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Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2.

Witt CC, Witt SH, Lerche S, Labeit D, Back W, Labeit S - EMBO J. (2007)

Bottom Line: The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues.Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis.Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany.

ABSTRACT
The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

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Related in: MedlinePlus

MuRF1 and MuRF2 interact with a shared set of myocellular proteins. (A) YTH screens with full-length MuRF1 and MuRF2 baits of both human cardiac (‘heart') and skeletal cDNA libraries (‘SKM') fished a total of 87 genes. The table summarizes those 35 prey clones identified independently in both MuRF1 and MuRF2 screens and thus predicted to interact with both MuRF1+2: 13 prey clone inserts code for sarcomeric proteins (4 of which are components of the Z-disk), 10 code for transcriptional regulators (2 of which are also associated with the Z-disk), 5 genes are involved in mitochondrial ATP production, and 6 genes participate in translation initiation and elongation. Numbers indicate independently identified prey clones in respective screens. M=interaction was found by mating. An SRF prey clone fished with the MuRF1 bait could not be confirmed by mating, as in our hands the 3′ UTR and not the coding sequence of SRF activated yeast growth during mating with MuRF1 and 2. (B) The interaction of selected proteins derived from the above-mentioned genes was studied in vitro by pull-downs using expressed MuRF1/MuRF2 Bcc (B-Box+coiled-coil domain) and MuRF1cc (coiled-coil domain) constructs (see also Supplementary Figure S1 and methods). MuRF1cc and MuRF1Bcc (arrows) co-eluted together with CARP, EEF1G, GFM1 MBP fusion proteins. Below: left—MuRF1cc co-eluted with myozenin-1/calsarcin-2, and MRP-L41/Pig3 MBP-fusion proteins; right—MuRF2Bcc co-eluted together with CARP, EEFG1, GFM1 MBP fusion proteins; controls—MBP plus MuRF1cc, Bcc, MuRF2Bcc, respectively, or fusion proteins only.
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f3: MuRF1 and MuRF2 interact with a shared set of myocellular proteins. (A) YTH screens with full-length MuRF1 and MuRF2 baits of both human cardiac (‘heart') and skeletal cDNA libraries (‘SKM') fished a total of 87 genes. The table summarizes those 35 prey clones identified independently in both MuRF1 and MuRF2 screens and thus predicted to interact with both MuRF1+2: 13 prey clone inserts code for sarcomeric proteins (4 of which are components of the Z-disk), 10 code for transcriptional regulators (2 of which are also associated with the Z-disk), 5 genes are involved in mitochondrial ATP production, and 6 genes participate in translation initiation and elongation. Numbers indicate independently identified prey clones in respective screens. M=interaction was found by mating. An SRF prey clone fished with the MuRF1 bait could not be confirmed by mating, as in our hands the 3′ UTR and not the coding sequence of SRF activated yeast growth during mating with MuRF1 and 2. (B) The interaction of selected proteins derived from the above-mentioned genes was studied in vitro by pull-downs using expressed MuRF1/MuRF2 Bcc (B-Box+coiled-coil domain) and MuRF1cc (coiled-coil domain) constructs (see also Supplementary Figure S1 and methods). MuRF1cc and MuRF1Bcc (arrows) co-eluted together with CARP, EEF1G, GFM1 MBP fusion proteins. Below: left—MuRF1cc co-eluted with myozenin-1/calsarcin-2, and MRP-L41/Pig3 MBP-fusion proteins; right—MuRF2Bcc co-eluted together with CARP, EEFG1, GFM1 MBP fusion proteins; controls—MBP plus MuRF1cc, Bcc, MuRF2Bcc, respectively, or fusion proteins only.

Mentions: A total of 87 genes were identified as MuRF1 or MuRF2 interacting prey clones that coded for myofibrillar proteins (18, including 11 Z-disk proteins), transcriptional regulators (11), translation factors (4), and component of the mitochondrial proteome (including ATP-synthesis (9)). Of these 87 genes, a set of 35 genes was fished with both MuRF1 and MuRF2 baits and was further confirmed by mating studies. The group of ligands shared by MuRF1 and MuRF2 included a set of four myofibrillar Z-disk proteins and the transcriptional regulators CARP, myozenin1/calsarcin2, FHL2 (also associated with Z-disk region; for review, see Clark et al, 2002) (Figure 3A). To further test whether the Yeast Two-Hybrid (YTH) prey clones code indeed for MuRF1 and MuRF2 binding proteins, we performed in vitro pull-down studies using expressed CARP, myozenin1/calsarcin2 (two molecules selected as known transcriptional regulators of muscle gene expression), MRP-L41/pig3 (selected as a member of the mitochondrial ribosomal group, also being implicated in growth control; see Yoo et al, 2005), and EEF1G/EF-1γ (selected as a sophisticatedly regulated component of the translation machinery; see Belle et al, 1995) as well as its mitochondrial counterpart GFM1. Results indicated that a central MuRF1 fragment that comprises the MuRF1 residues 109–315 (‘MuRF1Bcc'; see Supplementary Figure 1A) is both sufficient and required for interaction with CARP, EEF1G, GFM1, myozenin1/calsarcin-2, and pig3/MRP-L41 (Figure 3B). Similarly, expressed MuRF2Bcc interacted in vitro with CARP, EEF1G, and GFM1 (Figure 3B). Finally, YTH mating suggested that MuRF3Bcc does not interact with CARP, myozenin-1/calsarcin-2, and pig3/MRP-L41 (data not shown).


Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2.

Witt CC, Witt SH, Lerche S, Labeit D, Back W, Labeit S - EMBO J. (2007)

MuRF1 and MuRF2 interact with a shared set of myocellular proteins. (A) YTH screens with full-length MuRF1 and MuRF2 baits of both human cardiac (‘heart') and skeletal cDNA libraries (‘SKM') fished a total of 87 genes. The table summarizes those 35 prey clones identified independently in both MuRF1 and MuRF2 screens and thus predicted to interact with both MuRF1+2: 13 prey clone inserts code for sarcomeric proteins (4 of which are components of the Z-disk), 10 code for transcriptional regulators (2 of which are also associated with the Z-disk), 5 genes are involved in mitochondrial ATP production, and 6 genes participate in translation initiation and elongation. Numbers indicate independently identified prey clones in respective screens. M=interaction was found by mating. An SRF prey clone fished with the MuRF1 bait could not be confirmed by mating, as in our hands the 3′ UTR and not the coding sequence of SRF activated yeast growth during mating with MuRF1 and 2. (B) The interaction of selected proteins derived from the above-mentioned genes was studied in vitro by pull-downs using expressed MuRF1/MuRF2 Bcc (B-Box+coiled-coil domain) and MuRF1cc (coiled-coil domain) constructs (see also Supplementary Figure S1 and methods). MuRF1cc and MuRF1Bcc (arrows) co-eluted together with CARP, EEF1G, GFM1 MBP fusion proteins. Below: left—MuRF1cc co-eluted with myozenin-1/calsarcin-2, and MRP-L41/Pig3 MBP-fusion proteins; right—MuRF2Bcc co-eluted together with CARP, EEFG1, GFM1 MBP fusion proteins; controls—MBP plus MuRF1cc, Bcc, MuRF2Bcc, respectively, or fusion proteins only.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2168395&req=5

f3: MuRF1 and MuRF2 interact with a shared set of myocellular proteins. (A) YTH screens with full-length MuRF1 and MuRF2 baits of both human cardiac (‘heart') and skeletal cDNA libraries (‘SKM') fished a total of 87 genes. The table summarizes those 35 prey clones identified independently in both MuRF1 and MuRF2 screens and thus predicted to interact with both MuRF1+2: 13 prey clone inserts code for sarcomeric proteins (4 of which are components of the Z-disk), 10 code for transcriptional regulators (2 of which are also associated with the Z-disk), 5 genes are involved in mitochondrial ATP production, and 6 genes participate in translation initiation and elongation. Numbers indicate independently identified prey clones in respective screens. M=interaction was found by mating. An SRF prey clone fished with the MuRF1 bait could not be confirmed by mating, as in our hands the 3′ UTR and not the coding sequence of SRF activated yeast growth during mating with MuRF1 and 2. (B) The interaction of selected proteins derived from the above-mentioned genes was studied in vitro by pull-downs using expressed MuRF1/MuRF2 Bcc (B-Box+coiled-coil domain) and MuRF1cc (coiled-coil domain) constructs (see also Supplementary Figure S1 and methods). MuRF1cc and MuRF1Bcc (arrows) co-eluted together with CARP, EEF1G, GFM1 MBP fusion proteins. Below: left—MuRF1cc co-eluted with myozenin-1/calsarcin-2, and MRP-L41/Pig3 MBP-fusion proteins; right—MuRF2Bcc co-eluted together with CARP, EEFG1, GFM1 MBP fusion proteins; controls—MBP plus MuRF1cc, Bcc, MuRF2Bcc, respectively, or fusion proteins only.
Mentions: A total of 87 genes were identified as MuRF1 or MuRF2 interacting prey clones that coded for myofibrillar proteins (18, including 11 Z-disk proteins), transcriptional regulators (11), translation factors (4), and component of the mitochondrial proteome (including ATP-synthesis (9)). Of these 87 genes, a set of 35 genes was fished with both MuRF1 and MuRF2 baits and was further confirmed by mating studies. The group of ligands shared by MuRF1 and MuRF2 included a set of four myofibrillar Z-disk proteins and the transcriptional regulators CARP, myozenin1/calsarcin2, FHL2 (also associated with Z-disk region; for review, see Clark et al, 2002) (Figure 3A). To further test whether the Yeast Two-Hybrid (YTH) prey clones code indeed for MuRF1 and MuRF2 binding proteins, we performed in vitro pull-down studies using expressed CARP, myozenin1/calsarcin2 (two molecules selected as known transcriptional regulators of muscle gene expression), MRP-L41/pig3 (selected as a member of the mitochondrial ribosomal group, also being implicated in growth control; see Yoo et al, 2005), and EEF1G/EF-1γ (selected as a sophisticatedly regulated component of the translation machinery; see Belle et al, 1995) as well as its mitochondrial counterpart GFM1. Results indicated that a central MuRF1 fragment that comprises the MuRF1 residues 109–315 (‘MuRF1Bcc'; see Supplementary Figure 1A) is both sufficient and required for interaction with CARP, EEF1G, GFM1, myozenin1/calsarcin-2, and pig3/MRP-L41 (Figure 3B). Similarly, expressed MuRF2Bcc interacted in vitro with CARP, EEF1G, and GFM1 (Figure 3B). Finally, YTH mating suggested that MuRF3Bcc does not interact with CARP, myozenin-1/calsarcin-2, and pig3/MRP-L41 (data not shown).

Bottom Line: The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues.Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis.Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anesthesiology and Intensive Care, Universitätsklinikum Mannheim, Mannheim, Germany.

ABSTRACT
The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.

Show MeSH
Related in: MedlinePlus