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Nat Commun ; 12(1): 4028, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188059


CNNM/CorB proteins are a broadly conserved family of integral membrane proteins with close to 90,000 protein sequences known. They are associated with Mg2+ transport but it is not known if they mediate transport themselves or regulate other transporters. Here, we determine the crystal structure of an archaeal CorB protein in two conformations (apo and Mg2+-ATP bound). The transmembrane DUF21 domain exists in an inward-facing conformation with a Mg2+ ion coordinated by a conserved π-helix. In the absence of Mg2+-ATP, the CBS-pair domain adopts an elongated dimeric configuration with previously unobserved domain-domain contacts. Hydrogen-deuterium exchange mass spectrometry, analytical ultracentrifugation, and molecular dynamics experiments support a role of the structural rearrangements in mediating Mg2+-ATP sensing. Lastly, we use an in vitro, liposome-based assay to demonstrate direct Mg2+ transport by CorB proteins. These structural and functional insights provide a framework for understanding function of CNNMs in Mg2+ transport and associated diseases.

Proteínas de Transporte de Cátions/metabolismo , Hydrogenophilaceae/metabolismo , Magnésio/metabolismo , Methanomicrobiaceae/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Medição da Troca de Deutério , Simulação de Dinâmica Molecular , Conformação Proteica , Domínios Proteicos
Elife ; 102021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33661099


Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.

PLoS Negl Trop Dis ; 15(1): e0009052, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33471793


Intestinal helminth infection can impair host resistance to co-infection with enteric bacterial pathogens. However, it is not known whether helminth drug-clearance can restore host resistance to bacterial infection. Using a mouse helminth-Salmonella co-infection system, we show that anthelmintic treatment prior to Salmonella challenge is sufficient to restore host resistance to Salmonella. The presence of the small intestine-dwelling helminth Heligmosomoides polygyrus at the point of Salmonella infection supports the initial establishment of Salmonella in the small intestinal lumen. Interestingly, if helminth drug-clearance is delayed until Salmonella has already established in the small intestinal lumen, anthelmintic treatment does not result in complete clearance of Salmonella. This suggests that while the presence of helminths supports initial Salmonella colonization, helminths are dispensable for Salmonella persistence in the host small intestine. These data contribute to the mechanistic understanding of how an ongoing or prior helminth infection can affect pathogenic bacterial colonization and persistence in the mammalian intestine.

Coinfecção/microbiologia , Coinfecção/parasitologia , Resistência à Doença/fisiologia , Helmintíase/complicações , Enteropatias Parasitárias/complicações , Nematospiroides dubius/fisiologia , Salmonella/fisiologia , Animais , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal , Intestinos/microbiologia , Intestinos/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Salmonella/complicações , Salmonella typhi
Front Immunol ; 11: 557960, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33178185


Conflicting data has emerged regarding a role for eosinophils in IgA production, with some reports that eosinophils support both secretory and circulating IgA levels during homeostasis. Previous studies have compared antibody levels between wildtype and eosinophil-deficient mice, but these mice were obtained from different commercial vendors and/or were not littermates. Thus, the possibility remains that extrinsic environmental factors, rather than an intrinsic lack of eosinophils, are responsible for the reports of reduced IgA in eosinophil-deficient mice. Here we used wild-type and eosinophil-deficient (ΔdblGATA) mice that were purchased from a single vendor, subsequently bred in-house and either co-housed as adults, co-reared from birth or raised as littermates. We found no differences in the levels of secretory IgA or in the numbers of small intestinal IgA-producing plasma cells between wild-type and ΔdblGATA mice, demonstrating that under controlled steady-state conditions eosinophils are not essential for the maintenance of secretory IgA in the intestinal tract. While we found that levels of IgM and IgE were significantly elevated in the serum of ΔdblGATA mice compared to co-reared or co-housed wild-type mice, no significant differences in these or other circulating antibody isotypes were identified between genotypes in littermate-controlled experiments. Our results demonstrate that eosinophils are not required to maintain secretory or circulating IgA production and the absence of eosinophils does not impact circulating IgG1, IgG2b, IgM, or IgE levels during homeostasis. These findings emphasize the importance of optimally controlling rearing and housing conditions throughout life between mice of different genotypes.

Eosinófilos/imunologia , Eosinófilos/metabolismo , Imunoglobulina A/imunologia , Animais , Biomarcadores , Citocinas/metabolismo , Citometria de Fluxo , Imunoglobulina A/sangue , Imunoglobulina A Secretora/imunologia , Camundongos , Membrana Mucosa/imunologia , Membrana Mucosa/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo
Structure ; 28(7): 830-846.e9, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433991


Liver receptor homolog-1 (LRH-1; NR5A2) is a nuclear receptor that regulates a diverse array of biological processes. In contrast to dimeric nuclear receptors, LRH-1 is an obligate monomer and contains a subtype-specific helix at the C terminus of the DNA-binding domain (DBD), termed FTZ-F1. Although detailed structural information is available for individual domains of LRH-1, it is unknown how these domains exist in the intact nuclear receptor. Here, we developed an integrated structural model of human full-length LRH-1 using a combination of HDX-MS, XL-MS, Rosetta computational docking, and SAXS. The model predicts the DBD FTZ-F1 helix directly interacts with ligand binding domain helix 2. We confirmed several other predicted inter-domain interactions via structural and functional analyses. Comparison between the LRH-1/Dax-1 co-crystal structure and the integrated model predicted and confirmed Dax-1 co-repressor to modulate LRH-1 inter-domain dynamics. Together, these data support individual LRH-1 domains interacting to influence receptor structure and function.

Simulação de Dinâmica Molecular , Receptores Citoplasmáticos e Nucleares/química , Sítios de Ligação , DNA/química , DNA/metabolismo , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo