Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition of integrin αvß6.

Integrin αvß6 is a cell surface arginine-glycine-aspartic acid (RGD)-specific heterodimeric glycoprotein that is only expressed on epithelia during processes of tissue remodelling, including cancer. The specificity and molecular nature of interactions toward this integrin are poorly understood and new insights into such processes are important to cell biologists and pharmaceutical drug discovery. This study demonstrates the application of quantitative two-dimensional saturation transfer (Q2DSTD) NMR to obtain precise details of peptide interactions with integrin αvß6 and their correlation to specificity for the integrin. This approach highlights subtle but significant differences in ligand contact by three related 21-mer peptides: FMDV2, an αvß6 specific peptide and DBD1 and LAP2T1 peptides that bind many αv integrins in addition to αvß6. FMDV2 and DBD1 differ only by the cyclisation of DBD1; a process that removes αvß6 specificity. Q2DSTD NMR demonstrates these peptides experience significantly different interactions with the integrin; FMDV contacts primarily through four residues: 6Leu, 10Leu, 12Val and 13Leu, whereas DBD1 and LAP2T1 have more widespread contacts across their sequences. Q2DSTD NMR combined two-dimensional STD with quantitation by considering the relaxation of the ligand (CRL) to provide precise ligand contact information. This study also examines the role of CRL in the Q2DSTD process and how quantitation modifies STD data and unravels epitope-mapping variability to provide precise results that differentiate interactions at the atomic level for each peptide.

Store-operated Ca2+ entry controls ameloblast cell function and enamel development.

Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.

Pharmacological profiling of the TRPV3 channel in recombinant and native assays.

BACKGROUND AND PURPOSE: Transient receptor potential vanilloid subtype 3 (TRPV3) is implicated in nociception and certain skin conditions. As such, it is an attractive target for pharmaceutical research. Understanding of endogenous TRPV3 function and pharmacology remains elusive as selective compounds and native preparations utilizing higher throughput methodologies are lacking. In this study, we developed medium-throughput recombinant and native cellular assays to assess the detailed pharmacological profile of human, rat and mouse TRPV3 channels. EXPERIMENTAL APPROACH: Medium-throughput cellular assays were developed using a Ca(2+) -sensitive dye and a fluorescent imaging plate reader. Human and rat TRPV3 pharmacology was examined in recombinant cell lines, while the mouse 308 keratinocyte cell line was used to assess endogenous TRPV3 activity. KEY RESULTS: A recombinant rat TRPV3 cellular assay was successfully developed after solving a discrepancy in the published rat TRPV3 protein sequence. A medium-throughput, native, mouse TRPV3 keratinocyte assay was also developed and confirmed using genetic approaches. Whereas the recombinant human and rat TRPV3 assays exhibited similar agonist and antagonist profiles, the native mouse assay showed important differences, namely, TRPV3 activity was detected only in the presence of potentiator or during agonist synergy. Furthermore, the native assay was more sensitive to block by some antagonists. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate similarities but also notable differences in TRPV3 pharmacology between recombinant and native systems. These findings offer insights into TRPV3 function and these assays should aid further research towards developing TRPV3 therapies.