Revealing the Mechanism of Protein Degradation in Postmortem Meat: The Role of Phosphorylation and Ubiquitination

Abstract

The aim of this study was to investigate the possible effects of phosphorylation and ubiquitination on the degradation of myofibrillar proteins in mutton with different tenderness. The longissimus thoracis lumborum muscles were chosen and divided into tender and tough groups (n = 9), and then stored at 4 °C for 1 h, 12 h, 1 d, 3 d, and 5 d postmortem. Shear force, pH, myofibril fragmentation index, AMPK activity, E3 ubiquitin ligase abundance, protein phosphorylation, and the ubiquitination levels of muscle samples were measured. The results demonstrated that the meat of samples in the tender group had a higher degradation of desmin and a lower phosphorylation level of desmin at 1 d compared with the tough group. The ubiquitination level of desmin, AMPK activity, and E3 ubiquitin ligase abundance in the tender group were noticeably higher than those in the tough group at 12 h. There was a negative correlation between the shear force and desmin degradation. The desmin degradation was negatively correlated with desmin phosphorylation and ubiquitination levels. The phosphorylation level of desmin was positively correlated with its ubiquitination. In summary, this study suggests that AMPK and E3 ubiquitin ligase concurrently play significant roles in regulating meat tenderness by regulating phosphorylation and ubiquitination in meat postmortem.

Keywords: AMPK; E3 ubiquitin ligase; meat tenderness; protein posttranslational modifications.

Figure 1

The phosphorylation level of the myofibrillar proteins in the tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) An image of Pro-Q diamond staining for phosphorylated myofibrillar proteins. (B) An image of SYPRO Ruby staining for total proteins. (C) The phosphorylation level of myofibrillar protein with different tenderness. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a–c represent significant differences between different times within the same group (p < 0.05).

Figure 2

The ubiquitination level of the myofibrillar proteins in the tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) An image of the Western blot of ubiquitinated myofibrillar proteins in samples of different tenderness. (B) The ubiquitination level of the myofibrillar proteins with different tenderness. (C) The ubiquitination level of the myofibrillar protein with different tenderness. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a–c represent significant differences between different times within the same group (p < 0.05).

Figure 3

The degradation of desmin in tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) An image of a Western blot showing the abundance of desmin. (B) The degradation of desmin in samples of different tenderness. (n = 9). Note: x, y represent significant differences between different groups at the same time points (p < 0.05). a–d represent significant differences between different times within the same group (p < 0.05).

Figure 4

The phosphorylation level of desmin in tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) An image of the Western blot of the phosphorylation level of desmin. (B) The phosphorylation level of desmin in samples with different tenderness. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a–c represent significant differences between different times within the same group (p < 0.05).

Figure 5

The ubiquitination level of desmin in tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) The image of Western blot of ubiquitination level of desmin. (B) The ubiquitination level of desmin in samples with different tenderness. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a–c represent significant differences between different times within the same group (p < 0.05).

Figure 6

The activity of AMPK in the tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (A) An image of the Western blot of AMPK with different tenderness. (B) The activity of AMPK in samples of different tenderness. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a and b represent significant differences between different times within the same group (p < 0.05).

Figure 7

The abundance of MuRF1 in the tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a and b represent significant differences between different times within the same group (p < 0.05).

Figure 8

The abundance of MAFbx in the tender and tough groups of mutton muscle stored at 4 °C for 5 d postmortem. (n = 9). Note: x and y represent significant differences between different groups at the same time points (p < 0.05). a and b represent significant differences between different times within the same group (p < 0.05).

Figure 9

Correlation analysis of shear force, degradation of desmin, phosphorylation and ubiquitination levels of degradation and enzyme activity of mutton muscle. Note: Red color represents positive correlation, blue color represents negative correlation. Each rectangle indicates r-value (Pearson correlation coefficient for a pair of variables) and p-value.