Cardio-Renal syndrome (CRS) is a common and complex clinical condition in which multiple causative factors are involved. The time window between renal insult and development of acute kidney injury ...(AKI) in acute heart failure (AHF) can be varied in different patients and AKI often is diagnosed too late, only when the effects of the insult become evident with a loss or decline of renal function. For this reason, pharmaceutical interventions for AKI that have been shown to be renoprotective or beneficial when tested in experimental conditions do not display similar results in the clinical setting. In most cases patients with AHF are admitted with clinical signs and symptoms of congestion and fluid overload. Loop diuretics, typically used to induce an enhanced diuresis in these congested patients, often are associated with a subsequent significant decrease in glomerular filtration rate and cause a creatinine increase that is apparent within 72 hours. Early detection of AKI is not possible with the use of serum creatinine and there is a need for a timely diagnostic tool able to address renal damage while it is happening. We need to define the diagnosis of both AHF and AKI in the early phases of CRS type 1 by coupling a kidney damage marker such as neutrophil gelatinase-associated lipocalin (NGAL) with B-type natriuretic peptide (BNP). Indeed, it would be ideal to make available a panel including whole blood or plasma cardiac and renal biomarkers building specific, pathophysiologically based, molecular profiles. Based on current knowledge and consensus, we can use kidney damage biomarkers such as plasma NGAL for an early diagnosis of AKI. However, differences in individual patient values and uncertainties about the ideal cut-off values may currently limit the application of these biomarkers. We propose that NGAL may increase its usefulness in the diagnosis and prevention of CRS if a curve of plasma values rather than a single plasma measurement is determined. To apply the concept of measuring an NGAL curve in AHF patients, however, assay performance in the lower-range values becomes a critical factor. For this reason, we propose the use of the new extended-range plasma NGAL assay that may contribute to remarkably improve the sensitivity of AKI diagnosis in AHF and lead to more effective intervention strategies.
Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation ...loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (ΔF508) in the first nucleotide-binding domain (NBD1), ...which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of ΔF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ΔF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of ΔF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the ΔF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.
Lipid A modification with 4-amino-4-deoxy-L-arabinose confers on certain pathogenic bacteria, such as
Salmonella, resistance to cationic antimicrobial peptides, including those derived from the ...innate immune system. ArnB catalysis of amino group transfer from glutamic acid to the 4″-position of a UDP-linked ketopyranose molecule to form UDP-4-amino-4-deoxy-L-arabinose represents a key step in the lipid A modification pathway. Structural and functional studies of the ArnB aminotransferase were undertaken by combining X-ray crystallography with biochemical analyses. High-resolution crystal structures were solved for two native forms and one covalently inhibited form of
S. typhimurium ArnB. These structures permitted identification of key residues involved in substrate binding and catalysis, including a rarely observed nonprolyl
cis peptide bond in the active site.
The α subunit of bacterial luciferase unfolds and refolds reversibly by a three-state mechanism in urea-containing buffer. It has been proposed that the three-state unfolding of the α subunit arises ...from a stepwise unfolding of a C-terminal folding domain at lower concentrations of urea, followed by unfolding of the N-terminal domain at higher concentrations of urea (Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136−16145). The location of an anion binding site in the proposed N-terminal folding domain allowed the folding mechanism to be probed in the context of the intact polypeptide. Anions preferentially stabilized the N-terminal domain in a concentration-dependent manner. The polyvalent anions sulfate and phosphate were found to be more stabilizing than monovalent chloride ion. Cations did not show a similar stabilizing effect, demonstrating that the stabilization was due to the anions alone. The purified N-terminal domain prepared by limited proteolysis and anion exchange chromatography was found to refold cooperatively with a midpoint approximately that of the second unfolding transition of the α subunit. Phosphate ion stabilized this fragment to roughly the same extent as it did the α subunit. The results presented are consistent with the proposed two-domain folding model and demonstrate that anion binding to the N-terminal folding domain stabilizes the α subunit of bacterial luciferase.
Bacterial luciferase is a heterodimeric (αβ) enzyme composed of homologous subunits. When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the α subunit accumulates to high levels. The ...α subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit Sinclair, J. F., Waddle, J. J., Waddill, W. F., and Baldwin, T. O. (1993) Biochemistry 32, 5036−5044. Analytical ultracentrifugation of the α subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 ± 4.0 μM at 18 °C in 50 mM phosphate and 100 mM NaCl, pH 7.0. The α subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism. The first transition occurred over the range of 0−2 M urea with an associated free-energy change of 2.24 ± 0.25 kcal/mol at 18 °C in 50 mM phosphate buffer, pH 7.0. The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 ± 0.75 kcal/mol. The intermediate species, populated maximally at ca. 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states. The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried. The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca. 40% of its native secondary structure. N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the α subunit became protease labile over the urea concentration range at which the intermediate was maximally populated. These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain. Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the α subunit with those of the αβ heterodimer suggested that the unfolding pathway of the α subunit is the same, whether it is in the form of the free subunit or in the heterodimer.
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain ...(NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion
of residue Phe-508 (ÎF508) in the first nucleotide-binding domain ...(NBD1), which results in a severe reduction in the population
of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed
this to aberrant folding of ÎF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which
fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the ÎF508 mutation. Crystal structures show minimal changes in
protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in
the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility
that the primary effect of ÎF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference
with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site
mutations that suppress the trafficking defect caused by the ÎF508 mutation, suggesting that these suppressors might function
indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures
also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both
adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.
Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain ...(NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.
Departments of 1 Physiology, 3 Exercise and Sport Science, and 5 Anatomy and Cell Biology, East Carolina University, Greenville, North Carolina; 2 Departments of Nutritional Sciences and Internal ...Medicine, and Harry S. Truman Veterans Affairs Hospital, University of Missouri, Columbia, Missouri; and 4 Department of Physical Medicine and Rehabilitation, University of Michigan, Ann Arbor, Michigan
Submitted 14 September 2006
; accepted in final form 21 February 2007
Elevated oxidative capacity, such as occurs via endurance exercise training, is believed to protect against the development of obesity and diabetes. Rats bred both for low (LCR)- and high (HCR)-capacity endurance running provide a genetic model with inherent differences in aerobic capacity that allows for the testing of this supposition without the confounding effects of a training stimulus. The purpose of this investigation was to determine the effects of a high-fat diet (HFD) on weight gain patterns, insulin sensitivity, and fatty acid oxidative capacity in LCR and HCR male rats in the untrained state. Results indicate chow-fed LCR rats were heavier, hypertriglyceridemic, less insulin sensitive, and had lower skeletal muscle oxidative capacity compared with HCR rats. Upon exposure to an HFD, LCR rats gained more weight and fat mass, and their insulin resistant condition was exacerbated, despite consuming similar amounts of metabolizable energy as chow-fed controls. These metabolic variables remained unaltered in HCR rats. The HFD increased skeletal muscle oxidative capacity similarly in both strains, whereas hepatic oxidative capacity was diminished only in LCR rats. These results suggest that LCR rats are predisposed to obesity and that expansion of skeletal muscle oxidative capacity does not prevent excess weight gain or the exacerbation of insulin resistance on an HFD. Elevated basal skeletal muscle oxidative capacity and the ability to preserve liver oxidative capacity may protect HCR rats from HFD-induced obesity and insulin resistance.
fatty acid; lipid metabolism; liver; heart; skeletal muscle
Address for reprint requests and other correspondence: R. M. Lust, Brody School of Medicine, 600 Moye Blvd., 6N-96 Brody Bldg., East Carolina Univ., Greenville, NC 27834 (e-mail: lustr{at}ecu.edu )
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