In this study, the Longissimus dorsi muscle of Qinchuan cattle was injected with the proteasome inhibitor MG-132 immediately postmortem and then stored at 4 ℃. The effect of the ubiquitin-proteasome pathway (UPP) on protein degradation as well as changes in the proteasome activity, ubiquitin content and microstructure of the muscle during postmortem storage was explored in order to provide theoretical support for precise postmortem regulation of beef quality. With the extension of storage time, proteasome activity was lower and the contents of total soluble protein and ubiquitin were higher in the MG-132 group than in the control group. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the band intensity of total soluble proteins between 40 and 250 kDa was greater in the MG-132 group than in the control group; muscle structure was better preserved in the MG-132 group, and the Z line and the boundary between light and dark bands were clearer than those in the control group. The contents of total soluble protein and ubiquitin showed a significantly positive correlation (P < 0.05). In conclusion, postmortem injection of MG-132 inhibited the proteasome activity and the degradation of ubiquitinated proteins in the UPP in Qinchuan beef, which in turn altered protein degradation and attenuated muscle damage. This suggests that the UPP has a potential role in meat quality formation; the proteasome not only degrades proteins by itself alone to destroy beef myofibrillar structure, but also influences postmortem beef protein degradation through mediating the UPP, ultimately affecting postmortem beef quality.

In this study, the Longissimus dorsi muscle of Qinchuan beef stored at 4 ℃ after slaughter was determined for its quality indicators, energy level and proteasome activity. Meanwhile, differentially expressed proteins (DEPs) associated with proteasomes were identified. Bioinformatics methods were used to analyze the response mechanism of proteasomes to the quality and energy metabolism of postmortem beef. The results showed that pH decreased and then increased with increasing storage time, b* and myofibrillar fragmentation index (MFI) increased, L^*, a^*, centrifugal loss, cooking loss and shear force increased first and then decreased, the contents of the basic energy substances ATP, ADP and AMP decreased, and the relative activity of the 20S proteasome decreased significantly (P < 0.05). The correlation analysis showed that the activity of the 20S proteasome was significantly negatively correlated with b* (P < 0.05), positively correlated with ADP content (P < 0.05), positively correlated with AMP content (P < 0.01), and negatively correlated with MFI (P < 0.01), indicating a close relationship between proteasome activity and energy metabolism as well as beef quality. Using 4D label-free quantitative (LFQ) proteomics, eight differentially expressed proteasome subunits and 27 DEPs were identified. Gene Ontology (GO) annotation analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these proteasome subunits and related DEPs had molecular functions such as endopeptidase activity, actin binding, microfilament motor activity, and were involved in biological processes such as ubiquitin-dependent proteolytic metabolism, skeletal muscle contraction, muscle contraction, and glycolysis, thus causing changes in the quality of Qinchuan beef. During the early postmortem period, the proteasomes regulated the biological pathways by consuming energy substances to catabolize proteins, and during the later period, when the energy substances were depleted, the proteasomes catabolized proteins in postmortem beef through the energy provided by glycolysis and non-ATP-dependent proteasome subunits, which eventually affected the quality of Qinchuan beef.

In this study, a phylogenetic tree was constructed using bovine heat shock protein A6 (HSPA6) sequences and those of other organisms, and bioinformatic methods were used to analyze the basic physicochemical properties and hydrophilicity of bovine HSPA6 protein. Meanwhile, a protein interaction network was constructed to investigate the structural and functional properties of the protein encoded by the HSPA6 gene. The results showed that bovine HSPA6 protein had high similarity in amino acid sequence with those of sheep, Yangtze finless porpoise and other mammals. The molecular mass of bovine HSPA6 protein was 70570.64 u, the theoretical isoelectric point was 5.66, and it was an acidic hydrophilic protein without transmembrane structure or signal peptide. Bovine HSPA6 protein may have 11 phosphorylation sites with score greater than 0.900 and N-glycosylation sites at the terminal bases, and it was a relatively stable protein with a secondary structure consisting mainly of 40.38% α-helix and 33.65% random coil, containing two major structural domains, the N-terminal nucleotide-binding domain and the C-terminal peptide-binding domain, which played a role in the cytoplasm. The constructed protein network showed that bovine HSPA6 protein mainly interacted with BAG1, DNAJA4, DNAJB1 and DNAJC2, and was involved in the activity of adenylate exchange factors, ATPase activity and chaperone binding, indicating that the HSPA6 protein exerted a potential function in biological processes such as energy metabolism in the bovine organism. These multiple bioinformatic analyses provide a theoretical basis for an in-depth investigation of the mechanism of the effect of bovine HSPA6 protein on meat quality.