Background: The muscle ring finger (MuRF) protein is an E3 ubiquitin ligase that plays a pivotal role in the maintenance of striated muscle homeostasis. MuRF1 is well known for its role in mediating muscle atrophy, while MuRF2 and MuRF3 have been shown to contribute to microtubule stabilization, thereby influencing muscle differentiation and function. Despite their structural similarity, the cooperative and opposing functions of MuRF2 and MuRF3 remain poorly understood. The objective of this study was to identify and characterize novel MuRF interaction partners and to elucidate their roles in regulating muscle cell function.
Methods and Results: The identification of MuRF3-associated proteins was achieved through the implementation of stable isotope labeling with amino acids in cell culture (SILAC) in conjunction with affinity purification and quantitative mass spectrometry (AP-MS). This proteomic approach revealed sorting nexin 5 (SNX5)—a key component of the retromer complex involved in retrograde vesicular trafficking—as a novel interacting partner for both MuRF2 and MuRF3. Coimmunoprecipitation confirms the physical interaction between MuRF2/MuRF3 and SNX5, with distinct colocalization observed on early endosomes aligned along microtubules. Domain mapping experiments have demonstrated that MuRF2 promotes SNX5 ubiquitination and degradation via the ubiquitin–proteasome system, whereas MuRF3 counteracts this process, stabilizing SNX5. Mass spectrometry further identified the protein kinase A (PKA) regulatory subunit RI-α as a cargo component of SNX5-positive early endosomes in myocytes. In order to ascertain the functional significance of SNX5 in muscle cells, CRISPR-Cas9 knockout and RNA interference (siRNA)-mediated knockdown studies were conducted. The loss of SNX5 resulted in decreased RI-α stability, leading to constitutive activation of PKA. This hyperactivation suppressed the HDAC5–MEF2–myostatin signaling axis, thereby impairing myogenic differentiation. Conversely, SNX5 preserved RI-α integrity, thereby maintaining controlled PKA activity required for proper myogenic progression. Furthermore, SNX5 modulated PKA-dependent degradation of HDAC5, which influenced MEF2 activity and myostatin expression during differentiation.
Conclusion: MuRF2 and MuRF3 have been shown to exert opposing regulatory effects on the stability and retrograde trafficking of SNX5, thereby modulating PKA signaling and myogenic differentiation (Smith et al., 2022). Specifically, MuRF2 has been shown to promote SNX5 degradation, while MuRF3 has been demonstrated to preserve its stability, thereby ensuring balanced PKA activity. SNX5 has been identified as playing a pivotal role in stabilizing RI-α within the early endosomes, thereby facilitating the regulation of normal myogenic signaling. These findings serve to broaden the functional scope of MuRF proteins beyond the scope of proteasomal degradation and establish SNX5 as a key regulator of endosome-associated PKA signaling in muscle cells. It is evident that a comprehensive understanding of the MuRF2–MuRF3–SNX5–PKA axis is pivotal in order to gain novel insights into the complex dynamics of muscle homeostasis. This in turn identifies potential therapeutic targets for muscle-related diseases.
