Altered mechanical loads and skeletal muscle phenotype

描述（由申请方提供）：肌球蛋白重链（MyHC）是骨骼肌纤维表型和功能的典型标志物，因为它是最大无负荷缩短速度（Vmax）的主要决定因素。目前，很少有人知道如何减少机械负荷的综合生物反应[后肢非承重（NWB）]导致纤维型转变和改变的MyHC基因的转录调控。我们先前的转基因缺失-诱变和蛋白质-DNA相互作用研究鉴定了一种负元件（d β NRE-S; -332至-311），其结合仅在NWB-比目鱼核提取物中鉴定的两种不同蛋白质。通过亲和层析和EMSA分析，我们已经鉴定并证实了负作用单链DNA结合转录因子Puralpha和Purbeta代表了我们在NWB-Soleus核提取物中鉴定的富集的结合活性。我们发现，Puralpha和Purbeta减少betaMyHC报告基因在肌细胞中的转录，和dbetaNRE-S元件赋予的最小胸苷激酶启动子的Pur-dependent减少激活。通过免疫共沉淀，我们还表明两种Pur蛋白与Sp3蛋白相互作用;我们所示的有助于NWB的转录因子降低了β-MyHC基因转录。由于Puralpha和Purbeta已被证明在降低心脏aMyHC和血管平滑肌α-肌动蛋白基因转录中发挥作用，我们预计我们的研究结果将在基础和NWB条件下对骨骼肌、心脏和平滑肌基因网络的调控以及所有表达Pur蛋白的ceN中的基因调控具有全球相关性。因此，该提案的主要目标是通过执行以下目标来破译Pur蛋白在调节骨骼肌表型中的生理作用：1）研究转基因Pur蛋白过表达的表型后果（Puralpha和Purbeta）在成年小鼠骨骼肌中，2）通过酵母双杂交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，和3）通过瞬时共转染测定、C2 C12肌管染色质免疫沉淀和GST相互作用测定来研究Puralpha、Purbeta和Sp3蛋白之间的协同相互作用。北方和西方分析将评估Pur蛋白过表达对全肌肉表型的影响，这将与全肌肉和单纤维功能分析相关。这项工作预计将确定潜在的蛋白质靶点的治疗，旨在提供对策，改变肌肉表型和衰弱的功能丧失引起的机械负荷改变疾病，太空飞行或延长卧床休息。

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.