Supplementary Material for: Expression of the Sodium/Calcium/Potassium Exchanger, NCKX4, in Ameloblasts

Transcellular calcium transport is an essential activity in mineralized tissue formation, including dental hard tissues. In many organ systems, this activity is regulated by membrane-bound sodium/calcium (Na⁺/Ca²⁺) exchangers, which include the NCX and NCKX [sodium/calcium-potassium (Na⁺/Ca²⁺-K⁺) exchanger] proteins. During enamel maturation, when crystals expand in thickness, Ca²⁺ requirements vastly increase but exactly how Ca²⁺ traffics through ameloblasts remains uncertain. Previous studies have shown that several NCX proteins are expressed in ameloblasts, although no significant shifts in expression were observed during maturation which pointed to the possible identification of other Ca²⁺ membrane transporters. NCKX proteins are encoded by members of the solute carrier gene family, Slc24a, which include 6 different proteins (NCKX1–6). NCKX are bidirectional electrogenic transporters regulating Ca²⁺ transport in and out of cells dependent on the transmembrane ion gradient. In this study we show that all NCKX mRNAs are expressed in dental tissues. Real-time PCR indicates that of all the members of the NCKX group, NCKX4 is the most highly expressed gene transcript during the late stages of amelogenesis. In situ hybridization and immunolocalization analyses clearly establish that in the enamel organ, NCKX4 is expressed primarily by ameloblasts during the maturation stage. Further, during the mid-late maturation stages of amelogenesis, the expression of NCKX4 in ameloblasts is most prominent at the apical poles and at the lateral membranes proximal to the apical ends. These data suggest that NCKX4 might be an important regulator of Ca²⁺ transport during amelogenesis

Supplementary Material for: Rate-limiting enzymes in cardiometabolic health and aging in humans

Introduction. Rate-limiting enzymes (RLEs) are innate slow points in metabolic pathways, and many function in bioprocesses related to nutrient sensing. Many RLEs carry causal mutations relevant to inherited metabolic disorders. Because the activity of RLEs in cardiovascular health is poorly characterized, our objective was to assess their involvement in cardiometabolic health and disease, and where altered biophysical and biochemical functions can promote disease. Methods. A dataset of 380 human RLEs was compared to protein and gene datasets for factors likely to contribute to cardiometabolic disease, including proteins showing significant age-related altered expression in blood, and genetic loci with variants that associate with common cardiometabolic phenotypes. The biochemical reactions catalyzed by RLEs were evaluated for metabolites enriched in RLE subsets associating with various cardiometabolic phenotypes. Most significance tests were based on Z-score enrichment converted to P values with a normal distribution function. Results. Of 380 RLEs analyzed, 112 function in mitochondria, and 53 are assigned to inherited metabolic disorders. There was a depletion of RLE proteins known as aging biomarkers. At the gene level, RLEs were assessed for common genetic variants that associated with important cardiometabolic traits of LDL-cholesterol or any of the five outcomes pertinent to metabolic syndrome. This revealed several RLEs with links to cardiometabolic traits, from a minimum of 26 for HDL-cholesterol to a maximum of 45 for plasma glucose. Analysis of these GWAS-linked RLEs for enrichment of the molecular constituents of the catalyzed reactions disclosed a number of significant phenotype-metabolite links. These included blood pressure with acetate (P = 2.2x10-4) and NADP+ (P = 0.0091), plasma HDL-cholesterol and triglyceride with diacylglycerol (P = 2.6x10-5, 6.4x10-5, respectively) and diolein (P = 2.2x10-6, 5.9x10-6), and waist circumference with D-glucosamine 6-phosphate (P = 1.8x10-4). Conclusion. In the context of cardiometabolic health, aging and disease, these results highlight key diet-derived metabolites that are central to specific rate-limited processes that are linked to cardiometabolic health. These metabolites include acetate and diacylglycerol, pertinent to blood pressure and triglycerides, respectively, as well as diacylglycerol and HDL-cholesterol