Final Report Summary - EUGINDAT (European Genomics Initiative on Disorders of plasma membrane Amino acid Transporters)
EUGINDAT's main goal was the study of the rare Primary inherited aminoacidurias (PIA) (cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria and dicarboxylic aminoaciduria).
EUGINDAT had four horizontal activities:
- a clinic platform, aimed at generating a PIA database;
- an animal model platform for the generation of mouse models (knock out and ENU-mutated animals) of PIA and relevant amino acid and peptide transporters for renal reabsorption;
- a 3D protein structure platform for the resolution of amino acid and peptide transporter structures (2D and 3D crystallisation);
- a genetic platform for the identification of mutations causing PIA, modulator genes of PIA and polymorphisms related to renal reabsorption of amino acids and peptides.
Vertical activities include pathophysiological studies and the development of new treatments for cystinuria and lysinuric protein intolerance. The achievements in the genetic, molecular biology and clinical information include the following:
- Generation of a database on PIAs (PIA-Database).
- Fourth release of PIA-Database: The PIA-DATABASE was made publicly available on http://www.piaeugindat.eu/ in March 2007. It contains 208 patients with cystinuria and 24 patients with LPI. Patients in the PIA-DATABASE are coded to protect intimacy. Orphanet is evaluating the opportunity of taking the paternity of PIA-Database (maintenance cost - privacy issue - data property - decision about studies to be proposed, etc.) after EUGINDAT.
- An in depth description of the genotype and phenotype of non-related 164 cystinuria patients from the International Cystinuria Consortium (ICC). In total, EUGINDAT has identified 64 and 16 new mutations causing cystinuria and LPI respectively. The oligomerisation of the rBAT/b0,+AT transporter might have an impact on cystinuria. The heavy subunit rBAT dictates oligomerisation of the heteromeric amino acid transporters.
- The b0,+AT knock-out mice present cystinuria with lithiasis. Using this animal model, identification of a thiol drug with better in vivo efficiency to dissolve cystine calculi than Thiola and D-penicillamine.
- Establishment of a personalised medical management of cystinuria children.
- Identification of end-stage renal disease as a novel complication of lysinuric protein intolerance.
The achievements in the identification and characterisation of new proteins involved in renal re-absorption of amino acids include:
- identification and functional characterisation of several new amino acid transporters (human LAT4, mouse arpAT, mouse B0AT1, mouse SIT1);
- identification of transporter TAT1 as an efflux pathway for aromatic amino acids in proximal tubule and small intestinal epithelial cells;
- identification of collectrin as a key regulator of renal amino acid uptake with a potential impact on Hartnup disorder.
The achievements in the generation of genetically modified mouse models include: EUGINDAT partners developed more than 10 knockout or ENU-mutated mouse models of relevant amino acid and peptide transporters for renal reabsorption and intestinal abruption. This includes several models for cystinuria and LPI, and has been used to demonstrate the role of PEPT2 in the synthesis of glutathione and Pdk1 in the regulation of several renal amino acid transporters. Phenotyping of the generated models will continue for the next months and years.
The achievements in the structural biology of renal re-absorption of amino acids and peptides area include the following:
- The crystal structure of the 4F2hc-ectodomain (4F2hc-ED) has been solved at a 2.1 A resolution (PDB IDs 2DH2 and 2DH3). 4F2hc is the heavy subunit, among other amino acid transporters, of y+LAT1, which is mutated in Lysinuric protein intolerance (LPI). The structure reveals a tendency to homodimerisation and provides a model for the electrostatic interaction of the ectodomain with the plasma membrane. In vivo experiments confirmed the position of the ectodomain on the plasma membrane. This structure will help the resolution of the structure of 4F2hc-association HATs and has implications on cell adhesion, fusion and transformation.
- SteT (YkbA) from Bacillus subtilis and YdgR from Escherichia coli has been established as the first prokaryotic models of the mammalian light subunits of the heteromeric amino acid transporters and the proton-dependent oligopeptide transporter respectively. These studies represent the first 3D information of these types of transporters.
EUGINDAT had four horizontal activities:
- a clinic platform, aimed at generating a PIA database;
- an animal model platform for the generation of mouse models (knock out and ENU-mutated animals) of PIA and relevant amino acid and peptide transporters for renal reabsorption;
- a 3D protein structure platform for the resolution of amino acid and peptide transporter structures (2D and 3D crystallisation);
- a genetic platform for the identification of mutations causing PIA, modulator genes of PIA and polymorphisms related to renal reabsorption of amino acids and peptides.
Vertical activities include pathophysiological studies and the development of new treatments for cystinuria and lysinuric protein intolerance. The achievements in the genetic, molecular biology and clinical information include the following:
- Generation of a database on PIAs (PIA-Database).
- Fourth release of PIA-Database: The PIA-DATABASE was made publicly available on http://www.piaeugindat.eu/ in March 2007. It contains 208 patients with cystinuria and 24 patients with LPI. Patients in the PIA-DATABASE are coded to protect intimacy. Orphanet is evaluating the opportunity of taking the paternity of PIA-Database (maintenance cost - privacy issue - data property - decision about studies to be proposed, etc.) after EUGINDAT.
- An in depth description of the genotype and phenotype of non-related 164 cystinuria patients from the International Cystinuria Consortium (ICC). In total, EUGINDAT has identified 64 and 16 new mutations causing cystinuria and LPI respectively. The oligomerisation of the rBAT/b0,+AT transporter might have an impact on cystinuria. The heavy subunit rBAT dictates oligomerisation of the heteromeric amino acid transporters.
- The b0,+AT knock-out mice present cystinuria with lithiasis. Using this animal model, identification of a thiol drug with better in vivo efficiency to dissolve cystine calculi than Thiola and D-penicillamine.
- Establishment of a personalised medical management of cystinuria children.
- Identification of end-stage renal disease as a novel complication of lysinuric protein intolerance.
The achievements in the identification and characterisation of new proteins involved in renal re-absorption of amino acids include:
- identification and functional characterisation of several new amino acid transporters (human LAT4, mouse arpAT, mouse B0AT1, mouse SIT1);
- identification of transporter TAT1 as an efflux pathway for aromatic amino acids in proximal tubule and small intestinal epithelial cells;
- identification of collectrin as a key regulator of renal amino acid uptake with a potential impact on Hartnup disorder.
The achievements in the generation of genetically modified mouse models include: EUGINDAT partners developed more than 10 knockout or ENU-mutated mouse models of relevant amino acid and peptide transporters for renal reabsorption and intestinal abruption. This includes several models for cystinuria and LPI, and has been used to demonstrate the role of PEPT2 in the synthesis of glutathione and Pdk1 in the regulation of several renal amino acid transporters. Phenotyping of the generated models will continue for the next months and years.
The achievements in the structural biology of renal re-absorption of amino acids and peptides area include the following:
- The crystal structure of the 4F2hc-ectodomain (4F2hc-ED) has been solved at a 2.1 A resolution (PDB IDs 2DH2 and 2DH3). 4F2hc is the heavy subunit, among other amino acid transporters, of y+LAT1, which is mutated in Lysinuric protein intolerance (LPI). The structure reveals a tendency to homodimerisation and provides a model for the electrostatic interaction of the ectodomain with the plasma membrane. In vivo experiments confirmed the position of the ectodomain on the plasma membrane. This structure will help the resolution of the structure of 4F2hc-association HATs and has implications on cell adhesion, fusion and transformation.
- SteT (YkbA) from Bacillus subtilis and YdgR from Escherichia coli has been established as the first prokaryotic models of the mammalian light subunits of the heteromeric amino acid transporters and the proton-dependent oligopeptide transporter respectively. These studies represent the first 3D information of these types of transporters.