{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,11,23]],"date-time":"2025-11-23T20:03:11Z","timestamp":1763928191293,"version":"3.45.0"},"reference-count":41,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T00:00:00Z","timestamp":1760227200000},"content-version":"vor","delay-in-days":11,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["FI965\/5\u20101","FI965\/10\u20101"],"award-info":[{"award-number":["FI965\/5\u20101","FI965\/10\u20101"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["J cachexia sarcopenia muscle"],"published-print":{"date-parts":[[2025,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Muscle RING finger (MuRF) proteins are striated muscle\u2010specific E3 ubiquitin ligases essential for muscle homeostasis. Whereas MuRF1 is well known for its role in muscle atrophy, MuRF2 and MuRF3 contribute to microtubule stabilization, influencing muscle differentiation and function. Their cooperative functions in regulating myogenesis are unclear. This study aimed to identify novel MuRF2 and MuRF3 interaction partners and investigate their function in myogenic differentiation.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>Interaction partners of MuRF2 and MuRF3 were identified using stable isotope labelling with amino acids in cell culture (SILAC), followed by affinity purification and quantitative mass spectrometry (AP\u2010MS). Mechanistic analyses included co\u2010immunoprecipitation, domain mapping, ubiquitination assays, protein stability measurements and endosome isolation. Myogenic differentiation was evaluated by immunocytochemistry, qRT\u2010PCR and western blotting. Functional effects were assessed using CRISPR\u2010Cas9\u2010mediated knockout and siRNA silencing.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      We identified sorting nexin 5 (SNX5), a BAR and PX domain\u2010containing retromer component involved in retrograde vesicular transport, as a novel MuRF2 and MuRF3 binding partner. Both coiled\u2010coil domains of MuRF3 were required for SNX5 binding, and the BAR domain of SNX5 mediated interaction with MuRF2 and MuRF3. Immunofluorescence staining demonstrated MuRF3\u2013SNX5 interaction and colocalization on early endosomes along microtubules in myocytes. MuRF2 promoted ubiquitination of SNX5 at lysines 290 and 324, leading to proteasomal degradation, whereas MuRF3 counteracted this effect. Mass spectrometry revealed the protein kinase A regulatory subunit (PKA\u2010RI\u2010\u03b1) as cargo of SNX5\u2010coated early endosomes in myocytes. SNX5 knockout (SNX5\u2010KO) reduced RI\u2010\u03b1 stability in myocytes, enhanced PKA activity and increased HDAC5 degradation via the autophagy\u2010lysosomal pathway, leading to MEF2\u2010mediated upregulation of myostatin. SNX5\u2010KO impaired myogenesis, with significant reductions in myogenin\/\n                      <jats:italic>Myog<\/jats:italic>\n                      (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.005), myomaker\/\n                      <jats:italic>Mymk<\/jats:italic>\n                      (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.01), myomerger\/\n                      <jats:italic>Mymx<\/jats:italic>\n                      (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.005) and MyHC isoforms\n                      <jats:italic>Myh2<\/jats:italic>\n                      and\n                      <jats:italic>Myh4<\/jats:italic>\n                      (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.01). Myostatin treatment mimicked the SNX5\u2010KO phenotype, reducing fast\u2010twitch MyHC isoforms\n                      <jats:italic>Myh1<\/jats:italic>\n                      ,\n                      <jats:italic>Myh2<\/jats:italic>\n                      ,\n                      <jats:italic>Myh3<\/jats:italic>\n                      and\n                      <jats:italic>Myh4<\/jats:italic>\n                      (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.05 for all) and significantly lowering Myomaker, Myomerger and MyHC expression throughout differentiation (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.05 for all). Morphologically, myostatin\u2010treated cells were shorter and thinner and had fewer nuclei. Quantification showed reduced differentiation and fusion indices (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.001) and fewer nuclei per myosin\u2010positive cell (\n                      <jats:italic>p<\/jats:italic>\n                      \u2009&lt;\u20090.01).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>MuRF2 and MuRF3 exert opposing effects on SNX5\u2010mediated retrograde transport, influencing PKA signalling and myogenic differentiation. SNX5 stabilizes RI\u2010\u03b1 within early endosomes, facilitating ordered myogenic differentiation. Our findings expand the known functions of MuRF proteins beyond proteasomal degradation and identify SNX5 as a key regulator of PKA activity in muscle cells. These insights may provide novel therapeutic targets for muscle\u2010related disorders.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/jcsm.70103","type":"journal-article","created":{"date-parts":[[2025,10,13]],"date-time":"2025-10-13T04:54:27Z","timestamp":1760331267000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Novel MuRF2 Target SNX5 Regulates PKA Activity Through Stabilization of RI\u2010\u03b1 and Controls Myogenic Differentiation"],"prefix":"10.1002","volume":"16","author":[{"given":"Ning","family":"Li","sequence":"first","affiliation":[{"name":"DZHK (German Center for Cardiovascular Research), partner site Greifswald  Greifswald Germany"},{"name":"Department of Internal Medicine B, Cardiology University Medicine Greifswald  Greifswald 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Universit\u00e4tsmedizin Berlin Max Delbr\u00fcck Center for Molecular Medicine in the Helmholtz Association  Berlin Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elke","family":"Hammer","sequence":"additional","affiliation":[{"name":"DZHK (German Center for Cardiovascular Research), partner site Greifswald  Greifswald Germany"},{"name":"Interfaculty Institute for Genetics and Functional Genomics University Medicine Greifswald  Greifswald Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Britta","family":"Fielitz","sequence":"additional","affiliation":[{"name":"DZHK (German Center for Cardiovascular Research), partner site Greifswald  Greifswald Germany"},{"name":"Department of Internal Medicine B, Cardiology University Medicine Greifswald  Greifswald Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Uwe","family":"V\u00f6lker","sequence":"additional","affiliation":[{"name":"DZHK (German Center for Cardiovascular 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