C6ORF32, AN HDAC6-BINDING PROTEIN THAT REGULATES MYOBLAST DIFFERENTIATION

描述（由申请人提供）：骨骼肌由成肌细胞融合形成的多核合胞体组成。通过微阵列研究发现，在人成肌细胞融合早期，当肌管含有2-5个肌核时，C6ORF32的表达上调（Cerletti et al., 2006）。C6ORF32表达下调会导致成肌细胞融合受阻，这是由于肌原蛋白表达的显著降低（Yoon等，2007）。通过计算分析，预测的C6ORF32蛋白只包含一个已知功能域，即假定的hdac结合域。发现C6ORF32结合HDAC6，这是一种以促进细胞运动功能而闻名的IIb类HDAC。我们假设HDAC6和C6ORF32在功能上有联系，HDAC6和C6ORF32之间的相互作用通过降低HDAC6的去乙酰化活性来积极调节肌源性分化。研究骨骼肌中HDAC6和C6ORF32之间的调控联系，将在体外和体内进行研究，具体目的如下：1)确定C6ORF32与HDAC6的关键结合区域，并证明这两种蛋白在体内发生结合；2)确定HDAC6是否通过调节C6ORF32乙酰化/去乙酰化调控成肌细胞分化；3)评价C6ORF32是否通过调节HDAC6活性调控成肌细胞分化；4)通过影响HDAC6功能，确定体内C6ORF32表达的消融是否会导致肌肉发育、功能和再生缺陷。这些研究将为了解未来针对C6ORF32和HDAC6之间的调节相互作用的药物治疗是否能够增强肌原性修复奠定基础。

DESCRIPTION (provided by applicant): Skeletal muscle is comprised of multinucleated syncytia formed by the fusion of myoblasts. Through microarray studies, it was found that expression of C6ORF32 is upregulated early during human myoblast fusion, when myotubes contain between 2-5 myonuclei (Cerletti et al., 2006). Downregulation of C6ORF32 expression causes a block in myoblast fusion due to a significant decrease in myogenin expression (Yoon et al., 2007). By computational analyses, the predicted C6ORF32 protein contains only one domain of known function, a putative HDAC-binding domain. It was found that C6ORF32 binds to HDAC6, a class IIb HDAC known for its function in promoting cell motility. It is hypothesized that HDAC6 and C6ORF32 are functionally connected, and the interaction between HDAC6 and C6ORF32 positively regulates myogenic differentiation by reducing the de-acetylation activity of HDAC6. The regulatory connection between HDAC6 and C6ORF32 in skeletal muscle will be studied both in vitro and in vivo via the following specific aims: 1) Define the critical binding region of C6ORF32 to HDAC6, and demonstrate that binding of these two proteins occurs in vivo; 2) Determine if HDAC6 regulates myoblast differentiation by modulating C6ORF32 acetylation/de-acetylation; 3) Evaluate if C6ORF32 regulates myoblast differentiation by modulating HDAC6 activity; 4) Determine if ablation of C6ORF32 expression in vivo yields to defects in muscle development, function and regeneration by affecting HDAC6 function. These studies will constitute the groundwork for understanding whether future pharmacological therapies targeting the regulatory interaction between C6ORF32 and HDAC6 are amenable to enhance myogenic repair.