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Metalloprotease activation of myostatin, and methods of modulating myostatin activity

A technology of somatostatin and metalloprotease, applied in the field of metalloprotease activation of myostatin and regulating the activity of myostatin, can solve the problems of different and harmful effects, etc.

Inactive Publication Date: 2006-11-29
THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, these proteins can also act on various other proteins, therefore, agents that target and inhibit these proteases such as signal peptidases are likely to have different and deleterious effects if administered to living organisms

Method used

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  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity
  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity
  • Metalloprotease activation of myostatin, and methods of modulating myostatin activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

BMP-1 / TLD metalloproteinase family cleaves myostatin propeptide

[0087] This example demonstrates that members of the bone morphogenetic protein 1 / Tolloid (BMP-1 / TLD) family of metalloproteases cleave myostatin propeptide.

[0088] 500 ng of purified myostatin propeptide or purified latent myostatin complex containing propeptide and C-terminal dimer (Lee and McPherron, supra, 2001) was mixed with 100 ng of purified BMP-1 , mTLD, mTLL-1 or mTLL-2 (Scott et al., Devel. Biol. 213:283-300, 1999, which is incorporated herein by reference) were incubated overnight at 37°C. Reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by western blot analysis with antiserum against myostatin propeptide (Lee and McPherron, supra, 2001).

[0089] Isolated propeptide proteolytic cleavage products were detected in every reaction containing one of the four proteases, but not in control reactions containing no protease. Furthermor...

Embodiment 2

Cleavage of myostatin propeptide by metalloproteases activates latent myostatin

[0090] This example demonstrates that cleavage of myostatin propeptide by BMP-1 / TLD metalloproteases activates latent myostatin.

[0091] Purified myostatin propeptide and C-terminal dimer complexes were incubated with mTLL-1 and then examined using a reporter gene assay that specifically detects myostatin activity. with pGL3-(CAGA) 12 A204 rhabdomyosarcoma cells were transfected with a luciferase reporter construct comprising a luciferase coding sequence linked to a TGF-β-responsive CAGA sequence from the promoter of the TGF-β-inducible PAI-1 gene (Thies et al., supra, 2001). Transfected cells were contacted with untreated propeptide / C-terminal dimer complexes or with complexes that had been pre-incubated with mTLL-1. Incubation of complexes with mTLL-1 significantly increased the amount of luciferase activity detected in reporter cell assays, whereas complexes treated with mTLL-1 alone or wit...

Embodiment 3

Peptide substrates for members of the Tolloid family

[0093] Synthesize a series of peptides according to the sequence of myostatin propeptide, they are 10, 20, 30, 40 or 50 amino acid residues, each series includes three peptides, they all contain the corresponding BMP-1 / TLD The location of the cleavage site of the metalloprotease (in the wild-type peptide SEQ ID NOS: 9, 12, 15, 18 and 21, the amino acid residue "RD" is shown in bold, below). Peptides were synthesized in which the arginine residue at the P1 position immediately upstream of the cleavage site was changed to a glutamine residue (SEQ ID NOS: 10, 13, 16, 19 and 22; see bold), where Peptides were synthesized with aspartic acid changed to alanine at the P1' position immediately downstream of the cleavage site (SEQ ID NOS: 11, 14, 17, 20 and 23; see bold). The sequences of the peptides are listed below:

[0094] 50-mer (50-mer)

[0095]

KDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIIT

MPT (SEQ I...

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Abstract

It has been determined that metalloprotease cleavage of a myostatin pro peptide results in activation of a latent inactive myostatin to an active form. Accordingly, methods of identifying agents that modulate metalloprotease mediated activation of myostatin are provided, as are agents identified using such methods. Also provided are methods of modulating muscle growth in an organism by increasing or decreasing metalloprotease mediated cleavage of a myostatin pro peptide.

Description

[0001] This invention was made with government support, in part, through grants HD35887, AR47746, and GM63471 from the National Institutes of Health. The US Government has certain rights in this invention. technical field [0002] The present invention generally relates to the metalloprotease regulation of myostatin (myostatin) activity, more specifically, relates to the method for regulating the activity of myostatin by using an agonist or antagonist of metalloproteinase BMP-1 / TLD family, comprising For example, methods of modulating muscle development in an organism, the invention also relates to methods of identifying agonists and antagonists of such metalloproteases, and agonists and antagonists thus identified. Background technique [0003] Myostatin is a member of the transforming growth factor beta (TGF-β) family that is essential for the normal regulation of skeletal muscle growth. Myostatin is a secreted protein that is expressed specifically during embryonic devel...

Claims

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Application Information

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IPC IPC(8): A61K33/00A61K38/00A61K35/00A01N61/00C12N9/64A61K38/46C07K1/00C12Q1/37
Inventor S－J·李A·C·麦克弗洛D·S·格林斯潘W·N·帕帕诺N·沃尔夫曼K·汤姆金森
Owner THE JOHN HOPKINS UNIV SCHOOL OF MEDICINE
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