analysis confirmed sustained mitochondrial improvements in both treatment paradigms.
CONCLUSIONS: DWORF overexpression enhances SR Ca2+ dynamics, improves mitochondrial energetics, and attenuates pathological remodeling and heart failure progression in response to pressure overload. These findings support DWORF as a promising therapeutic target for heart failure.

Keywords: calcium; cardiomyopathy, dilated; genetic therapy; mitochondria; myocytes, cardiac

DOI: https://doi.org/10.1161/CIRCRESAHA.125.326550

Exp Eye Res. 2025 Sep 03. pii: S0014-4835(25)00372-0. [Epub ahead of print] 110601

A novel protein encoded by hsa_circ_0068626 contributes to age-related cataract via the p62/Keap1/Nrf2 signaling pathway-mediated ferroptosis.

Yuxin Chen, Huajian Li, Lei Wu, Xi Zou, Yue Zhang, Rongrong Huang, Yong Wang.

This study investigates whether circular RNAs (circRNAs) modulate ferroptosis in lens epithelial cells (LECs) during age-related cataract (ARC) pathogenesis via novel encoded proteins. Initial circRNA-sequencing identified hsa_circ_0068626 (circTFRC) as significantly upregulated in ARC, predominantly localized to the cytoplasm through nuclear-cytoplasmic fractionation and fluorescence in situ hybridization (FISH). Functional assays revealed that circTFRC depletion impaired LECs proliferation and viability, while overexpression exacerbated ferroptosis, evidenced by elevated intracellular reactive oxygen species (ROS) and Fe2+ level via fluorescence probes and flow cytometry. Mechanistically, circTFRC harbored an open reading frame (ORF) and internal ribosome entry site (IRES), enabling translation of the circTFRC-236aa protein, confirmed by polysome profiling and custom antibody detection. Western blot analyses demonstrated that circTFRC-236aa activated the p62/Keap1/Nrf2 axis, correlating with GPX4 suppression and ferroptosis. Transmission electron microscopy further visualized mitochondrial morphological abnormalities consistent with ferroptotic stress. Collectively, these findings establish circTFRC as a pro-ferroptotic regulator in ARC, where its encoded circTFRC-236aa drives pathological progression via p62/Keap1/Nrf2 pathway activation, offering a novel therapeutic target for mitigating ARC-associated LECs damage.

Keywords: Age-related cataract; circular RNAs; ferroptosis; lens epithelial cells; translation

DOI: https://doi.org/10.1016/j.exer.2025.110601