Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.

Pathogen-specific TLR2 Protein Activation Programs Macrophages to Induce Wnt-beta-Catenin Signaling

Innate immunity recognizes and resists various pathogens; however, the mechanisms regulating pathogen versus non-pathogen discrimination are still imprecisely understood. Here, we demonstrate that pathogen-specific activation of TLR2 upon infection with Mycobacterium bovis BCG, in comparison with other pathogenic microbes, including Salmonella typhimurium and Staphylococcus aureus, programs macrophages for robust up-regulation of signaling cohorts of Wnt-beta-catenin signaling. Signaling perturbations or genetic approaches suggest that infection-mediated stimulation of Wnt-beta-catenin is vital for activation of Notch1 signaling. Interestingly, inducible NOS (iNOS) activity is pivotal for TLR2-mediated activation of Wnt-beta-catenin signaling as iNOS(-/-) mice demonstrated compromised ability to trigger activation of Wnt-beta-catenin signaling as well as Notch1-mediated cellular responses. Intriguingly, TLR2-driven integration of iNOS/NO, Wnt-beta-catenin, and Notch1 signaling contributes to its capacity to regulate the battery of genes associated with T(Reg) cell lineage commitment. These findings reveal a role for differential stimulation of TLR2 in deciding the strength of Wnt-beta-catenin signaling, which together with signals from Notch1 contributes toward the modulation of a defined set of effector functions in macrophages and thus establishes a conceptual framework for the development of novel therapeutics.

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions.

Biologically triggered exploding protein based microcapsules for drug delivery

Biologically triggered exploding microcapsules were synthesized by layer-by-layer assembly of biopolymers. The microcapsules showed controlled rupturing behaviour upon exposure to a pathologically relevant biomolecule, trypsin. These microcapsules offer significant potential for clinical applications.

Distinct Roles of FANCO/RAD51C Protein in DNA Damage Signaling and Repair Implications for Fanconi Anemia and Breast Cancer Susceptibility

RAD51C, a RAD51 paralog, has been implicated in homologous recombination (HR), and germ line mutations in RAD51C are known to cause Fanconi anemia (FA)-like disorder and breast and ovarian cancers. The role of RAD51C in the FA pathway of DNA interstrand cross-link (ICL) repair and as a tumor suppressor is obscure. Here, we report that RAD51C deficiency leads to ICL sensitivity, chromatid-type errors, and G(2)/M accumulation, which are hallmarks of the FA phenotype. We find that RAD51C is dispensable for ICL unhooking and FANCD2 monoubiquitination but is essential for HR, confirming the downstream role of RAD51C in ICL repair. Furthermore, we demonstrate that RAD51C plays a vital role in the HR-mediated repair of DNA lesions associated with replication. Finally, we show that RAD51C participates in ICL and double strand break-induced DNA damage signaling and controls intra-S-phase checkpoint through CHK2 activation. Our analyses with pathological mutants of RAD51C that were identified in FA and breast and ovarian cancers reveal that RAD51C regulates HR and DNA damage signaling distinctly. Together, these results unravel the critical role of RAD51C in the FA pathway of ICL repair and as a tumor suppressor.

Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT-IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH3 (methyl) stretching bands at 2872 and 2960 cm1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm1, which originates from stretching vibrations of CH2 (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH2 stretching are concentration-dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH2 groups, we hypothesized that CH2 vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre-treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH2 vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl-lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH2 stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Free Energy Barriers for Escape of Water Molecules from Protein Hydration Layer

Free energy barriers separating interfacial water molecules from the hydration layer at the surface of a protein to the bulk are obtained by using the umbrella sampling method of free energy calculation. We consider hydration layer of chicken villin head piece (HP-36) which has been studied extensively by molecular dynamics simulations. The free energy calculations reveal a strong sensitivity to the secondary structure. In particular, we find a region near the junction of first and second helix that contains a cluster of water molecules which are slow in motion, characterized by long residence times (of the order of 100 ps or more) and separated by a large free energy barrier from the bulk water. However, these ``slow'' water molecules constitute only about 5-10% of the total number of hydration layer water molecules. Nevertheless, they play an important role in stabilizing the protein conformation. Water molecules near the third helix (which is the important helix for biological function) are enthalpically least stable and exhibit the fastest dynamics. Interestingly, barrier height distributions of interfacial water are quite broad for water surrounding all the three helices (and the three coils), with the smallest barriers found for those near the helix-3. For the quasi-bound water molecules near the first and second helices, we use well-known Kramers' theory to estimate the residence time from the free energy surface, by estimating the friction along the reaction coordinate from the diffusion coefficient by using Einstein relation. The agreement found is satisfactory. We discuss the possible biological function of these slow, quasi-bound (but transient) water molecules on the surface.

Synthesis of beta-arabinofuranoside glycolipids, studies of their binding to surfactant protein-A and effect on sliding motilities of M. smegmatis

Surfactant protein A (SP-A), which is a lung innate immune system component, is known to bind glycolipids present at the cell surface of a mycobacterial pathogen. Lipoarabinomannan (LAM), a component of mycobacterial thick, waxy cell wall, is one of the glycolipid ligands for SP-A. In order to assess binding of synthetic glycolipids with SP-A and the glycosidic linkage preferences for the interaction, beta-arabinofuranoside trisaccharide glycolipids constituted with beta-(1 -> 2), beta-(1 -> 3) and beta-(1 -> 2), beta-(1 -> 5) linkages relevant to LAM were synthesized through chemical glycosylations. The efficacies of synthetic glycolipids to interact with SP-A were assessed by using the surface plasmon resonance (SPR) technique, from which association-dissociation rate constants and equilibrium binding constants were derived. The equilibrium binding constants of the interaction of two constitutionally varying beta-arabinofuranoside glycolipids with SP-A were found to be in the millimolar range. A comparison of the results with few alpha-anomeric arabinofuranoside glycolipids showed that glycolipids with beta-anomeric linkages were having relatively lower equilibrium binding constants than those with alpha-anomeric linkages in binding to the protein, whereas oligosaccharides alone, without lipidic chains, exhibited higher equilibrium binding constants. Further, the synthetic compounds inhibited the growth of mycobacteria and affected sliding motilities of the bacteria, although to an extent relatively lesser than that of synthetic compounds constituted with alpha-anomeric linkages.

Structure and Mechanistic Insights into Novel Iron-mediated Moonlighting Functions of Human J-protein Cochaperone, Dph4

J-proteins are obligate cochaperones of Hsp70s and stimulate their ATPase activity via the J-domain. Although the functions of J-proteins have been well understood in the context of Hsp70s, their additional co-evolved ``physiological functions'' are still elusive. We report here the solution structure and mechanism of novel iron-mediated functional roles of human Dph4, a type III J-protein playing a vital role in diphthamide biosynthesis and normal development. The NMR structure of Dph4 reveals two domains: a conserved J-domain and a CSL-domain connected via a flexible linker-helix. The linker-helix modulates the conformational flexibility between the two domains, regulating thereby the protein function. Dph4 exhibits a unique ability to bind iron in tetrahedral coordination geometry through cysteines of its CSL-domain. The oxidized Fe-Dph4 shows characteristic UV-visible and electron paramagnetic resonance spectral properties similar to rubredoxins. Iron-bound Dph4 (Fe-Dph4) also undergoes oligomerization, thus potentially functioning as a transient ``iron storage protein,'' thereby regulating the intracellular iron homeostasis. Remarkably, Fe-Dph4 exhibits vital redox and electron carrier activity, which is critical for important metabolic reactions, including diphthamide biosynthesis. Further, we observed that Fe-Dph4 is conformationally better poised to perform Hsp70-dependent functions, thus underlining the significance of iron binding in Dph4. Yeast Jjj3, a functional ortholog of human Dph4 also shows a similar iron-binding property, indicating the conserved nature of iron sequestration across species. Taken together, our findings provide invaluable evidence in favor of additional co-evolved specialized functions of J-proteins, previously not well appreciated.

Hydration dynamics of protein molecules in aqueous solution: Unity among diversity

Dielectric dispersion and NMRD experiments have revealed that a significant fraction of water molecules in the hydration shell of various proteins do not exhibit any slowing down of dynamics. This is usually attributed to the presence of the hydrophobic residues (HBR) on the surface, although HBRs alone cannot account for the large amplitude of the fast component. Solvation dynamics experiments and also computer simulation studies, on the other hand, repeatedly observed the presence of a non-negligible slow component. Here we show, by considering three well-known proteins (lysozyme, myoglobin and adelynate kinase), that the fast component arises partly from the response of those water molecules that are hydrogen bonded with the backbone oxygen (BBO) atoms. These are structurally and energetically less stable than those with the side chain oxygen (SCO) atoms. In addition, the electrostatic interaction energy distribution (EIED) of individual water molecules (hydrogen bonded to SCO) with side chain oxygen atoms shows a surprising two peak character with the lower energy peak almost coincident with the energy distribution of water hydrogen bonded to backbone oxygen atoms (BBO). This two peak contribution appears to be quite general as we find it for lysozyme, myoglobin and adenylate kinase (ADK). The sharp peak of EIED at small energy (at less than 2 k(B)T) for the BBO atoms, together with the first peak of EIED of SCO and the HBRs on the protein surface, explain why a large fraction (similar to 80%) of water in the protein hydration layer remains almost as mobile as bulk water Significant slowness arises only from the hydrogen bonds that populate the second peak of EIED at larger energy (at about 4 k(B)T). Thus, if we consider hydrogen bond interaction alone, only 15-20% of water molecules in the protein hydration layer can exhibit slow dynamics, resulting in an average relaxation time of about 5-10 ps. The latter estimate assumes a time constant of 20-100 ps for the slow component. Interestingly, relaxation of water molecules hydrogen bonded to back bone oxygen exhibit an initial component faster than the bulk, suggesting that hydrogen bonding of these water molecules remains frustrated. This explanation of the heterogeneous and non-exponential dynamics of water in the hydration layer is quantitatively consistent with all the available experimental results, and provides unification among diverse features.

Dependence of Protein Adsorption on Wetting Behavior of UHMWPE-HA-Al2O3-CNT Hybrid Biocomposites

Ultrahigh-molecular-weight polyethylene (UHMWPE) is used as an articulating surface in total hip and knee joint replacement. In order to enhance long-term durability/wear resistance properties, UHMWPE-based polymer-ceramic hybrid composites are being developed. Surface properties such as wettability and protein adsorption alter with reinforcement or with change in surface chemistry. From this perspective, the wettability and protein adsorption behavior of compression-molded UHMWPE-hydroxyapatite (HA)-aluminum oxide (Al2O3)-carbon nanotube (CNT) composites were analyzed in conjunction with surface roughness. The combined effect of Al2O3 and CNT shows enhancement of the contact angle by similar to 37A degrees compared with the surface of the UHMWPE matrix reinforced with HA. In reference to unreinforced UHMWPE, protein adsorption density also increased by similar to 230% for 2 wt.%HA-5 wt.%Al2O3-2 wt.%CNT addition to UHMWPE. An important conclusion is that the polar and dispersion components of the surface free energy play a significant role in wetting and protein adsorption than do the total free energy or chemistry of the surface. The results of this study have major implications for the biocompatibility of these newly developed biocomposites.

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

Cascaded walks in protein sequence space: use of artificial sequences in remote homology detection between natural proteins

Over the past two decades, many ingenious efforts have been made in protein remote homology detection. Because homologous proteins often diversify extensively in sequence, it is challenging to demonstrate such relatedness through entirely sequence-driven searches. Here, we describe a computational method for the generation of `protein-like' sequences that serves to bridge gaps in protein sequence space. Sequence profile information, as embodied in a position-specific scoring matrix of multiply aligned sequences of bona fide family members, serves as the starting point in this algorithm. The observed amino acid propensity and the selection of a random number dictate the selection of a residue for each position in the sequence. In a systematic manner, and by applying a `roulette-wheel' selection approach at each position, we generate parent family-like sequences and thus facilitate an enlargement of sequence space around the family. When generated for a large number of families, we demonstrate that they expand the utility of natural intermediately related sequences in linking distant proteins. In 91% of the assessed examples, inclusion of designed sequences improved fold coverage by 5-10% over searches made in their absence. Furthermore, with several examples from proteins adopting folds such as TIM, globin, lipocalin and others, we demonstrate that the success of including designed sequences in a database positively sensitized methods such as PSI-BLAST and Cascade PSI-BLAST and is a promising opportunity for enormously improved remote homology recognition using sequence information alone.

Subdiffusion in hair bundle dynamics: The role of protein conformational fluctuations

The detection of sound signals in vertebrates involves a complex network of different mechano-sensory elements in the inner ear. An especially important element in this network is the hair bundle, an antenna-like array of stereocilia containing gated ion channels that operate under the control of one or more adaptation motors. Deflections of the hair bundle by sound vibrations or thermal fluctuations transiently open the ion channels, allowing the flow of ions through them, and producing an electrical signal in the process, eventually causing the sensation of hearing. Recent high frequency (0.1-10 kHz) measurements by Kozlov et al. Proc. Natl. Acad. Sci. U. S. A. 109, 2896 (2012)] of the power spectrum and the mean square displacement of the thermal fluctuations of the hair bundle suggest that in this regime the dynamics of the hair bundle are subdiffusive. This finding has been explained in terms of the simple Brownian motion of a filament connecting neighboring stereocilia (the tip link), which is modeled as a viscoelastic spring. In the present paper, the diffusive anomalies of the hair bundle are ascribed to tip link fluctuations that evolve by fractional Brownian motion, which originates in fractional Gaussian noise and is characterized by a power law memory. The predictions of this model for the power spectrum of the hair bundle and its mean square displacement are consistent with the experimental data and the known properties of the tip link. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4768902]

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.