RESUMEN
The homogeneously glycosylated 76-amino acid adiponectin collagenous domains (ACDs) with all of the possible 15 glycoforms have been chemically and individually synthesized using stereoselective glycan synthesis and chemical peptide ligation. The following biological and pharmacological studies enabled correlating glycan pattern to function in the inhibition of cancer cell growth as well as the regulation of systemic energy metabolism. In particular, hAdn-WM6877 was tested in detail with different mouse models and it exhibited promising in vivo antitumor, insulin sensitizing, and hepatoprotective activities. Our studies demonstrated the possibility of using synthetic glycopeptides as the adiponectin downsized mimetic for the development of novel therapeutics to treat diseases associated with deficient adiponectin.
Asunto(s)
Adiponectina/síntesis química , Adiponectina/metabolismo , Adiponectina/química , Glicosilación , Humanos , Modelos Moleculares , Estructura MolecularRESUMEN
The effects of current treatment strategies for ischemic stroke are weakened by ischemia-reperfusion (I/R) injury. Effective treatments targeting I/R injury are still insufficient. Adiponectin (APN), a fat-derived hormone, has a wide range of antioxidative and anti-inflammatory effects. However, the application of APN to the central nervous system is restricted by its limited blood-brain barrier permeability. Therefore, an adiponectin peptide (APNp) was chemically synthesized on the basis of the functional area in the APN structure. The present study was carried out to explore the effect and the underlying mechanism of APNp on I/R injury. A transient middle cerebral artery occlusion (tMCAO) model with C57BL/6 J mice was used, and an in vitro oxygen-glucose deprivation and reintroduction (OGD-R) model with primary astrocytes was induced. The results showed that APNp decreased the cerebral infarction volume, alleviated brain edema, improved neurological function and had antioxidative, anti-inflammatory, and antiapoptotic effects against cerebral I/R injury. In addition, APNp upregulated the phosphorylation of AMPK and GSK-3ß, promoted the nuclear translocation of Nrf2 and increased the expression of Trx1. The protective effect of APNp was abolished by compound C, a selective AMPK inhibitor, and PX-12, a selective Trx inhibitor. Moreover, APNp decreased the protein level of TXNIP and suppressed the activation of the NLRP3 inflammasome in astrocytes, which were also reversed by compound C and PX-12. These findings suggest that APNp, as a potential substitute for adiponectin, has a great potential for clinical application in the treatment of acute brain ischemia.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adiponectina/uso terapéutico , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Estrés Oxidativo/efectos de los fármacos , Daño por Reperfusión/metabolismo , Adiponectina/síntesis química , Adiponectina/farmacología , Animales , Células Cultivadas , Inflamasomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Distribución Aleatoria , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/tratamiento farmacológicoRESUMEN
Chemoselective peptide ligation methods have provided synthetic access to numerous proteins, including those bearing native post-translational modifications and unnatural labels. This protocol outlines the chemical synthesis of proteins using a recently discovered reaction (diselenide-selenoester ligation (DSL)) in a rapid, additive-free manner. After ligation, the products can be chemoselectively deselenized to produce native peptide and protein products. We describe methods for the synthesis of suitably functionalized peptide diselenide and peptide selenoester fragments via Fmoc-solid-phase peptide synthesis (SPPS) protocols, fusion of these fragments by DSL, and the chemoselective deselenization of the ligation products to generate native synthetic proteins. We demonstrate the method's utility through the total chemical synthesis of the post-translationally modified collagenous domain of the hormone adiponectin via DSL-deselenization at selenocystine (the oxidized form of selenocysteine) and the rapid preparation of two tick-derived thrombin-inhibiting proteins by DSL-deselenization at ß-selenoaspartate and γ-selenoglutamate. This method should find widespread use for the rapid synthesis of proteins, including cases in which other peptide ligation methods cannot be used (or cannot be used efficiently), e.g., at sterically hindered or deactivated acyl donors. The method's speed and efficiency may render it useful in the generation of synthetic protein libraries. Each protein discussed can be synthesized within 15 working days from resin loading and can be readily produced by practitioners with master's-level experience in organic chemistry. Each synthesis using these protocols was performed independently by two labs (one academic and one industrial), which attained comparable yields of the protein products.
Asunto(s)
Proteínas/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Adiponectina/síntesis química , Cistina/análogos & derivados , Cistina/química , Compuestos de Organoselenio/químicaRESUMEN
Adiponectin has many beneficial effects on cardiovascular and obesity-related disorders. It is part of a class of proteins that contains short collagenous domains, along with surfactant proteins A and D, and complement protein C1q. This class of biomacromolecules requires post-translational modifications to form biologically active assemblies. By introducing a set of post-translational modifying enzymes into Escherichia coli , we have created a prokaryotic expression system that functionally assembles adiponectin, as assessed by the ability of produced adiponectin multimers to suppress human endothelial cell apoptosis. This study represents the first example of the assembly of functional high order multimers of any member of this class of proteins outside of eukaryotic cells. Furthermore, the results give fundamental insight into the process of assembly such as the necessity and sufficiency of various post-translational steps for functional assembly. We expect that fine-tuning of the expression system will allow for efficient production and functional assembly of biomolecules that assemble via short collagenous domains.