Myostatin and myostatin binders

[Biggerstronger]

Does anyone have any information on myostatin binders? Such as any in-vitro or even in-vivo studies, availability, or even use. I know I've seen some myostatin inhibitors being sold at different places, but I assumed that those were probably just BS.

(Sorry, I know this is off-topic...but I thought it would be more appropriate here)

[einstein1905]

Nothing being sold as an oral will bind myostatin (GDF-8). Their have been in vivo studies.....complete myostatin KO animals (cattle, mice, rats, etc) and also some RNAi work, which conditionally "silences GDF-8 expression, so the effects are transient.....I don't believe there has been any publication of the RNAi stuff (against myostatin mRNA) in vivo though.
Do a pubmed search for either or both myostatin and GDF-8.

GASP-1 is a protein that has been alleged to antagonize myostatin.

[rambo]

There is no product on the market, nor can I foresee one in the future that can bind GDF-8 orally.

[Biggerstronger]

Well I was originally thinking about follistatin. As its been known to bind with myostatin from what I've read.

Do you know anything about follistatin?

[Biggerstronger]

Recombinant Human Follistatin is available from a certain research company. I believe they hold the patent on it.

[Biggerstronger]

HUMAN Cytokine/Growth Factor/Chemokines Sample Spec sheets (subject to change without notice):


--------------------------------------------------------------------------------
DATA SHEET:Human Follistatin
Follistatin belongs to a group of structurally-diverse diffusible proteins that binds to TGF-beta ligands and inhibit their activity by hindering access to signaling receptors. This naturally occurring antagonist binds to Activin, BMP-2,-4,-6,-7, Myostatin, GDF-11, and TGF-beta1. Follistatin is expressed in the pituitary, ovaries, decidual cells of the endometrium, and in some other tissues. This recombinant human Follistatin is a 31.5 kDa protein containing 288 amino acids including 36 cysteine residues.

Package Size: 20 micrograms $xxx

1 X 500ug=$x,xxx

1mg=$x,xxx

Mini-Pack:$xx/5ug

-inquire for custom sizes/bulk packaging

Supplied: The sterile filtered solution was lyophilized from 10mM sodium Phosphate pH7.5 + 75mM NaCl with no additives

Source: E. coli

Purity: >95% by SDS-Page and HPLC analysis Endotoxin <1EU/ug

Reconstitution: The lyophilized human Follistatin should be reconstituted in sterile DI water to a concentration of 0.1-1mg/ml. Allow to set at least 30 minutes at 4-8 DEG C, Mix well. If desired, centrifuge for 1-2 minutes at 1000 rpm to concentrate material in vial. . This solution can be diluted into other buffered solutions and stored at 4 DEG C for up to 1 week or stored at -20 DEG C for future use.

Note: If this material is not to be used for coating or labeling (or other applications requiring carrier free material), the addition of 0.1% BSA to further dilution buffers or dilution in sterile culture media is recommended to enhance stability and minimize absorption to vial. Ideally, maintain stock >10ug/ml.

Storage: The lyophilized powder is stable at room temperature for a few weeks but it is best stored desiccated at -20 Deg C. Reconstituted material should be stored in working aliquots at -20DEG C. AVOID FREQUENT FREEZE THAW CYCLES.

AA Sequence:

GNCWLRQAKN GRCQVLYKTE LSKEECCSTG RLSTSWTEED VNDNTLFKWM

IFNGGAPNCI PCKETCENVD CGPGKKCRMN KKNKPRCVCA PDCSNITWKG

PVCGLDGKTY RNECALLKAR CKEQPELEVQ YQGRCKKTCR DVFCPGSSTC

VVDQTNNAYC VTCNRICPEP ASSEQYLCGN DGVTYSSACH LRKATCLLGR

SIGLAYEGKC IKAKSCEDIQ CTGGKKCLWD FKVGRGRCSL CDELCPDSKS

DEPVCASDNA TYASECAMKE AACSSGVLLE VKHSGSCN


Reactivity: Determined by its ability to neutralize Activin A inhibitory effect of murine MPC-11 cells. The expected ED50 us 0.1-0.4ug/ml in the presence of 7.5ng/ml Activin A.



Precautions: For In vitro research Use Only. Not for use in or on humans or animals or for diagnostics. It is the responsibility of the user to comply with all local/state and Federal rules in the use of this product. We are not responsible for any patent infringements that might result with the use of or derivation of this product.

(Note: I removed any references to prices)

[einstein1905]

You can't use something that'll bind all the TGF-beta members. To inhibit TGF-beta 1 would be detrimental.

The follistatin being sold is just for in vitro work.

To neutralize TGFb-1 would be almost a guarantee for developing skin cancer and likely lung cancer, as TGFb-1 is crucial, directly and indirectly, at controlling many aspects of cell division.

[Biggerstronger]

That leads me into my other thought...would the follistatin be only available to the muscle that it's injected into or would it be absorbed into the blood stream to be carried into other muscles?

This is sounding dangerous...

[Biggerstronger]

I wonder if it would be possible to add a amino chain to the compound that would resist binding to certain growth factors, like they did with IGF1

[einstein1905]

Follistatin binds TGFb family members by binding the creatively named follistatin binding domains on them, so there isn't any real specificity.
The best bet would be to use anti-GDF-8 antibodies, which would just "titrate" GDF-8 in a dose-dependent manner. There was myogrow that was allegedly 3-4 different ssRNA species that were complementary to myostatin mRNA, so it would bind the complementary strands of RNA to form dsRNA, which the body quickly degrades.....this effectively "silences" gene expression of myostatin. It was a weak idea....the RNAi is a sound method, but you'd be far better off using a plasmid that encoded the respective RNA species that would complement myostatin mRNA....you would then get multiple copies of the RNAi species as opposed to just the number of copies injected. The specificity of RNAi is almost perfect.

The problem with anti-GDF-8 Abs is the potential for an autoimmune response, depending on the species the Ab were produced in. Ab-mediated degradation would ensue, and various peptide fragments would be displayed via MHCII.....the Abs from a different species would likely cause a localized "danger response" upregulating costimulatory molecules and allowing the body to have great potential for developing an immune response against many different regions of the myostatin that had been bound and degraded......since myostatin shares many homologous regions with other TGFb members, this could be very serious.

[rambo]

Follistatin binds TGFb family members by binding the creatively named follistatin binding domains on them, so there isn't any real specificity.
The best bet would be to use anti-GDF-8 antibodies, which would just "titrate" GDF-8 in a dose-dependent manner. There was myogrow that was allegedly 3-4 different ssRNA species that were complementary to myostatin mRNA, so it would bind the complementary strands of RNA to form dsRNA, which the body quickly degrades.....this effectively "silences" gene expression of myostatin. It was a weak idea....the RNAi is a sound method, but you'd be far better off using a plasmid that encoded the respective RNA species that would complement myostatin mRNA....you would then get multiple copies of the RNAi species as opposed to just the number of copies injected. The specificity of RNAi is almost perfect.

The problem with anti-GDF-8 Abs is the potential for an autoimmune response, depending on the species the Ab were produced in. Ab-mediated degradation would ensue, and various peptide fragments would be displayed via MHCII.....the Abs from a different species would likely cause a localized "danger response" upregulating costimulatory molecules and allowing the body to have great potential for developing an immune response against many different regions of the myostatin that had been bound and degraded......since myostatin shares many homologous regions with other TGFb members, this could be very serious.

You are sooooo my hero.

[Biggerstronger]

Wait...Can you explain something?

myostatin shares many homologous regions with other TGFb members

So you're saying myostatin is structurely very similar to all other tgfb's and if you're body thinks that the myostatin abs is a virus your body would attack not only myostatin but all tgfb's.

What controls the production of myostatin? Wouldn't there have to be some sort of negative feedback loop otherwise your body would eventually waste away? (I.E. Old age, Muscle wasting diseases: aids, muscular dystrophy)


OR

Is myostatin production somehow related to the production of other tgf's?

Also does TGFb's have anything to do with white or red blood cell production...immune system functions?

[einstein1905]

Wait...Can you explain something?

So you're saying myostatin is structurely very similar to all other tgfb's and if you're body thinks that the myostatin abs is a virus your body would attack not only myostatin but all tgfb's.

What controls the production of myostatin? Wouldn't there have to be some sort of negative feedback loop otherwise your body would eventually waste away?

There is great potential for the body to form an immune response to myostatin if anti-myostatin Abs are used........the immune response will be mounted against regions of the degraded peptide (~13aa regions)...since many TGFb family members share many homologous regions with myostatin, if the region(s) of myostatin against which the immune system forms a response happen to be regions of shared homology with any other TGFb members, they will be just as susceptible to an immune response.

The body would recognize the antibody:myostatin complex as foreign (also the Abs themselves too)...since the body has recognized a "foreign" antigen in a certain environment, many components of the immune system will upregulate many costimulatory molecules, which facilitate the development of an adaptive immune response to, ideally, the forein antigen, but also to any proteins that are degraded in the local environment.....this is the classical pattern of development of autoimmunity......It's almost guaranteed that a foreign antigen be present to prime the local immune environment.

Certainly, the regulation of myostatin is, as with all genes, controlled by the net effect of positive and negative regulators. Gh actually has a negative impact of myostatin expression

[Biggerstronger]

Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains
Jennifer J. Hill, Yongchang Qiu, Rodney M. Hewick and Neil M. Wolfman
Department of Protein Chemistry and Proteomics (J.J.H., Y.Q., R.M.H.), and Department of Musculoskeletal Sciences (N.M.W.), Wyeth Research, Cambridge, Massachusetts 02140

Address all correspondence and requests for reprints to: Jennifer J. Hill, Department of Protein Chemistry and Proteomics, Wyeth Research, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140. E-mail: [email protected].

Myostatin, a member of the TGFß superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFß superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFß binding proteins.


Differential Response to Exogenous and Endogenous Myostatin in Myoblasts Suggests that Myostatin Acts as an Autocrine Factor in Vivo
Ramón Ríos1, Susana Fernández-Nocelos, Isabel Carneiro, Víctor M. Arce and Jesús Devesa
Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, A Coruña 15782, Spain

Address all correspondence and requests for reprints to: Víctor M. Arce, M.D., Ph.D., Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, San Francisco 1, 15782 Santiago de Compostela, Spain. E-mail: [email protected].

Myostatin is a member of the TGF-ß superfamily that is essential for proper regulation of skeletal muscle growth. As do other TGF-ß superfamily members, myostatin signals into the cell via a receptor complex that consists of two distinct transmembrane proteins, known as the type I and type II receptors. Vertebrates have seven distinct type I receptors, each of which can mix and match with one of five type I receptors to mediate signals for all the TGF-ß family ligands. Accumulating evidence indicates that myostatin shares its pair of receptors with activin, and therefore, the question arises about how specificity in signaling is achieved. Our hypothesis is that a mechanism has to exist to restrict myostatin actions to the muscle cells. To investigate this possibility, we compared the effect of endogenous myostatin (myostatin overexpressed by myoblasts) and exogenous myostatin (recombinant myostatin added to the culture medium) in cultured myoblasts. As opposed to exogenous myostatin, endogenous myostatin induced the transcription of a reporter vector in cultured myoblasts. Notably, the myostatin concentrations that failed to induce a response in myoblasts were effective in MCF-7 cells (human mammary carcinoma) and in HepG2 cells (human hepatic carcinoma). Based on our observations, we propose that a mechanism exists that differentially regulates the bioavailability of endogenous and exogenous myostatin to muscle cells. This is consistent with a model in which myostatin actions are exerted in vivo in an autocrine fashion

Activation of latent myostatin by the BMP-1/tolloid family of metalloproteinases
Neil M. Wolfman * , Alexandra C. McPherron , William N. Pappano ¶, Monique V. Davies ||, Kening Song **, Kathleen N. Tomkinson *, Jill F. Wright *, Liz Zhao **, Suzanne M. Sebald , Daniel S. Greenspan ¶ and Se-Jin Lee

*Department of Inflammation, ||Antibody Technology Group, and **Department of Cardiovascular and Metabolic Diseases, Wyeth Research, 200 CambridgePark Drive, Cambridge, MA 02140; Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and Departments of ¶Biomolecular Chemistry and Pathology and Laboratory Medicine, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX and approved October 6, 2003 (received for review August 4, 2003)


Myostatin is a transforming growth factor family member that acts as a negative regulator of skeletal muscle growth. Myostatin circulates in the blood of adult mice in a noncovalently held complex with other proteins, including its propeptide, which maintain the C-terminal dimer in a latent, inactive state. This latent form of myostatin can be activated in vitro by treatment with acid; however, the mechanisms by which latent myostatin is activated in vivo are unknown. Here, we show that members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteinases can cleave the myostatin propeptide in this complex and can thereby activate latent myostatin. Furthermore, we show that a mutant form of the propeptide resistant to cleavage by BMP-1/TLD proteinases can cause significant increases in muscle mass when injected into adult mice. These findings raise the possibility that members of the BMP-1/TLD family may be involved in activating latent myostatin in vivo and that molecules capable of inhibiting these proteinases may be effective agents for increasing muscle mass for both human therapeutic and agricultural applications.

[Biggerstronger]

Since GASP-1 seems like it's the only direct myostatin binder has anyone used it yet?

[einstein1905]

Since GASP-1 seems like it's the only direct myostatin binder has anyone used it yet?

"Thus, GASP-1 represents a novel class of inhibitory TGFß binding proteins."
GASP-1 binds both mature and "pro"myostatin, yes, but it hasn't been stated that it does NOT bind the other TGFb family members. There was a research company that was selling GASP-1 (or so they claimed), but they're now out of business. The same dangers still hold true for using GASP-1......inhibition of other TGFb family members.

[johnsomebody]

Wow, this is really interesting.

Aren't they using myostatin antibodies to reduce myostatin levels in current experiments? I'm wondering if it's ever been tried on humans yet -it's been used in mice, from what I recall reading.

[Biggerstronger]

Natural TGF-ß Antagonists
Once liberated from latent complexes, ligands of the TGF-ß family may be intercepted by a host of diffusible proteins which bind the ligands with varying degrees of affinity and inhibit their access to signaling receptors. The expression of some of these natural antagonists is positively regulated by their TGF-ß binding partners. Reverse diffusion of the antagonists, i.e. from target cells towards localized sources of competent ligands, creates signaling gradients in the extracellular milieu (4). The interplay between ligands of the TGF-ß family and their natural antagonists has major biological significance during developmental processes, in which the response of cells can vary considerably depending upon the local concentration of the signaling molecule. The growing list of TGF-ß natural antagonists includes noggin, chordin, chordin-like proteins, follistatin (FS), FS-related proteins, sclerostin, and the DAN/cerberus family of proteins (Table 1).

Table 1: Antagonists of TGF-ß Ligands



Natural TGF-ß Antagonists Structural Features Contained in the Antagonist Polypeptide (M.W.)
Known TGF-ß Binding Partners

Noggin Unique Noggin cysteine-knot domain (26 kDa) BMP-2,-4,-5,-6,-7, -13/GDF-6, -14/GDF-5
Chordin 4 CR/VWC (Chordin) domains, 3 SOG repeats (102 kDa) BMP-2,-4,-7
Chordin-like/neuralin/ventroptin 3 Chordin domains (51 kDa) BMP-4,-5,-6
Follistatin 3 Cysteine-rich Follistain (FS) and 3 kazal domains (38 kDa) Activin, BMP-2,-4,-6,-7, Myostatin/GDF-8, GDF-11, TGF-ß1
Follistatin-like related gene (FLRG) 2 FS and 2 kazal domains (28 kDa) Activin, Myostatin/GDF-8, GDF-11, TGF-ß1


(*Note I highlighted this antagonist, because it regulates muscle growth)

Antagonist / Structural Features of Antagonist / Known TGFB Binding Partner

GASP-1 /1 Wap, 1 FS, 1 kazal, 1 IG-like, 2 kunitz, 1 netrin domains (63 kDa) / Myostatin/GDF-8, GDF-11

Follistatin-related protein (FSRP) 1 FS, 1 CR/VWRC, 2 EF-hand domains (35 kDa) Activin, BMP-2,-6,-7
DAN Unique DAN cysteine-knot domain (19 kDa) BMP-2,-4,-7, -14/GDF-5
Cerberus DAN-like cysteine-knot domain (30 kDa) BMP-2,-4,-7, Activin, Nodal
Gremlin DAN-like cysteine-knot domain (21 kDa) BMP-2,-4,-7
Sclerostin /SOST Unique Sclerostin cysteine-knot domain (24 kDa) BMP-5,-6
Decorin Multiple leucine-rich repeats (40 kDa) TGF-ß1, -2
alpha-2 macroglobulin Multiple proteinase inhibitor domains (163 kDa) TGF-ß1, -2, Activin, Inhibin

A novel member in this structurally-diverse group of proteins is GDF-associated serum protein-1 (GASP-1). GASP-1 has been identified in normal mouse and human serum as a myostatin-associated protein. The primary structure of GASP-1 reveals multiple putative protease inhibitory domains, and a cysteine-rich FS-like domain (5). Most TGF-ß natural antagonists are capable of interacting with more than one ligand, but display discriminating preferences for different ligands. For example, follistatin, which binds activin in a nearly irreversible manner, displays high-affinity binding for BMP-7, and a lower affinity for BMP-2 and -4, and a lower affinity for BMP-7.


– Part II of this article will be pubished in our upcoming 2004 catalog.

References:

1. D. Onichtchouk et al., Nature, Vol. 401, 480-485 (1999)
2. S.L. Lee and A.C. McPherron, Proc. Nat. Acad. Sci., Vol. 98, 9306-9311 (2001)
3. J. Massague and Y-G. Chen, Genes and Development, Vol. 14, 627-644 (2000)
4. T.L. Gumienny and R.W. Padgett, Trends in Endocrinology and Metabolism, Vol. 13, 295-299 (2002)
5. J.J. Hill et al. Molecular Endocrinology, Vol. 17, 1144-1154 (2003)

[Geebee]

Myostatin has been blocked on several people in US labs and the results are not bad, but they are not awsome. It's terribly expensive. Few people got it yet because of the price, but not because of results. As far as I know the Czech Republic has tried it too. The problem is homeostasis : treatment has to be long enough for homeostasis to adapt to the new BW, otherwise, everything will get back to normal.
You probably know that the best pros had their genes tweaked in the West Indies (they are supposed to be "on holidays") ; I don't know more about it. I was wondering if this 'gene tweaking' wasn't just myostatin blocking.

[Biggerstronger]

blocking myostatin (as a source of muscle growth) is a semi-new discovery.

Do you have any in-vivo studies of blocking myostatin in humans?

[Geebee]

Biggerstronger, I have several (half a dozen a year ago, probably many more now) ; but I promised I wouldn't give details and I always keep my confidentiality promisses (fortunately for classified !!).