Molecular regulation of protein turnover in skeletal muscle

项目摘要/摘要 美国人口继续老龄化，数千万人可能会受到与衰老相关的肌肉的影响 在人生的某个阶段出现萎缩和虚弱(即肌肉功能障碍)。这代表着一个巨大的未得到满足的 医疗需要，因为衰老相关的肌肉功能障碍缺乏有效的治疗，损害独立性， 延长住院时间，增加全因死亡率。蛋白平衡受损是导致衰老的主要因素。 在哺乳动物身上。骨骼肌纤维是长寿细胞，因此特别容易受到蛋白质毒性的影响。 压力和依赖于有效的蛋白质周转(即，功能失调的蛋白质和耦合的细胞器的降解 正确的蛋白质合成)以保持功能。事实上，衰老相关肌肉中的蛋白质周转是有缺陷的 功能障碍，但所涉及的机制仍未被充分了解，阻碍了潜在的识别 这种情况的治疗靶点。这里提出的研究将有助于通过以下方式解决这个问题 研究ULK2蛋白，我们最近证明它是维持有效的 骨骼肌中的蛋白质周转。我们的后续研究发现，ULK2缺陷的肌肉有几个 使人想起衰老肌肉的特征，例如蛋白质降解受损，表现为 不可溶的p62相关泛素化蛋白聚集体，增加eIF2α(S51)的磷酸化，这限制了 蛋白质合成，以及萎缩和虚弱。相反，增强的ULK2表达似乎 增强衰老肌肉的力量。这些观察结果有力地支持了ULK2活动不足的模型 与衰老相关的肌肉功能障碍有关，ULK2依赖的信号通路可能是治疗的靶点。 对于这一问题，我们已经在骨骼肌ULK2上确定了3个独特的磷酸化位点，对其进行了桥式调节 它的功能，并将一个保守的ULK磷酸化基序定位到FIP200上，潜在地调节其 与p62的相互作用。这种相互作用已被证明是自噬小体形成所必需的 泛素化的蛋白质聚集体及其随后的降解。此外，我们确定ULK2 与GCN1相互作用，抑制其对eIF2GCN2α激酶GCN2的刺激。我们建议的研究将 在这些重要的初步发现的基础上，使用小鼠模型来解决成人和老年人的三个特定目标 肌肉。在目标1中，我们将确定ULK2功能的获得和丧失如何调节蛋白质周转和肌肉 函数与其并列逻辑ULK1的增益和损耗进行比较。在目标2中，我们将研究突变形式 ULK2和FIP200通过阻断或模拟它们的磷酸化来确定它们在调节蛋白质中的作用 退化。在目标3中，我们将通过GCN1功能的获得和丧失来确定其在ULK2介导的过程中的作用 蛋白质合成的调节。通过这些研究，我们希望增进我们对 调节骨骼肌蛋白质周转的机制，潜在地确定衰老治疗的新靶点- 相关的肌肉功能障碍。

PROJECT SUMMARY/ABSTRACT The US population continues to age and tens of millions of people will likely be affected by aging-related muscle atrophy and weakness (i.e., muscle dysfunction) at some point in life. This represents an enormous unmet medical need because aging-related muscle dysfunction lacks effective therapy, compromises independence, prolongs hospitalization and increases all-cause mortality. Impaired proteostasis is a major contributor to aging in mammals. Skeletal muscle fibers are long-living cells thereby being particularly susceptible to proteotoxic stress and relying on efficient protein turnover (i.e., degradation of dysfunctional proteins and organelles coupled to proper protein synthesis) to remain functional. Indeed, protein turnover is defective in aging-related muscle dysfunction, but the mechanisms involved remain insufficiently understood hindering the identification of potential therapeutic targets for this condition. The research proposed here would help to address this issue by investigating the protein ULK2, which we have recently demonstrated to be required for maintenance of effective protein turnover in skeletal muscle. Our follow-up studies uncovered that ULK2 deficient muscles had several features reminiscent of aging muscle, such as impaired protein degradation evidenced by accumulation of insoluble p62-associated ubiquitinated protein aggregates, increased eIF2α (S51) phosphorylation, which limits protein synthesis, as well as atrophy and weakness. Conversely, enhanced ULK2 expression appears to increase strength in aged muscle. These observations strongly support a model where insufficient ULK2 activity contributes to aging-related muscle dysfunction and that ULK2-dependent signaling may be targeted for therapy. To this matter, we have identified 3 unique phosphorylation sites at skeletal muscle ULK2 pontentially modulating its function and have mapped a conserved ULK phoshorylation motif to FIP200 potentially modulating its interaction with p62. This interaction has been shown to be required for autophagosome formation at ubiquitinated protein aggregates and their subsequent degradation. In addition, we determined that ULK2 interacts with GCN1 pontentially inhibiting its stimulation of the eIF2α kinase GCN2. Our proposed studies will build upon these important initial findings and use mouse models to address 3 specific aims in adult and aged muscle. In Aim 1, we will establish how gain and loss of function of ULK2 modulate protein turnover and muscle function in comparison with gain and loss of function of its paralog ULK1. In Aim 2, we will study mutated forms of ULK2 and FIP200 that either block or mimic their phosphorylation to establish their role in regulating protein degradation. In Aim 3, we will use gain and loss of function of GCN1 to establish its role in ULK2-mediated regulation of protein synthesis. Through these studies, we hope to advance our understanding on the mechanisms regulating skeletal muscle protein turnover, potentially identifying new targets for therapy in aging- related muscle dysfunction.