ABSTRACT
Mutations in the human EYA1 gene have been associated with several human diseases including branchio-oto (BO) and branchio-oto-renal (BOR) syndrome, as well as congenital cataracts and ocular anterior segment anomalies. BOR patients suffer from severe malformations of the ears, branchial arches and kidneys. The phenotype of Eya1-heterozygous mice resembles the symptoms of human patients suffering from BOR syndrome. The Eya1 gene encodes a multifunctional protein that acts as a protein tyrosine phosphatase and a transcriptional coactivator. It has been shown that Eya1 interacts with Six transcription factors, which are also required for nuclear translocation of the Eya1 protein. We investigated the effects of seven disease-causing Eya1 missense mutations on Eya1 protein function, in particular cellular localization, ability to interact with Six proteins, and protein stability. We show here that the BOR-associated Eya1 missense mutations S454P, L472R, and L550P lead to enhanced proteasomal degradation of the Eya1 protein in mammalian cells. Moreover, Six proteins lead to a significant stabilization of Eya1, which is caused by Six-mediated protection from proteasomal degradation. In case of the mutant L550P, loss of interaction with Six proteins leads to rapid protein degradation. Our observations suggest that protein destabilization constitutes a novel disease causing mechanism for Eya1.
Subject(s)
Branchio-Oto-Renal Syndrome/genetics , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Tyrosine Phosphatases/genetics , Animals , COS Cells , Cell Line, Tumor , Cell Nucleus/metabolism , Chlorocebus aethiops , Homeodomain Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mutation, Missense , Nuclear Proteins/metabolism , Protein Binding , Protein Stability , Protein Transport , Protein Tyrosine Phosphatases/metabolism , Proteolysis , UbiquitinationABSTRACT
The eyes absent (Eya) genes encode a family of proteins that combine the functions of transcriptional cofactors, signal transducers and enzymes, namely protein tyrosine phosphatases. The latter activity resides in the highly conserved C-terminal Eya domain (ED). Here, we investigated the substrate specificity of the Arabidopsis thaliana homologue (AtEya) by using low-molecular-weight compounds and synthetic phosphotyrosine (pY)-containing peptides that correspond either to phosphorylation sites in proteins or to peptides that were selected through the screening of a combinatorial peptide library. AtEya displayed modest peptide substrate specificity and was sensitive to charges adjacent to pY. In general, the presence of acidic residues on the N-terminal side of the phosphorylation site was critical for catalysis, whereas basic amino acids seemed to be preferred with respect to high-affinity binding. We also detected significant acyl phosphatase activity of AtEya; this suggests that Eya proteins might have further substrates in vivo. In addition, we analysed the phosphatase activity of a number of variants of the mouse Eya1 protein that harbours single point mutations that were associated with branchio-oto-renal syndrome (BOR), branchio-oto syndrome (BO) and ocular defects, respectively, in humans. While BOR mutations led to a significantly reduced phosphatase activity, BO mutants as well as those that are associated with ocular defects only displayed activity that was similar to wild-type levels.
Subject(s)
Arabidopsis Proteins/metabolism , Branchio-Oto-Renal Syndrome/genetics , Eye Abnormalities/genetics , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutant Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Point Mutation , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Animals , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Branchio-Oto-Renal Syndrome/enzymology , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Kinetics , Mice , Mutant Proteins/chemistry , Mutant Proteins/genetics , Nuclear Proteins/chemistry , Peptide Library , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Phenylalanine/metabolism , Phosphorylation , Protein Structure, Tertiary , Protein Tyrosine Phosphatases/chemistry , Substrate SpecificityABSTRACT
BACKGROUND: Worldwide surveillance of antimicrobial resistance among urinary tract pathogens is useful to determine important trends and geographical variation for common Gram negative and positive species. We initiated this study to learn the drug resistance pattern and its location (genomic or plasmid-borne) among the strains of Proteus mirabilis, one of the most common causative agents of urinary tract infection (UTI). MATERIAL/METHODS: A total of 200 urine samples were collected from UTI patients, followed by isolation and identification of Proteus milabilis strains. Antibiotic sensitivity and resistance analysis was performed by the disc diffusion method, employing multiple antibiotic discs. The sensitivity was monitored by visualizing the zone of inhibition around the disc. Plasmid isolation was performed by the method of Kado and Liu. Transfer of the R-Plasmid to recipient E.coli C600 cells was achieved using the method of Lederberg and Cohen. RESULTS: Clinical study revealed that this infection is more common in young pregnant women. Various strains of Proteus mirabilis isolated during the course of the study were found to show multiple antibiotic resistance, which was further characterized as plasmid-borne drug resistance. CONCLUSIONS: Proteus mirabilis is one of the important causative agents of urinary tract infection in young women especially during the state of pregnancy. Multiple antibiotic resistance markers of Proteus strains were identified as plasmid mediated.
