Altered mechanical loads and skeletal muscle phenotype

描述(申请人提供)：肌球蛋白重链(MyHC)是骨骼肌纤维表型和功能的典型标志，因为它是最大无负荷缩短速度(Vmax)的主要决定因素。目前，对机械负荷减少[后肢非负重(NWB)]的综合生物学反应如何导致纤维类型转移和MyHC基因转录调控的改变，人们知之甚少。我们之前的转基因缺失-突变和蛋白质-DNA相互作用研究发现了一个负元件(dbetaNRE-S；-332至-311)，它结合了仅在NWB-比目鱼核提取液中发现的两种不同的蛋白质。通过亲和层析和EMSA分析，我们已经鉴定并证实了负作用的单链DNA结合转录因子Purpha和Purbeta代表了我们在NWB-Soleus核提取液中鉴定的丰富的结合活性。我们发现，Purpha和Purbeta降低了肌肉细胞中MyHC报告基因的转录，并且dbetaNRE-S元件赋予了Pur依赖的最小胸苷激酶启动子的激活减少。通过免疫共沉淀，我们还发现两种Pur-蛋白都与Sp3-蛋白相互作用；我们发现的与NWB相关的转录因子降低了β-MyHC基因的转录。由于Purpha和Purbeta已被证明在心脏aMyHC和血管平滑肌α-肌动蛋白基因转录下降中起作用，我们预计我们的发现将对基础和NWB条件下骨骼肌、心肌和平滑肌基因网络的调控以及所有pur蛋白表达的CEN的基因调控具有全球相关性。因此，该建议的主要目的是通过实现以下目的来破译Pur-蛋白在调节骨骼肌表型中的生理作用：1)研究转基因Pur-蛋白(Purpha和Purbeta)在成年小鼠骨骼肌中过表达的表型后果；2)利用酵母2-杂交组和经典蛋白质组学分离和研究肌肉特异性Pur相互作用蛋白(S)；3)通过瞬时共转染实验、C2C12肌管染色质免疫沉淀和GST-相互作用实验，研究Pura、Purbeta和Sp3-蛋白之间的协同作用。Northern和Western分析将评估Pur蛋白过度表达对整个肌肉表型的影响，这将与整体肌肉和单纤维功能分析相关。这项工作有望确定潜在的治疗蛋白质靶点，旨在为疾病、太空飞行或长时间卧床引起的机械负荷变化引起的肌肉表型改变和衰弱功能丧失提供对策。

DESCRIPTION (provided by applicant): Myosin heavy chain (MyHC) is a quintessential marker of skeletal muscle fiber phenotype and function since it is a major determinate of maximum unloaded velocity of shortening (Vmax). Presently, little is known about how the integrative biological response to decreased mechanical loading [hindlimb non-weight bearing (NWB)] leads to fiber-type shifts and altered transcriptional regulation of MyHC genes. Our previous transgenic deletion- mutagenesis and protein-DNA interaction studies identified a negative element (dbetaNRE-S; -332 to -311) that binds two different proteins identified only in NWB-soleus nuclear extract. By affinity chromatography and EMSA analysis we have identified and confirmed that the negative acting single-stranded DNA-binding transcription factors, Puralpha and Purbeta, represent the enriched binding activity we identified in NWB-Soleus nuclear extract. We show that Puralpha and Purbeta decrease betaMyHC reporter gene transcription in muscle cells, and that the dbetaNRE-S element confers Pur-dependent decreased activation of a minimal thymidine kinase promoter. By co- immunoprecipitation we also show that both Pur-proteins interact with Sp3-proteins; transcription factors shown by us to contribute to NWB decreased betaMyHC gene transcription. Since Puralpha and Purbeta have been shown to play a role in decreased cardiac aMyHC and vascular smooth muscle alpha-actin gene transcription, we expect that our findings will have global relevance to the regulation of skeletal, cardiac and smooth muscle gene networks under basal and NWB conditions, and gene regulation in all Pur-protein expressing ceNs. Thus, the primary goal of this proposal is to decipher the physiological role of the Pur-proteins in regulating skeletal muscle phonotype by performing the following aims: 1) to study the phenotypic consequences of transgenic overexpression of Pur- proteins (Puralpha and Purbeta) in adult mouse skeletal muscle, 2) to isolate and study muscle-specific Pur-interacting protein(s) by yeast 2-hybrid and classical proteomics, and 3) to investigate the cooperative interaction between Puralpha, Purbeta, and Sp3-proteins via transient co-transfection assays, chromatin immunoprecipitation with C2C12 myotubes, and GST-interaction assays. Northern and western analysis will assess the impact of Pur-protein overexpression on whole muscle phenotype, which will be correlated to whole muscle and single fiber functional analysis. This work is expected to identify potential protein targets for therapies aimed at providing countermeasures against altered muscle phenotype and debilitating loss of function induced by altered mechanical loads resulting from disease, space flight or extended bed rest.