Both WFIKKN1 and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144-1154). Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.     

Targeted Approach to Distinguish and Determine Absolute Levels of GDF8 and GDF11 in Mouse Serum

Growth differentiation factor 11 (GDF11) is a TGF‐β superfamily circulating factor that regulates cardiomyocyte size in rodents, sharing 90% amino acid sequence identity in the active domains with myostatin (GDF8)—the major determinant of skeletal muscle mass. Conflicting data on age‐related changes in circulating levels have been reported mainly due to the lack of specific detection methods. More recently, liquid chromatography tandem mass spectrometry (LC‐MS/MS) based assay showed that the circulating levels of GDF11 do not change significantly throughout human lifespan, but GDF8 levels decrease with aging in men. Here a novel detection method is demonstrated based on parallel reaction monitoring LC‐MS/MS assay combined with immunoprecipitation to reliably distinguish GDF11 and GDF8 as well as determine their endogenous levels in mouse serum. The data indicate that both GDF11 and GDF8 circulating levels significantly decline with aging in female mice.     

WFIKKN1 and WFIKKN2: “Companion” proteins regulating TGFB activity

The WFIKKN (WAP, Follistatin/kazal, Immunoglobulin, Kunitz and Netrin domain-containing) protein family is composed of two multidomain proteins: WFIKKN1 and WFIKKN2. They were formed by domain shuffling and are likely present in deuterostoms. The WFIKKN (also called GASP) proteins are well known for their function in muscle and skeletal tissues, namely, inhibition of certain members of the transforming growth factor beta (TGFB) superfamily such as myostatin (MSTN) and growth and differentiation factor 11 (GDF11). However, the role of the WFIKKN proteins in other tissues is still poorly understood in spite of evidence suggesting possible action in the inner ear, brain and reproduction. Further, several recent studies based on next generation technologies revealed differential expression of WFIKKN1 and WFIKKN2 in various tissues suggesting that their function is not limited to MSTN and GDF11 inhibition in musculoskeletal tissue. In this review, we summarize current knowledge about the WFIKKN proteins and propose that they are “companion” proteins for various growth factors by providing localized and sustained presentation of TGFB proteins to their respective receptors, thus regulating the balance between the activation of Smad and non-Smad pathways by TGFB.     

WFIKKN1 and WFIKKN2 bind growth factors TGFβ1, BMP2 and BMP4 but do not inhibit their signalling activity

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (K(d) ～ 10(-6) m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFβ1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space.     

Biological functions of the WAP domain-containing multidomain proteins WFIKKN1 and WFIKKN2

WFIKKN1 and WFIKKN2 are two closely related multidomain proteins consisting of a WAP (whey acidic protein)-, a follistatin-, an immunoglobulin-, two Kunitz-type protease inhibitor-domains and an NTR domain (netrin domain). Recent experiments have shown that both WFIKKN1 and WFIKKN2 bind myostatin and GDF11 (growth and differentiation factor 11) with high affinity and are potent antagonists of these growth factors. Structure-function studies on WFIKKN proteins have revealed that their interactions with GDF8 and GDF11 are mediated primarily by the follistatin and NTR domains.     

A laser ablation ICP‐MS based method for multiplexed immunoblot analysis: applications to manganese‐dependent protein dynamics of photosystem II in barley (Hordeum vulgare L.)

Manganese (Mn) constitutes an essential co‐factor in the oxygen‐evolving complex of photosystem II (PSII). Consequently, Mn deficiency reduces photosynthetic efficiency and leads to changes in PSII composition. In order to study these changes, multiplexed protein assays are advantageous. Here, we developed a multiplexed antibody‐based assay and analysed selected PSII subunits in barley (Hordeum vulgare L.). A selection of antibodies were labelled with specific lanthanides and immunoreacted with thylakoids exposed to Mn deficiency after western blotting. Subsequently, western blot membranes were analysed by laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS), which allowed selective and relative quantitative analysis via the different lanthanides. The method was evaluated against established liquid chromatography electrospray ionization tandem mass spectrometry (LC‐ESI‐MS/MS) methods, based on data‐dependent acquisition (DDA) and selected reaction monitoring (SRM). Manganese deficiency resulted in a general decrease in PSII protein abundances, an effect that was shown to be reversible upon Mn re‐supplementation. Specifically, the extrinsic proteins PsbP and PsbQ showed Mn‐dependent changes in abundances. Similar trends in the response to Mn deficiency at the protein level were observed when comparing DDA, SRM and LA‐ICP‐MS results. A biologically important exception to this trend was the loss of PsbO in the SRM analysis, which highlights the necessity of validating protein changes by more than one technique. The developed method enables a higher number of proteins to be multiplexed in comparison to existing immunoassays. Furthermore, multiplexed protein analysis by LA‐ICP‐MS provides an analytical platform with high throughput appropriate for screening large collections of plants.     

Growth differentiation factor 11: A new hope for the treatment of cardiovascular diseases

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor-β superfamily that has garnered significant attention due to its anti-cardiac aging properties. Many studies have revealed that GDF11 plays an indispensable role in the onset of cardiovascular diseases (CVDs). Consequently, it has emerged as a potential target and novel therapeutic agent for CVD treatment. However, currently, no literature reviews comprehensively summarize the research on GDF11 in the context of CVDs. Therefore, herein, we comprehensively described GDF11’s structure, function, and signaling in various tissues. Furthermore, we focused on the latest findings concerning its involvement in CVD development and its potential for clinical translation as a CVD treatment. We aim to provide a theoretical basis for the prospects and future research directions of the GDF11 application regarding CVDs.     

Heterotypy in the N-terminal region of growth/differentiation factor 5 (GDF5) mature protein during teleost evolution

Heterotypy is now recognized as a generative force in the formationof new proteins through modification of existing proteins. Wereport that heterotypy in the N-terminal region of the maturegrowth/differentiation factor 5 (GDF5) protein occurred duringevolution of teleosts. N-terminal length variation of GDF5 wasfound among teleost interfamilies and interorders but not withinteleost families or among tetrapods. We further show that increaseof proline and glutamine to the N-terminal region of matureGDF5 occurred in Eurypterygii, the higher lineage of teleosts.Because the basic amino acids, believed to control diffusion,are conserved in this region across all species examined, wesuggest that the N-terminal elongation of the mature GDF5 proteinduring evolution has altered the protein diffusion in Eurypterygii,leading to high concentrations of the protein in the joint ofthe pharyngeal skeleton, the location of cartilage formationduring development.     

Dynamics of expression of growth differentiation factor 15 in normal and PIN development in the mouse

Growth differentiation factor (GDF15) is a distant member of the transforming growth factor-beta superfamily, a diverse group of structurally related proteins that exert multiple effects on cell fate such as on cell growth and differentiation but little is known about GDF15 in these processes. Previously we observed the mature GDF15 to be associated with human prostate carcinogenesis hence prompting us to study GDF15 further. Here we report gdf15 expression both at the RNA and protein levels, in normal prostatic tissues of wild type (wt) and prostatic intraepithelial neoplasia (PIN) of transgenic (Tg) 12T-7s model mice during embryonic, postnatal, and adult prostate formation up to 15 weeks after birth. Dynamic changes in expression, at both the mRNA and protein level, correlated with cell proliferation and differentiation during distinct phases of normal mouse prostate development and alterations in the dynamics of gdf15 expression correlated with the changes in development resulting in PIN formation. Most notably mature gdf15 protein was significantly elevated during hyperplasia and PIN development. Changes in the protein levels did not always correlate well with the mRNA levels. This was more prominent during PIN than during normal prostate development suggesting that this may also be an indicator of disturbed regulation of gdf15 in PIN. We propose that gdf15 is a growth factor with dual function either promoting proliferation or growth arrest and differentiation due most likely to differences in cellular differentiation. Because of the differentiation defect in PIN its epithelium no longer responds to gdf15 by cellular growth arrest as does the normal epithelium and gdf may even stimulate proliferation. The data supports our hypothesis that GDF15 plays a role in the early stages of human prostate cancer.     

Combining selected reaction monitoring with discovery proteomics in limited biological samples

Simultaneous quantification of multiple proteins by selected reaction monitoring (SRM) has several applications in cell signaling studies including embryo proteomics. However, concerns have recently been raised over the specificity of SRM assays due to possible ion redundancy and/or sequence similarity of selected peptide with multiple non‐related proteins. In this Viewpoint article, we discuss some simple measures that can increase our confidence in the accuracy of SRM scans used in proteomic experiments. At least in embryonic samples from porcine species, these measures were found to be useful in validating MS‐identified differentially expressed proteins. Among the nine proteins analyzed by SRM assay, all the proteins that were found to be up‐ or down‐regulated in MS experiment were also faithfully up‐ or down‐regulated in SRM assay.     

Growth differentiation factor 11 promotes differentiation of MSCs into endothelial‐like cells for angiogenesis

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor‐β super family. It has multiple effects on development, physiology and diseases. However, the role of GDF11 in the development of mesenchymal stem cells (MSCs) is not clear. To explore the effects of GDF11 on the differentiation and pro‐angiogenic activities of MSCs, mouse bone marrow–derived MSCs were engineered to overexpress GDF11 (MSC^GDF11) and their capacity for differentiation and paracrine actions were examined both in vitro and in vivo. Expression of endothelial markers CD31 and VEGFR2 at the levels of both mRNA and protein was significantly higher in MSC^GDF11 than control MSCs (MSC^Vector) during differentiation. More tube formation was observed in MSC^GDF11 as compared with controls. In an in vivo angiogenesis assay with Matrigel plug, MSC^GDF11 showed more differentiation into CD31⁺ endothelial‐like cells and better pro‐angiogenic activity as compared with MSC^Vector. Mechanistically, the enhanced differentiation by GDF11 involved activation of extracellular‐signal‐related kinase (ERK) and eukaryotic translation initiation factor 4E (EIF4E). Inhibition of either TGF‐β receptor or ERK diminished the effect of GDF11 on MSC differentiation. In summary, our study unveils the function of GDF11 in the pro‐angiogenic activities of MSCs by enhancing endothelial differentiation via the TGFβ‐R/ERK/EIF4E pathway.     

Antibody‐based profiling of cerebrospinal fluid within multiple sclerosis

Antibody suspension bead arrays have proven to enable multiplexed and high‐throughput protein profiling in unfractionated plasma and serum samples through a direct labeling approach. We here describe the development and application of an assay for protein profiling of cerebrospinal fluid (CSF). While setting up the assay, systematic intensity differences between sample groups were observed that reflected inherent sample specific total protein amounts. Supplementing the labeling reaction with BSA and IgG diminished these differences without impairing the apparent sensitivity of the assay. We also assessed the effects of heat treatment on the analysis of CSF proteins and applied the assay to profile 43 selected proteins by 101 antibodies in 339 CSF samples from a multiple sclerosis (MS) cohort. Two proteins, GAP43 and SERPINA3 were found to have a discriminating potential with altered intensity levels between sample groups. GAP43 was detected at significantly lower levels in secondary progressive MS compared to early stages of MS and the control group of other neurological diseases. SERPINA3 instead was detected at higher levels in all MS patients compared to controls. The developed assay procedure now offers new possibilities for broad‐scale protein profiling of CSF within neurological disorders.     

Integrated Seed Proteome and Phosphoproteome Analyses Reveal Interplay of Nutrient Dynamics,Carbon–Nitrogen Partitioning,and Oxidative Signaling in Chickpea

Nutrient dynamics in storage organs is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. Although sink organ developmental events have been identified, understanding of translational and post‐translational regulation of reserve synthesis, accumulation, and utilization in legumes is limited. To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, an integrated proteomics and phosphoproteomics study in six sequential seed developmental stages in chickpea is performed. MS/MS analyses identify 109 unique nutrient‐associated proteins (NAPs) involved in metabolism, storage and biogenesis, and protein turnover. Differences and similarities in 60 nutrient‐associated phosphoproteins (NAPPs) containing 93 phosphosites are compared with NAPs. Data reveal accumulation of carbon–nitrogen metabolic and photosynthetic proteoforms during seed filling. Furthermore, enrichment of storage proteoforms and protease inhibitors is associated with cell expansion and seed maturation. Finally, combined proteoforms network analysis identifies three significant modules, centered around malate dehydrogenase, HSP70, triose phosphate isomerase, and vicilin. Novel clues suggest that ubiquitin–proteasome pathway regulates nutrient reallocation. Second, increased abundance of NAPs/NAPPs related to oxidative and serine/threonine signaling indicates direct interface between redox sensing and signaling during seed development. Taken together, nutrient signals act as metabolic and differentiation determinant governing storage organ reprogramming.     

The proregion of mouse BMP15 regulates the cooperative interactions of BMP15 and GDF9

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are secreted by the mammalian oocyte and are essential for ovarian follicular development, ovulation, and fertility. However, the secreted forms of the BMP15 and GDF9 proteins and the nature of cooperative molecular interactions between BMP15 and GDF9 previously reported have not been fully characterized. In this study, we found that recombinant mouse BMP15 and GDF9 are secreted as cleaved mature and proregion proteins, with BMP15 also secreted as uncleaved promature protein. Noncovalent interactions were identified between the mature and proregion proteins of each growth factor. Moreover, GDF9 mature protein was found to coimmunoprecipitate with the BMP15 proregion, suggestive of a heteromeric association between BMP15 and GDF9. Mouse GDF9 was found to exist mostly as a dimer of mature protein, in both the presence and absence of BMP15. In contrast, BMP15 formed mostly multimers of proregion and mature protein when combined with GDF9, providing further evidence for heteromeric interaction. Mouse BMP15 was found to act cooperatively with GDF9 in a rat granulosa cell thymidine incorporation bioassay and to signal through the BMPR2 and ACVR1B/TGFBR1/ACVR1C receptor-mediated pathways. Immunoneutralization experiments using GDF9 mature protein antibody indicated that these cooperative interactions are species specific. Additionally, immunoneutralization with proregion antibodies highlighted the involvement of the BMP15 proregion in BMP15/GDF9 cooperative interactions. Taken together, these findings support a novel hypothesis where the extracellular cooperative interactions of recombinant mouse BMP15 and GDF9 are multimeric, involving the proregion of BMP15, and may well be species specific.     

Platform for establishing interlaboratory reproducibility of selected reaction monitoring-based mass spectrometry peptide assays

Mass spectrometry (MS) is an attractive alternative to quantification of proteins by immunoassays, particularly for protein biomarkers of clinical relevance. Reliable quantification requires that the MS-based assays are robust, selective, and reproducible. Thus, the development of standardized protocols is essential to introduce MS into clinical research laboratories. The aim of this study was to establish a complete workflow for assessing the transferability and reproducibility of selected reaction monitoring (SRM) assays between clinical research laboratories. Four independent laboratories in North America, using identical triple-quadrupole mass spectrometers (Quantum Ultra, Thermo), were provided with standard protocols and instrumentation settings to analyze unknown samples and internal standards in a digested plasma matrix to quantify 51 peptides from 39 human proteins using a multiplexed SRM assay. The interlaboratory coefficient of variation (CV) was less than 10% for 25 of 39 peptides quantified (12 peptides were not quantified based upon hydrophobicity) and exhibited CVs less than 20% for the remaining peptides. In this report, we demonstrate that previously developed research platforms for SRM assays can be improved and optimized for deployment in clinical research environments.     

Verification of a biomarker discovery approach for detection of Down syndrome in amniotic fluid via multiplex selected reaction monitoring (SRM) assay

Prenatal screening test for Down syndrome (DS) can be improved by discovery of novel biomarkers. A multiplex selected reaction monitoring (SRM) assay was developed to test previously identified thirteen candidate proteins in amniotic fluid (AF). One unique peptide was selected for each protein based on discovery data, while three MS/MS transitions were selected based on intelligent SRM results. For one of the candidates, matrix metalloproteinase-2 (MMP2), ELISA was also performed to validate SRM results in AF and to test serum samples. Comparison of AF samples from DS versus controls via SRM assay revealed five proteins that were differentially expressed. Bile salt-activated lipase, mucin-13, carboxypeptidase A1, and dipeptidyl peptidase 4 showed a decrease in DS-affected AF, and MMP2 showed an increase, in comparison to controls (P<0.05). Discovery-based spectral counting ratios and SRM ratios showed a strong correlation, and MMP2 ELISA further confirmed the validity of the SRM data. Potential implications of differentially expressed proteins during fetal development are proposed. Our data also shows that SRM can provide a high-throughput and accurate platform for biomarker verification.     

The novel coccidian micronemal protein MIC11 undergoes proteolytic maturation by sequential cleavage to remove an internal propeptide

Host cell invasion is a key step in the life cycle of the intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis. Attachment and invasion by this parasite is dependent on secretion of proteins from the micronemes, cigar-shaped organelles found in the apical end of the parasite. Although many of these proteins contain adhesive motifs suggestive of a role in parasite attachment, a growing subset of microneme proteins (MICs) do not possess adhesive sequences implying that they have alternative roles. We have identified a novel 16 kDa microneme protein, TgMIC11, that is conserved among several coccidian parasites. As it traffics through the secretory system, TgMIC11 is modified by two successive proteolytic events to remove an internal propeptide, resulting in the mature protein that consists of an alpha-chain and beta-chain tethered by a single disulfide bond. Dual staining immunofluorescence confirmed that TgMIC11 localises to the apical micronemes and, like other micronemal proteins, it is also secreted in a calcium dependent manner. This is the first microneme protein characterised to date in the phylum Apicomplexa that possesses this unique structure and undergoes maturation by removal of an internal propeptide.     

Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein-1: a novel protein with protease inhibitor and follistatin domains

Myostatin, a member of the TGFbeta 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 TGFbeta 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 TGFbeta binding proteins.