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1.
Mol Pharmacol ; 96(2): 233-246, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31189666

RESUMO

The orphan G-protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery, where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-ß5, leading to the regulation of neighboring GPCR-induced Go protein activity. GPR158 also interacts with αo, although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain that are putatively involved in G-protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-ß5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in αo binding. Using a Gαo-ßγ bioluminescence resonance energy transfer (BRET) sensor, we found that GPR158 decreases the BRET signal as observed upon G-protein activation; however, no constitutive activity of GPR158 could be detected through the measurement of various G-protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of Gαo by the VCPWE motifs, possibly leading to its dissociation from ßγ Such action of GPR158 is expected to prolong the ßγ activity, as also observed with some activators of G-protein signaling. Taken together, our data revealed a complex functional scaffolding or signaling role for GPR158 controlling Go through an original mechanism.


Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Regulação da Expressão Gênica , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Receptores Acoplados a Proteínas G/genética
2.
Biochemistry ; 57(18): 2679-2693, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29609464

RESUMO

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.


Assuntos
Flavoproteínas/química , Manganês/química , Ribonucleotídeo Redutases/química , Superóxidos/química , Catálise , Domínio Catalítico , Flavobacterium/química , Flavobacterium/enzimologia , Flavoproteínas/metabolismo , Ferro/química , Oxirredução , Oxigênio/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismo , Tirosina/química
3.
Biochimie ; 94(2): 461-70, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21889567

RESUMO

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension "D-E-P") and SRTX-i3 (25 aa, including extension "V-N-R-N"). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ET(A) and ET(B) receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca(2+) measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ET(B) receptor known to date.


Assuntos
Endotelina-1/metabolismo , Peptídeos/síntese química , Receptores de Endotelina/agonistas , Venenos de Víboras , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Cálcio/metabolismo , Cricetinae , Injeções Intravenosas , Transporte de Íons/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/toxicidade , Ligação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Receptores de Endotelina/metabolismo , Relação Estrutura-Atividade , Taxa de Sobrevida , Transfecção , Vasoconstritores/síntese química , Vasoconstritores/toxicidade , Venenos de Víboras/síntese química , Venenos de Víboras/toxicidade
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