Biomedical subjects

Rashmi S Hegde

Publications and source records attributed to Rashmi S Hegde.

11 recordsLinked to original sources

PubMed2006

Branchio-oto-renal syndrome associated mutations in Eyes Absent 1 result in loss of phosphatase activity.

The Eyes Absent (Eya) proteins are tyrosine phosphatases and transcriptional activators involved in cell-fate determination and organ development. Mutations in the gene encoding Eya homologue 1 have been implicated in the multi-organ developmental disorder branchio-oto-renal syndrome (BOR) and in ocular defects. Here we report that BOR-associated mutations lead to a loss of phosphatase activity in Eya1 proteins, while mutations associated with ocular defects yield Eya1 proteins with near normal levels of phosphatase activity. Furthermore we demonstrate that the N-terminal domain attenuates the catalytic activity of Eya suggesting a mechanism of regulation.

Amino Acid Sequence↗

PubMed2006

Indirect readout of DNA sequence by papillomavirus E2 proteins depends upon net cation uptake.

The papillomavirus E2 proteins bind with high affinity to palindromic DNA sequences consisting of two highly conserved four base-pair sequences flanking a variable "spacer" of identical length (ACCG NNNN CGGT). While intimate contacts are observed between the bound proteins and conserved DNA in the available co-crystal structures, no contact is seen between the proteins and the spacer DNA. The ability of human papillomavirus strain 16 (HPV-16) E2 and bovine papillomavirus strain 1 (BPV-1) E2 to discriminate among binding sites with different spacer sequences is dependent on their sensitivity to the unique conformational and/or dynamic properties of the spacer DNA in a process termed "indirect readout". Differential sequence-specific K(+) uptake in low ionic strength solutions lacking Mg(2+) is observed upon E2 protein binding to sites containing the AATT, TTAA or ACGT spacer sequences. In contrast, the cation displacement typical of protein-DNA complex formation is observed at high K(+) concentrations or in the presence of Mg(2+). These results are interpreted to reflect the sequence-specific stabilization of bent DNA conformations by cations localized within the narrowed minor grooves of the protein-bound DNA and the intrinsic structure and flexibility of the DNA target. Mg(2+) differentially affects the binding of the HPV-16 E2 DNA binding domain (HPV16-E2/D) and the BPV-1 E2 DNA binding domain (BPV1-E2/D) to sites bearing different spacer sequences. This study suggests that monovalent and divalent cations contribute to the discrimination of DNA structure and flexibility that could in turn contribute to the specificity with which HPV16-E2/D and BPV1-E2/D mediate DNA replication and gene transcription.

Animals↗

PubMed2005

Analyses of variant acid beta-glucosidases: effects of Gaucher disease mutations.

Acid beta-glucosidase (GCase) is a 497-amino acid, membrane-associated lysosomal exo-beta-glucosidase whose defective activity leads to the Gaucher disease phenotypes. To move toward a structure/function map for disease mutations, 52 selected single amino acid substitutions were introduced into GCase, expressed in an insect cell system, purified, and characterized for basic kinetic, stability, and activator response properties. The variant GCases from Gaucher disease patients and selected variant GCases from the mouse had decreased relative k(cat) and differential effects on active site binding and/or attachment of mechanism-based covalent (conduritol B epoxide) or reversible (deoxynojirimycin derivatives) inhibitors. A defect in negatively charged phospholipid activation was present in the majority of variant GCases but was increased in two, N370S and V394L. Deficits in saposin C enhancement of k(cat) were present in variant GCases involving residues 48-122, whereas approximately 2-fold increases were obtained with the L264I GCase. About 50% of variant GCases each had wild-type or increased sensitivity to in vitro cathepsin D digestion. Mapping of these properties onto the crystal structures of GCase indicated wide dispersion of functional properties that can affect catalytic function and stability. Site-directed mutagenesis of cysteine residues showed that the disulfide bonds, Cys(4)-Cys(16) and Cys(18)-Cys(23), and a free Cys(342) were essential for activity; the free Cys(126) and Cys(248) were not. Relative k(cat) was highly sensitive to a His substitution at Arg(496) but not at Arg(495). These studies and high phylogenetic conservation indicate localized and general structural effects of Gaucher disease mutations that were not obvious from the nature of the amino acid substitution, including those predicted to be nondisruptive (e.g. Val --> Leu). These results provide initial studies for the engineering of variant GCases and, potentially, molecular chaperones for therapeutic use.

Amino Acid Sequence↗

PubMed2005

Expression of a novel Ski-like gene in Xenopus development.

Members of the Ski/Sno family of gene products contain a characteristic peptide domain involved in protein-protein or protein-DNA interaction. Here, we characterize the developmental expression of xDawg, in Xenopus laevis, of a new gene, related to the Ski/Sno family of transcription regulators. The Ski/Sno domain of xDawg is predicted to present an electropositive surface, consistent with a role in DNA binding. This gene is expressed in the marginal zone of early gastrulae, and in the brain, sensory vesicles, and cranial neural crest of neurula and tailbud embryos.

Amino Acid Sequence↗

PubMed2005

Characterization of a plant, tyrosine-specific phosphatase of the aspartyl class.

The Arabidopsis thaliana homologue of the Eyes Absent genes (AtEYA) encodes a protein corresponding to the C-terminal conserved domain of the animal Eyes Absent proteins. We show here that AtEYA is a tyrosine-specific phosphatase that hydrolyzes its substrates in a metal-dependent reaction analogous to the phosphoserine phosphatases of the haloacid dehalogenase (HAD) family. The animal Eyes Absent proteins are a novel family of dual-function enzymes: they are transcription factors as well as phosphatases. They also represent a new mechanistic class of tyrosine phosphatases (PTPs) that do not have the Cys-containing signature motif. In contrast, AtEYA is only a tyrosine phosphatase and has no transactivation domain. Using the reaction mechanism of other HAD family enzymes as a model, we have conducted mutational analyses on AtEYA to query the roles of conserved residues. This analysis confirms the importance of the putative nucleophile, the general acid, and the metal-binding residues. Additionally, an inhibitory profile that is diagnostic of this new class of protein tyrosine phosphatases is described. The results of these studies on AtEYA reveal that while the animal and plant Eyes Absent proteins catalyze the same dephosphorylation reaction, the details of their specificity and active site environment, as well as their biological roles, are distinct.

Animals↗

PubMed2004

Predicting indirect readout effects in protein-DNA interactions.

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding.

Base Sequence↗

PubMed2004

Crystal structure of the dachshund homology domain of human SKI.

The nuclear protooncoprotein SKI negatively regulates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. It directly interacts with the Smads and, by various mechanisms, represses the transcription of TGF-beta-responsive genes. SKI is a multidomain protein that includes a domain bearing high sequence similarity with the retinal determination protein Dachshund (the Dachshund homology domain, DHD). The SKI-DHD has been implicated in SMAD-2/3, N-CoR, SKIP, and PML-RARalpha binding. The 1.65 A crystal structure of the Dachshund homology domain of human SKI is reported here. The SKI-DHD adopts a mixed alpha/beta structure which includes features found in the forkhead/winged-helix family of DNA binding proteins, although SKI-DHD is not a DNA binding domain. Residues that form a contiguous surface patch on SKI-DHD are conserved within the Ski/Sno family and with Dachshund, suggesting that this domain may mediate intermolecular interactions common to these proteins.

Amino Acid Sequence↗

PubMed2004

The P domain of norovirus capsid protein forms dimer and binds to histo-blood group antigen receptors.

Noroviruses (NVs) are the most important pathogen of epidemic nonbacterial gastroenteritis. The recent finding that NVs recognize human histo-blood group antigens (HBGAs) as receptors provided a new approach to study the pathogenesis of NVs. Using computational and site-directed mutagenesis approaches, our investigators previously identified a plausible binding pocket in the P domain of the NV capsids. In this study, we further characterize the role of the P domain in the interaction with human HBGA receptors using three NV strains representing three binding patterns. Our results show that the isolated P domain, although it did not form virus-like particles (VLPs), formed dimers, and the dimers bound HBGAs with the same patterns as those of the intact viral capsids. In contrast, the S domain, which formed small, thin-layer VLPs, did not bind A, B, or H HBGAs. A chimera containing the S domain of VA387 and the P domain of MOH revealed a binding pattern of the P donor strain (MOH). Deletion experiments revealed that an intact P domain is necessary for receptor binding. The P domain dimers are stable over a broad range of pH (2 to 11) or under strong denaturing conditions. Taken together, our results suggest that the P domain of NV contains essential elements for strain-specific binding to receptors. Further study of the P domain will provide useful information about the virus-receptor interaction. The high yield and easy production of the recombinant P protein in the Escherichia coli expression system will provide a simple approach to this goal.

ABO Blood-Group System↗

PubMed2003

Eyes absent represents a class of protein tyrosine phosphatases.

The Eyes absent proteins are members of a conserved regulatory network implicated in the development of the eye, muscle, kidney and ear. Mutations in the Eyes absent genes have been associated with several congenital disorders including the multi-organ disease bronchio-oto-renal syndrome, congenital cataracts and late-onset deafness. On the basis of previous analyses it has been shown that Eyes absent is a nuclear transcription factor, acting through interaction with homeodomain-containing Sine oculis (also known as Six) proteins. Here we show that Eyes absent is also a protein tyrosine phosphatase. It does not resemble the classical tyrosine phosphatases that use cysteine as a nucleophile and proceed by means of a thiol-phosphate intermediate. Rather, Eyes absent is the prototype for a class of protein tyrosine phosphatases that use a nucleophilic aspartic acid in a metal-dependent reaction. Furthermore, the phosphatase activity of Eyes absent contributes to its ability to induce eye formation in Drosophila.

Amino Acid Sequence↗

PubMed2002

Structure of the retinal determination protein Dachshund reveals a DNA binding motif.

The Dachshund proteins are essential components of a regulatory network controlling cell fate determination. They have been implicated in eye, limb, brain, and muscle development. These proteins cannot be assigned to any recognizable structural or functional class based on amino acid sequence analysis. The 1.65 A crystal structure of the most conserved domain of human DACHSHUND is reported here. The protein forms an alpha/beta structure containing a DNA binding motif similar to that found in the winged helix/forkhead subgroup of the helix-turn-helix family. This unexpected finding alters the previously proposed molecular models for the role of Dachshund in the eye determination pathway. Furthermore, it provides a rational framework for future mechanistic analyses of the Dachshund proteins in several developmental contexts.

Amino Acid Motifs↗

PubMed2001

The papillomavirus E2 proteins: structure, function, and biology.

Nearly twenty years after the first high-resolution crystal structures of specific protein-DNA complexes were determined, the stereo-chemical basis for protein-DNA recognition remains an active area of investigation. One outstanding question is, how are proteins able to detect noncontacted sequences in their binding sites? The papillomavirus E2 proteins represent a particularly suitable group of proteins in which to examine the mechanisms of "indirect readout." Coordinated structural and thermodynamic studies of the E2-DNA interaction conducted over the past five years are summarized in this review. The data support a model in which the electrostatic properties of the individual E2 proteins correlate with their affinities for intrinsically flexible or rigidly prebent DNA targets.

Models, Molecular↗