N-myristoylated proteins, key components in intracellular signal transduction systems enabling rapid and flexible cell responses

N-myristoylation, one of the co- or post-translational modifications of proteins, has so far been regarded as necessary for anchoring of proteins to membranes. Recently, we have revealed that Nα-myristoylation of several brain proteins unambiguously regulates certain protein–protein interactions that may affect signaling pathways in brain. Comparison of the amino acid sequences of myristoylated proteins including those in other organs suggests that this regulation is involved in signaling pathways not only in brain but also in other organs. Thus, it has been shown that myristoylated proteins in cells regulate the signal transduction between membranes and cytoplasmic fractions. An algorithm we have developed to identify myristoylated proteins in cells predicts the presence of hundreds of myristoylated proteins. Interestingly, a large portion of the myristoylated proteins thought to take part in signal transduction between membranes and cytoplasmic fractions are included in the predicted myristoylated proteins. If the proteins functionally regulated by myristoylation, a posttranslational protein modification, were understood as cross-talk points within the intracellular signal transduction system, known signaling pathways could thus be linked to each other, and a novel map of this intracellular network could be constructed. On the basis of our recent results, this review will highlight the multifunctional aspects of protein N-myristoylation in brain

Heart-type Fatty acid-binding protein in Acute Myocardial infarction Evaluation (FAME): Background and design of a diagnostic study in primary care

<p>Abstract</p> <p>Background</p> <p>Currently used biomarkers for cardiac ischemia are elevated in blood plasma after a delay of several hours and therefore unable to detect acute coronary syndrome (ACS) in a very early stage. General practitioners (GPs), however, are often confronted with patients suspected of ACS within hours after onset of complaints. This ongoing study aims to evaluate the added diagnostic value beyond clinical assessment for a rapid bedside test for heart-type fatty-acid binding protein (H-FABP), a biomarker that is detectable as soon as one hour after onset of ischemia.</p> <p>Methods</p> <p>Participating GPs perform a blinded H-FABP rapid bedside test (Cardiodetect^®) in patients with symptoms suggestive of ACS such as chest pain or discomfort at rest. All patients, whether referred to hospital or not, undergo electrocardiography (ECG) and venapunction for a plasma troponin test within 12–36 hours after onset of complaints. A final diagnosis will be established by an expert panel consisting of two cardiologists and one general practitioner (blinded to the H-FABP test result), using all available patient information, also including signs and symptoms. The added diagnostic value of the H-FABP test beyond history taking and physical examination will be determined with receiver operating characteristic curves derived from multivariate regression analysis.</p> <p>Conclusion</p> <p>Reasons for presenting the design of our study include the prevention of publication bias and unacknowledged alterations in the study aim, design or data-analysis. To our knowledge this study is the first to assess the diagnostic value of H-FABP <it>outside </it>a hospital-setting. Several previous hospital-based studies showed the potential value of H-FABP in diagnosing ACS. Up to now however it is unclear whether these results are equally promising when the test is used in primary care. The first results are expected in the end of 2008.</p

Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Role of the Subunits Interactions in the Conformational Transitions in Adult Human Hemoglobin: an Explicit Solvent Molecular Dynamics Study

Hemoglobin exhibits allosteric structural changes upon ligand binding due to the dynamic interactions between the ligand binding sites, the amino acids residues and some other solutes present under physiological conditions. In the present study, the dynamical and quaternary structural changes occurring in two unligated (deoxy-) T structures, and two fully ligated (oxy-) R, R2 structures of adult human hemoglobin were investigated with molecular dynamics. It is shown that, in the sub-microsecond time scale, there is no marked difference in the global dynamics of the amino acids residues in both the oxy- and the deoxy- forms of the individual structures. In addition, the R, R2 are relatively stable and do not present quaternary conformational changes within the time scale of our simulations while the T structure is dynamically more flexible and exhibited the T\rightarrow R quaternary conformational transition, which is propagated by the relative rotation of the residues at the {\alpha}1{\beta}2 and {\alpha}2{\beta}1 interface.Comment: Reprinted (adapted) with permission from J. Phys. Chem. B DOI:10.1021/jp3022908. Copyright (2012) American Chemical Societ

Molecular Modeling Study for Inhibition Mechanism of Human Chymase and Its Application in Inhibitor Design

Human chymase catalyzes the hydrolysis of peptide bonds. Three chymase inhibitors with very similar chemical structures but highly different inhibitory profiles towards the hydrolase function of chymase were selected with the aim of elucidating the origin of disparities in their biological activities. As a substrate (angiotensin-I) bound crystal structure is not available, molecular docking was performed to dock the substrate into the active site. Molecular dynamics simulations of chymasecomplexes with inhibitors and substrate were performed to calculate the binding orientation of inhibitors and substrate as well as to characterize conformational changes in the active site. The results elucidate details of the 3D chymase structure as well as the importance of K40 in hydrolase function. Binding mode analysis showed that substitution of a heavier Cl atom at the phenyl ring of most active inhibitor produced a great deal of variation in its orientation causing the phosphinate group to interact strongly with residue K40. Dynamics simulations revealed the conformational variation in region of V36-F41upon substrate and inhibitor binding induced a shift in the location of K40 thus changing its interactions with them. Chymase complexes with the most activecompound and substrate were used for development of a hybrid pharmacophore model which was applied in databases screening. Finally, hits which bound well at the active site, exhibited key interactions and favorable electronic properties were identified as possible inhibitors for chymase. This study not only elucidates inhibitorymechanism of chymase inhibitors but also provides key structural insights which will aid in the rational design of novel potent inhibitors of the enzyme. In general, the strategy applied in the current study could be a promising computational approach and may be generally applicable to drug design for other enzymes

Folding of the apolipoprotein A1 driven by the salt concentration as a possible mechanism to improve cholesterol trapping

The folding of the cholesterol trapping apolipoprotein A1 in aqueous solution at increasing ionic strength is studied using atomically detailed molecular dynamics simulations. We calculate various structural properties to characterize the conformation of the protein, such as the radius of gyration, the radial distribution function and the end to end distance. Additionally we report information using tools specifically tailored for the characterization of proteins, such as the mean smallest distance matrix and the Ramachandran plot. We find that two qualitatively different configurations of this protein are preferred, one where the protein is extended, and one where it forms loops or closed structures. It is argued that the latter promote the association of the protein with cholesterol and other fatty acids.Comment: 14 pages, 6 figures. To appear in "Selected Topics of Computational and Experimental Fluid Mechanics", Springer, J. Klapp, G. Ru\'iz, A. Medina, A. L\'opez & L. Di G. Sigalotti (eds.), 201

Structural and Thermodynamic Approach to Peptide Immunogenicity

In the conventional paradigm of humoral immunity, B cells recognize their cognate antigen target in its native form. However, it is well known that relatively unstable peptides bearing only partial structural resemblance to the native protein can trigger antibodies recognizing higher-order structures found in the native protein. On the basis of sound thermodynamic principles, this work reveals that stability of immunogenic proteinlike motifs is a critical parameter rationalizing the diverse humoral immune responses induced by different linear peptide epitopes. In this paradigm, peptides with a minimal amount of stability (ΔGX<0 kcal/mol) around a proteinlike motif (X) are capable of inducing antibodies with similar affinity for both peptide and native protein, more weakly stable peptides (ΔGX>0 kcal/mol) trigger antibodies recognizing full protein but not peptide, and unstable peptides (ΔGX>8 kcal/mol) fail to generate antibodies against either peptide or protein. Immunization experiments involving peptides derived from the autoantigen histidyl-tRNA synthetase verify that selected peptides with varying relative stabilities predicted by molecular dynamics simulations induce antibody responses consistent with this theory. Collectively, these studies provide insight pertinent to the structural basis of immunogenicity and, at the same time, validate this form of thermodynamic and molecular modeling as an approach to probe the development/evolution of humoral immune responses

Variational Methods for Biomolecular Modeling

Structure, function and dynamics of many biomolecular systems can be characterized by the energetic variational principle and the corresponding systems of partial differential equations (PDEs). This principle allows us to focus on the identification of essential energetic components, the optimal parametrization of energies, and the efficient computational implementation of energy variation or minimization. Given the fact that complex biomolecular systems are structurally non-uniform and their interactions occur through contact interfaces, their free energies are associated with various interfaces as well, such as solute-solvent interface, molecular binding interface, lipid domain interface, and membrane surfaces. This fact motivates the inclusion of interface geometry, particular its curvatures, to the parametrization of free energies. Applications of such interface geometry based energetic variational principles are illustrated through three concrete topics: the multiscale modeling of biomolecular electrostatics and solvation that includes the curvature energy of the molecular surface, the formation of microdomains on lipid membrane due to the geometric and molecular mechanics at the lipid interface, and the mean curvature driven protein localization on membrane surfaces. By further implicitly representing the interface using a phase field function over the entire domain, one can simulate the dynamics of the interface and the corresponding energy variation by evolving the phase field function, achieving significant reduction of the number of degrees of freedom and computational complexity. Strategies for improving the efficiency of computational implementations and for extending applications to coarse-graining or multiscale molecular simulations are outlined.Comment: 36 page

Effects of Single Nucleotide Polymorphisms on Human N-Acetyltransferase 2 Structure and Dynamics by Molecular Dynamics Simulation

BACKGROUND: Arylamine N-acetyltransferase 2 (NAT2) is an important catalytic enzyme that metabolizes the carcinogenic arylamines, hydrazine drugs and chemicals. This enzyme is highly polymorphic in different human populations. Several polymorphisms of NAT2, including the single amino acid substitutions R64Q, I114T, D122N, L137F, Q145P, R197Q, and G286E, are classified as slow acetylators, whereas the wild-type NAT2 is classified as a fast acetylator. The slow acetylators are often associated with drug toxicity and efficacy as well as cancer susceptibility. The biological functions of these 7 mutations have previously been characterized, but the structural basis behind the reduced catalytic activity and reduced protein level is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We performed multiple molecular dynamics simulations of these mutants as well as NAT2 to investigate the structural and dynamical effects throughout the protein structure, specifically the catalytic triad, cofactor binding site, and the substrate binding pocket. None of these mutations induced unfolding; instead, their effects were confined to the inter-domain, domain 3 and 17-residue insert region, where the flexibility was significantly reduced relative to the wild-type. Structural effects of these mutations propagate through space and cause a change in catalytic triad conformation, cofactor binding site, substrate binding pocket size/shape and electrostatic potential. CONCLUSIONS/SIGNIFICANCE: Our results showed that the dynamical properties of all the mutant structures, especially in inter-domain, domain 3 and 17-residue insert region were affected in the same manner. Similarly, the electrostatic potential of all the mutants were altered and also the functionally important regions such as catalytic triad, cofactor binding site, and substrate binding pocket adopted different orientation and/or conformation relative to the wild-type that may affect the functions of the mutants. Overall, our study may provide the structural basis for reduced catalytic activity and protein level, as was experimentally observed for these polymorphisms

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available