K153R polymorphism in myostatin gene increases the rate of promyostatin activation by furin

Recent studies demonstrated an association between the K153R polymorphism in the myostatin gene with extreme longevity, lower muscle strength and obesity but the molecular basis of these associations has not been clarified. Here, we show that the K153R mutation significantly increases the rate of proteolysis of promyostatin by furin, but has no effect on the activity of the latent complex or the cleavage of the latent complex by bone morphogenetic protein 1 (BMP-1). The increased rate of activation of K153R mutant promyostatin may explain why this polymorphism is associated with obesity, lower muscle strength and extension of lifespan

Wnt fehérjék és Wnt receptorok = Interaction of Wnt proteins with receptors and antagonists

Arginine-scanning mutagenezis és CD spektroszkópia segítségével meghatároztuk a Wnt inhibitory factor-1 WIF-doménjének Wnt-kötőhelyét. Kimutattuk, hogy a Wnt-kötőhely egyik szubrégiója (melynek kialakításában a Ile25, Phe27 és Phe42 aminosavak vesznek részt) a Wnt fehérjék palmitoil csoportját köti, a kötőhely másik szubrégiója (melynek kialakításában a Tyr13, Trp15 és Leu32 aminosavak vesznek részt) a Wnt-k aminosav-oldalláncainak kötésében játszik fontos szerepet. Az a megfigyelésünk, hogy a Tyr13, Trp15 és Leu32 mutációja a WIF-domén Wnt5a affinitásának növekedéséhez, ugyanakkor a Wnt3a affinitásának csökkenéséhez vezet, azt jelzi, hogy ez a szubrégió fontos szerepet játszik a domén Wnt specifitásának meghatározásában. A Wnt specifitást meghatározó aminosavak azonosításával olyan WIF mutánsokat állíthatunk elő, melyekkel lehetőség nyílik a daganatos betegségekben kulcsszerepet játszó Wnt fehérjék szelektív gátlására. | We have localized the Wnt-binding site of the WIF-domain of Wnt inhibitory factor-1 by structure-guided arginine-scanning mutagenesis in combination with surface plasmon resonance assays. We have shown that the subregion of the Wnt-binding site defined by Ile25, Phe27 and Phe42 may bind the palmitoyl group of Wnt-s, the other subregion defined by residues Tyr13, Trp15 and Leu32, however, is critical for interactions with amino acid side-chains of Wnts. Our observation that substitution of these residues of WIF resulted in an increased affinity for Wnt5a, but decreased affinity for Wnt3a suggests that these residues may define the specificity spectrum of WIF for Wnts. These results hold promise for the more specific targeting of Wnt family members with WIF variants in various forms of cancer

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin

The NTR domain of WFIKKN1 protein has been shown to have significant affinity for the prodomain regions of promyostatin and latent myostatin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that WFIKKN1 inhibits the release of myostatin from promyostatin and/or latent myostatin. WFIKKN1 was found to have no effect on processing of promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, however, was significantly enhanced in the presence of WFIKKN1 and this enhancer activity was superstimulated by heparin. Unexpectedly, WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 contributes most significantly to the observed enhancer activity. Analysis of a pro-TGF-β -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible to BMP1. In view of this observation, the most plausible explanation for the BMP1-enhancer activity of the KKN1 fragment is that it shifts a conformational equilibrium of latent myostatin from the closed circular structure of the homodimer to a more open form, making the cleavage sites more accessible to BMP1. On the other hand, the observation that the enhancer activity of KKN1 is superstimulated in the presence of heparin is explained by the fact KKN1, latent myostatin, and BMP1 have affinity for heparin and these interactions with heparin increase the local concentrations of the reactants thereby facilitating the action of BMP1. Enzymes: Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies

Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondas et al. (2008) J Biol Chem 283, 23677–23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor- binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2

Reassessing Domain Architecture Evolution of Metazoan Proteins: Major Impact of Gene Prediction Errors

In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI’s GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI’s GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI’s GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA differences in as much as it increases the proportion of terminal over internal DA differences. Here we have shown that in the case of RefSeq, EnsEMBL and NCBI’s GNOMON predicted protein sequences of Metazoan species, the contribution of gene prediction errors to domain architecture differences of orthologs is comparable to or greater than those due to true gene rearrangements. We have also demonstrated that domain architecture comparison may serve as a useful tool for the quality control of gene predictions and may thus guide the correction of sequence errors. Our findings caution that earlier genome-scale studies based on comparison of predicted (frequently mispredicted) protein sequences may have led to some erroneous conclusions about the evolution of novel domain architectures of multidomain proteins. A reassessment of the DA evolution of orthologous and paralogous proteins is presented in an accompanying paper [1]

Correction: Nagy, A., et al. Reassessing Domain Architecture Evolution of Metazoan Proteins: Major Impact of Gene Prediction Errors. Genes 2011, 2, 449-501.

We found some errors in the published versions of Figure S2, Figure S3 and Figure S8 of our paper [1]. The correct Figures are presented below