Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway.

MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN-/- . The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3ß pathway and highly consistent with the lower glycogen content in gluteus of MSTN-/- . Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth.

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of fat-1 transgenic cattle.

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO2 enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC-MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.

A lncRNA promotes myoblast proliferation by up-regulating GH1.

Long noncoding RNAs (lncRNAs) are key regulatory factors for gene expression in a variety of biological processes; however, the role of lncRNAs in muscle formation and development is poorly understood, particularly in cattle. Here, we identified a highly expressed lncRNA in muscle, lncYYW, by high-throughput sequencing in bovine longissimus, scapular, intercostal, and gluteus muscles. The expression of lncYYW increased gradually during myoblast differentiation. Overexpression of lncYYW increased the number of cells in the DNA synthesis (S) stage of the cell cycle and upregulated the expression of two well-established myogenic markers, myogenin and myosin heavy chain. A microarray analysis showed that lncYYW positively regulates the expression of growth hormone 1 and its downstream genes, AKT1 and PIK3CD, in bovine myoblasts. This discovery provides a good foundation for further study of the mechanism of action of lncYYW during bovine myoblast development. Taken together, our results reveal a novel lncRNA associated with bovine myoblast proliferation and differentiation. This lncRNA will play a crucial and critical role in future studies of bovine muscle development.

A yeast two-hybrid assay reveals CMYA1 interacting proteins.

CMYA1 is a protein that plays an important role in muscle development and contains a highly conserved Xin repeats region. However, the function of the Xin-repeats in CMYA1 is unknown, and there is little information regarding proteins that interact with the CMYA1/Xin-repeats in the bovine system. In this study, we generated a high-quality bovine muscle cDNA library and performed a yeast two-hybrid screen twice using both CMYA1 and the Xin-repeats as bait. There were 27 candidate-interacting proteins identified in this screen. Three of the 27 proteins (RPL35A, RPL21 and EIF3G) interacted with both CMYA1 and the Xin-repeats, and this interaction was further confirmed using one-to-one Y2H mating. These results showed that the three candidate proteins interacted with CMYA1/Xin-repeats and indicated that the Xin-repeats is a key region of CMYA1 required for protein interaction. In conclusion, our results provide new targets on the bovine CMYA1/Xin-repeats interacting proteins, and these findings provide an important reference for the study of how bovine muscle development is regulated.