Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics.

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

Identification and characterization of Fbxl22, a novel skeletal muscle atrophy-promoting E3 ubiquitin ligase.

Muscle-specific E3 ubiquitin ligases have been identified in muscle atrophy-inducing conditions. The purpose of the current study was to explore the functional role of F-box and leucine-rich protein 22 (Fbxl22), and a newly identified splice variant (Fbxl22-193), in skeletal muscle homeostasis and neurogenic muscle atrophy. In mouse C2C12 muscle cells, promoter fragments of the Fbxl22 gene were cloned and fused with the secreted alkaline phosphatase reporter gene to assess the transcriptional regulation of Fbxl22. The tibialis anterior muscles of male C57/BL6 mice (12-16 wk old) were electroporated with expression plasmids containing the cDNA of two Fbxl22 splice variants and tissues collected after 7, 14, and 28 days. Gastrocnemius muscles of wild-type and muscle-specific RING finger 1 knockout (MuRF1 KO) mice were electroporated with an Fbxl22 RNAi or empty plasmid and denervated 3 days posttransfection, and tissues were collected 7 days postdenervation. The full-length gene and novel splice variant are transcriptionally induced early (after 3 days) during neurogenic muscle atrophy. In vivo overexpression of Fbxl22 isoforms in mouse skeletal muscle leads to evidence of myopathy/atrophy, suggesting that both are involved in the process of neurogenic muscle atrophy. Knockdown of Fbxl22 in the muscles of MuRF1 KO mice resulted in significant additive muscle sparing 7 days after denervation. Targeting two E3 ubiquitin ligases appears to have a strong additive effect on protecting muscle mass loss with denervation, and these findings have important implications in the development of therapeutic strategies to treat muscle atrophy.

Inhibition of protein phosphatase methylesterase 1 dysregulates MAP kinase signaling and attenuates muscle cell differentiation.

Protein phosphatase methylesterase 1 has been identified as a novel gene in skeletal muscle that is upregulated in response to neurogenic atrophy in mice. Western blot analysis confirms that Ppme1 is expressed during both muscle cell proliferation and differentiation. Additionally, the Ppme1 promoter is active in muscle cells, while mutation of a conserved E-box element prevents full induction of the Ppme1 reporter gene, suggesting that Ppme1 is transcriptionally regulated by myogenic regulatory factors. Interestingly, immunofluorescence analysis indicates that Ppme1 is localized to both the cytoplasm and the nucleus, while cell fractionation shows that Ppme1 is found only in the cytoplasm. Functional studies reveal that inhibition of Ppme1 using ABL127 or AMZ30 attenuates muscle cell differentiation. Interestingly, inhibition of Ppme1 by ABL127 led to a significant increase in AP-1 reporter activity, as well as, increases in ERK1/2, c-Jun, Ppme1, and PP2A protein levels in differentiating muscle cells. In contrast, AMZ30 treated cells showed a significant decrease in AP-1 reporter activity and a decrease in ERK1/2 and p38 phosphorylation levels. Finally, co-immunoprecipitation studies show that ABL127, but not AMZ30, causes disruption of the endogenous interaction between Ppme1 and PP2A. The data in this study show for the first time that Ppme1 is expressed in skeletal muscle and is upregulated in response to neurogenic atrophy. Furthermore, these findings suggest that Ppme1 may act as a sentinel of the MAP kinase signaling pathway and may indirectly regulate the ERK1/2 and p38 branches via a non-canonical mechanism leading to inhibition of muscle cell differentiation.

Zinc finger protein 593 is upregulated during skeletal muscle atrophy and modulates muscle cell differentiation.

Skeletal muscle atrophy is a debilitating condition that can arise due to aging, cancer, corticosteroid use, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study analyzed gene expression patterns in gastrocnemius muscle following sciatic nerve transection and found for the first time that Zinc Finger Protein 593 (Zfp593) is expressed in skeletal muscle and is induced in response to denervation. Quantitative PCR and Western blot analyses confirmed that Zfp593 is expressed in both proliferating myoblasts and differentiated myotubes. To assess sub-cellular location, GFP-tagged Zfp593 was expressed in C2C12 cells and found to localize to the nucleus. The Zfp593 protein possesses a putative zinc finger domain and is believed to function as a modulator of the Oct-2 transcription factor. Interestingly, ectopic expression of Zfp593 did not affect the ability of Oct-1 or Oct-2 to inhibit an Oct reporter gene in muscle cells. Finally, Zfp593 overexpression in cultured muscle cells resulted in significant repression of muscle cell differentiation and attenuation of ERK1/2 and p38 phosphorylation, but did not vitiate protein synthesis. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that it is induced in response to neurogenic atrophy furthers our understanding of the molecular genetic events of muscle wasting.

Opportunities and perspectives for utilisation of co-products in the meat industry.

Meat co-products are the non-meat components arising from meat processing/fabrication and are generated in large quantities on a daily basis. Co-products are considered as low added-value products, and in general it is difficult for industries to divert efforts into increasing their value. While many of these products can be edible those not used for human consumption or pet food is usually processed to be used as animal feed, fertilizer or fuel. However, to a large extent meat co-products are an excellent source of high nutritive value protein, minerals and vitamins and hence may be better diverted to contribute to alleviate the increasing global demand for protein. In this review the current uses, legislation and potential techniques for meat co-products processing are reviewed with the aim of showing a route to improve meat industry sustainability, profitability and better usage of available resources.

Optimization of protein recovery from bovine lung by pH shift process using response surface methodology.

BACKGROUND: Response surface methodology (RSM) was used in a sequential manner to optimize solubilization and precipitation conditions in the recovery of protein from bovine lung using pH shift. RESULTS: Separate D-optimal designs were employed for protein solubilization and precipitation. Independent variables investigated for protein solubilization were time (10-120 min), temperature (4-20 °C), pH (8.0-11.0) and solvent/sample ratio (2.5-10). Variables for protein precipitation were time (0-60 min) and pH (4.25-6.00). Soluble protein yields ranged from 323 to 649 g kg-1 and the quadratic model for protein solubilization revealed a coefficient of determination R2 of 0.9958. Optimal conditions for maximum protein solubility were extraction time 140 min, temperature 19 °C, pH 10.8 and solvent/sample ratio 13.02. Protein precipitation yields varied from 407 to 667 g kg-1 , giving a coefficient of determination R2 of 0.9335. Optimal conditions for maximum protein precipitation were pH 5.03 and 60 min. Based on the RSM model, solubilization conditions were manipulated to maximize protein solubilization under reduced water and alkaline usage. These conditions were also validated. CONCLUSION: Models for solubilization and precipitation using bovine and porcine lung were validated; predicted and actual yields were in good agreement, showing cross-species applicability of the results. © 2017 Society of Chemical Industry.

Harnessing the Potential of Blood Proteins as Functional Ingredients: A Review of the State of the Art in Blood Processing.

Blood is generated in very large volumes as a by-product in slaughterhouses all around the world. On the one hand, blood generation presents a serious environmental issue because of its high pollutant capacity; however, on the other hand, blood has the potential to be collected and processed to generate high-added-value food ingredients based on its exceptional nutritive value and its excellent functional properties. In this paper, we review the current state of the art for blood processing, from collection to final recovery of protein isolates, the functional properties of blood, impact of processing on functional properties, and potential applications as food ingredients. Furthermore, future challenges are outlined for this underutilized and abundant product from the meat industry.