Mitochondrial Ca(2+) efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na(+)-dependent mechanism mediates mitochondrial Ca(2+) efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na(+)/Ca(2+) exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca(2+) and Na(+) fluorescent imaging, we demonstrate that mitochondrial Na(+)-dependent Ca(2+) efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca(2+) transport was inhibited, moreover, by CGP-37157 and exhibited Li(+) dependence, both hallmarks of mitochondrial Na(+)-dependent Ca(2+) efflux. Finally, NCLX-mediated mitochondrial Ca(2+) exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na(+)/Ca(2+) exchanger.

https://www.pnas.org/content/pnas/107/1/436.full.pdf

NCLX is an essential component of mitochondrial Na+/Ca2+ exchange.

Human skin consists of a stratified, cellular epidermis and an underlying dermis of connective tissue [1–5]. The dermal–epidermal junction is undulating in section; ridges of the epidermis, known as rete ridges, project into the dermis. The junction provides mechanical support for the epidermis and acts as a partial barrier against exchange of cells and large molecules. Below the dermis is a fatty layer, the panniculus adiposus, usually designated as ‘subcutaneous’. This is separated from the rest of the body by a vestigial layer of striated muscle, the panniculus carnosus. There are two main kinds of human skin. Glabrous skin (non-hairy skin), found on the palms and soles, is grooved on its surface by continuously alternating ridges and sulci, in individually unique configurations known as dermatoglyphics. It is characterized by a thick epidermis divided into several well-marked layers, including a compact stratum corneum, by the presence of encapsulated sense organs within the dermis, and by a lack of hair follicles and sebaceous glands. Hair-bearing skin (Fig. 3.1), on the other hand, has both hair follicles and sebaceous glands but lacks encapsulated sense organs. There is also wide variation between different body sites. For example, the scalp with its large hair follicles may be contrasted with the forehead, which has only small vellus-producing follicles, albeit associated with large sebaceous glands. The axilla is notable because it has apocrine glands in addition to the eccrine sweat glands, which are found throughout the body. Regional variation is further considered below. Chapter 3

Anatomy and Organization of Human Skin

The genetic basis underlying normal variation in the pigmentary traits of skin, hair and eye colour has been the subject of intense research directed at understanding the diversity seen both between and within human populations. A combination of approaches have been used including comparative genomics of candidate genes and the identification of regions of the human genome under positive selection, together with genome-wide and specific allele association studies. Independent selection for different pigmentation gene sets has been found between Asian, European and African populations. Several genome-wide association studies for pigmentation have now been conducted and identified single nucleotide polymorphism (SNP) markers in known, TYR, TYRP1, OCA2, SLC45A2, SLC24A5, MC1R, ASIP, KITLG and previously unknown SLC24A4, IRF4, TPCN2, candidate genes. The contribution of SNP polymorphisms present in populations from South Asia have been tested and alleles found at TYR, SLC45A2 and SLC24A5 can largely account for differences between those of darkest and lightest skin reflectance using a simple additive model. Skin and hair colour associations in Europeans are found within a range of pigmentation gene alleles, whereas blue-brown eye colour can be explained by a single SNP proposed to regulate OCA2 expression. Functional testing of variant alleles has begun to connect phenotype correlations with biological differences. Variant MC1R alleles show direct correlations between the biochemical signalling properties of the encoded receptor and the red-hair fair skin pigmentation phenotype. Direct testing of a range of clonal melanocyte cultures derived from donor skin tissue characterized for three causal SNPs within SLC45A2, SLC24A5 and OCA2 has assessed their impact on melanin content and tyrosinase enzyme activity. From a culmination of genetic and functional studies, it is apparent that a number of genes impacting melanosome biogenesis or the melanin biosynthetic pathway are candidates to explain the diversity seen in human pigmentation.

/pdf/molecular-genetics-of-human-pigmentation-diversity-1mdzg0bil1.pdf

Molecular genetics of human pigmentation diversity

Acidic calcium stores open for business: expanding the potential for intracellular Ca2+ signaling

Skin pigmentation is one of the most overt human physical traits with consequences on susceptibility to skin cancer. The variations in skin pigmentation are dependent on geographic location and population ethnicity. Skin colouration is mainly due to the pigmentation substance melanin, produced in specialized organelles (melanosomes) within dendritic melanocytes, and transferred to neighbouring keratinocytes. The two types of melanin synthesized in well defined chemical reactions are the protective dark coloured eumelanin and the sulphur containing light red-yellow pheomelanin. The events leading to the synthesis of melanin are controlled by signalling cascades that involve a host of genes encoding ligands, receptors, transcription factors, channel transporters and many other crucial molecules. Several variants within the genes involved in pigmentation have been associated with high risk phenotypes like fair skin, brown-red hair and green-blue eyes. Many of those variants have also been implicated in the risk of various skin cancers. The variants within the key pigmentation gene, melanocortin-receptor 1 (MC1R), in particular have been ubiquitously linked with high risk traits and skin cancers involving both pigmentary and non-pigmentary functions and likely interaction with variants in other genes. Many of the variants in other genes, functional in pigmentation pathway, have also been associated with phenotypic variation and risk of skin cancers. Those genes include agouti signalling protein (ASIP), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), oculocutaneous albinism II (OCA2), various solute carrier genes and transporters. Most of those associations have been confirmed in genome wide association studies that at the same time have also identified new loci involved in phenotypic variation and skin cancer risk. In conclusion, the genetic variants within the genes involved in skin pigmentation besides influencing phenotypic traits are important determinants of risk of several skin cancers. However, ultimate risk of skin cancer is dependent on interplay between genetic and host factors.

Genetics of pigmentation in skin cancer--a review.

http://www.jbc.org/content/early/2007/12/28/jbc.M707521200.full.pdf

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women.

Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction.

Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody–based TR-FIA, and gas chromatography–mass spectrometry showed correlations ( r , 0.911–0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavone-rich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied.

Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status.

/pdf/monoclonal-antibody-based-time-resolved-fluorescence-pbip2cwrfs.pdf

Monoclonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study

The invention provides a method of identifying compounds that either increase or decrease skin and/or hair pigmentation, the method comprising determining the ability of a test compound to modulate NCKX-mediated calcium ion movement across a membrane (e.g. NCKX5). The method may comprise the steps of exposing a membrane comprising a NCKX molecule or variant, fusion or derivative thereof to a test compound and measuring either directly or indirectly the calcium ion concentration on one or both sides of the membrane. The invention also relates to kits, nucleic acid molecules, polypeptides and cells useful in the method of the invention.

Richard M. Ogborne

Papers

SLC24A5 Encodes a trans-Golgi Network Protein with Potassium-dependent Sodium-Calcium Exchange Activity That Regulates Human Epidermal Melanogenesis

Monoclonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study

Method of screening for compounds that alter skin and/or hair pigmentation