Despite decades of speculation that inhibiting endogenous insulin degradation might treat type-2 diabetes1, 2, and the identification of IDE (insulin-degrading enzyme) as a diabetes susceptibility gene3, 4, the relationship between the activity of the zinc metalloprotein IDE and glucose homeostasis remains unclear. Although Ide−/− mice have elevated insulin levels, they exhibit impaired, rather than improved, glucose tolerance that may arise from compensatory insulin signalling dysfunction5, 6. IDE inhibitors that are active in vivo are therefore needed to elucidate IDE’s physiological roles and to determine its potential to serve as a target for the treatment of diabetes. Here we report the discovery of a physiologically active IDE inhibitor identified from a DNA-templated macrocycle library. An X-ray structure of the macrocycle bound to IDE reveals that it engages a binding pocket away from the catalytic site, which explains its remarkable selectivity. Treatment of lean and obese mice with this inhibitor shows that IDE regulates the abundance and signalling of glucagon and amylin, in addition to that of insulin. Under physiological conditions that augment insulin and amylin levels, such as oral glucose administration, acute IDE inhibition leads to substantially improved glucose tolerance and slower gastric emptying. These findings demonstrate the feasibility of modulating IDE activity as a new therapeutic strategy to treat type-2 diabetes and expand our understanding of the roles of IDE in glucose and hormone regulation.
Mucosal-associated invariant T (MAIT) cells are an evolutionarily conserved αβ T-cell lineage that express a semi-invariant T-cell receptor (TCR) restricted to the MHC related-1 (MR1) protein. MAIT cells are dependent upon MR1 expression and exposure to microbes for their development and stimulation, yet these cells can exhibit microbial-independent stimulation when responding to MR1 from different species. We have used this microbial-independent, crossspecies reactivity of MAIT cells to define the molecular basis of MAIT-TCR/MR1 engagement and present here a 2.85 Å complex structure of a human MAIT-TCR bound to bovine MR1. The MR1 binding groove is similar in backbone structure to classical peptide-presenting MHC class I molecules (MHCp), yet is partially occluded by large aromatic residues that form cavities suitable for small ligand presentation. The docking of the MAIT-TCR on MR1 is perpendicular to the MR1 surface and straddles the MR1 α1 and α2 helices, similar to classical αβ TCR engagement of MHCp. However, the MAIT-TCR contacts are dominated by the α-chain, focused on the MR1 α2 helix. TCR β-chain contacts are mostly through the variable CDR3β loop that is positioned proximal to the CDR3α loop directly over the MR1 open groove. The elucidation of the MAIT TCR/ MR1 complex structure explains how the semi-invariant MAIT-TCR engages the nonpolymorphic MR1 protein, and sheds light onto ligand discrimination by this cell type. Importantly, this structure also provides a critical link in our understanding of the evolution of αβ T-cell recognition of MHC and MHC-like ligands.M ucosal-associated invariant T (MAIT) cells are a highly conserved T-cell subset found in most mammalian species (1-4). In humans, they can constitute up to 10% of circulating double-negative T cells, although they are much less frequent in mice (1,5,6). Most MAIT cells lack expression of the CD4 or CD8 coreceptors, although many MAIT cells express the αα form of the CD8 coreceptor (1). In humans, these cells are found at moderate frequency in the intestine and represent up to ∼50% of T cells in the liver (7). The cells exhibit an effector-memory phenotype and express the CD161 receptor (6). Their presence as mature effector cells in the periphery is dependent on B cells and the gut commensal flora (6, 8). Stimulated human MAIT cells can express both proinflammatory cytokines (IFN-γ, TNF-α, and IL-17) and cytolytic effectors (granzyme B) (7, 9, 10). MAIT cells are known best for their reactivity against various microorganisms from both bacterial and fungal origin (9, 10). These microorganisms include several important human pathogens, such as Mycobacterium tuberculosis, Salmonella typhimurium, and Staphylococcus aureus. Indeed, a significant proportion of the nonclassically restricted responding T cells in M. tuberculosisinfected individuals were determined to be of the MAIT lineage (9). MAIT cells have also demonstrated autoreactivity and have been associated with various autoimmune disorders (11, 12); they have also been found in both k...
Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic β cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained β-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.
MR1-restricted Mucosal Associated Invariant T (MAIT) cells represent a sub-population of αβ T cells with innate-like properties and limited TCR diversity. MAIT cells are of interest due to their reactivity against bacterial and yeast species, suggesting they play a role in defense against pathogenic microbes. Despite the advances in understanding MAIT cell biology, the molecular and structural basis behind their ability to detect MR1-antigen complexes is unclear. Here we present our structural and biochemical characterization of MAIT TCR engagement of MR1 presenting an E. coli-derived stimulatory ligand, rRL-6-CH2OH previously found in Salmonella typhimurium. We show a clear enhancement of MAIT TCR binding to MR1 due to presentation of this ligand. Our structure of a MAIT TCR/MR1/rRL-6-CH2OH complex shows an evolutionarily conserved binding orientation, with a clear role for both the CDR3α and CDR3β loops in recognition of the rRL-6-CH2OH stimulatory ligand. We also present two additional xeno-reactive MAIT TCR/MR1 complexes that recapitulate the docking orientation documented previously despite having variation in the CDR2β and CDR3β loop sequences. Our data supports a model by which MAIT TCRs engage MR1 in a conserved fashion, their binding affinities modulated by the nature of the MR1 presented antigen or diversity introduced by alternate Vβ usage or CDR3β sequences.
Understanding the interactions between small molecules and proteins can be approached from different perspectives and is important for the advancement of basic science and drug development. Chemists often use bioactive small molecules, such as natural products or synthetic compounds, as probes to identify therapeutically relevant protein targets. Biochemists and biologists often begin with a specific protein and seek to identify the endogenous metabolites that bind to it. These interests have led to the development of methodology that relies heavily on synthetic and analytical chemistry to identify protein-small molecule and protein-metabolite interactions. Here, we survey these strategies, highlighting key findings, to demonstrate the value of these approaches in answering important chemical and biological questions.
Genetic studies of rare diseases can identify genes of unknown function that strongly impact human physiology. Prolyl endopeptidase-like (PREPL) is an uncharacterized member of the prolyl peptidase family that was discovered because of its deletion in humans with hypotonia-cystinuria syndrome (HCS). HCS is characterized by a number of physiological changes including diminished growth and neonatal hypotonia or low muscle tone. HCS patients have deletions in other genes as well, making it difficult to tease apart the specific role of PREPL. Here, we develop a PREPL null (PREPL−/−) mouse model to address the physiological role of this enzyme. Deletion of exon 11 from the Prepl gene, which encodes key catalytic amino acids, leads to a loss of PREPL protein as well as lower Prepl mRNA levels. PREPL−/− mice have a pronounced growth phenotype, being significantly shorter and lighter than their wild type (PREPL+/+) counterparts. A righting assay revealed that PREPL−/− pups took significantly longer than PREPL+/+ pups to right themselves when placed on their backs. This deficit indicates that PREPL−/− mice suffer from neonatal hypotonia. According to these results, PREPL regulates growth and neonatal hypotonia in mice, which supports the idea that PREPL causes diminished growth and neonatal hypotonia in humans with HCS. These animals provide a valuable asset in deciphering the underlying biochemical, cellular and physiological pathways that link PREPL to HCS, and this may eventually lead to new insights in the treatment of this disease.
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