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Genetic analysis of the role of proteolysis in the activation of latent myostatin.

Lee SJ - PLoS ONE (2008)

Bottom Line: We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases.Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members.These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. sjlee@jhmi.edu

ABSTRACT
Myostatin is a secreted protein that normally acts to limit skeletal muscle growth. As a result, there is considerable interest in developing agents capable of blocking myostatin activity, as such agents could have widespread applications for the treatment of muscle degenerative and wasting conditions. Myostatin normally exists in an inactive state in which the mature C-terminal portion of the molecule is bound non-covalently to its N-terminal propeptide. We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases. Here, I show that mice engineered to carry a germline point mutation rendering the propeptide protease-resistant exhibit increases in muscle mass approaching those seen in mice completely lacking myostatin. Mice homozygous for the point mutation have increased muscling even though their circulating levels of myostatin protein are dramatically increased, consistent with an inability of myostatin to be activated from its latent state. Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members. These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.

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Generation of mice carrying a Mstn point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in Mstn alleles containing a single LoxP site (in intron 1) either with (MstnD76A) or without (MstnLoxP) the point mutation. (b) Northern analysis of Mstn RNA expression in mutant mice. Muscle RNA isolated from Mstn−/−, wild type, and MstnD76A/D76A mice was electrophoresed, blotted, and hybridized with a Mstn probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, Mstn−/−, and MstnD76A/D76A mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain [2] or the propeptide [15]. In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells [15].
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pone-0001628-g001: Generation of mice carrying a Mstn point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in Mstn alleles containing a single LoxP site (in intron 1) either with (MstnD76A) or without (MstnLoxP) the point mutation. (b) Northern analysis of Mstn RNA expression in mutant mice. Muscle RNA isolated from Mstn−/−, wild type, and MstnD76A/D76A mice was electrophoresed, blotted, and hybridized with a Mstn probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, Mstn−/−, and MstnD76A/D76A mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain [2] or the propeptide [15]. In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells [15].

Mentions: To determine whether this mechanism for activating latent myostatin functions in vivo, I generated mice in which the aspartate to alanine change in the propeptide was introduced into the germline. As shown in Figure 1a, the D76A point mutation was engineered into a myostatin genomic construct, which was used for homologous targeting in embryonic stem cells. Following injection of the targeted embryonic stem cells into blastocysts, I obtained mice that transmitted the mutant allele through the germline. These mice were then crossed to EIIa-cre transgenic mice [20] to remove the neo cassette that had been placed into the first intron. In order to rule out the possibility that any observed effects might result from the LoxP site remaining following removal of the neo cassette, I also generated mice carrying this LoxP site but which were wild type at aspartate 76. Both of these lines were backcrossed at least 6 times to C57 BL/6 mice prior to analysis, and all analysis was carried out on both female and male mice at 10 weeks of age.


Genetic analysis of the role of proteolysis in the activation of latent myostatin.

Lee SJ - PLoS ONE (2008)

Generation of mice carrying a Mstn point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in Mstn alleles containing a single LoxP site (in intron 1) either with (MstnD76A) or without (MstnLoxP) the point mutation. (b) Northern analysis of Mstn RNA expression in mutant mice. Muscle RNA isolated from Mstn−/−, wild type, and MstnD76A/D76A mice was electrophoresed, blotted, and hybridized with a Mstn probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, Mstn−/−, and MstnD76A/D76A mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain [2] or the propeptide [15]. In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells [15].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2237902&req=5

pone-0001628-g001: Generation of mice carrying a Mstn point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in Mstn alleles containing a single LoxP site (in intron 1) either with (MstnD76A) or without (MstnLoxP) the point mutation. (b) Northern analysis of Mstn RNA expression in mutant mice. Muscle RNA isolated from Mstn−/−, wild type, and MstnD76A/D76A mice was electrophoresed, blotted, and hybridized with a Mstn probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, Mstn−/−, and MstnD76A/D76A mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain [2] or the propeptide [15]. In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells [15].
Mentions: To determine whether this mechanism for activating latent myostatin functions in vivo, I generated mice in which the aspartate to alanine change in the propeptide was introduced into the germline. As shown in Figure 1a, the D76A point mutation was engineered into a myostatin genomic construct, which was used for homologous targeting in embryonic stem cells. Following injection of the targeted embryonic stem cells into blastocysts, I obtained mice that transmitted the mutant allele through the germline. These mice were then crossed to EIIa-cre transgenic mice [20] to remove the neo cassette that had been placed into the first intron. In order to rule out the possibility that any observed effects might result from the LoxP site remaining following removal of the neo cassette, I also generated mice carrying this LoxP site but which were wild type at aspartate 76. Both of these lines were backcrossed at least 6 times to C57 BL/6 mice prior to analysis, and all analysis was carried out on both female and male mice at 10 weeks of age.

Bottom Line: We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases.Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members.These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. sjlee@jhmi.edu

ABSTRACT
Myostatin is a secreted protein that normally acts to limit skeletal muscle growth. As a result, there is considerable interest in developing agents capable of blocking myostatin activity, as such agents could have widespread applications for the treatment of muscle degenerative and wasting conditions. Myostatin normally exists in an inactive state in which the mature C-terminal portion of the molecule is bound non-covalently to its N-terminal propeptide. We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases. Here, I show that mice engineered to carry a germline point mutation rendering the propeptide protease-resistant exhibit increases in muscle mass approaching those seen in mice completely lacking myostatin. Mice homozygous for the point mutation have increased muscling even though their circulating levels of myostatin protein are dramatically increased, consistent with an inability of myostatin to be activated from its latent state. Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members. These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.

Show MeSH
Related in: MedlinePlus