11 research outputs found
A Structure-Based Approach for Detection of Thiol Oxidoreductases and Their Catalytic Redox-Active Cysteine Residues
Cysteine (Cys) residues often play critical roles in proteins, for example, in
the formation of structural disulfide bonds, metal binding, targeting proteins
to the membranes, and various catalytic functions. However, the structural
determinants for various Cys functions are not clear. Thiol oxidoreductases,
which are enzymes containing catalytic redox-active Cys residues, have been
extensively studied, but even for these proteins there is little understanding
of what distinguishes their catalytic redox Cys from other Cys functions.
Herein, we characterized thiol oxidoreductases at a structural level and
developed an algorithm that can recognize these enzymes by (i) analyzing amino
acid and secondary structure composition of the active site and its similarity
to known active sites containing redox Cys and (ii) calculating accessibility,
active site location, and reactivity of Cys. For proteins with known or modeled
structures, this method can identify proteins with catalytic Cys residues and
distinguish thiol oxidoreductases from the enzymes containing other catalytic
Cys types. Furthermore, by applying this procedure to Saccharomyces
cerevisiae proteins containing conserved Cys, we could identify the
majority of known yeast thiol oxidoreductases. This study provides insights into
the structural properties of catalytic redox-active Cys and should further help
to recognize thiol oxidoreductases in protein sequence and structure
databases
Mutations in DSTYK and dominant urinary tract malformations.
ABSTRACT
Introduction
Congenital abnormalities of the kidney of the urinary tract are the most common cause of pediatric kidney failure. These disorders are highly heterogeneous, and their etiology is poorly understood.
Methods
We performed genome-wide linkage analysis and whole-exome sequencing in a family with autosomal dominant congenital abnormalities of the kidney of the urinary tract (7 affected family members). We also performed sequence analysis in 311 unrelated patients, as well as histologic and functional studies.
Results
Linkage analysis identified five regions of the genome that were shared among all affected family members. Exome sequencing identified a single rare deleterious variant within these linkage intervals, a heterozygous splice-site mutation in dual serine/threonine and tyrosine protein kinase (DSTYK). This variant, which resulted in aberrant gene product splicing, was present in all affected family members. Additional independent DSTYK mutations, including nonsense and splice-site mutations, were detected among 7/311 unrelated patients. DSTYK is highly expressed in the maturing epithelia of all major organs, localizing to cell membranes. Knockdown in zebrafish resulted in multi-organ developmental defects, resembling loss of fibroblast growth factor (FGF) signaling. Consistent with this finding, DSTYK colocalizes with FGF receptors in the ureteric bud and metanephric mesenchyme. Finally, DSTYK knockdown in human embryonic kidney cells inhibited FGF-stimulated ERK-phosphorylation, the principal signal downstream of receptor tyrosine kinases.
Conclusions
We detected DSTYK mutations in 2.2% of patients with congenital abnormalities of the kidney and urinary tract whom we studied, suggesting that DSTYK is a major determinant of human urinary tract development, downstream of FGF signaling
Defining the Molecular Character of the Developing and Adult Kidney Podocyte
BACKGROUND: The podocyte is a remarkable cell type, which encases the capillaries of the kidney glomerulus. Although mesodermal in origin it sends out axonal like projections that wrap around the capillaries. These extend yet finer projections, the foot processes, which interdigitate, leaving between them the slit diaphragms, through which the glomerular filtrate must pass. The podocytes are a subject of keen interest because of their key roles in kidney development and disease. METHODOLOGY/PRINCIPAL FINDINGS: In this report we identified and characterized a novel transgenic mouse line, MafB-GFP, which specifically marked the kidney podocytes from a very early stage of development. These mice were then used to facilitate the fluorescent activated cell sorting based purification of podocytes from embryos at E13.5 and E15.5, as well as adults. Microarrays were then used to globally define the gene expression states of podocytes at these different developmental stages. A remarkable picture emerged, identifying the multiple sets of genes that establish the neuronal, muscle, and phagocytic properties of podocytes. The complete combinatorial code of transcription factors that create the podocyte was characterized, and the global lists of growth factors and receptors they express were defined. CONCLUSIONS/SIGNIFICANCE: The complete molecular character of the in vivo podocyte is established for the first time. The active molecular functions and biological processes further define their unique combination of features. The results provide a resource atlas of gene expression patterns of developing and adult podocytes that will help to guide further research of these incredible cells