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1.
FEBS Open Bio ; 12(5): 1075-1086, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35182051

RESUMO

Nitric oxide synthase 3 (NOS3) is a major vasoprotective enzyme that catalyzes the conversion of l-arginine to nitric oxide (NO) in response to a significant number of signaling pathways. Here, we provide evidence that NOS3 interactions with MAP kinases have physiological relevance. Binding interactions of NOS3 with c-Jun N-terminal kinase (JNK1α1 ), p38α, and ERK2 were characterized using optical biosensing with full-length NOS3 and NOS3 specific peptides and phosphopeptides. Like p38α and ERK2, JNK1α1 exhibited high-affinity binding to full-length NOS3 (KD 15 nm). Rate constants exhibited fast-on, slow-off binding (kon = 4106 m-1 s-1 ; koff = 6.2 × 10-5 s-1 ). Further analysis using synthetic NOS3 peptides revealed two MAP kinase binding sites unique to NOS3. p38α evinced similar affinity with both NOS3 binding sites. For ERK2 and JNK1α1, the affinity at the two sites differed. However, NOS3 peptides with a phosphate at either S114 or S633 did not meaningfully interact with the kinases. Immunoblotting revealed that each kinase phosphorylated NOS3 with a unique pattern. JNK1α1 predominantly phosphorylated NOS3 at S114, ERK2 at S600, and p38α phosphorylated both residues. In vitro production of NO was unchanged by phosphorylation at these sites. In human microvascular endothelial cells, endogenous interactions of all the MAP kinases with NOS3 were captured using proximity ligation assay in resting cells. Our results underscore the importance of MAP kinase interactions, identifying two unique NOS3 interaction sites with potential for modulation by MAP kinase phosphorylation (S114) and other signaling inputs, like protein kinase A (S633).


Assuntos
Células Endoteliais , Proteínas Quinases Ativadas por Mitógeno , Sítios de Ligação , Células Endoteliais/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Peptídeos/metabolismo , Fosforilação
2.
Cell Rep ; 28(4): 909-922.e6, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340153

RESUMO

Pemphigus vulgaris (PV) is an autoimmune disease characterized by blistering sores on skin and mucosal membranes, caused by autoantibodies primarily targeting the cellular adhesion protein, desmoglein-3 (Dsg3). To better understand how Dsg3-specific autoantibodies develop and cause disease in humans, we performed a cross-sectional study of PV patients before and after treatment to track relevant cellular responses underlying disease pathogenesis, and we provide an in-depth analysis of two patients by generating a panel of mAbs from single Dsg3-specific memory B cells (MBCs). Additionally, we analyzed a paired sample from one patient collected 15-months prior to disease diagnosis. We find that Dsg3-specific MBCs have an activated phenotype and show signs of ongoing affinity maturation and clonal selection. Monoclonal antibodies (mAbs) with pathogenic activity primarily target epitopes in the extracellular domains EC1 and EC2 of Dsg3, though they can also bind to the EC4 domain. Combining antibodies targeting different epitopes synergistically enhances in vitro pathogenicity.


Assuntos
Doenças Autoimunes/imunologia , Pênfigo/imunologia , Análise de Célula Única , Anticorpos Monoclonais/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Desmogleína 3/química , Desmogleína 3/imunologia , Células Germinativas/metabolismo , Humanos , Memória Imunológica , Ligação Proteica , Domínios Proteicos , Hipermutação Somática de Imunoglobulina/genética
3.
Mol Biol Cell ; 30(12): 1390-1405, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30943110

RESUMO

Desmogleins (Dsgs) are cadherin family adhesion molecules essential for epidermal integrity. Previous studies have shown that desmogleins associate with lipid rafts, but the significance of this association was not clear. Here, we report that the desmoglein transmembrane domain (TMD) is the primary determinant of raft association. Further, we identify a novel mutation in the DSG1 TMD (G562R) that causes severe dermatitis, multiple allergies, and metabolic wasting syndrome. Molecular modeling predicts that this G-to-R mutation shortens the DSG1 TMD, and experiments directly demonstrate that this mutation compromises both lipid raft association and desmosome incorporation. Finally, cryo-electron tomography indicates that the lipid bilayer within the desmosome is ∼10% thicker than adjacent regions of the plasma membrane. These findings suggest that differences in bilayer thickness influence the organization of adhesion molecules within the epithelial plasma membrane, with cadherin TMDs recruited to the desmosome via the establishment of a specialized mesoscale lipid raft-like membrane domain.


Assuntos
Desmossomos/metabolismo , Microdomínios da Membrana/metabolismo , Sequência de Aminoácidos , Animais , Desmogleínas/química , Desmogleínas/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Lipoilação , Camundongos , Modelos Biológicos , Mutação/genética , Domínios Proteicos
4.
Elife ; 72018 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-29999492

RESUMO

Desmosomes are adhesive junctions composed of two desmosomal cadherins: desmocollin (Dsc) and desmoglein (Dsg). Previous studies demonstrate that E-cadherin (Ecad), an adhesive protein that interacts in both trans (between opposing cells) and cis (on the same cell surface) conformations, facilitates desmosome assembly via an unknown mechanism. Here we use structure-function analysis to resolve the mechanistic roles of Ecad in desmosome formation. Using AFM force measurements, we demonstrate that Ecad interacts with isoform 2 of Dsg via a conserved Leu-175 on the Ecad cis binding interface. Super-resolution imaging reveals that Ecad is enriched in nascent desmosomes, supporting a role for Ecad in early desmosome assembly. Finally, confocal imaging demonstrates that desmosome assembly is initiated at sites of Ecad mediated adhesion, and that Ecad-L175 is required for efficient Dsg2 and desmoplakin recruitment to intercellular contacts. We propose that Ecad trans interactions at nascent cell-cell contacts initiate the recruitment of Dsg through direct cis interactions with Ecad which facilitates desmosome assembly.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Desmogleína 2/metabolismo , Desmossomos/metabolismo , Multimerização Proteica , Células HEK293 , Humanos , Microscopia de Força Atômica , Microscopia Confocal , Microscopia de Fluorescência , Ligação Proteica , Mapeamento de Interação de Proteínas
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