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1.
Elife ; 92020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32515349

RESUMO

Activin A functions in BMP signaling in two ways: it either engages ACVR1B to activate Smad2/3 signaling or binds ACVR1 to form a non-signaling complex (NSC). Although the former property has been studied extensively, the roles of the NSC remain unexplored. The genetic disorder fibrodysplasia ossificans progressiva (FOP) provides a unique window into ACVR1/Activin A signaling because in that disease Activin can either signal through FOP-mutant ACVR1 or form NSCs with wild-type ACVR1. To explore the role of the NSC, we generated 'agonist-only' Activin A muteins that activate ACVR1B but cannot form the NSC with ACVR1. Using one of these muteins, we demonstrate that failure to form the NSC in FOP results in more severe disease pathology. These results provide the first evidence for a biological role for the NSC in vivo and pave the way for further exploration of the NSC's physiological role in corresponding knock-in mice.


Assuntos
Receptores de Ativinas Tipo I/metabolismo , Ativinas/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Miosite Ossificante/genética , Transdução de Sinais/genética , Receptores de Ativinas Tipo I/genética , Ativinas/genética , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Técnicas de Introdução de Genes , Camundongos , Camundongos Transgênicos , Mutação , Miosite Ossificante/patologia
2.
Proc Natl Acad Sci U S A ; 116(31): 15505-15513, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31315975

RESUMO

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.


Assuntos
Ativinas/química , Ativinas/metabolismo , Complexos Multiproteicos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Humanos , Camundongos , Modelos Moleculares , Complexos Multiproteicos/química , Ratos , Fator de Crescimento Transformador beta/metabolismo
3.
Sci Transl Med ; 7(303): 303ra137, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-26333933

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by episodically exuberant heterotopic ossification (HO), whereby skeletal muscle is abnormally converted into misplaced, but histologically normal bone. This HO leads to progressive immobility with catastrophic consequences, including death by asphyxiation. FOP results from mutations in the intracellular domain of the type I BMP (bone morphogenetic protein) receptor ACVR1; the most common mutation alters arginine 206 to histidine (ACVR1(R206H)) and has been thought to drive inappropriate bone formation as a result of receptor hyperactivity. We unexpectedly found that this mutation rendered ACVR1 responsive to the activin family of ligands, which generally antagonize BMP signaling through ACVR1 but cannot normally induce bone formation. To test the implications of this finding in vivo, we engineered mice to carry the Acvr1(R206H) mutation. Because mice that constitutively express Acvr1[R206H] die perinatally, we generated a genetically humanized conditional-on knock-in model for this mutation. When Acvr1[R206H] expression was induced, mice developed HO resembling that of FOP; HO could also be triggered by activin A administration in this mouse model of FOP but not in wild-type controls. Finally, HO was blocked by broad-acting BMP blockers, as well as by a fully human antibody specific to activin A. Our results suggest that ACVR1(R206H) causes FOP by gaining responsiveness to the normally antagonistic ligand activin A, demonstrating that this ligand is necessary and sufficient for driving HO in a genetically accurate model of FOP; hence, our human antibody to activin A represents a potential therapeutic approach for FOP.


Assuntos
Receptores de Ativinas Tipo I/genética , Ativinas/metabolismo , Mutação , Miosite Ossificante/genética , Receptores de Ativinas Tipo I/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteína 1A de Ligação a Tacrolimo/metabolismo
4.
Cell Metab ; 6(5): 376-85, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17983583

RESUMO

Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1(-/-) mice are treated with DEX, the MuRF1(-/-) animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.


Assuntos
Dexametasona/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Leupeptinas/farmacologia , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Ligação Proteica , Isoformas de Proteínas/metabolismo , Interferência de RNA , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
5.
Mol Cell ; 12(6): 1403-11, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14690595

RESUMO

ARF GTPases are activated by guanine nucleotide exchange factors (GEFs) of the Sec7 family that promote the exchange of GDP for GTP. Brefeldin A (BFA) is a fungal metabolite that binds to the ARF1*GDP*Sec7 complex and blocks GEF activity at an early stage of the reaction, prior to guanine nucleotide release. The crystal structure of the ARF1*GDP*Sec7*BFA complex shows that BFA binds at the protein-protein interface to inhibit conformational changes in ARF1 required for Sec7 to dislodge the GDP molecule. Based on a comparative analysis of the inhibited complex, nucleotide-free ARF1*Sec7 and ARF1*GDP, we suggest that, in addition to forcing nucleotide release, the ARF1-Sec7 binding energy is used to open a cavity on ARF1 to facilitate the rearrangement of hydrophobic core residues between the GDP and GTP conformations. Thus, the Sec7 domain may act as a dual catalyst, facilitating both nucleotide release and conformational switching on ARF proteins.


Assuntos
Fator 1 de Ribosilação do ADP/química , Brefeldina A/química , Fatores de Troca do Nucleotídeo Guanina/química , Guanosina Difosfato/metabolismo , Estrutura Quaternária de Proteína , Inibidores da Síntese de Proteínas/química , Fator 1 de Ribosilação do ADP/metabolismo , Sítios de Ligação , Brefeldina A/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Substâncias Macromoleculares , Modelos Moleculares , Inibidores da Síntese de Proteínas/metabolismo
6.
Nature ; 419(6904): 271-7, 2002 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-12239560

RESUMO

COPII-coated vesicles form on the endoplasmic reticulum by the stepwise recruitment of three cytosolic components: Sar1-GTP to initiate coat formation, Sec23/24 heterodimer to select SNARE and cargo molecules, and Sec13/31 to induce coat polymerization and membrane deformation. Crystallographic analysis of the Saccharomyces cerevisiae Sec23/24-Sar1 complex reveals a bow-tie-shaped structure, 15 nm long, with a membrane-proximal surface that is concave and positively charged to conform to the size and acidic-phospholipid composition of the COPII vesicle. Sec23 and Sar1 form a continuous surface stabilized by a non-hydrolysable GTP analogue, and Sar1 has rearranged from the GDP conformation to expose amino-terminal residues that will probably embed in the bilayer. The GTPase-activating protein (GAP) activity of Sec23 involves an arginine side chain inserted into the Sar1 active site. These observations establish the structural basis for GTP-dependent recruitment of a vesicular coat complex, and for uncoating through coat-controlled GTP hydrolysis.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Proteínas de Membrana/química , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Cristalografia por Raios X , Dimerização , Proteínas Ativadoras de GTPase , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular
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