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Major Achievements of the Eugindat Project

Genetic, Molecular Biology and Clinical information

• Generation of a database on Primary Inherited Aminoacidurias (PIA-DATABASE): Fourth release of PIA-DATABASE was made publicly available (http://www.piaeugindat.eu/) in March 2007. PIA-DATABASE contains 208 patients with cystinuria and 24 patients with LPI. Patients in 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. (http://www.orpha.net/actor/Orphanews/2007/070718.html).
• An in depth description of the genotype and phenotype of non-related 164 cystinuria patients from the International Cystinuria Consortium (ICC) (Publication 3). In total EUGINDAT has identified 64 and 16 new mutations causing cystinuria and LPI respectively (Publications 3, 4 and unpublished results).
• Oligomerization of the rBAT/b0,+AT transporter might have an impact on cystinuria. The heavy subunit rBAT dictates oligomerization of the heteromeric amino acid transporters (Publication 18).
• The b0,+AT knock-out mice present cystinuria with lithiasis. Using this animal model (Publication 20), identification of a thiol drug with better in vivo efficiency to dissolve cystine calculi than Thiola and D-penicillamine.
• Establishment of a personalized medical management of cystinuria children (Publication 41).
• Identification of end-stage renal disease as a novel complication of lysinuric protein intolerance (Publication 35).

Identification and characterization of new proteins involved in renal reabsorption of amino acids

• Identification and functional characterization of several new amino acid transporters (human LAT4, mouse arpAT, mouse B0AT1, mouse SIT1) (Publications 5, 6, 7, 9, 11).
• Identification of transporter TAT1 as an efflux pathway for aromatic amino acids in proximal tubule and small intestinal epithelial cells (Publications 10 and 39).
• Identification of collectrin as a key regulator of renal amino acid uptake with a potential impact on Hartnup Disorder (Publication 37).

Generation of genetically modified mouse models

• 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 (Publication 20) and LPI (Publication 25), and have been used to demonstrate the role of PEPT2 in the synthesis of glutathione (Publication 24) and Pdk1 in the regulation of several renal amino acid transporters (Publication 36). Phenotyping of the generated models will continue for the next months and years.

Structural biology of renal reabsorption of amino acids and peptides

• The crystal structure of the 4F2hc-ectodomain (4F2hc-ED) has been solved at 2.1 Å resolution (PDB IDs 2DH2 and 2DH3) (Figure 3). 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 homodimerization 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 (Publication 21).
• 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 (Publications 19 and 23). These studies represent the first three-dimensional information of these types of transporters.