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Kaushik Dutta

Publications and source records attributed to Kaushik Dutta.

15 recordsLinked to original sources
PubMed2006

NMR structure of stem-loop D from human rhinovirus-14.

The 5'-cloverleaf of the picornavirus RNA genome is essential for the assembly of a ribonucleoprotein replication complex. Stem-loop D (SLD) of the cloverleaf is the recognition site for the multifunctional viral protein 3Cpro. This protein is the principal viral protease, and its interaction with SLD also helps to position the viral RNA-dependent RNA polymerase (3Dpol) for replication. Human rhinovirus-14 (HRV-14) is distinct from the majority of picornaviruses in that its SLD forms a cUAUg triloop instead of the more common uYACGg tetraloop. This difference appears to be functionally significant, as 3Cpro from tetraloop-containing viruses cannot bind the HRV-14 SLD. We have determined the solution structure of the HRV-14 SLD using NMR spectroscopy. The structure is predominantly an A-form helix, but with a central pyrimidine-pyrimidine base-paired region and a significantly widened major groove. The stabilizing hydrogen bonding present in the uYACGg tetraloop was not found in the cUAUg triloop. However, the triloop uses different structural elements to present a largely similar surface: sequence and underlying architecture are not conserved, but key aspects of the surface structure are. Important structural differences do exist, though, and may account for the observed cross-isotype binding specificities between 3Cpro and SLD.

Carbohydrates↗
PubMed2006

Impact of Type 2 diabetes and aging on cardiomyocyte function and O-linked N-acetylglucosamine levels in the heart.

Increased levels of O-linked attachment of N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic proteins are implicated in the development of diabetic cardiomyopathy and are regulated by O-GlcNAc transferase (OGT) expression and its substrate UDP-GlcNAc. Therefore, the goal of this study was to determine whether the development of diabetes in the Zucker diabetic fatty (ZDF) rat, a model of Type 2 diabetes, results in defects in cardiomyocyte mechanical function and, if so, whether this is associated with increased levels of O-GlcNAc and increased OGT expression. Six-week-old ZDF rats were hyperinsulinemic but normoglycemic, and there were no differences in cardiomyocyte mechanical function, UDP-GlcNAc, O-GlcNAc, or OGT compared with age-matched lean control rats. Cardiomyocytes isolated from 22-wk-old hyperglycemic ZDF rats exhibited significantly impaired relaxation, compared with both age-matched lean control and 6-wk-old ZDF groups. There was also a significant increase in O-GlcNAc levels in high-molecular-mass proteins in the 22-wk-old ZDF group compared with age-matched lean control and 6-wk-old ZDF groups; this was associated with increased UDP-GlcNAc levels but not increased OGT expression. Surprisingly, there was a significant decrease in overall O-GlcNAc levels between 6 and 22 wk of age in lean, ZDF, and Sprague-Dawley rats that was associated with decreased OGT expression. These results support the notion that an increase in O-GlcNAc on specific proteins may contribute to impaired cardiomyocyte function in diabetes. However, this study also indicates that in the heart the level of O-GlcNAc on proteins appears to be differentially regulated by age and diabetes.

Acetylglucosamine↗
PubMed2006

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis.

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.

Binding Sites↗
PubMed2006

Solution structure and folding characteristics of the C-terminal SH3 domain of c-Crk-II.

Crk-II is a signaling adaptor protein that is involved in many cellular processes including apoptosis, proliferation, and differentiation. It has a modular domain architecture consisting of an Src homology 2 domain (SH2) followed by two Src homology 3 (SH3) domains. The structures and ligand-binding properties of the SH2 and the middle SH3 domains are well-characterized. Several studies suggest that the C-terminal SH3 domain plays an important regulatory role in the protein; however, no structural information is available on this domain, and relatively little is known about its binding partners. In the current work, we have solved the solution NMR structure of the C-terminal SH3 domain. The domain adopts the standard SH3 fold comprising a five-stranded beta barrel. In agreement with alignment and modeling studies, the structure indicates that the canonical-binding surface of the SH3 domain is unusually polar and suggests that this domain may not bind typical PXXP ligands or that it may bind them with reduced affinity. Thermodynamic and kinetic studies show that the domain folds in a reversible two-state manner and that the stability of the fold is similar to that observed for other SH3 domains. These studies offer some insight into the likely structural and thermodynamic consequences of point mutations in the cSH3 domain that are known to deregulate Crk-II function. Our results set the stage for a better understanding the role of the cSH3 domain in the context of the full-length protein.

Amino Acid Sequence↗
PubMed2006

Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR).

We describe a high-throughput in-cell nuclear magnetic resonance (NMR)-based method for mapping the structural changes that accompany protein-protein interactions (STINT-NMR). The method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring the protein interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution.

Adaptor Proteins, Signal Transducing↗
PubMed2006

NMR solution structure and backbone dynamics of domain III of the E protein of tick-borne Langat flavivirus suggests a potential site for molecular recognition.

Flaviviruses cause many human diseases, including dengue fever, yellow fever, West Nile viral encephalitis, and hemorrhagic fevers, and are transmitted to their vertebrate hosts by infected mosquitoes and ticks. Domain III of the envelope protein (E-D3) is considered to be the primary viral determinant involved in the virus-host-cell receptor interaction, and thus represents an excellent target for antiviral drug development. Langat (LGT) virus is a naturally attenuated BSL-2 TBE virus and is a model for the pathogenic BSL-3 and BSL-4 viruses in the serogroup. We have determined the solution structure of LGT-E-D3 using heteronuclear NMR spectroscopy. The backbone dynamics of LGT-E-D3 have been investigated using 15N relaxation measurements. A detailed analysis of the solution structure and dynamics of LGT-E-D3 suggests potential residues that could form a surface for molecular recognition, and thereby represent a target site for antiviral therapeutics design.

Amino Acid Sequence↗
PubMed2005

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats.

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).

Animals↗
PubMed2004

Dynamic influences on a high-affinity, high-specificity interaction involving the C-terminal SH3 domain of p67phox.

An analysis of the backbone dynamics of the C-terminal Src homology 3 (SH3) domain of p67(phox), p67(phox)SH3(C), in complex with a 32-residue high-affinity (K(d) = 24 nM) peptide, Pf, from the C-terminal region of p47(phox) is presented. This paper represents the first detailed analysis of the backbone dynamics and the ligand-induced changes therein of a high-affinity, high-specificity interaction involving an SH3 domain. The dynamic features are compared with those in the high-affinity, highly specific interaction between the SH3 domain of C-terminal Src kinase (Csk-SH3) and a proline-rich peptide from proline-enriched phosphatase (PEP). Both systems share common dynamic features especially in the canonical PxxP motif recognition surface where slow micro- to millisecond time scale dynamics persist on complex formation especially in several residues that are implicated in ligand recognition and in stabilizing the SH3 fold. These residues are highly conserved in SH3 domains. Ile505, which lies outside the PxxP recognition motif on p67(phox)SH3(C) and is key in conferring high specificity to the p67(phox)SH3(C)/Pf interaction, becomes more disordered upon complex formation. This behavior is similar to that seen in the residues that constitute the specificity surface in Csk-SH3.

Amino Acids↗
PubMed2003

Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations.

The apoptosis-associated Par-4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimer's and Huntington's diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par-4 C-terminus, which is responsible for Par-4 self-association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface. Analysis of circular dichroism spectra reveals that a dramatic alteration of the folding/unfolding equilibrium of this peptide can be effected through directed-point mutagenesis, confirming that the two acidic residues are indeed key to the pH-dependent folding behavior of the Par-4 coiled coil, and further suggesting that alleviation of charge repulsion through exposure to either a low pH microenvironment or an electrostatically complementary environment may be necessary for efficient folding of the Par-4 C-terminus.

Amino Acid Sequence↗
PubMed2002

The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded.

The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N-terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1-D and 2-D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these findings, we propose a model that helps to explain previously enigmatic observations.

Amino Acid Sequence↗
PubMed2002

Calcium coordination studies of the metastatic Mts1 protein.

Mts1, also known as S100A4, is an 11 kDa calcium-binding protein strongly linked to metastasis. As a member of the S100 protein family, Mts1 is predicted to contain four alpha-helices and two calcium-binding loops, the second of which forms a canonical EF hand, while the first is a pseudo-EF hand, using two extra residues and principally backbone carbonyls rather than side chain oxygens to coordinate calcium. Here we follow chemical shift changes which occur in Mts1 upon titration of calcium. The results are consistent with calcium coordination by the EF hands described above. Filling of the first (pseudo) EF hand occurs at a lower calcium concentration than does filling of the second (canonical) EF hand. Concurrent with filling of site I, resonances from much of helix 4 vanish while the chemical shifts of a possibly nascent helical segment immediately C-terminal to helix 4 increase in helical character. Other smaller changes are seen, including a change in the linker joining helix 2 and helix 3. Since binding of effector molecules to S100 proteins has been shown to involve the C-terminus and linker regions, these calcium-induced changes have implications for the role of Mts1 in metastasis.

Amino Acid Sequence↗
PubMed2002

Depressed PKA activity contributes to impaired SERCA function and is linked to the pathogenesis of glucose-induced cardiomyopathy.

We have previously described a cardiomyopathy induced by culturing ventricular myocytes from normal adult rats in a medium containing high concentrations of glucose, which recapitulates cellular changes associated with early onset diabetic cardiomyopathy. This investigation was designed to evaluate cellular mechanisms that could contribute to slowed cytosolic Ca(2+) removal and myocyte relaxation in glucose-induced cardiomyopathy. Isolated ventricular myocytes were cultured overnight in medium containing normal glucose (n=5.5mM) or high glucose (HG=25.5mM). Cytosolic Ca(2+) removal was monitored with fluo-3 and myocyte mechanics with video-edge detection. Electrically stimulated Ca(2+) transients were prolonged in HG cells (A(T/PK)=215+/-7ms, n=41) compared to N myocytes (A(T/PK)=173+/-5ms, n=34). By pharmacological and ionic manipulations, Ca(2+) removal attributable to SERCA was slower in the HG group (A(D/PK)=290+/-17ms,n =41) compared to N (A(D/PK)=219+/-10, n=34), whereas NCX function was similar in both groups of cells. Total PKA activity was depressed in HG myocytes by 56% compared to N cells. beta-adrenergic receptor stimulation with ISO (10(-7)M) normalized myocyte relaxation, Ca(2+) transients and PKA activity in HG myocytes. Furthermore, inhibition of PKA with H89 (10(-5)M) depressed peak fractional shortening (PS) and slowed relengthening (A(R/PK)) to a greater extent in N (-50% for PS and 92% for A(R/PK)) than in HG cells (-25% for PS and 48% A(R/PK)). Depressed cytosolic Ca(2+) removal was not, however, associated with changes in basal levels of phosphorylated PLB, nor levels of SERCA, NCX or PLB proteins. We conclude that cellular mechanisms associated with the early onset glucose-induced cardiomyocyte dysfunction involves alterations in Ca(2+) regulation, which may be a common manifestation of other forms of cardiomyopathies.

Animals↗