RESUMO
BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.
Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Hipersensibilidade Alimentar/tratamento farmacológico , Imunoglobulina E/imunologia , Receptores de IgE/imunologia , Anafilaxia/imunologia , Animais , Antialérgicos/farmacologia , Anticorpos Monoclonais/farmacologia , Feminino , Hipersensibilidade Alimentar/imunologia , Imunoglobulina G/imunologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/imunologia , Receptores de IgE/genética , Quinase Syk/imunologiaRESUMO
BACKGROUND: Anaphylaxis is classically mediated by allergen cross-linking of IgE bound to the α chain of FcεRI, the mast cell/basophil high affinity IgE receptor. Allergen cross-linking of the IgE/FcεRI complex activates these cells, inducing release of disease-causing mediators, cytokines, and enzymes. We previously demonstrated that IgE-mediated anaphylaxis could be safely prevented in wild-type BALB/c mice by rapid desensitization with anti-mouse FcεRIα mAb. OBJECTIVE: This study sought to use humanized mice to extend these results to humans. METHODS: We actively immunized huFcεRIα/F709 mice, which express human (hu) instead of mouse FcεRIα and a mutant IL-4 receptor that lacks inhibitory function. We passively immunized huFcεRIα mice, as well as human cord blood-reconstituted reNSGS mice, which are immune-deficient, produce mast cell-stimulating human cytokines, and develop numerous human mast cells. For desensitization, we used anti-huFcεRIα mAbs that bind FcεRIα regardless of its association with IgE (noncompeting mAbs), and/or mAbs that compete with IgE for huFcεRIα binding (competing mAbs). Anaphylaxis was induced by intravenous injection of antigen or anti-huIgE mAb. RESULTS: Anti-huFcεRIα mAb rapid desensitization was safer and more effective than allergen rapid desensitization and suppressed anaphylaxis more rapidly than omalizumab or ligelizumab. Rapid desensitization of naïve, IgE-sensitized huFcεRIα mice and huFcεRIα/F709 mice that were egg-allergic with anti-FcεRIα mAbs safely removed >98% of IgE from peritoneal mast cells and completely suppressed IgE-mediated anaphylaxis. Rapid desensitization of reNSGS mice with anti-FcεRIα mAbs also safely removed â¼98% of mast cell IgE and prevented IgE-mediated anaphylaxis. CONCLUSIONS: Rapid desensitization with anti-FcεRIα mAbs may be a safe, effective, and practical way to prevent IgE-mediated anaphylaxis.
Assuntos
Anafilaxia/imunologia , Anticorpos Monoclonais/farmacologia , Dessensibilização Imunológica/métodos , Receptores de IgE/antagonistas & inibidores , Anafilaxia/prevenção & controle , Animais , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Myostatin, a member of the TGF-ß family of ligands, is a strong negative regulator of muscle growth. As such, it is a prime therapeutic target for muscle wasting disorders. Similar to other TGF-ß family ligands, myostatin is neutralized by binding one of a number of structurally diverse antagonists. Included are the antagonists GASP-1 and GASP-2, which are unique in that they specifically antagonize myostatin. However, little is known from a structural standpoint describing the interactions of GASP antagonists with myostatin. Here, we present the First low resolution solution structure of myostatin-free and myostatin-bound states of GASP-1 and GASP-2. Our studies have revealed GASP-1, which is 100 times more potent than GASP-2, preferentially binds myostatin in an asymmetrical 1:1 complex, whereas GASP-2 binds in a symmetrical 2:1 complex. Additionally, C-terminal truncations of GASP-1 result in less potent myostatin inhibitors that form a 2:1 complex, suggesting that the C-terminal domains of GASP-1 are the primary mediators for asymmetric complex formation. Overall, this study provides a new perspective on TGF-ß antagonism, where closely related antagonists can utilize different ligand-binding strategies.
Assuntos
Proteínas de Transporte/metabolismo , Miostatina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células CHO , Cromatografia em Gel , Cricetinae , Cricetulus , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Ligação Proteica , Espalhamento de Radiação , Ressonância de Plasmônio de Superfície , UltracentrifugaçãoRESUMO
Bone morphogenetic proteins (BMPs) are antagonized through the action of numerous extracellular protein antagonists, including members from the differential screening-selected gene aberrative in neuroblastoma (DAN) family. In vivo, misregulation of the balance between BMP signaling and DAN inhibition can lead to numerous disease states, including cancer, kidney nephropathy, and pulmonary arterial hypertension. Despite this importance, very little information is available describing how DAN family proteins effectively inhibit BMP ligands. Furthermore, our understanding for how differences in individual DAN family members arise, including affinity and specificity, remains underdeveloped. Here, we present the structure of the founding member of the DAN family, neuroblastoma suppressor of tumorigenicity 1 (NBL1). Comparing NBL1 to the structure of protein related to Dan and Cerberus (PRDC), a more potent BMP antagonist within the DAN family, a number of differences were identified. Through a mutagenesis-based approach, we were able to correlate the BMP binding epitope in NBL1 with that in PRDC, where introduction of specific PRDC amino acids in NBL1 (A58F and S67Y) correlated with a gain-of-function inhibition toward BMP2 and BMP7, but not GDF5. Although NBL1(S67Y) was able to antagonize BMP7 as effectively as PRDC, NBL1(S67Y) was still 32-fold weaker than PRDC against BMP2. Taken together, this data suggests that alterations in the BMP binding epitope can partially account for differences in the potency of BMP inhibition within the DAN family.
Assuntos
Proteína Morfogenética Óssea 2/antagonistas & inibidores , Proteína Morfogenética Óssea 7/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intercelular/química , Mutação de Sentido Incorreto , Proteínas/química , Substituição de Aminoácidos , Animais , Proteína Morfogenética Óssea 2/química , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 7/química , Proteína Morfogenética Óssea 7/genética , Células CHO , Proteínas de Ciclo Celular , Cricetinae , Cricetulus , Citocinas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutagênese , Estrutura Terciária de Proteína , Proteínas/genética , Relação Estrutura-AtividadeRESUMO
Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast Saccharomyces cerevisiae select a site for polarized growth in a specific pattern depending on cell type. Haploid a and α cells bud in the axial budding pattern, which depends on a transient marker and requires proteins Bud3, Bud4, Axl1 and Axl2. Here, we report that Bud4 functions as a platform that mediates the ordered assembly of the axial landmark at the division site during M and early G1 phase. Whereas Bud4 associates with Bud3 in all cell types and in the absence of Axl1 or Axl2, Bud4 interacts with Axl1 and Axl2 mainly in haploid cells and only in the presence of all other components of the landmark. Bud4 can bind to GTP or GDP, and a GTP-binding-defective Bud4 fails to interact with Axl1 in vitro. The same bud4 mutation leads to mis-localization of Axl1 and disrupts the axial budding pattern, indicating that GTP binding to Bud4 is important for its role in bud-site selection. We also show the cell-type-specific association of the axial landmark with Bud5, a GDP/GTP exchange factor for Rsr1. Despite their expression in all cell types, Bud4 and Axl2 associate with Bud5 specifically in haploid cells and in the presence of Axl1, whose expression is limited to a and α cells. Together, our findings suggest that Bud4 plays a critical role in the assembly of the axial landmark and its link to the Rsr1 GTPase module.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Sequência de Aminoácidos , Ciclo Celular , Proteínas de Ciclo Celular/genética , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Citocinese/fisiologia , Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/genéticaRESUMO
Background and Aims: We previously identified small molecules predicted to reverse an ileal gene signature for future Crohn's Disease (CD) strictures. Here we used a new human intestinal organoid (HIO) model system containing macrophages to test a lead candidate, eicosatetraynoic acid (ETYA). Methods: Induced pluripotent stem cell lines (iPSC) were derived from CD patients and differentiated into macrophages and HIOs. Macrophages and macrophage:HIO co-cultures were exposed to lipopolysaccharide (LPS) with and without ETYA pre-treatment. Cytospin and flow cytometry characterized macrophage morphology and activation markers, and RNA sequencing defined the global pattern of macrophage gene expression. TaqMan Low Density Array, Luminex multiplex assay, immunohistologic staining, and sirius red polarized light microscopy were performed to measure macrophage cytokine production and HIO pro-fibrotic gene expression and collagen content. Results: iPSC-derived macrophages exhibited morphology similar to primary macrophages and expressed inflammatory macrophage cell surface markers including CD64 and CD68. LPS-stimulated macrophages expressed a global pattern of gene expression enriched in CD ileal inflammatory macrophages and matrisome secreted products, and produced cytokines and chemokines including CCL2, IL1B, and OSM implicated in refractory disease. ETYA suppressed CD64 abundance and pro-fibrotic gene expression pathways in LPS stimulated macrophages. Co-culture of LPS-primed macrophages with HIO led to up-regulation of fibroblast activation genes including ACTA2 and COL1A1 , and an increase in HIO collagen content. ETYA pre-treatment prevented pro-fibrotic effects of LPS-primed macrophages. Conclusions: ETYA inhibits pro-fibrotic effects of LPS-primed macrophages upon co-cultured HIO. This model may be used in future untargeted screens for small molecules to treat refractory CD.
RESUMO
Inflammatory Bowel Disease ( IBD ) is a chronic and often debilitating autoinflammatory condition, with an increasing incidence in children. Standard-of-care therapies lead to sustained transmural healing and clinical remission in fewer than one-third of patients. For children, TNFα inhibition remains the only FDA-approved biologic therapy, providing an even greater urgency to understanding mechanisms of response. Genome-wide association studies ( GWAS ) have identified 418 independent genetic risk loci contributing to IBD, yet the majority are noncoding and their mechanisms of action are difficult to decipher. If causal, they likely alter transcription factor ( TF ) binding and downstream gene expression in particular cell types and contexts. To bridge this knowledge gap, we built a novel resource: multiome-seq (tandem single-nuclei ( sn )RNA-seq and chromatin accessibility ( snATAC )-seq) of intestinal tissue from pediatric IBD patients, where anti-TNF response was defined by endoscopic healing. From the snATAC-seq data, we generated a first-time atlas of chromatin accessibility (putative regulatory elements) for diverse intestinal cell types in the context of IBD. For cell types/contexts mediating genetic risk, we reasoned that accessible chromatin will co-localize with genetic disease risk loci. We systematically tested for significant co-localization of our chromatin accessibility maps and risk variants for 758 GWAS traits. Globally, genetic risk variants for IBD, autoimmune and inflammatory diseases are enriched in accessible chromatin of immune populations, while other traits (e.g., colorectal cancer, metabolic) are enriched in epithelial and stromal populations. This resource opens new avenues to uncover the complex molecular and cellular mechanisms mediating genetic disease risk.
RESUMO
TGF-ß family ligands are involved in a variety of critical physiological processes. For instance, the TGF-ß ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-ß family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.
Assuntos
Proteínas Relacionadas à Folistatina/química , Modelos Moleculares , Miostatina/química , Animais , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Proteínas Relacionadas à Folistatina/genética , Proteínas Relacionadas à Folistatina/metabolismo , Humanos , Miostatina/genética , Miostatina/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Perturbagen analysis of Crohn's disease (CD) ileal gene expression data identified small molecules including eicosatetraynoic acid (ETYA), which may exert an antifibrotic effect. We developed a patient-specific human intestinal organoid (HIO) model system to test small molecule regulation of mitochondrial and wound-healing functions implicated in stricturing behavior. METHODS: HIOs were made from CD induced pluripotent stem cells with and without a loss-of-function haplotype in the DUOX2 gene implicated in ileal homeostasis and characterized under basal conditions and following exposure to butyrate and ETYA using RNA sequencing, flow cytometry, and immunofluorescent and polarized light microscopy. Mitochondrial activity was measured using high-resolution respirometry and tissue stiffness using atomic force microscopy. RESULTS: HIOs expressed core mitochondrial and extracellular matrix (ECM) genes and enriched biologic functions implicated in CD ileal strictures; ECM gene expression was suppressed by both butyrate and ETYA, with butyrate also suppressing genes regulating epithelial proliferation. Consistent with this, butyrate, but not ETYA, exerted a profound effect on HIO epithelial mitochondrial function, reactive oxygen species production, and cellular abundance. Butyrate and ETYA suppressed HIO expression of alpha smooth muscle actin expressed by myofibroblasts, type I collagen, and collagen protein abundance. HIOs exhibited tissue stiffness comparable to normal human ileum; this was reduced by chronic ETYA exposure in HIOs carrying the DUOX2 loss-of-function haplotype. CONCLUSIONS: ETYA regulates ECM genes implicated in strictures and suppresses collagen content and tissue stiffness in an HIO model. HIOs provide a platform to test personalized therapeutics, including small molecules prioritized by perturbagen analysis.
A subset of pediatric Crohn's disease patients develop intestinal strictures requiring surgery. The microbial metabolite butyrate and eicosatetraynoic acid regulate pathways implicated in stricture formation in a human intestinal organoid model system, which may be used to test new therapies.
Assuntos
Doença de Crohn , Butiratos/metabolismo , Butiratos/farmacologia , Colágeno/metabolismo , Constrição Patológica/metabolismo , Doença de Crohn/genética , Oxidases Duais/metabolismo , Matriz Extracelular/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mitocôndrias/metabolismo , Organoides/metabolismoRESUMO
Polarized cell growth requires the coupling of a defined spatial site on the cell cortex to the apparatus that directs the establishment of cell polarity. In the budding yeast Saccharomyces cerevisiae, the Ras-family GTPase Rsr1p/Bud1p and its regulators select the proper site for bud emergence on the cell cortex. The Rho-family GTPase Cdc42p and its associated proteins then establish an axis of polarized growth by triggering an asymmetric organization of the actin cytoskeleton and secretory apparatus at the selected bud site. We explored whether a direct linkage exists between the Rsr1p/Bud1p and Cdc42p GTPases. Here we show specific genetic interactions between RSR1/BUD1 and particular cdc42 mutants defective in polarity establishment. We also show that Cdc42p coimmunoprecipitated with Rsr1p/Bud1p from yeast extracts. In vitro studies indicated a direct interaction between Rsr1p/Bud1p and Cdc42p, which was enhanced by Cdc24p, a guanine nucleotide exchange factor for Cdc42p. Our findings suggest that Cdc42p interacts directly with Rsr1p/Bud1p in vivo, providing a novel mechanism by which direct contact between a Ras-family GTPase and a Rho-family GTPase links the selection of a growth site to polarity establishment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Polaridade Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Ciclo Celular/genética , Clonagem Molecular , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína/genética , Proteínas Proto-Oncogênicas/genética , Saccharomyces cerevisiae/genética , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
In the budding yeast Saccharomyces cerevisiae, selection of the bud site determines the axis of polarized cell growth and eventual oriented cell division. Bud sites are selected in specific patterns depending on cell type. These patterns appear to depend on distinct types of marker proteins in the cell cortex; in particular, the bipolar budding of diploid cells depends on persistent landmarks at the birth-scar-distal and -proximal poles that involve the proteins Bud8p and Bud9p, respectively. Rax1p and Rax2p also appear to function specifically in bipolar budding, and we report here a further characterization of these proteins and of their interactions with Bud8p and Bud9p. Rax1p and Rax2p both appear to be integral membrane proteins. Although commonly used programs predict different topologies for Rax2p, glycosylation studies indicate that it has a type I orientation, with its long N-terminal domain in the extracytoplasmic space. Analysis of rax1 and rax2 mutant budding patterns indicates that both proteins are involved in selecting bud sites at both the distal and proximal poles of daughter cells as well as near previously used division sites on mother cells. Consistent with this, GFP-tagged Rax1p and Rax2p were both observed at the distal pole as well as at the division site on both mother and daughter cells; localization to the division sites was persistent through multiple cell cycles. Localization of Rax1p and Rax2p was interdependent, and biochemical studies showed that these proteins could be copurified from yeast. Bud8p and Bud9p could also be copurified with Rax1p, and localization studies provided further evidence of interactions. Localization of Rax1p and Rax2p to the bud tip and distal pole depended on Bud8p, and normal localization of Bud8p was partially dependent on Rax1p and Rax2p. Although localization of Rax1p and Rax2p to the division site did not appear to depend on Bud9p, normal localization of Bud9p appeared largely or entirely dependent on Rax1p and Rax2p. Taken together, the results indicate that Rax1p and Rax2p interact closely with each other and with Bud8p and Bud9p in the establishment and/or maintenance of the cortical landmarks for bipolar budding.
Assuntos
Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Divisão Celular , Membrana Celular/metabolismo , Polaridade Celular , Proliferação de Células , Clonagem Molecular , Citoplasma/metabolismo , Proteínas Fúngicas/fisiologia , Deleção de Genes , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
Myostatin, a member of the transforming growth factor (TGF)-ß family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-ß family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-ß family.
Assuntos
Ativinas/antagonistas & inibidores , Substâncias de Crescimento/farmacologia , Miostatina/antagonistas & inibidores , Simulação por Computador , Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Bibliotecas de Moléculas PequenasRESUMO
Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-ß family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-ß family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.
Assuntos
Ativinas/química , Proteínas Relacionadas à Folistatina/química , Folistatina/química , Miostatina/química , Ativinas/antagonistas & inibidores , Animais , Linhagem Celular , Folistatina/metabolismo , Proteínas Relacionadas à Folistatina/genética , Proteínas Relacionadas à Folistatina/metabolismo , Células HEK293 , Humanos , Camundongos , Miostatina/antagonistas & inibidores , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The heritability of B cell chronic lymphocytic leukemia (CLL) is relatively high; however, no predisposing mutation has been convincingly identified. We show that loss or reduced expression of death-associated protein kinase 1 (DAPK1) underlies cases of heritable predisposition to CLL and the majority of sporadic CLL. Epigenetic silencing of DAPK1 by promoter methylation occurs in almost all sporadic CLL cases. Furthermore, we defined a disease haplotype, which segregates with the CLL phenotype in a large family. DAPK1 expression of the CLL allele is downregulated by 75% in germline cells due to increased HOXB7 binding. In the blood cells from affected family members, promoter methylation results in additional loss of DAPK1 expression. Thus, reduced expression of DAPK1 can result from germline predisposition, as well as epigenetic or somatic events causing or contributing to the CLL phenotype.