Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor.

The human sweet taste receptor is a heterodimeric receptor composed of two distinct G-protein-coupled receptors (GPCRs), TAS1R2 and TAS1R3. The TAS1R2 and TAS1R3 subunits are members of a small family of class C GPCRs whose members share the same architecture, comprising a Venus Flytrap (VFT) module linked to the seven transmembrane domains (TMDs) by a short cysteine-rich region (CRR). The VFT module of TAS1R2 contains the primary binding site for most of the sweet-tasting compounds, including natural sugars and artificial and natural sweeteners. However, cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the VFT, CRR and TMD of TAS1R3 interact with some sweeteners, including the sweet-tasting protein brazzein. The aim of this study was to better understand the contribution of TAS1R2-VFT in the binding of sweet stimuli. To achieve this, we heterologously expressed human TAS1R2-VFT (hTAS1R2-VFT) in Escherichia coli. Circular dichroism spectroscopic studies revealed that hTAS1R2-VFT was properly folded with evidence of secondary structures. Using size-exclusion chromatography coupled with light scattering, we found that hTAS1R2-VFT behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that hTAS1R2-VFT is capable of binding sweet stimuli with Kd values, in agreement with physiological detection. Furthermore, we investigated whether the impact of point mutations, already shown to have deleterious effects on cellular assays, could impact the ability of hTAS1R2-VFT to bind sweet ligands. As expected, the ligand affinities of hTAS1R2-VFT were drastically reduced through the introduction of single amino acid substitutions (D278A and E382A) known to abolish the response of the full-length TAS1R2/TAS1R3 receptor. This study demonstrates the feasibility of producing milligram quantities of hTAS1R2-VFT to further characterize the mechanism of binding interaction and perform structural studies.

New insights into the substrate specificity of macrophage elastase MMP-12.

Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

Functional roles of the sweet taste receptor in oral and extraoral tissues.

PURPOSE OF REVIEW: This review summarizes and discusses the current knowledge about the physiological roles of the sweet taste receptor in oral and extraoral tissues. RECENT FINDINGS: The expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, including the gut, pancreas, bladder, brain and, more recently, bone and adipose tissues. In the gut, this receptor has been suggested to be involved in luminal glucose sensing, the release of some satiety hormones, the expression of glucose transporters, and the maintenance of glucose homeostasis. More recently, the sweet taste receptor was proposed to regulate adipogenesis and bone biology. SUMMARY: The perception of sweet taste is mediated by the T1R2/T1R3 receptor, which is expressed in the oral cavity, wherein it provides input on the caloric and macronutrient contents of ingested food. This receptor recognizes all the chemically diverse compounds perceived as sweet by human beings, including natural sugars and sweeteners. Importantly, the expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, wherein it has been proposed to regulate metabolic processes. This newly recognized role of the sweet taste receptor makes this receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions, such as diabetes and hyperlipidemia.