Structural Basis of Non-Latent Signaling by the Anti-Müllerian Hormone Procomplex.

Most TGFß family ligands exist as procomplexes consisting of a prodomain noncovalently bound to a growth factor (GF); Whereas some prodomains confer latency, the Anti-Müllerian Hormone (AMH) prodomain maintains a remarkably high affinity for the GF yet remains active. Using single particle EM methods, we show the AMH prodomain consists of two subdomains: a vestigial TGFß prodomain-like fold and a novel, helical bundle GF-binding domain, the result of an exon insertion 450 million years ago, that engages both receptor epitopes. When associated with the prodomain, the AMH GF is distorted into a strained, open conformation whose closure upon bivalent binding of AMHR2 displaces the prodomain through a conformational shift mechanism to allow for signaling.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Activin E is a TGFß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming Growth Factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, similar to ActC. Collectively, our results establish ActE as an ALK7 ligand, thereby providing a link between genetic and in vivo studies of ActE as a regulator of adipose tissue.

Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF related protein (CTRP) family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE, and that larger species are dispensable for BMP inhibition. Additionally, we used an in-silico approach to identify a helix, termed the ligand binding domain (LBD), that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the LBD is crucial for activity through luciferase assays and surface plasmon resonance (SPR) analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers.

BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle.

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-ß family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development.

Follistatin Forms a Stable Complex With Inhibin A That Does Not Interfere With Activin A Antagonism.

Inhibins are transforming growth factor-ß family heterodimers that suppress follicle-stimulating hormone (FSH) secretion by antagonizing activin class ligands. Inhibins share a common ß chain with activin ligands. Follistatin is another activin antagonist, known to bind the common ß chain of both activins and inhibins. In this study, we characterized the antagonist-antagonist complex of inhibin A and follistatin to determine if their interaction impacted activin A antagonism. We isolated the inhibin A:follistatin 288 complex, showing that it forms in a 1:1 stoichiometric ratio, different from previously reported homodimeric ligand:follistatin complexes, which bind in a 1:2 ratio. Small angle X-ray scattering coupled with modeling provided a low-resolution structure of inhibin A in complex with follistatin 288. Inhibin binds follistatin via the shared activin ß chain, leaving the α chain free and flexible. The inhibin A:follistatin 288 complex was also shown to bind heparin with lower affinity than follistatin 288 alone or in complex with activin A. Characterizing the inhibin A:follistatin 288 complex in an activin-responsive luciferase assay and by surface plasmon resonance indicated that the inhibitor complex readily dissociated upon binding type II receptor activin receptor type IIb, allowing both antagonists to inhibit activin signaling. Additionally, injection of the complex in ovariectomized female mice did not alter inhibin A suppression of FSH. Taken together, this study shows that while follistatin binds to inhibin A with a substochiometric ratio relative to the activin homodimer, the complex can dissociate readily, allowing both proteins to effectively antagonize activin signaling.

GDF11 and aging biology - controversies resolved and pending.

Since the exogenous administration of GDF11, a TGF-ß superfamily member, was reported to have beneficial effects in some models of human disease, there have been many research studies in GDF11 biology. However, many studies have now confirmed that exogenous administration of GDF11 can improve physiology in disease models, including cardiac fibrosis, experimental stroke, and disordered metabolism. GDF11 is similar to GDF8 (also called Myostatin), differing only by 11 amino acids in their mature signaling domains. These two proteins are now known to be biochemically different both in vitro and in vivo. GDF11 is much more potent than GDF8 and induces more strongly SMAD2 phosphorylation in the myocardium compared to GDF8. GDF8 and GDF11 prodomain are only 52% identical and are cleaved by different Tolloid proteases to liberate the mature signaling domain from inhibition of the prodomain. Here, we review the state of GDF11 biology, highlighting both resolved and remaining controversies.

Molecular Mechanisms of AMH Signaling.

Anti-Müllerian Hormone (AMH) is a secreted glycoprotein hormone with critical roles in reproductive development and regulation. Its chemical and mechanistic similarities to members of the Transforming Growth Factor ß (TGF-ß) family have led to its placement within this signaling family. As a member of the TGF-ß family, AMH exists as a noncovalent complex of a large N-terminal prodomain and smaller C-terminal mature signaling domain. To produce a signal, the mature domain will bind to the extracellular domains of two type I and two type II receptors which results in an intracellular SMAD signal. Interestingly, as will be discussed in this review, AMH possesses several unique characteristics which set it apart from other ligands within the TGF-ß family. In particular, AMH has a dedicated type II receptor, Anti-Müllerian Hormone Receptor Type II (AMHR2), making this interaction intriguing mechanistically as well as therapeutically. Further, the prodomain of AMH has remained largely uncharacterized, despite being the largest prodomain within the family. Recent advancements in the field have provided valuable insight into the molecular mechanisms of AMH signaling, however there are still many areas of AMH signaling not understood. Herein, we will discuss what is known about the biochemistry of AMH and AMHR2, focusing on recent advances in understanding the unique characteristics of AMH signaling and the molecular mechanisms of receptor engagement.

The orphan ligand, activin C, signals through activin receptor-like kinase 7.

Activin ligands are formed from two disulfide-linked inhibin ß (Inhß) subunit chains. They exist as homodimeric proteins, as in the case of activin A (ActA; InhßA/InhßA) or activin C (ActC; InhßC/InhßC), or as heterodimers, as with activin AC (ActAC; InhßA:InhßC). While the biological functions of ActA and activin B (ActB) have been well characterized, little is known about the biological functions of ActC or ActAC. One thought is that the InhßC chain functions to interfere with ActA production by forming less active ActAC heterodimers. Here, we assessed and characterized the signaling capacity of ligands containing the InhßC chain. ActC and ActAC activated SMAD2/3-dependent signaling via the type I receptor, activin receptor-like kinase 7 (ALK7). Relative to ActA and ActB, ActC exhibited lower affinity for the cognate activin type II receptors and was resistant to neutralization by the extracellular antagonist, follistatin. In mature murine adipocytes, which exhibit high ALK7 expression, ActC elicited a SMAD2/3 response similar to ActB, which can also signal via ALK7. Collectively, these results establish that ActC and ActAC are active ligands that exhibit a distinct signaling receptor and antagonist profile compared to other activins.

Visceral adipose tissue remodeling in pancreatic ductal adenocarcinoma cachexia: the role of activin A signaling.

Pancreatic ductal adenocarcinoma (PDAC) patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue. Activin A levels were measured in serum and analyzed in tumor specimens of both a cohort of Stage IV PDAC patients and the genetically engineered KPC mouse model. Our data revealed that serum activin A levels were significantly elevated in Stage IV PDAC patients in comparison to age-matched non-cancer patients. Little is known about the role of activin A in adipose tissue wasting in the setting of PDAC cancer cachexia. We established a correlation between elevated activin A and remodeling of visceral adipose tissue. Atrophy and fibrosis of visceral adipose tissue was examined in omental adipose tissue of Stage IV PDAC patients and gonadal adipose tissue of an orthotopic mouse model of PDAC. Remarkably, white visceral adipose tissue from both PDAC patients and mice exhibited decreased adipocyte diameter and increased fibrotic deposition. Strikingly, expression of thermogenic marker UCP1 in visceral adipose tissues of PDAC patients and mice remained unchanged. Thus, we propose that activin A signaling could be relevant to the acceleration of visceral adipose tissue wasting in PDAC-associated cachexia.

Structures of activin ligand traps using natural sets of type I and type II TGFß receptors.

The 30+ unique ligands of the TGFß family signal by forming complexes using different combinations of type I and type II receptors. Therapeutically, the extracellular domain of a single receptor fused to an Fc molecule can effectively neutralize subsets of ligands. Increased ligand specificity can be accomplished by using the extracellular domains of both the type I and type II receptor to mimic the naturally occurring signaling complex. Here, we report the structure of one "type II-type I-Fc" fusion, ActRIIB-Alk4-Fc, in complex with two TGFß family ligands, ActA, and GDF11, providing a snapshot of this therapeutic platform. The study reveals that extensive contacts are formed by both receptors, replicating the ternary signaling complex, despite the inherent low affinity of Alk4. Our study shows that low-affinity type I interactions support altered ligand specificity and can be visualized at the molecular level using this platform.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

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.

Characterization of tolloid-mediated cleavage of the GDF8 procomplex.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the larger TGFß superfamily of signaling ligands. GDF8 has been well characterized as a negative regulator of muscle mass. After synthesis, GDF8 is held latent by a noncovalent complex between the N-terminal prodomain and the signaling ligand. Activation of latent GDF8 requires proteolytic cleavage of the prodomain at residue D99 by a member of the tolloid family of metalloproteases. While tolloid proteases cleave multiple substrates, they lack a conserved consensus sequence. Here, we investigate the tolloid cleavage site of the GDF8 prodomain to determine what residues contribute to tolloid recognition and subsequent proteolysis. Using sequential alanine mutations, we identified several residues adjacent to the scissile bond, including Y94, that when mutated, abolish tolloid-mediated activation of latent GDF8. Using the astacin domain of Tll1 (Tolloid Like 1) we determined that prodomain mutants were more resistant to proteolysis. Purified latent complexes harboring the prodomain mutations, D92A and Y94A, impeded activation by tolloid but could be fully activated under acidic conditions. Finally, we show that co-expression of GDF8 WT with prodomain mutants that were tolloid resistant, suppressed GDF8 activity. Taken together our data demonstrate that residues towards the N-terminus of the scissile bond are important for tolloid-mediated activation of GDF8 and that the tolloid-resistant version of the GDF8 prodomain can function dominant negative to WT GDF8.

FSTL3-Neutralizing Antibodies Enhance Glucose-Responsive Insulin Secretion in Dysfunctional Male Mouse and Human Islets.

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.

Heparin-mediated dimerization of follistatin.

Heparin and heparan sulfate (HS) are highly sulfated polysaccharides covalently bound to cell surface proteins, which directly interact with many extracellular proteins, including the transforming growth factor-ß (TGFß) family ligand antagonist, follistatin 288 (FS288). Follistatin neutralizes the TGFß ligands, myostatin and activin A, by forming a nearly irreversible non-signaling complex by surrounding the ligand and preventing interaction with TGFß receptors. The FS288-ligand complex has higher affinity than unbound FS288 for heparin/HS, which accelerates ligand internalization and lysosomal degradation; however, limited information is available for how FS288 interactions with heparin affect ligand binding. Using surface plasmon resonance (SPR) we show that preincubation of FS288 with heparin/HS significantly decreased the association kinetics for both myostatin and activin A with seemingly no effect on the dissociation rate. This observation is dependent on the heparin/HS chain length where small chain lengths less than degree of polymerization 10 (dp10) did not alter association rates but chain lengths >dp10 decreased association rates. In an attempt to understand the mechanism for this observation, we uncovered that heparin induced dimerization of follistatin. Consistent with our SPR results, we found that dimerization only occurs with heparin molecules >dp10. Small-angle X-ray scattering of the FS288 heparin complex supports that FS288 adopts a dimeric configuration that is similar to the FS288 dimer in the ligand-bound state. These results indicate that heparin mediates dimerization of FS288 in a chain-length-dependent manner that reduces the ligand association rate, but not the dissociation rate or antagonistic activity of FS288.

Characterization of the different oligomeric states of the DAN family antagonists SOSTDC1 and SOST.

The DAN (differential screening-selected gene aberrative in neuroblastoma) family are a group of secreted extracellular proteins which typically bind to and antagonize BMP (bone morphogenetic protein) ligands. Previous studies have revealed discrepancies between the oligomerization state of certain DAN family members, with SOST (a poor antagonist of BMP signaling) forming a monomer while Grem1, Grem2, and NBL1 (more potent BMP antagonists) form non-disulfide linked dimers. The protein SOSTDC1 (Sclerostin domain containing protein 1) is sequentially similar to SOST, but has been shown to be a better BMP inhibitor. In order to determine the oligomerization state of SOSTDC1 and determine what effect dimerization might have on the mechanism of DAN family antagonism of BMP signaling, we isolated the SOSTDC1 protein and, using a battery of biophysical, biochemical, and structural techniques, showed that SOSTDC1 forms a highly stable non-covalent dimer. Additionally, this SOSTDC1 dimer was shown, using an in vitro cell based assay system, to be an inhibitor of multiple BMP signaling growth factors, including GDF5, while monomeric SOST was a very poor antagonist. These results demonstrate that SOSTDC1 is distinct from paralogue SOST in terms of both oligomerization and strength of BMP inhibition.

Structural perspective of BMP ligands and signaling.

The Bone Morphogenetic Proteins (BMPs) are the largest class signaling molecules within the greater Transforming Growth Factor Beta (TGFß) family, and are responsible for a wide array of biological functions, including dorsal-ventral patterning, skeletal development and maintenance, as well as cell homeostasis. As such, dysregulation of BMPs results in a number of diseases, including fibrodysplasia ossificans progressiva (FOP) and pulmonary arterial hypertension (PAH). Therefore, understanding BMP signaling and regulation at the molecular level is essential for targeted therapeutic intervention. This review discusses the recent advances in the structural and biochemical characterization of BMPs, from canonical ligand-receptor interactions to co-receptors and antagonists. This work aims to highlight how BMPs differ from other members of the TGFß family, and how that information can be used to further advance the field. Lastly, this review discusses several gaps in the current understanding of BMP structures, with the aim that discussion of these gaps will lead to advancements in the field.