Sotatercept analog suppresses inflammation to reverse experimental pulmonary arterial hypertension.

Sotatercept is an activin receptor type IIA-Fc (ActRIIA-Fc) fusion protein that improves cardiopulmonary function in patients with pulmonary arterial hypertension (PAH) by selectively trapping activins and growth differentiation factors. However, the cellular and molecular mechanisms of ActRIIA-Fc action are incompletely understood. Here, we determined through genome-wide expression profiling that inflammatory and immune responses are prominently upregulated in the lungs of a Sugen-hypoxia rat model of severe angio-obliterative PAH, concordant with profiles observed in PAH patients. Therapeutic treatment with ActRIIA-Fc-but not with a vasodilator-strikingly reversed proinflammatory and proliferative gene expression profiles and normalized macrophage infiltration in diseased rodent lungs. Furthermore, ActRIIA-Fc normalized pulmonary macrophage infiltration and corrected cardiopulmonary structure and function in Bmpr2 haploinsufficient mice subjected to hypoxia, a model of heritable PAH. Three high-affinity ligands of ActRIIA-Fc each induced macrophage activation in vitro, and their combined immunoneutralization in PAH rats produced cardiopulmonary benefits comparable to those elicited by ActRIIA-Fc. Our results in complementary experimental and genetic models of PAH reveal therapeutic anti-inflammatory activities of ActRIIA-Fc that, together with its known anti-proliferative effects on vascular cell types, could underlie clinical activity of sotatercept as either monotherapy or add-on to current PAH therapies.

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.

Functionally diverse heteromeric traps for ligands of the transforming growth factor-ß superfamily.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily are important targets for therapeutic intervention but present challenges because they signal combinatorially and exhibit overlapping activities in vivo. To obtain agents capable of sequestering multiple TGF-ß superfamily ligands with novel selectivity, we generated soluble, heterodimeric ligand traps by pairing the extracellular domain (ECD) of the native activin receptor type IIB (ActRIIB) alternately with the ECDs of native type I receptors activin receptor-like kinase 4 (ALK4), ALK7, or ALK3. Systematic analysis of these heterodimeric constructs by surface plasmon resonance, and comparison with their homodimeric counterparts, revealed that each type I receptor partner confers a distinct ligand-binding profile to the heterodimeric construct. Additional characterization in cell-based reporter gene assays confirmed that the heterodimeric constructs possessed different profiles of signaling inhibition in vitro, which translated into altered patterns of pharmacological activity when constructs were administered systemically to wild-type mice. Our results detail a versatile platform for the modular recombination of naturally occurring receptor domains, giving rise to inhibitory ligand traps that could aid in defining the physiological roles of TGF-ß ligand sets or be directed therapeutically to human diseases arising from dysregulated TGF-ß superfamily signaling.

ActRIIB:ALK4-Fc alleviates muscle dysfunction and comorbidities in murine models of neuromuscular disorders.

Patients with neuromuscular disorders suffer from a lack of treatment options for skeletal muscle weakness and disease comorbidities. Here, we introduce as a potential therapeutic agent a heterodimeric ligand-trapping fusion protein, ActRIIB:ALK4-Fc, which comprises extracellular domains of activin-like kinase 4 (ALK4) and activin receptor type IIB (ActRIIB), a naturally occurring pair of type I and II receptors belonging to the TGF-ß superfamily. By surface plasmon resonance (SPR), ActRIIB:ALK4-Fc exhibited a ligand binding profile distinctly different from that of its homodimeric variant ActRIIB-Fc, sequestering ActRIIB ligands known to inhibit muscle growth but not trapping the vascular regulatory ligand bone morphogenetic protein 9 (BMP9). ActRIIB:ALK4-Fc and ActRIIB-Fc administered to mice exerted differential effects - concordant with SPR results - on vessel outgrowth in a retinal explant assay. ActRIIB:ALK4-Fc induced a systemic increase in muscle mass and function in wild-type mice and in murine models of Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and disuse atrophy. Importantly, ActRIIB:ALK4-Fc improved neuromuscular junction abnormalities in murine models of DMD and presymptomatic ALS and alleviated acute muscle fibrosis in a DMD model. Furthermore, in combination therapy ActRIIB:ALK4-Fc increased the efficacy of antisense oligonucleotide M12-PMO on dystrophin expression and skeletal muscle endurance in an aged DMD model. ActRIIB:ALK4-Fc shows promise as a therapeutic agent, alone or in combination with dystrophin rescue therapy, to alleviate muscle weakness and comorbidities of neuromuscular disorders.

Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity.

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.

Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

Phosphorylation of Bacillus subtilis gene regulator AbrB modulates its DNA-binding properties.

AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.

Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain.

Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.

Activity of the Bacillus thuringiensis NprR-NprX cell-cell communication system is co-ordinated to the physiological stage through a complex transcriptional regulation.

NprR is a quorum sensor of the RNPP family found in bacteria of the Bacillus cereus group. In association with its cognate peptide NprX, NprR controls the expression of genes essential for survival and sporulation of Bacillus thuringiensis during its necrotrophic development in insects. Here, we report that the nprR-nprX genes are not autoregulated and are co-transcribed from a σ(A) -dependent promoter (PA ) located upstream from nprR. The transcription from PA starts at the onset of the stationary phase and is controlled by two transcriptional regulators: CodY and PlcR. The nutritional repressor CodY represses nprR-nprX transcription during the exponential growth phase and the quorum sensor PlcR activates nprR-nprX transcription at the onset of stationary phase. We show that nprX is also transcribed independently of nprR from two promoters, PH and PE , dependent on the sporulation-specific sigma factors, σ(H) and σ(E) respectively. Both promoters ensure nprX transcription during late stationary phase while transcription from PA has decreased. These results show that the activity of the NprR-NprX quorum sensing system is tightly co-ordinated to the physiological stage throughout the developmental process of the Bacillus.

Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR.

The quorum-sensing regulator PlcR is the master regulator of most known virulence factors in Bacillus cereus. It is a helix-turn-helix (HTH)-type transcription factor activated upon binding of its cognate signaling peptide PapR on a tetratricopeptide repeat-type regulatory domain. The structural and functional properties of PlcR have defined a new family of sensor regulators, called the RNPP family (for Rap, NprR, PrgX, and PlcR), in Gram-positive bacteria. To fully understand the activation mechanism of PlcR, we took a closer look at the conformation changes induced upon binding of PapR and of its target DNA, known as PlcR-box. For that purpose we have determined the structures of the apoform of PlcR (Apo PlcR) and of the ternary complex of PlcR with PapR and the PlcR-box from the plcA promoter. Comparison of the apoform of PlcR with the previously published structure of the PlcR-PapR binary complex shows how a small conformational change induced in the C-terminal region of the tetratricopeptide repeat (TPR) domain upon peptide binding propagates via the linker helix to the N-terminal HTH DNA-binding domain. Further comparison with the PlcR-PapR-DNA ternary complex shows how the activation of the PlcR dimer allows the linker helix to undergo a drastic conformational change and subsequent proper positioning of the HTH domains in the major groove of the two half sites of the pseudopalindromic PlcR-box. Together with random mutagenesis experiments and interaction measurements using peptides from distinct pherogroups, this structural analysis allows us to propose a molecular mechanism for this functional switch.

Saposins utilize two strategies for lipid transfer and CD1 antigen presentation.

Transferring lipid antigens from membranes into CD1 antigen-presenting proteins represents a major molecular hurdle necessary for T-cell recognition. Saposins facilitate this process, but the mechanisms used are not well understood. We found that saposin B forms soluble saposin protein-lipid complexes detected by native gel electrophoresis that can directly load CD1 proteins. Because saposin B must bind lipids directly to function, we found it could not accommodate long acyl chain containing lipids. In contrast, saposin C facilitates CD1 lipid loading in a different way. It uses a stable, membrane-associated topology and was capable of loading lipid antigens without forming soluble saposin-lipid antigen complexes. These findings reveal how saposins use different strategies to facilitate transfer of structurally diverse lipid antigens.

Cadherin-11 regulates fibroblast inflammation.

Fibroblasts are important participants in inflammation. Although not leukocytes, their capacity to produce cytokines, chemokines, and other inflammatory factors locally in tissues suggests that they can contribute to inflammatory diseases. For example, fibroblasts in a rheumatoid arthritis (RA) joint are a dominant source of IL-6 and RANKL in the synovium, both of which are therapeutic targets for inflammation and bone erosion. Previously, we found that fibroblasts can be targeted by mAb directed against cadherin-11 (cad-11), a mesenchymal cadherin that fibroblasts selectively express. Targeting cad-11 significantly reduced inflammation as assessed by joint swelling and clinical inflammation scores. However, the mechanism by which anti-cad-11 reduced inflammation was not known. Here, we show that cad-11 engagement induces synovial fibroblasts to secret proinflammatory cytokines including IL-6. Cad-11 engagement strongly synergized with TNF-α and IL-1ß in the induction of IL-6. Importantly, cad-11 activated MAP kinases and NF-κB for IL-6 induction. IL-6 levels in ankles of inflamed joints were reduced in cad-11 mutant mice compared to wild-type mice with inflammatory arthritis. Thus, we suggest that cad-11 modulates synovial fibroblasts to evoke inflammatory factors that may contribute to the inflammatory process in RA.

Structural characterization of Spo0E-like protein-aspartic acid phosphatases that regulate sporulation in bacilli.

Spore formation is an extreme response of many bacterial species to starvation. In the case of pathogenic species of Bacillus and Clostridium, it is also a component of disease transmission. Entry into the pathway of sporulation in Bacillus subtilis and its relatives is controlled by an expanded two-component system in which starvation signals lead to the activation of sensor kinases and phosphorylation of the master sporulation response regulator Spo0A. Accumulation of threshold concentrations of Spo0A approximately P heralds the commitment to sporulation. Countering the activities of the sensor kinases are phosphatases such as Spo0E, which dephosphorylate Spo0A approximately P and inhibit sporulation. Spo0E-like protein-aspartic acid-phosphate phosphatases, consisting of 50-90 residues, are conserved in sporeforming bacteria and unrelated in sequence to proteins of known structure. Here we determined the structures of the Spo0A approximately P phosphatases BA1655 and BA5174 from Bacillus anthracis using nuclear magnetic resonance spectroscopy. Each is composed of two anti-parallel alpha-helices flanked by flexible regions at the termini. The signature SQELD motif (SRDLD in BA1655) is situated in the middle of helix alpha2 with its polar residues projecting outward. BA5174 is a monomer, whereas BA1655 is a dimer. The four-helix bundle structure in the dimer is reminiscent of the phosphotransferase Spo0B and the chemotaxis phosphatase CheZ, although in contrast to these systems, the subunits in BA1655 are in head-to-tail rather than head-to-head apposition. The implications of the structures for interactions between the phosphatases and their substrate Spo0A approximately P are discussed.

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis.

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 A resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

Substrate and dioxygen binding to the endospore coat laccase from Bacillus subtilis.

The CotA laccase from the endospore coat of Bacillus subtilis has been crystallized in the presence of the non-catalytic co-oxidant 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and the structure was determined using synchrotron radiation. The binding site for this adduct is well defined and indicates how ABTS, in conjunction with laccases, could act as an oxidative mediator toward non-phenolic moieties. In addition, a dioxygen moiety is clearly defined within the solvent channel oriented toward one of the T3 copper atoms in the trinuclear center.