Shaping biological knowledge: applications in proteomics.

The central dogma of molecular biology has provided a meaningful principle for data integration in the field of genomics. In this context, integration reflects the known transitions from a chromosome to a protein sequence: transcription, intron splicing, exon assembly and translation. There is no such clear principle for integrating proteomics data, since the laws governing protein folding and interactivity are not quite understood. In our effort to bring together independent pieces of information relative to proteins in a biologically meaningful way, we assess the bias of bioinformatics resources and consequent approximations in the framework of small-scale studies. We analyse proteomics data while following both a data-driven (focus on proteins smaller than 10 kDa) and a hypothesis-driven (focus on whole bacterial proteomes) approach. These applications are potentially the source of specialized complements to classical biological ontologies.

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity.

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.

Luminal heterodimeric amino acid transporter defective in cystinuria.

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+-independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are approximately 48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+-dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L-type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.

Overxpression of Bcl-2 inhibits UVA-mediated immediate apoptosiinrat 6 fibroblasts: evidence for the involvement of Bcl-2 as an antioxidant.

We examined the effect of broad spectrum UVA (320-380 nm) and UVB (290-320 nm) radiation on the induction of apoptosis in the rat 6 fibroblast cell line (R6). UVA, but not UVB, induces apoptosis in this cell line. The morphological changes and DNA ladders associated with apoptosis occurred within the first 4 h after UVA irradiation, a phenomenon referred to as "immediate" apoptosis. From previous studies, it is known that Bcl-2 inhibits most types of apoptotic cell death. Overexpression of mouse Bcl-2 in the R6 fibroblasts inhibited the UVA-induced immediate apoptosis. The induction of the heme oxygenase 1 (HO-1) gene by UVA is a general response to oxidative stress. As a marker of oxidative stress, we monitored the effect of Bcl-2 overexpression on the level of HO-1 mRNA accumulation after UVA irradiation. The results showed that the overexpression of Bcl-2 in the R6 fibroblasts strongly reduces the level of HO-1 induction from 12.5- to 4.9-fold. We propose that Bcl-2 expression inhibits UVA-induced immediate apoptosis via an antioxidant pathway, suppressing either the generation or effects of specific UVA-mediated reactive oxygen species.