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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 and analysis of mice carrying a loss-of-function mutation in the Tll2 gene.(a) Gene targeting strategy. Locations of exons 6–9 are shown as black boxes, and LoxP sites are denoted by triangles. (b) Northern analysis of Tll2 expression levels. Twenty micrograms of poly A-selected brain RNA isolated from either wild type or Tll2−/− mice were electrophoresed, blotted, and hybridized with a Tll2 probe corresponding to exons 1–3. The blot was re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Muscle weight increases in Tll2−/− mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).
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pone-0001628-g003: Generation and analysis of mice carrying a loss-of-function mutation in the Tll2 gene.(a) Gene targeting strategy. Locations of exons 6–9 are shown as black boxes, and LoxP sites are denoted by triangles. (b) Northern analysis of Tll2 expression levels. Twenty micrograms of poly A-selected brain RNA isolated from either wild type or Tll2−/− mice were electrophoresed, blotted, and hybridized with a Tll2 probe corresponding to exons 1–3. The blot was re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Muscle weight increases in Tll2−/− mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).

Mentions: To determine whether TLL-2 plays a role in regulating myostatin latency in vivo, I generated mice carrying a targeted deletion of the Tll2 gene. The Tll2 gene consists of 21 exons, with the protease domain being encoded in exons 4–8. As shown in Figure 3a, I generated a construct in which a LoxP site and a LoxP/neo cassette were introduced into introns 6 and 7, respectively. Following homologous recombination in embryonic stem cells and transfer of the targeted cells into blastocysts, several chimeric mice were obtained that transmitted the targeted allele through the germline. Offspring from the chimeric mice were then mated with EIIa-cre transgenic mice to generate mice in which exon 7 had been completely deleted. The deletion allele was then backcrossed six times onto a C57 BL/6 genetic background prior to analysis. Mice homozygous for the deletion were viable and fertile, and all analysis was carried out on 10-week old mice.


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

Lee SJ - PLoS ONE (2008)

Generation and analysis of mice carrying a loss-of-function mutation in the Tll2 gene.(a) Gene targeting strategy. Locations of exons 6–9 are shown as black boxes, and LoxP sites are denoted by triangles. (b) Northern analysis of Tll2 expression levels. Twenty micrograms of poly A-selected brain RNA isolated from either wild type or Tll2−/− mice were electrophoresed, blotted, and hybridized with a Tll2 probe corresponding to exons 1–3. The blot was re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Muscle weight increases in Tll2−/− mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2237902&req=5

pone-0001628-g003: Generation and analysis of mice carrying a loss-of-function mutation in the Tll2 gene.(a) Gene targeting strategy. Locations of exons 6–9 are shown as black boxes, and LoxP sites are denoted by triangles. (b) Northern analysis of Tll2 expression levels. Twenty micrograms of poly A-selected brain RNA isolated from either wild type or Tll2−/− mice were electrophoresed, blotted, and hybridized with a Tll2 probe corresponding to exons 1–3. The blot was re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Muscle weight increases in Tll2−/− mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).
Mentions: To determine whether TLL-2 plays a role in regulating myostatin latency in vivo, I generated mice carrying a targeted deletion of the Tll2 gene. The Tll2 gene consists of 21 exons, with the protease domain being encoded in exons 4–8. As shown in Figure 3a, I generated a construct in which a LoxP site and a LoxP/neo cassette were introduced into introns 6 and 7, respectively. Following homologous recombination in embryonic stem cells and transfer of the targeted cells into blastocysts, several chimeric mice were obtained that transmitted the targeted allele through the germline. Offspring from the chimeric mice were then mated with EIIa-cre transgenic mice to generate mice in which exon 7 had been completely deleted. The deletion allele was then backcrossed six times onto a C57 BL/6 genetic background prior to analysis. Mice homozygous for the deletion were viable and fertile, and all analysis was carried out on 10-week old mice.

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