RESUMEN
BACKGROUND: Activins and bone morphogenetic proteins (BMPs) play critical, sometimes opposing roles, in multiple physiological and pathological processes and diseases. They signal to distinct Smad branches; activins signal mainly to Smad2/3, while BMPs activate mainly Smad1/5/8. This gives rise to the possibility that competition between the different type I receptors through which activin and BMP signal for common type II receptors can provide a mechanism for fine-tuning the cellular response to activin/BMP stimuli. Among the transforming growth factor-ß superfamily type II receptors, ACVR2A/B are highly promiscuous, due to their ability to interact with different type I receptors (e.g., ALK4 vs. ALK2/3/6) and with their respective ligands [activin A (ActA) vs. BMP9/2]. However, studies on complex formation between these full-length receptors situated at the plasma membrane, and especially on the potential competition between the different activin and BMP type I receptors for a common activin type II receptor, were lacking. RESULTS: We employed a combination of IgG-mediated patching-immobilization of several type I receptors in the absence or presence of ligands with fluorescence recovery after photobleaching (FRAP) measurements on the lateral diffusion of an activin type II receptor, ACVR2A, to demonstrate the principle of competition between type I receptors for ACVR2. Our results show that ACVR2A can form stable heteromeric complexes with ALK4 (an activin type I receptor), as well as with several BMP type I receptors (ALK2/3/6). Of note, ALK4 and the BMP type I receptors competed for binding ACVR2A. To assess the implications of this competition for signaling output, we first validated that in our cell model system (U2OS cells), ACVR2/ALK4 transduce ActA signaling to Smad2/3, while BMP9 signaling to Smad1/5/8 employ ACVR2/ALK2 or ACVR2/ALK3. By combining ligand stimulation with overexpression of a competing type I receptor, we showed that differential complex formation of distinct type I receptors with a common type II receptor balances the signaling to the two Smad branches. CONCLUSIONS: Different type I receptors that signal to distinct Smad pathways (Smad2/3 vs. Smad1/5/8) compete for binding to common activin type II receptors. This provides a novel mechanism to balance signaling between Smad2/3 and Smad1/5/8.
Asunto(s)
Activinas , Factor de Crecimiento Transformador beta , Activinas/química , Activinas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Ligandos , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.
Asunto(s)
Hormona Antimülleriana/química , Hormona Antimülleriana/metabolismo , Receptores de Péptidos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Activinas/química , Secuencia de Aminoácidos , Proteínas Morfogenéticas Óseas/química , Cristalografía por Rayos X , Modelos Moleculares , Receptores de Péptidos/química , Receptores de Factores de Crecimiento Transformadores beta/química , Homología Estructural de ProteínaRESUMEN
Diabetes is caused by insufficient insulin production from pancreatic beta cells or insufficient insulin action, leading to an inability to control blood glucose. While a wide range of treatments exist to alleviate the symptoms of diabetes, therapies addressing the root cause of diabetes through replacing lost beta cells with functional cells remain an object of active pursuit. We previously demonstrated that genetic deletion of Fstl3, a critical regulator of activin activity, enhanced beta cell number and glucose-responsive insulin production. These observations suggested the hypothesis that FSTL3 neutralization could be used to therapeutically enhance beta cell number and function in humans. To pursue this possibility, we developed an FSTL3-neutralizing antibody, FP-101, and characterized its ability to prevent or disrupt FSTL3 from complexing with activin or related ligands. This antibody was selective for FSTL3 relative to the closely related follistatin, thereby reducing the chance for off-target effects. In vitro assays with FP-101 and activin revealed that FP-101-mediated neutralization of FSTL3 can enhance both insulin secretion and glucose responsiveness to nonfunctional mouse and human islets under conditions that model diabetes. Thus, FSTL3 neutralization may provide a novel therapeutic strategy for treating diabetes through repairing dysfunctional beta cells.
Asunto(s)
Anticuerpos Neutralizantes/química , Proteínas Relacionadas con la Folistatina/genética , Folistatina/química , Eliminación de Gen , Células Secretoras de Insulina/metabolismo , Activinas/química , Animales , Glucemia/análisis , Glucosa/metabolismo , Células HEK293 , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Islotes Pancreáticos/citología , Ligandos , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Activins belong to the transforming growth factor (TGF)-ß family of multifunctional cytokines and signal via the activin receptors ALK4 or ALK7 to activate the SMAD2/3 pathway. In some cases, activins also signal via the bone morphogenetic protein (BMP) receptor ALK2, causing activation of the SMAD1/5/8 pathway. In this study, we aimed to dissect how activin A and activin B homodimers, and activin AB and AC heterodimers activate the two main SMAD branches. We compared the activin-induced signaling dynamics of ALK4/7-SMAD2/3 and ALK2-SMAD1/5 in a multiple myeloma cell line. Signaling via the ALK2-SMAD1/5 pathway exhibited greater differences between ligands than signaling via ALK4/ALK7-SMAD2/3. Interestingly, activin B and activin AB very potently activated SMAD1/5, resembling the activation commonly seen with BMPs. As SMAD1/5 was also activated by activins in other cell types, we propose that dual specificity is a general mechanism for activin ligands. In addition, we found that the antagonist follistatin inhibited signaling by all the tested activins, whereas the antagonist cerberus specifically inhibited activin B. Taken together, we propose that activins may be considered dual specificity TGF-ß family members, critically affecting how activins may be considered and targeted clinically.
Asunto(s)
Receptores de Activinas Tipo I/metabolismo , Activinas/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Activinas/química , Línea Celular Tumoral , Humanos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
Activins are members of the transforming growth factor-ß (TGF-ß) superfamily of signaling proteins and were originally identified as components of follicular fluid. The proteins are now known to play critical roles in numerous normal and pathological processes and conditions, but less is clear about the relationships between their gene organization and protein variant expression and structure. The four human and mouse activin (Act) genes, termed INHßA, INHßB, INHßC and INHßE, differ in exon numbers. Human INHßA is the most complex with 7 exons and elicits production of three Act A variants (Act A X1, X2 and X3) differing in their pro-region, as we showed previously. Here we further analyzed the mouse INHßA gene and found that its 4 exons encode for a single open reading frame (mouse Act A), corresponding to the shortest human Act A X3 variant. Activins are synthesized and secreted as large complexes made of a long pro-region and a short mature C- terminal ligand and are known to interact with the heparan sulfate (HS) chains of cell surface and matrix proteoglycans. Human Act A X1 and X2 variants do have a HS-binding domain (HBD) with Cardin/Weintraub traits in their pro-region, while the X3 variant does not as shown previously. We found that the mouse Act A lacks a HBD as well. However, we identified a typical HBD in the pro-region of both mouse and human Act B, and synthetic peptides containing that domain interacted with immobilized HS and cell surface with nanomolar affinity. In sum, human and mouse Act A genes elicit expression of different variant sets, while there is concordance in Act B protein expression, reflecting possible evolutionary diversity in function of, and responses to, these signaling proteins in the two species.
Asunto(s)
Activinas/metabolismo , Variación Genética , Heparitina Sulfato/metabolismo , Proteínas Mutantes/metabolismo , Activinas/química , Activinas/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Ratones , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Unión Proteica , Conformación Proteica , Homología de SecuenciaRESUMEN
OBJECTIVES: The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. RESULTS: In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5 kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. CONCLUSIONS: In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.
Asunto(s)
Activinas/metabolismo , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Periplasma/metabolismo , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/metabolismo , Sistema de Translocación de Arginina Gemela/metabolismo , Activinas/química , Activinas/genética , Membrana Celular/enzimología , Dicroismo Circular , Escherichia coli/genética , Halobacterium salinarum/genética , Haloferax volcanii/genética , Humanos , Pliegue de Proteína , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
Activin A is a member of the transforming growth factor-beta (TGF-ß) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-ß-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.
Asunto(s)
Activinas/genética , Escherichia coli/genética , Periplasma/metabolismo , Señales de Clasificación de Proteína , Activinas/química , Activinas/aislamiento & purificación , Cromatografía de Afinidad , Humanos , Células K562 , Estructura Secundaria de Proteína , alfa-Amilasas/metabolismoRESUMEN
Activins regulate numerous processes including inflammation and are synthesized as precursors consisting of a long N-terminal pro-region and a mature protein. Genomic human databases currently list three activin A (Act A) variants termed X1, X2 and X3. The X3 variant is the shortest, lacks N-terminal segments present in X1 and X2, and has been the focus of most past literature. Here, we asked whether these variants are expressed by human cells and tissues and what structural features are contained within their pro-regions. Human monocytic-like cells THP1 and U937 expressed X1 and X2 variants after exposure to phorbol ester or granulocyte-macrophage colony-stimulating factor, while X2 transcripts were present in placenta. Expression vectors encoding full length X2 or X3 variants resulted in production and secretion of biologically active Act A from cultured cells. Previous studies reported a putative HS-binding domain (HBD) in the X3 pro-region. Here, we identified a novel HBD with consensus HS-binding motifs near the N-terminal end of X1 and X2 pro-regions. Peptides encompassing this new domain interacted with substrate-bound HS with nanomolar affinity, while peptides from putative X3 HBD did not. In good agreement, full length X2 pro-region interacted with heparin-agarose, while the X3 pro-region did not. In sum, our study reveals that Act A variants are expressed by inflammatory cells and placenta and yield biological activity. The high affinity HBD in X1 and X2 pro-region and its absence in X3 could greatly influence overall Act A distribution, availability and activity in physiological and pathological circumstances.
Asunto(s)
Activinas/metabolismo , Secuencias de Aminoácidos , Heparitina Sulfato/metabolismo , Conformación Proteica , Activinas/química , Activinas/genética , Secuencia de Aminoácidos , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Subunidades beta de Inhibinas/química , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/metabolismo , Modelos Moleculares , Ésteres del Forbol/farmacología , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células THP-1 , Células U937RESUMEN
TGFß family ligands, which include the TGFßs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFßs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFß and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFß, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFß signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFß family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.
Asunto(s)
Activinas/química , Activinas/metabolismo , Complejos Multiproteicos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Humanos , Ratones , Modelos Moleculares , Complejos Multiproteicos/química , Ratas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Bone morphogenetic protein (BMP)/carriers approved for orthopedic procedures achieve efficacy superior or equivalent to autograft bone. However, required supraphysiological BMP concentrations have been associated with potential local and systemic adverse events. Suboptimal BMP/receptor binding and rapid BMP release from approved carriers may contribute to these outcomes. To address these issues and improve efficacy, we engineered chimeras with increased receptor binding by substituting BMP-6 and activin A receptor binding domains into BMP-2 and optimized a carrier for chimera retention and tissue ingrowth. BV-265, a BMP-2/BMP-6/activin A chimera, demonstrated increased binding affinity to BMP receptors, including activin-like kinase-2 (ALK2) critical for bone formation in people. BV-265 increased BMP intracellular signaling, osteogenic activity, and expression of bone-related genes in murine and human cells to a greater extent than BMP-2 and was not inhibited by BMP antagonist noggin or gremlin. BV-265 induced larger ectopic bone nodules in rats compared to BMP-2 and was superior to BMP-2, BMP-2/6, and other chimeras in nonhuman primate bone repair models. A composite matrix (CM) containing calcium-deficient hydroxyapatite granules suspended in a macroporous, fenestrated, polymer mesh-reinforced recombinant human type I collagen matrix demonstrated improved BV-265 retention, minimal inflammation, and enhanced handling. BV-265/CM was efficacious in nonhuman primate bone repair models at concentrations ranging from 1/10 to 1/30 of the BMP-2/absorbable collagen sponge (ACS) concentration approved for clinical use. Initial toxicology studies were negative. These results support evaluations of BV-265/CM as an alternative to BMP-2/ACS in clinical trials for orthopedic conditions requiring augmented healing.
Asunto(s)
Activinas/química , Proteína Morfogenética Ósea 6/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Activinas/farmacología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 6/farmacología , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Humanos , Osteogénesis/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
In several retinal degenerative disease pathologies, such as dry age-related macular degeneration (AMD), the retinal pigment epithelium (RPE) cell monolayer becomes dysfunctional. Promising tissue engineering treatment approaches implant RPE cells on scaffolds into the subretinal space. However, these approaches are not without challenges. Two major challenges that must be addressed are RPE dedifferentiation and the inflammatory response to cell/scaffold implantation. Design and optimization of scaffold cues for the purpose of RPE transplantation remain relatively unexplored, specifically the mechanical properties of the scaffolds. Prior work from our group indicated that by varying substrate moduli significant differences could be induced in cell cytoskeleton structure, cellular activity, and expression of inflammatory markers. We hypothesized that Activin A would provide rescue effects for cells demonstrating dedifferentiated characteristics. Results demonstrated that for cells on low modulus scaffolds, the mechanical environment was the dominating factor and Activin A was unable to rescue these cells. However, Activin A did demonstrate rescue effects for cells on high modulus scaffolds. This finding indicates that when cultured on scaffolds with an appropriate modulus, exogenous factors, such as Activin A, can improve RPE cell expression, morphology, and activity, while an inappropriate scaffold modulus can have devastating effects on RPE survival regardless of chemical stimulation. These findings have broad implications for the design and optimization of scaffolds for long-term successful RPE transplantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2871-2880, 2018.
Asunto(s)
Activinas/farmacología , Supervivencia Celular/efectos de los fármacos , Proteínas Inmovilizadas/farmacología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Andamios del Tejido/química , Activinas/administración & dosificación , Activinas/química , Materiales Biocompatibles/química , Línea Celular , Células Cultivadas , Sistemas de Liberación de Medicamentos , Módulo de Elasticidad , Humanos , Hidrogeles/química , Proteínas Inmovilizadas/administración & dosificación , Proteínas Inmovilizadas/química , Ensayo de MaterialesRESUMEN
Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-ß. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-ß and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.
Asunto(s)
Miostatina/química , Activinas/química , Animales , Atrofia/patología , Diferenciación Celular , Dependovirus , Factor 2 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento/química , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Mutación , Miostatina/genética , Dominios Proteicos , Dispersión del Ángulo Pequeño , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Transforming growth factor-ß (TGF-ß) family members, which include TGF-ßs, activins and bone morphogenetic proteins, are pleiotropic cytokines that elicit cell type-specific effects in a highly context-dependent manner in many different tissues. These secreted protein ligands signal via single-transmembrane Type I and Type II serine/threonine kinase receptors and intracellular SMAD transcription factors. Deregulation in signaling has been implicated in a broad array of diseases, and implicate the need for intricate fine tuning in cellular signaling responses. One important emerging mechanism by which TGF-ß family receptor signaling intensity, duration, specificity and diversity are regulated and/or mediated is through cell surface co-receptors. Here, we provide an overview of the co-receptors that have been identified for TGF-ß family members. While some appear to be specific to TGF-ß family members, others are shared with other pathways and provide possible ways for signal integration. This review focuses on novel functions of TGF-ß family co-receptors, which continue to be discovered.
Asunto(s)
Activinas/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Activinas/química , Animales , Proteínas Morfogenéticas Óseas/química , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Receptores de Factores de Crecimiento Transformadores beta/química , Factor de Crecimiento Transformador beta/químicaRESUMEN
Luspatercept (ACE-536, ACVR2B-Fc), a fusion protein consisting of the extracellular domain of ActRIIB receptor and the Fc-part of human immunoglobulin G1 (IgG1), is currently under clinical development (Phase III). It stimulates the formation of red blood cells and hence may be misused by athletes for doping purposes in the future. Several antibody-based strategies for the detection of Luspatercept and other ACVR2B-Fc fusion proteins in human serum were evaluated (ELISA; IEF-, SDS-, and SAR-PAGE followed by Western blotting; immunoprecipitation). Two methods led to useful results: a commercial "soluble" ACTR-IIB ELISA, which also detected Luspatercept and other ACVR2B-Fc's, but showed no cross-reactivity with Sotatercept/ACVR2A-Fc's. The ELISA might be applied as fast screening tool (100 µL serum; limit of detection (LOD) ca 15.6 ng/mL). The second method uses a polyclonal ACVR2B-antibody for immunoprecipitation followed by SAR-PAGE and Western blotting with a monoclonal detection antibody (50 µL serum; LOD ca 1.0 ng/mL). It can be used for initial as well as for confirmatory testing. Due to the high doses (mg/kg) and long serum half-life of Luspatercept, both strategies may be useful in anti-doping control in the future. Copyright © 2017 John Wiley & Sons, Ltd.
Asunto(s)
Receptores de Activinas Tipo II/química , Receptores de Activinas Tipo II/metabolismo , Activinas/análisis , Anticuerpos Monoclonales/química , Fragmentos Fc de Inmunoglobulinas/análisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Activinas/química , Activinas/metabolismo , Western Blotting , Doping en los Deportes , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Límite de Detección , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Activin/Nodal/TGF-ß signaling pathway plays a major role in maintaining mouse epiblast stem cells (EpiSCs). The EpiSC-maintaining medium, which contains Activin A and bFGF, induces differentiation of mouse embryonic stem cells (ESCs) to EpiSCs. Here, we show that Activin A also has an ability to efficiently propagate ESCs without differentiation to EpiSCs when combined with a MEK inhibitor PD0325901. ESCs cultured in Activin+PD retained high-level expression of naive pluripotency-related transcription factors. Genomewide analysis showed that the gene expression profile of ESCs cultured in Activin+PD resembles that of ESCs cultured in 2i. ESCs cultured in Activin+PD also showed features common to the naive pluripotency of ESCs, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding shows a role of Activin/Nodal/TGF-ß signaling in stabilizing self-renewal gene regulatory networks in ESCs.
Asunto(s)
Activinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Activinas/química , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización WntRESUMEN
Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-ß family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-ß1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein.
Asunto(s)
Activinas/química , Activinas/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Folistatina/farmacología , Factor 2 de Diferenciación de Crecimiento/química , Factor 2 de Diferenciación de Crecimiento/metabolismo , Humanos , Modelos Moleculares , Dominios Proteicos , Factor de Crecimiento Transformador beta1/química , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Human embryonic stem cell (hESC)-derived endodermal cells are of interest for the development of cellular therapies to treat disorders such as liver failure. The soluble form of activin A (Act) has been widely used as an in vitro inducer of definitive endoderm (DE). In this study, we have developed a nanofibrous poly (É-caprolactone) substrate, biofunctionalized with Act, for directed differentiation of hESCs into DE. Bioconjugation of Act on nanofibrous meshes was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunostaining. In order to investigate the bioactivity of immobilized Act (iAct), hESCs were cultivated on the Act-conjugated nanofibers for five days. The nanofibers with covalent iAct significantly increased expression levels of the endodermal markers SOX17, FOXA2, and CXCR4, compared with physically adsorbed Act (aAct) or without Act (noAct). In addition, iAct retained its bioactivity after storage for five days in the absence of cell seeding. The capability of cultivated cells to generate the DE-derived lineage was evaluated through further differentiation of seeded cells into hepatocyte-like cells (HLCs). Interestingly, the iAct sample showed a higher level of hepatic markers compared to the aAct sample. We also demonstrated that iAct in the presence of soluble Act (sAct) could improve the conventional protocol to generate HLCs from hESCs. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2861-2872, 2016.
Asunto(s)
Activinas/metabolismo , Materiales Biocompatibles/metabolismo , Diferenciación Celular , Hepatocitos/citología , Células Madre Embrionarias Humanas/citología , Activinas/química , Materiales Biocompatibles/química , Línea Celular , Hepatocitos/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Nanofibras/química , Nanofibras/ultraestructuraRESUMEN
Since their original discovery as regulators of follicle-stimulating hormone (FSH) secretion and erythropoiesis, the TGF-ß family members activin and inhibin have been shown to participate in a variety of biological processes, from the earliest stages of embryonic development to highly specialized functions in terminally differentiated cells and tissues. Herein, we present the history, structures, signaling mechanisms, regulation, and biological processes in which activins and inhibins participate, including several recently discovered biological activities and functional antagonists. The potential therapeutic relevance of these advances is also discussed.