Mitochondrial Tu translation elongation factor (TUFM) has been demonstrated to be involved in cellular autophagy during the postmortem aging of Qinchuan beef. This study aimed to explore the relationship between TUFM expression and changes in energy metabolism during this process. Changes in the expression and content of TUFM, the contents of glucose (GLU), lactic acid (LA), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and the activities of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), triose phosphate isomerase (TPI), malate dehydrogenase (MDH), and cytochrome c oxidase (COX) in the Longissimus dorsi muscle of Qinchuan cattle were determined at different periods (0, 2, 12, 24, 48, 96, 144 and 192 h) of aging at 4 ℃. The results showed that during postmortem aging, the contents of GLU, ATP, ADP and AMP, and the activities of LDH, SDH and TPI exhibited a decreasing trend. Meanwhile, the expression level of TUFM, MDH activity, and the contents of LA and TUFM showed an initial increase followed by a decrease. The activity of COX exhibited an initial decrease followed by a temporary increase and then a decrease. The various indicators of energy metabolism and TUFM exerted their effects mainly at the early stage of postmortem aging. Pearson’s correlation analysis indicated that the expression level of TUFM was significantly positively correlated with MDH and LA but significantly negatively correlated with ATP, ADP, AMP, LDH, SDH, TPI, COX, and GLU at the early stage (P < 0.01). At the middle and late stages, TUFM expression was significantly positively correlated with all these indicators (GLU, LA, ATP, ADP, AMP, LDH, MDH, SDH, TPI and COX) (P < 0.01). In conclusion, during the early period of postmortem aging, the gradual increase of TUFM expression can provide energy to muscle cells for energy metabolism pathways, which may be possibly due to the positive involvement of TUFM in cellular autophagy. When energy is deficient during the middle and late periods, TUFM may inhibit cellular autophagy to prioritize energy use for important pathways (e.g., energy metabolism pathways) other than cellular autophagy, thereby maintaining cellular homeostasis. In summary, TUFM plays a dual role during the postmortem aging process, in regulating cellular autophagy to provide energy for metabolic pathways, thereby assisting in maintaining the duration of post-mortem energy metabolism.

In order to explore the mechanism of the tenderness change of the longissimus dorsa muscle of Qinchuan cattle during postmortem ageing, this study measured the changes in energy substances, pH and myofibril fragmentation index (MFI) during storage up to eight days, and it explored the effect of proteomic changes on beef tenderness by four-dimensional label-free quantification (4D-LFQ). It was found that the shear force value increased first and then decreased with storage time, reaching a maximum value of (157.94 ± 2.53) N on the 4^th day, indicating the worst tenderness; however, the opposite trend was observed for the pH, reaching a minimum value of 5.37 ± 0.03 on this day. The contents of the energy substances ATP, AMP and NADH showed a decreasing trend with storage time, and the decrease was the most significant from days zero to two, while the reverse trend was observed for MFI. Totally 11 differential proteins related to the tenderness were identified through correlation analysis. Among them, ATP5F1D, ATP5F1C, NDUFB5, NDUFA6, and SUCG1 regulated the changes in ATP content and pH by participating in the glycolysis process, causing muscle fiber cross-linking, muscle stiffness, and ultimately leading to decreased tenderness; PPP3R1, CAMK2D, HNRNPK, PSMD13, and CTSD regulated muscle cell apoptosis and proteolysis by participating in the muscle fiber membrane, the membrane protein complexes, the organelle inner membranes, and the calcium signaling pathway, resulting in an increase in MFI and ultimately contributing to improve beef tenderness.

In order to explore the effects of structural proteins on the quality changes of Longissimus dorsi muscle from Qinchuan cattle during storage, 4D-label free quantification (4D-LFQ) was used to analyze the proteomic changes of beef Longissimus dorsi muscle during different storage periods (0, 2, 4, 6 and 8 days). The changes in shear force, myofibrillar fragmentation index (MFI) and protein content were measured as well. The results showed that the shear force tended to increase and then decrease during the storage period of 8 days (P < 0.01), the increase being greater than the decrease. The MFI showed a significant upward trend with storage time (P < 0.01), increasing by up to 250.81% after 8 days of storage; however, the opposite trend was observed for the total soluble protein content (P < 0.05), decreasing by up to 34.60%. Structural proteins in muscle tissue were degraded with postmortem metabolic changes in muscle tissue, which possibly affected tenderness development and caused a significant decrease in the content of myofibrillar protein (P < 0.05). The content of myofibrillar protein decreased rapidly at first and then slowly, decreasing by 50.56% over the entire storage period. During the first four days, the abundance of four proteins (alpha-actin-1, myosin heavy chain 9, myosin regulatory light chain 2, and myosin regulatory light chain 12B) were changed by regulating the calcium ion-binding and cytoskeletal protein-binding pathways in the development of skeletal muscle tissue. During the 8 days of storage, the abundance of eight proteins (myosin regulatory light chain 2, myosin heavy chain 6, alpha-actin-1, actin, alpha cardiac muscle 1, myosin regulatory light chain 2, troponin Ⅰ 1, troponin Ⅰ 2, and myosin heavy chain 15) was changed by regulating the calcium ion-binding pathway in the development of muscle organ and striated muscle tissue. Moreover, the structural proteins were degraded as a result of the regulation of the physiological state of cells by myosin binding, calcium ion binding, cytoskeletal protein binding myofibrillar assembly, skeletal muscle tissue development, muscle organ development, and striated muscle tissue development, leading to an increase in the MFI, thus improving the tenderness.

After different degrees of oxidation in the Fenton system, the changes in the structure and emulsification properties of beef myofibrillar proteins were investigated and the relationship between them were evaluated. The results showed that with increasing H₂O₂ concentrations from 0 to 20 mmol/L, the oxidation of myofibrillar protein became more serious, the carbonyl content, dimer tyrosine content and surface hydrophobicity increased gradually, and the contents of total sulfhydryl and free sulfhydryl groups decreased gradually; the percentage of α-helix exhibited a downward trend and the percentage of β-sheet showed an upward trend; solubility, emulsification activity and emulsion stability all tended to first increase and then decrease, while the opposite trend was observed for turbidity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that oxidation induced protein cross-linking. Correlation analysis found that the contents of carbonyl, sulfhydryl and dimer tyrosine, surface hydrophobicity and secondary structure indicators had significant correlations with solubility, turbidity and emulsification properties. In summary, the oxidation degree of myofibrillar protein increased gradually with increasing H₂O₂ concentration in the Fenton oxidation system, causing changes in its structure and emulsification properties.

In order to investigate the effect of differential expression of heat shock protein family A (HSP70) member 6 (HSPA6) on the meat quality of Qinchuan cattle during postmortem maturation, the Longissimus dorsi muscle of Qinchuan cattle was stored at 4 ℃ and evaluated for meat quality at 0, 2, 4, 6 and 8 days postmortem. 4D-Label free quantification (4D-LFQ) was used to analyze the expression of HSPA6 and related differential proteins. The results showed that the expression of HSPA6 decreased with increasing storage time, the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and reduced form of nicotinamide-adenine dinucleotide (NADH) decreased, the myofibril fragmentation index (MFI) increased, and the shear force and centrifugal loss increased and then decreased. Correlation analysis showed that HSPA6 expression was positively correlated with the contents of ATP and NADH and shear force (P < 0.05) and negatively correlated with MFI and centrifugal loss (P < 0.01), indicating that HSPA6 degradation was closely related to energy metabolism and water retention in beef during storage. Proteomics identified 24 significantly differentially expressed proteins (DEPs) associated with HSPA6 expression, which could cause proteasome-mediated ubiquitin-dependent proteolytic metabolism and cellular proteolysis through carbohydrate derivative binding and purine nucleoside triphosphate binding, in turn causing metabolic disorders or imbalances. These DEPs could also control protein degradation and cell apoptosis, in turn affecting structural changes in muscle and reducing tissue energy levels, and ultimately affecting the tenderness, MFI and water retention of Qinchuan beef through resistance to cellular structural protein degradation.

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.

This study addressed the effect of adenosine 5’-diphosphate (ADP) ribosylation on the mitochondrial function and quality of Qinchuan cattle meat during early postmortem aging. The Longissimus dorsi muscle of Qinchuan cattle treated with 20 µmol/L rucaparib, a poly(ADP-ribose) polymerase 1 (PARP1) inhibitor, were evaluated for mitochondrial indices, myofibrillar fragmentation index (MFI), shear force, pH, and other quality indices after being stored for 0 h, 6 h, 12 h, 2 d, 4 d, and 8 d and the expression levels of PARP1 and desmin were detected using Western blot. The results showed that the content of reactive oxygen species (ROS) in the treated group was significantly lower than that in the control group (P < 0.05) during 0 h–8 d (except 12 h) after slaughter. The caspase-3 activity and MFI in the treated group were significantly lower than those in the control group (P < 0.05) during 0–12 h. Mitochondrial membrane potential during 2–4 d as well as succinate dehydrogenase (SDH) activity during 4–8 d were significantly higher in the treated group than in the control group (P < 0.05). These findings indicate that PARP1 inhibition, which characterizes ADP ribosylation, can slow down the decrease in mitochondrial membrane potential and increase SDH activity, preserving mitochondrial function to some extents and delaying the decrease in MFI and the degradation of desmin and thereby meat tenderization.

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.

In order to investigate the effect of mitochondrial Tu translation elongation factor (TUFM) expression on the water-holding capacity (WHC) of meat during the postmortem aging of Qinchuan cattle meat, changes in the pH, storage loss, centrifugal loss, cooking loss, and water distribution of the longissimus dorsi of Qinchuan cattle as well as its myofibrillar protein degradation were determined after different aging durations at 4 ℃. Besides, TUFM expression and content as well as beclin 1 expression were examined after 0, 96 and 192 h of aging. The results showed that myofibrillar protein was degraded during the postmortem aging process, and there was a close relationship between the expression of TUFM and beclin1 protein and the WHC of beef. Proteomics revealed consistent changing trends between the expression of TUFM and its content. In general, beclin1 protein expression, storage loss, centrifugal loss, and cooking loss initially increased and subsequently decreased, whereas the opposite was true for pH. Pearson’s correlation analysis showed that the expression of TUFM was highly significantly positively correlated with low-field nuclear magnetic resonance peak area ratio P_2b and beclin 1 protein expression (P < 0.01), significantly positively correlated with storage loss, centrifugal loss, and cooking loss (P < 0.05), highly significantly negatively correlated with P₂₁ (P < 0.01), and significantly negatively correlated with P₂₂ (P < 0.05), but had no significant correlation with pH (P > 0.05). Through proteomics, 23 differential proteins related to TUFM were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the differential proteins could mediate cellular autophagy by participating in energy metabolism through a variety of pathways. Pearson’s correlation analysis showed that five differential proteins (ATP5F1D, EEF1A2, GSPT1, NDUFB5, and SUCLG1) were significantly correlated with WHC (P < 0.05, P < 0.01). From these analyses, it is clear that there are six proteins including TUFM that affect the WHC of meat by affecting cellular autophagy positively or negatively through various pathways such as energy metabolism and oxygen transport.