Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Here, we report the solution structure of ZNF593, a protein identified in a functional study as a negative modulator of the DNA-binding activity of the Oct-2 transcription factor. ZNF593 contains a classic C(2)H(2) zinc finger domain flanked by about 40 disordered residues on each terminus. Although the protein contains a high degree of intrinsic disorder, the structure of the zinc finger domain was resolved by NMR spectroscopy without a need for N- or C-terminal truncations. The tertiary structure of the zinc finger domain is composed of a beta-hairpin that positions the cysteine side chains for zinc coordination, followed by an atypical kinked alpha-helix containing the two histidine side chain ligands. The structural topology of ZNF593 is similar to a fragment of the double-stranded RNA-binding protein Zfa and the C-terminal zinc finger of MBP-1, a human enhancer binding protein. The structure presented here will provide a guide for future functional studies of how ZNF593 negatively modulates the DNA-binding activity of Oct-2, a POU domain-containing transcription factor. Our work illustrates the unique capacity of NMR spectroscopy for structural analysis of folded domains in a predominantly disordered protein.
Project description:Human quinolinate phosphoribosyltransferase (EC 22.214.171.124) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.
Project description:Filamin A (FlnA) plays a critical role in cytoskeletal organization, cell motility and cellular signaling. FlnA utilizes different binding sites on a series of 24 immunoglobulin-like domains (Ig repeats) to interact with diverse cytosolic proteins and with cytoplasmic portions of membrane proteins. Mutations in a specific domain, Ig10 (FlnA-Ig10), are correlated with two severe forms of the otopalatodigital syndrome spectrum disorders Melnick-Needles syndrome and frontometaphyseal dysplasia. The crystal structure of FlnA-Ig10 determined at 2.44?Å resolution provides insight into the perturbations caused by these mutations.
Project description:We investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level.SNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively.Six of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes.There is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies.
Project description:Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.
Project description:Cortical thickness has been investigated since the beginning of the 20th century, but we do not know how similar the cortical thickness profiles among humans are. In this study, the local similarity of cortical thickness profiles was investigated using sliding window methods. Here, we show that approximately 5% of the cortical thickness profiles are similarly expressed among humans while 45% of the cortical thickness profiles show a high level of heterogeneity. Therefore, heterogeneity is the rule, not the exception. Cortical thickness profiles of somatosensory homunculi and the anterior insula are consistent among humans, while the cortical thickness profiles of the motor homunculus are more variable. Cortical thickness profiles of homunculi that code for muscle position and skin stimulation are highly similar among humans despite large differences in sex, education, and age. This finding suggests that the structure of these cortices remains well preserved over a lifetime. Our observations possibly relativize opinions on cortical plasticity.
Project description:Understanding the timing and character of the expansion of Homo sapiens out of Africa is critical for inferring the colonization and admixture processes that underpin global population history. It has been argued that dispersal out of Africa had an early phase, particularly ~130-90 thousand years ago (ka), that reached only the East Mediterranean Levant, and a later phase, ~60-50?ka, that extended across the diverse environments of Eurasia to Sahul. However, recent findings from East Asia and Sahul challenge this model. Here we show that H. sapiens was in the Arabian Peninsula before 85?ka. We describe the Al Wusta-1 (AW-1) intermediate phalanx from the site of Al Wusta in the Nefud desert, Saudi Arabia. AW-1 is the oldest directly dated fossil of our species outside Africa and the Levant. The palaeoenvironmental context of Al Wusta demonstrates that H. sapiens using Middle Palaeolithic stone tools dispersed into Arabia during a phase of increased precipitation driven by orbital forcing, in association with a primarily African fauna. A Bayesian model incorporating independent chronometric age estimates indicates a chronology for Al Wusta of ~95-86?ka, which we correlate with a humid episode in the later part of Marine Isotope Stage 5 known from various regional records. Al Wusta shows that early dispersals were more spatially and temporally extensive than previously thought. Early H. sapiens dispersals out of Africa were not limited to winter rainfall-fed Levantine Mediterranean woodlands immediately adjacent to Africa, but extended deep into the semi-arid grasslands of Arabia, facilitated by periods of enhanced monsoonal rainfall.
Project description:The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 A resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an alphabetabetaalpha four-layer topology. A metal ion residing between the central beta-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn(2+). Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn(2+) to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and beta-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. Overall design: One-condition experment, gene expression of 3A6