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1.  NMR insights into protein allostery 
Allosterism is one of nature's principal methods for regulating protein function. Allosterism utilizes ligand binding at one site to regulate the function of the protein by modulating the structure and dynamics of a distant binding site. In this review, we first survey solution NMR techniques and how they may be applied to the study of allostery. Subsequently, we describe several examples of application of NMR to protein allostery and highlight the unique insight provided by this experimental technique.
doi:10.1016/j.abb.2011.10.023
PMCID: PMC4086649  PMID: 22198279
Solution NMR; Dynamics; Allostery; Conformational Changes; Relaxation Dispersion; Silent Allostery
2.  USING SIMULATIONS TO PROVIDE THE FRAMEWORK FOR EXPERIMENTAL PROTEIN FOLDING STUDIES 
doi:10.1016/j.abb.2012.12.015
PMCID: PMC4084838  PMID: 23266569
Protein folding; Molecular dynamics; Folding/unfolding pathway; Protein dynamics
3.  Calcium sensitivity and myofilament lattice structure in titin N2B KO mice 
The cellular basis of the Frank-Starling “Law of the Heart” is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension-pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d10) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I11/I10) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I11/I10 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.
doi:10.1016/j.abb.2012.12.004
PMCID: PMC3636180  PMID: 23246787
4.  Structural and Functional Consequences of Cardiac Troponin C L57Q and I61Q Ca2+-Desensitizing Variants 
Archives of biochemistry and biophysics  2013;535(1):10.1016/j.abb.2013.02.006.
Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova and Davis (2004) J Biol Chem 279, 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca2+ binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca2+ binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca2+ affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca2+, but did decrease the interaction in the presence of Ca2+. In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca2+. This difference in the absence of Ca2+ suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca2+ binding induced by I61Q may result from destabilization of the Ca2+ binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca2+ binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca2+ binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.
doi:10.1016/j.abb.2013.02.006
PMCID: PMC3657564  PMID: 23454346
Troponin C; Troponin I; Calcium binding; Fluorescence spectroscopy; Molecular dynamic simulation
5.  Post-translational modifications of myofilament proteins involved in length-dependent prolongation of relaxation in rabbit right ventricular myocardium 
The phosphorylation state of several cardiac myofilament proteins changes with the level of stretch in intact, twitch-contracting cardiac muscles. It remains unclear which kinases are involved in the length-dependent phosphorylation of these proteins. We set out to investigate which kinases are involved after a step-wise change in cardiac muscle length. We hypothesize that myofilament protein phosphorylation by PKCβII and PKA alters contractile kinetics during length-dependent activation. Right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and stimulated to contract at 1 Hz. Twitch force recordings where taken at taut and optimal muscle lengths before and after administration of kinase inhibitors at 37 °C. PKCβII inhibition significantly decreased time from stimulation to peak force (TTP), time from peak force to 50% relaxation (RT50), and 90% relaxation (RT90) at optimal muscle length. This led to a loss in the length-dependent increase of RT50 and RT90 in the presence of the PKCβII inhibitor, whereas the length-dependent increase in RT50 and RT90 was seen in the controls. PKA inhibition using H-89 significantly decreased TTP at both taut and optimal muscle lengths. Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicates a role for PKCβII in the phosphorylation of tropomyosin and myosin light chain-2 (MLC2) and PKA for tropomyosin, troponin-I, MLC2, myosin binding protein-C, troponin-T (TnT) 3 and TnT4. Our data provide evidence for two signaling kinases acting upon myofilament proteins during length-dependent activation, and provide further insight for length-dependent myofilament function.
doi:10.1016/j.abb.2012.10.005
PMCID: PMC3640662  PMID: 23085150
Frank-Starling mechanism; Contraction; Kinetics; Protein kinase C; Protein kinase A; Phosphorylation; Rabbit
6.  Myofilament Incorporation and Contractile Function after Gene Transfer of Cardiac Troponin I Ser43/45Ala 
Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70-80% of endogenous cTnI replaced within 4 days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2 days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4 days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca2+ sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca2+ transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.
doi:10.1016/j.abb.2012.12.021
PMCID: PMC3640666  PMID: 23318976
Troponin; Myofilament; Contractile Proteins; Heart; Phosphorylation; Protein Kinase C
7.  Intercalated Disc Protein, mXinα, Suppresses p120-Catenin-Induced Branching Phenotype via Its Interactions with p120-Catenin and Cortactin 
The Xin repeat-containing proteins, Xinα (Xirp1) and Xinβ (Xirp2), localize to the intercalated discs (ICDs) of mammalian hearts. Mouse Xinα (mXinα) directly interacts with β-catenin and actin filaments, potentially coupling the N-cadherin/β-catenin complexes to the underlying actin cytoskeleton and modulating ICD integrity and function. Supporting this possibility, mXinα-null hearts develop ICD structural defects and cardiomyopathy with conduction defects. However, the underlying mechanisms leading to these defects remain unclear. Here, we showed that mXinα also interacted with p120-catenin and cortactin. Different from the β-catenin binding domain, there existed multiple p120-catenin binding sites on mXinα, while only the extreme N-terminus of mXinα containing a SH3-binding motif could interact with cortactin. In mouse heart, a significant fraction of cortactin was co-localized with N-cadherin to ICDs, whereas in mXinα-null heart, this fraction of cortactin was drastically reduced. Therefore, mXinα may modulate ICD integrity and function through its interactions with catenins and cortactin. Analyses of the in vivo consequence of p120-catenin and mXinα interaction revealed that force-expressed mXinα or its fragments significantly suppressed the p120-catenin-induced branching phenotypes. It is known that p120-catenin directly regulates Rho GTPases, leading to the branching phenotype. Thus, mXinα may sequester the p120-catenin from inhibiting RhoA activity and/or from activating Rac1 activity.
doi:10.1016/j.abb.2012.12.018
PMCID: PMC3640673  PMID: 23296090
8.  Length-dependent effects on cardiac contractile dynamics are different in cardiac muscle containing α- or β-myosin heavy chain 
doi:10.1016/j.abb.2012.10.011
PMCID: PMC3640676  PMID: 23111184
papillary muscles; contractile proteins; cardiac myosins; Contractility; Cardiac muscle; Cardiac proteins; Myofilaments; Myosin
9.  Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation 
Tropomyosin (Tm) is a central protein in the Ca2+ regulation of striated muscle. The Tm isoform undergoes phosphorylation at serine residue 283. While the biochemical and steady-state muscle function of muscle purified Tm phosphorylation have been explored, the effects of Tm phosphorylation on the dynamic properties of muscle contraction and relaxation are unknown. To investigate the kinetic regulatory role of Tm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants from insect cells. Purified Tm’s regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm’s, however disassociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to WT Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. These data demonstrate that Tm pseudo-phosphorylation slows deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle activation. This supports a role for Tm as a key protein in the regulation of muscle relaxation dynamics.
doi:10.1016/j.abb.2012.11.010
PMCID: PMC3640754  PMID: 23232082
Tropomyosin; phosphorylation; myofibril; relaxation; human; calcium
10.  Asymmetric Myosin Binding to the Thin Filament as Revealed by a Fluorescent Nanocircuit 
The interplay between myosin, actin, and striated muscle regulatory proteins involves complex cooperative interactions that propagate along the thin filament. A repeating unit of the tropomyosin dimer, troponin heterotrimer, and the actin protofilament heptamer is sometimes assumed to be able to bind myosin at any of its seven actins when activated even though the regulatory proteins are asymmetrically positioned along this repeating unit. Analysis of the impact of this asymmetry on actin and myosin interactions by sensitized emission luminescence resonance energy transfer spectroscopy and a unique fluorescent nanocircuit design reveals that the troponin affects the structure and function of myosin heads bound nearby in a different manner than myosin heads bound further away from the troponin. To test this hypothesis, a fluorescent nanocircuit reported the position of the myosin lever arm only when the myosin was bound adjacent to the troponin, or in controls, only when the myosin was bound distant from the troponin. Confirming the hypothesis, the myosin lever arm is predominantly in the prepowerstroke orientation when bound near troponin, but is predominantly in the postpowerstroke orientation when bound distant from troponin. These data are consistent with the hypothesis that troponin is responsible for the formation of myosin binding target zones along the thin filament.
doi:10.1016/j.abb.2012.12.014
PMCID: PMC3627744  PMID: 23274408
troponin bridge; target zone; muscle regulation; muscle contraction; lanthanide chelates; time-resolved fluorescence
11.  Structural and Kinetic Effects of Hypertrophic Cardiomyopathy Related Mutations R146G/Q and R163W on the Regulatory Switching Activity of Rat Cardiac Troponin I 
Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca2+ and myosin S1 dependent conformational transitions in rat cTn structure. Ca2+-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca2+-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR–actin interaction were consistent with increased passive tension.
doi:10.1016/j.abb.2012.12.007
PMCID: PMC3616153  PMID: 23246786
Cardiac troponin; hypertrophic cardiomyopathy; FRET; fly casting model; stopped-flow; thin filament regulation
12.  Dose Dependent Effects of Reactive Oxygen and Nitrogen Species on the Function of Neuronal Nitric Oxide Synthase 
Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dosedependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (≤ 50 μM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (≤ 50 μM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.
doi:10.1016/j.abb.2008.01.003
PMCID: PMC4073612  PMID: 18201545
neuronal nitric oxide synthase; nitric oxide; superoxide; peroxynitrite; hydroxyl; hydrogen peroxide; dose-dependent; uncoupling; tetrahydrobiopterin; monomerization
13.  Characterization of superoxide production from aldehyde oxidase: an important source of oxidants in biological tissues 
Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at ∼290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol/min/mg. Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 nmol/min/mg and 355 nmol/min/mg. In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased.
doi:10.1016/j.abb.2006.12.032
PMCID: PMC4073616  PMID: 17353002
Aldehyde oxidase; Xanthine oxidase; Superoxide; Hydrogen peroxide; Electron paramagnetic resonance; Spin trapping; Cytochrome c reduction; Reactive oxygen species; Free radicals; Oxygen consumption
14.  Reactive Oxygen and Nitrogen Species Regulate Inducible Nitric Oxide Synthase Function Shifting the Balance of Nitric Oxide and Superoxide Production 
Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen / nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH4)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH4 addition. Peroxynitrite (ONOO−) produced the greatest change in NO generation rate, ~95% decrease, and BH4 only partially restored this loss of activity. Superoxide (O2.−) greatly decreased NO generation, however, BH4 addition restored this activity. Hydroxyl radical (.OH) mildly decreases NO generation in a BH4-dependent manner. iNOS was resistant to H2O2 with only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced O2.− production from iNOS, while ONOO− had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO− induces irreversible enzyme inactivation and decreases both NO and O2.− production.
doi:10.1016/j.abb.2009.11.019
PMCID: PMC4073618  PMID: 19932078
inducible nitric oxide synthase; nitric oxide; superoxide; peroxynitrite; hydroxyl; hydrogen peroxide; dose-dependent; uncoupling; tetrahydrobiopterin; monomerization
15.  Regulation of the human cathepsin E gene by the constitutive androstane receptor 
Cathepsin E (CTSE) is an aspartic protease that has been linked to antigen processing and innate immunity. Elevated levels of CTSE expression have also been associated with several forms of cancer, including carcinomas exhibiting highly invasive character. In this study, we performed DNA microarray experiments, together with quantitative reverse transcriptase PCR analyses and enzymatic activity determinations to identify human CTSE as a novel target gene for regulation by the constitutive androstane receptor (CAR), a nuclear receptor activated by the liver tumor promoting agent, phenobarbital. In particular, two motifs within the 5′-flanking region of the human CTSE gene were identified as direct sites of interaction with CAR/RXRα heterodimers, a direct repeat-3 site at position −766 and a direct repeat-4 site at position −1407. Thus, these studies demonstrate CAR-mediated regulation of CTSE within primary hepatocyte cultures from several individual donors and suggest that elevated CTSE activity may play a functional role in the etiology of hepatocarcinogenesis.
doi:10.1016/j.abb.2007.08.001
PMCID: PMC4064465  PMID: 17888866
Cathepsin E; Constitutive androstane receptor; Hepatocytes; Primary hepatocytes; Liver; Human; Hepatocytes
16.  Evaluation of UGT protein interactions in human hepatocytes: Effect of siRNA down regulation of UGT1A9 and UGT2B7 on propofol glucuronidation in human hepatocytes☆ 
Previous experiments performed in recombinant systems have suggested that protein–protein interactions occur between the UGTs and may play a significant role in modulating enzyme activity. However, evidence of UGT protein–protein interactions either in vivo or in more physiologically relevant in vitro systems has yet to be demonstrated. In this study, we examined oligomerization and its ability to affect glucuronidation in plated human hepatocytes. siRNA down regulation experiments and activity studies were used to examine changes in metabolite formation of one UGT isoform due to down regulation of a second UGT isoform. Selective siRNA directed towards UGT1A9 or UGT2B7 resulted in significant and selective decreases in their respective mRNA levels. As expected, the metabolism of the UGT1A9 substrate propofol decreased with UGT1A9 down regulation. Interestingly, UGT1A9 activity, but not UGT1A9 mRNA expression, was also diminished when UGT2B7 expression was selectively inhibited, implying potential interactions between the two isoforms. Minor changes to UGT1A4, UGT2B4 and UGT2B7 activity were also observed when UGT1A9 expression was selectively down regulated. To our knowledge, this represents the first piece of evidence that UGT protein–protein interactions occur in human hepatocytes and suggests that expression levels of UGT2B7 may directly impact the glucuronidation activity of selective UGT1A9 substrates.
doi:10.1016/j.abb.2013.03.012
PMCID: PMC4059507  PMID: 23562620
UDP-Glucuronosyltransferase; siRNA; Protein interactions; Hepatocytes
17.  Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding 
Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca2+/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.
doi:10.1016/j.abb.2013.03.004
PMCID: PMC3779284  PMID: 23507581
Nitric oxide synthase; Calmodulin; Reductase; Flavoprotein; Electron transfer
18.  (-)-Epicatechin prevents TNFα-induced activation of signaling cascades involved in inflammation and insulin sensitivity in 3T3-L1 adipocytes 
Obesity is major public health concern worldwide and obese individuals exhibit a higher risk of chronic diseases such as type 2 diabetes. Inflammation plays a significant role in metabolic regulation and mounting evidence highlight the contribution of adipose tissue to systemic inflammatory state. Food extracts with a high content of (-)-epicatechin have been found to exert systemic anti-inflammatory actions, however the anti-inflammatory actions of (-)-epicatechin on adipose tissue remain to be determined. The aim of this study was to investigate the capacity of (-)-epicatechin to prevent tumor necrosis alpha (TNFα)-induced activation of cell signals involved in inflammation and insulin resistance (NF-κB, mitogen-activated protein kinases (MAPKs), AP-1, and peroxisome proliferator activated receptor γ (PPARγ)) in differentiated white adipocytes (3T3-L1). TNFα triggered the activation of transcription factors NF-κB and AP-1, and MAPKs ERK1/2, JNK, and p38. (-)-Epicatechin caused a dose (0.5-10 μM)-dependent decrease in TNFα-mediated JNK, ERK1/2, and p-38 phosphorylation, and nuclear AP-1-DNA binding. (-)-Epicatechin also inhibited TNFα-triggered activation of the NF-κB signaling cascade, preventing TNFα-mediated p65 nuclear transport and nuclear NF-κB-DNA binding. (-)-Epicatechin also attenuated the TNFα-mediated downregulation of PPARγ expression and decreased nuclear DNA binding. Accordingly, (-)-epicatechin inhibited TNFα-mediated altered transcription of genes (MCP-1, interleukin-6, TNFα, resistin, and protein-tyrosine phosphatase 1B) involved in inflammation and insulin signaling. In conclusion, (-)-epicatechin can attenuate TNFα-mediated triggering of signaling cascades involved in inflammation and insulin resistance. These findings could be of relevance in the dietary management of obesity and metabolic syndrome.
doi:10.1016/j.abb.2012.02.019
PMCID: PMC3992864  PMID: 22425757
Adipocytes; NF-κB; MAPK; PPAR; epicatechin; inflammation; insulin resistance
19.  Kinetic Mechanism of DNA Translocation by the RSC Molecular Motor 
ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.
doi:10.1016/j.abb.2013.01.011
PMCID: PMC3609899  PMID: 23399434
chromatin remodeling enzyme; RSC; DNA translocation; energy transduction; kinetic modeling
20.  Oncogenic Ras suppresses Cdk1 in a complex manner during the incubation of activated Xenopus egg extracts 
Archives of biochemistry and biophysics  2013;532(2):10.1016/j.abb.2013.01.006.
The activity of Cdk1 is the driving force for entry into M-phase during the cell cycle. Activation of Cdk1 requires synthesis and accumulation of cyclin B, binding of cyclin B to Cdk1, and removal of the inhibitory tyr-15-Cdk1 phosphorylation. It was previously shown that oncogenic Ras suppresses Cdk1 activation during the incubation of activated Xenopus egg extracts. However, how oncogenic Ras suppresses Cdk1 remained unclear. Using the histone H1 kinase assay to follow Cdk1 activity and Western blot analysis to assess levels of both cyclin B2 and phosphorylated-tyr-15-Cdk1, how oncogenic Ras suppresses Cdk1 is studied. The results indicate that oncogenic Ras suppresses Cdk1 via induction of persistent phosphorylation of tyr-15-Cdk1. Interestingly, the results reveal that, compared with cyclin B2 in control activated egg extracts, which increased, peaked and then declined during the incubation, oncogenic Ras induced continuous accumulation of cyclin B2. The results also indicate that oncogenic Ras induces continuous accumulation of cyclin B2 primarily through stabilization of cyclin B2, which is mediated by constitutive activation of the Raf-Mek-Erk-p90rsk pathway. Taken together, these results indicate that oncogenic Ras suppresses Cdk1 in a complex manner: It induces continuous accumulation of cyclin B2, but also causes persistent inhibitory phosphorylation of tyr-15-Cdk1.
doi:10.1016/j.abb.2013.01.006
PMCID: PMC3809123  PMID: 23376039
Signal transduction; oncogenic Ras; Cdk1; Cell cycle arrest; Xenopus egg extracts
21.  Transport of anti-IL-6 antigen binding fragments into cartilage and the effects of injury 
The efficacy of biological therapeutics against cartilage degradation in osteoarthritis is restricted by the limited transport of macromolecules through the dense, avascular extracellular matrix. The availability of biologics to cell surface and matrix targets is limited by steric hindrance of the matrix, and the microstructure of matrix itself can be dramatically altered by joint injury and the subsequent inflammatory response. We studied the transport into cartilage of a 48 kDa anti-IL-6 antigen binding fragment (Fab) using an in vitro model of joint injury to quantify the transport of Fab fragments into normal and mechanically injured cartilage. The anti-IL-6 Fab was able to diffuse throughout the depth of the tissue, suggesting that Fab fragments can have the desired property of achieving local delivery to targets within cartilage, unlike full-sized antibodies which are too large to penetrate beyond the cartilage surface. Uptake of the anti-IL-6 Fab was significantly increased following mechanical injury, and an additional increase in uptake was observed in response to combined treatment with TNFα and mechanical injury, a model used to mimic the inflammatory response following joint injury. These results suggest that joint trauma leading to cartilage degradation can further alter the transport of such therapeutics and similar-sized macromolecules.
doi:10.1016/j.abb.2012.12.020
PMCID: PMC3596461  PMID: 23333631
cartilage; TNFα; Fab; transport; injury; osteoarthritis
22.  Knockdown of Pyruvate Carboxylase or Fatty Acid Synthase Lowers Numerous Lipids and Glucose-Stimulated Insulin Release in Insulinoma Cells 
We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20–30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated insulin secretion.
doi:10.1016/j.abb.2013.01.002
PMCID: PMC3596477  PMID: 23357280
Insulinoma cells; shRNA; pyruvate carboxylase; fatty acid synthase; phospholipids; cholesterol esters; lipid remodeling
23.  Stimulation of Mouse Cyp1b1 during Adipogenesis: Characterization of Promoter Activation by the Transcription Factor Pax6 
Cytochrome P4501B1 (Cyp1b1) is expressed specifically in certain neural crest (NC) cells during embryogenesis. Mesenchymal progenitor cells that develop from NC cells are modeled here by mouse C3H10T1/2 and 3T3-L1 cells. Dexamethasone in combination with methylisobutylxanthine (DM) induces Cyp1b1 and a 6.7 kb mouse Cyp1b1 promoter-luciferase reporter in each cell type prior to adipogenesis. An 18 base sequence (at −6.11 kb) (PaxE) which was essential for this reporter stimulation in 3T3-L1 cells bound the transcription factor Pax6. This is shown by gel mobility shifts and sequence mutations. Heterologous vector expression of Pax6 in 3T3-L1 cells enhanced DM stimulated Cyp1b1 promoter activity through cooperation with two Sp1 sites in the proximal promoter region. Chromatin immunoprecipitation showed that DM stimulated binding of Pax6 adjacent to Sp1 in the proximal promoter more than in the PaxE region. The Cyp1b1 induction by DM in C3H10T1/2 cells was more rapid but independent of Pax6. The far upstream enhancer region (FUER) found in rat Cyp1b1 responded to DM but was inactive in the mouse promoter due to key sequence changes. The expression patterns of Pax6 and Cyp1b1 frequently overlap during mouse embryogenesis. The relationship between Pax6 and Cyp1b1 expression warrants further investigation, particularly in the NC.
doi:10.1016/j.abb.2013.01.007
PMCID: PMC3596501  PMID: 23376040
Cyp1b1; Adipogenesis; Pax6; Sp1; PPARγ; Embryo fibroblast
24.  Structural Studies of N-Terminal Mutants of Connexin 32 Using 1H NMR Spectroscopy 
The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with 1H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D 1H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.
doi:10.1016/j.abb.2012.05.027
PMCID: PMC3938314  PMID: 22705201
NMR; atomic resolution structure; ion channels; voltage dependent gating; connexins; structure-function; Protein Structure and Function
25.  X-ray Crystal Structure of the Cytochrome P450 2B4 Active Site Mutant F297A in Complex with Clopidogrel: Insights into Compensatory Rearrangements of the Binding Pocket† 
Prior X-ray crystal structures of cytochrome P450 2B4 revealed the pivotal role of rearrangement of the side chains of residues F206 and F297 in the active site in accommodating various inhibitors or substrates. To explore the role of these residues, 2B4 F206A and F297A were created by site-directed mutagenesis and characterized functionally. The structure of F297A with clopidogrel demonstrated the reorientation of the ligand such that the methyl ester group is oriented toward the heme, whereas the thiophene moiety now extends to the additional void in the F297A mutant. Most interestingly, movement of the I helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A-virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the real F297A mutant or a virtual mutant with the I-helix re-positioned oriented clopidogrel preferentially with either the methyl ester or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate and interact with the substrate in a distinct orientation while maintaining the overall closed protein conformation.
doi:10.1016/j.abb.2012.12.016
PMCID: PMC3580012  PMID: 23296089
Cytochrome P450; P450 2B4 F297A; Clopidogrel; X-ray Crystal Structure; Ligand Docking

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