Prevalent Mutations of Human Prion Protein: A Molecular Modeling and Molecular Dynamics Study

Abstract

Point mutations in the human prion protein gene, leading to amino acid substitutions in the human prion protein contribute to conversion of PrP^C to PrP^Sc and amyloid formation, resulting in prion diseases such as familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS), and fatal familial insomnia. We have investigated impressions of prevalent mutations including Q217R, D202N, F198S, on the human prion protein and compared the mutant models with wild types. Structural analyses of models were performed with molecular modeling and molecular dynamics simulation methods. According to our results, frequently occurred mutations are observed in conserved and fully conserved sequences of human prion protein and the most fluctuation values occur in the Helix 1 around residues 144–152 and C-terminal end of the Helix 2. Our analysis of results obtained from MD simulation clearly shows that this long-range effect plays an important role in the conformational fluctuations in mutant structures of human prion protein. Results obtained from molecular modeling such as creation or elimination of some hydrogen bonds, increase or decrease of the accessible surface area and molecular surface, loss or accumulation of negative or positive charges on specific positions, and altering the polarity and pKa values, show that amino acid point mutations, though not urgently change the stability of PrP, might have some local impacts on the protein interactions which are required for oligomerization into fibrillar species.     

1-Alkanols and membranes: A story of attraction

Although 1-alkanols have long been known to act as penetration enhancers and anesthetics, the mode of operation is not yet understood. In this study, long-time molecular dynamics simulations have been performed to investigate the effect of 1-alkanols of various carbon chain lengths onto the structure and dynamics of dimyristoylphosphatidylcholine bilayers. The simulations were complemented by microcalorimetry, continuous bleaching and film balance experiments. In the simulations, all investigated 1-alkanols assembled inside the lipid bilayer within tens of nanoseconds. Their hydroxyl groups bound preferentially to the lipid carbonyl group and the hydrocarbon chains stretched into the hydrophobic core of the bilayer. Both molecular dynamics simulations and experiments showed that all 1-alkanols drastically affected the bilayer properties. Insertion of long-chain 1-alkanols decreased the area per lipid while increasing the thickness of the bilayer and the order of the lipids. The bilayer elasticity was reduced and the diffusive motion of the lipids within the bilayer plane was suppressed. On the other hand, integration of ethanol into the bilayer enlarged the area per lipid. The bilayer became softer and lipid diffusion was enhanced.     

Membrane curvature and surface area per lipid affect the conformation and oligomeric state of HIV-1 fusion peptide: A combined FTIR and MD simulation study

Fourier-transformed infrared spectroscopy (FTIR) and molecular dynamics (MD) simulation results are presented to support our hypothesis that the conformation and the oligomeric state of the HIV-1 gp41 fusion domain or fusion peptide (gp41-FP) are determined by the membrane surface area per lipid (APL), which is affected by the membrane curvature. FTIR of the gp41-FP in the Aerosol-OT (AOT) reversed micellar system showed that as APL decreases from ∼ 50 to 35 Å² by varying the AOT/water ratio, the FP changes from the monomeric α-helical to the oligomeric β-sheet structure. MD simulations in POPE lipid bilayer systems showed that as the APL decreases by applying a negative surface tension, helical monomers start to unfold into turn-like structures. Furthermore, an increase in the applied lateral pressure during nonequilibrium MD simulations favored the formation of β-sheet structure. These results provide better insight into the relationship between the structures of the gp41-FP and the membrane, which is essential in understanding the membrane fusion process. The implication of the results of this work on what is the fusogenic structure of the HIV-1 FP is discussed.     

The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.     

Structural and Dynamical Properties of a Full-length HIV-1 Integrase: Molecular Dynamics Simulations

Abstract

The structural and dynamical properties of the complete full-length structure of HIV-1 integrase were investigated using Molecular Dynamics approach. Simulations were carried out for the three systems, core domain only (CORE), full-length structure without (FULL) and with a Mg²⁺ (FULL+ION) in its active site, aimed to investigate the difference in the molecular properties of the full-length models due to their different construction procedures as well as the effects of the two ends, C- and N-terminal, on those properties in the core domain. The full-length structure was prepared from the two experimental structures of two-domain fragment. The following properties were observed to differ significantly from the previous reports: (i) relative topology formed by an angle between the three domains; (ii) the cavity size defined by the catalytic triad, Asp64, Asp116, and Glul52; (iii) distances and solvation of the Mg²⁺; and (iv) conformation of the catalytic residues. In addition, the presence of the two terminal domains decreases the mobility of the central core domain significantly.     

利用原子力显微镜对A型流感病毒的形态学研究

利用透射电子显微镜(TEM)和原子力显微镜(AFM)观察流感病毒(H1N1),探讨AFM在病毒形态研究中的应用,为病毒形态学研究提供一种新型、简便、快捷的工具.TEM采用磷钨酸负染方法,AFM采用轻敲模式在大气常温下扫描成像,并对主要指标长度(直径)、Ra、Rq等进行测量.两种方法最终得到相似的形态学结果,流感病毒呈球状、丝状,并有一些形状介于两者之间.TEM提供了流感病毒二维图像,可见钉状突起,AFM则呈现了流感病毒三维图像,且可见病毒表面有凹凸不平的特征和边缘有齿轮状的突起,同时获得表面粗糙度等可以量化指标.与TEM观察相比,原子力显微镜是一种制样简单、观察直观的新型病毒形态学研究工具,其表征参数可以作为病毒形态学研究的量化指标.     

Activation of the hypoxia-inducible factor-1 in overloaded temporomandibular joint, and induction of osteoclastogenesis

Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats. Here we demonstrate for the first time that hypoxia-inducible factor-1α (Hif-1α) is activated in mature chondrocytes of temporomandibular joint-osteoarthritis (TMJ-OA) model rat by mechanical overload, and that activated Hif-1 in chondrocytes can induce osteoclastogenesis via repression of osteoprotegerin (Opg) expression.In rat TMJs, degeneration of the condylar cartilage became prominent in proportion to the duration of overloading. Hif-1α expression was observed specifically in mature and hypertrophic chondrocytes, and Hif-1α-positivity, level of Vegf expression, and tartrate-resistant acid phosphatase (TRAP)-positive cell numbers all increased in the same manner. When ATDC5 cells induced differentiation by insulin were cultured under hypoxia, Hif-1α induction was observed in mature stage, but not in immature stage. Inductions of Hif-1-target genes showed a similar expression pattern. In addition, expression of Opg decreased in hypoxia, and Hif-1α played a role, in part, in its regulation.     

A Molecular Dynamics Study of Human Defensins HBD-1 and HNP-3 in Water

Abstract

Mammalian defensins are crucial components of the innate immune system. They are characterized by three disulfide bridges and exhibit broad spectrum antibacterial activity. The spacing between the cysteines and disulfide connectivities in the two classes of defensins, the α- and β-forms, are different. The structural motif of 3 β-strands appears to be conserved in α and β-defensins despite differences in disulfide connectivities and spacing between cysteines. In this study, Molecular Dynamics Simulations (MDS) have been carried out to study the conformational behavior of α- and β-defensins with and without disulfide bridges. Our results indicate that β-strands in the C-terminal region of HBD-1 and HNP-3 do not unfold during the course of MDS. The segment adopting α-helix in HBD-1 unfolds early during the simulations. The backbone hydrogen bonds in HBD-1 and HNP-3 are broken during MDS. When the disulfide bonds are absent, the N-terminal β-strand unfolds by 20 ns but β-strands are observed in the C-terminal region of HNP-3. HBD-1, without disulfide bridges, unfolds to a greater extent during the course of the MDS. Examination of distances between sulfur atoms of cysteines without disulfide bridges during the simulations indicate that there is no specific preference for native disulfide bridges, which could be the reason for the experimental observation of non-native disulfide bridge formation during chemical synthesis of human α- and β-defensins. Since defensins with non-native disulfide bridges are biologically active, the exact three dimensional structures observed for native HBD-1 and HNP-3 does not appear to be essential for exhibiting antibacterial activity.     

Molecular dynamics studies of the transmembrane domain of gp41 from HIV-1

Helix-helix interactions in the putative three-helix bundle formation of the gp41 transmembrane (TM) domain may contribute to the process of virus-cell membrane fusion in HIV-1 infection. In this study, molecular dynamics is used to analyze and compare the conformations of monomeric and trimeric forms of the TM domain in various solvent systems over the course of 4 to 23-ns simulations. The trimeric bundles of the TM domain were stable as helices and remained associated in a hydrated POPE lipid bilayer for the duration of the 23-ns simulation. Several stable inter-chain hydrogen bonds, mostly among the three deprotonated arginine residues located at the center of each of the three TM domains, formed in a right-handed bundle embedded in the lipid bilayer. No such bonds were observed when the bundle was left-handed or when the central arginine residue in each of the three TM helices was replaced with isoleucine (R_I mutant), suggesting that the central arginine residues may play an essential role in maintaining the integrity of the three-helix bundle. These observations suggest that formation of the three-helix bundle of the TM domain may play a role in the trimerization of gp41, thought to occur during the virus-cell membrane fusion process.     

A CHO cell line engineered to express XBP1 and ERO1‐Lα has increased levels of transient protein expression

Transient gene expression (TGE) systems currently provide rapid and scalable (up to 100 L) methods for generating multigram quantities of recombinant heterologous proteins. Product titers of up to 1 g/L have been demonstrated in HEK293 cells 1 but reported yields from Chinese hamster ovary (CHO) cells are lower at ～300 mg/L. 2 We report on the establishment of an engineered CHOS cell line, which has been developed for TGE. This cell line has been engineered to express both X‐box binding protein (XBP‐1S) and endoplasmic reticulum oxidoreductase (ERO1‐Lα) and has been named CHOS‐XE. CHOS‐XE cells produced increased antibody (MAb) yields (5.3– 6.2 fold) in comparison to CHOS cells. Product quality was unchanged as assessed by size, charge, propensity to aggregate, major glycosylation species, and thermal stability. To further develop and test this TGE system, five commercial media were assessed, and one was shown to offer the greatest increase in antibody yields. With the addition of a commercial feed, MAb titers reached 875 mg/L. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:697–706, 2013     

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC₅₀ of benzimidazole (?)-1 is ～100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (?)/(+)-1 and (?)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (?)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (?) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (?) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.     

Molecular Dynamics Study of the Structure,Flexibility and Dynamics of Thermostable L1 Lipase at High Temperatures

Molecular Dynamics (MD) simulations have been used to understand how protein structure, dynamics, and flexibility are affected by adaptation to high temperature for several years. We report here the results of the high temperature MD simulations of Bacillus stearothermophilus L1 (L1 lipase). We found that the N-terminal moiety of the enzyme showed a high flexibility and dynamics during high temperature simulations which preceded and followed by clear structural changes in two specific regions; the small domain and the main catalytic domain or core domain of the enzyme. These two domains interact with each other through a Zn²⁺-binding coordination with Asp-61 and Asp-238 from the core domain and His-81 and His-87 from the small domain. Interestingly, the His-81 and His-87 were among the highly fluctuated and mobile residues at high temperatures. The results appear to suggest that tight interactions of Zn²⁺-binding coordination with specified residues became weak at high temperature which suggests the contribution of this region to the thermostability of the enzyme. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

Partial restoration of T‐cell function in aged mice by in vitro blockade of the PD‐1/ PD‐L1 pathway

Programmed cell death‐1 (PD‐1) is a newly characterized negative regulator of immune responses. The interaction of PD‐1 with its ligands (PD‐L1 and PD‐L2) inhibits T‐cell proliferation and cytokine production in young mice. Increased PD‐1 expression has been described during chronic infections, inducing chronic activation of the immune system to control it. As aging is associated with chronic immune activation, PD‐1 may contribute to age‐associated T‐cell dysfunction. Our data showed the following results in aged mice: (i) the number of PD‐1‐expressing T cells and the level of expression of PD‐Ls was increased on dendritic cell subsets and T cells; (ii) PD‐1⁺ T cells were exhausted effector memory T cells, as shown by their lower level of CD127, CD25 and CD28, as well as their limited proliferative and cytokine‐producing capacity; (iii) the expression of PD‐1 was up‐regulated after T‐cell receptor‐mediated activation of CD8⁺ T cells, but not of CD4⁺ T cells; (iv) blockade of the PD‐1/PD‐L1 pathway moderately improved the cytokine production of T cells from old mice but did not restore their proliferation; and (v) blockade of the PD‐1/PD‐L1 pathway did not restore function of PD‐1⁺ T cells; its effect appeared to be exclusively mediated by increased functionality of the PD‐1^? T cells. Our data thus suggest that blockade of the PD‐1/PD‐L1 is not likely to be efficient at restoring exhausted T‐cell responses in aged hosts, although improving the responses of PD‐1^? T cells may prove to be a helpful strategy in enhancing primary responses.     

消癌平诱导caspase-3活化动态过程的实时监测

消癌平是从萝摩科(Asclepiadaceae)植物通关藤(Marsdenia tenacissima)根部提取的药物,已经广泛应用于癌症和多种炎症的临床治疗,并且具有较好的疗效。将消癌平注射液与细胞培养液按照1∶1的比例混合后共同培养人肺腺癌(ASTC-a-1)细胞,通过CCK-8方法检测发现消癌平能够显著抑制细胞的增长。然后利用荧光共聚焦扫描显微镜和荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术在稳定表达了SCAT3质粒的单个ASTC-a-1细胞中实时动态监测消癌平处理后SCAT3的空间分布以及SCAT3被caspase-3切割的动态过程。实验结果表明:消癌平处理后20 min中SCAT3开始向细胞核以及细胞膜部位转移,约在100min左右SCAT3被迅速切割。     

Loop motion and base release in purine-specific nucleoside hydrolase: A molecular dynamics study

Although various Trypanosoma vivax purine-specific inosine–adenosine–guanosine nucleoside hydrolase (IAG-NH) crystal structures have been determined and the chemical reaction mechanism of substrate hydrolysis has been studied recently, the mechanistic details for the release of base and ribose are still unclear. Herein molecular dynamics (MD) simulations combined with umbrella sampling technique were utilized to explore the regulation mechanisms of key residues and loops 1 and 2 for the base release. Our results have indicated that the base release process is not the rate-limiting step in the entire hydrolysis process, and the very low barrier of ~ 5.6 kcal/mol can be washed out easily by the notable exothermicity from the substrate hydrolysis step. Moreover, the MD simulations have revealed that Glu82/Trp83 in loop 1 and His247/Arg252 in loop 2 are important to modulate the base release. The partial helix-to-coil change of loop 2 along with the base release process has been observed, showing good agreement with the IAG-NH crystal structures. The local binding site around the ribose after the base release is also discussed.     

Force field impact and spin-probe modeling in molecular dynamics simulations of spin-labeled T4 lysozyme

Several attempts have been made to compute electron paramagnetic resonance (EPR) spectra of biomolecules, using motional models or simulated trajectories to describe dynamics. Ideally, the simulated trajectories should capture fast (picosecond) snapshots of spin-probe rotations accurately, while being lengthy enough to ensure a proper Fourier integration of the time-domain signal. It is the interplay of the two criteria that poses computational challenges to the method. In this context, an analysis of the spin-probe and protein conformational sampling and equilibration, with different force fields and with explicit solvent, may be a useful attempt. The present work reports a comparative study of the effect of the molecular dynamics (MD) force field on conformational sampling and equilibration in two spin-labeled T4 lysozyme (T4L) variants, N40C and K48C. Ensembles of 10× 3 ns-trajectories per variant and per force field (OPLS/AMBER and AMBER99) are analyzed for a reliable assessment of convergence and sampling. It is found that subtle site-dependent differences in spin-probe rotations and torsions are more readily captured in the AMBER99 trajectories than in the OPLS/AMBER simulations. On the other hand, sampling and equilibration are found to be better with the OPLS/AMBER force field at equal trajectory lengths.Figure: Left panel: The spin-probe R1 ring and the spin-probe Euler angles , and . Middle panel: Illustration of the diffusion in a cone model for the spin-probe motion: snapshots of helix B and of the R1 ring in N40C, taken at 0.3 ps intervals from AMBER trajectory 1. Right panel: The N40C mutant with the spin label (solid mode), solvated in a cubic box.     

Explicit Solvent Molecular Dynamics Simulation of Duplex Formed by the Modified Oligonucleotide with Alternating Phosphate/Phosphonate Internucleoside Linkages and its Natural Counterpart

Abstract

Impact of the internucleoside linkage modification by inserting a methylene group on the ability of the modified oligonucleotide to hybridize with a natural DNA strand was studied by fully solvated molecular dynamics (MD) simulations. Three undecamer complexes were analyzed: natural dT₁₁.dA₁₁ duplex as a reference and two its analogs with alternating modified and natural linkages in the deoxyadenosine chain. The isopolar, non-isosteric modified linkages were of 5′-O-PO₂-CH₂-O-3′ (5′PC3′) or 5′-O-CH₂-PO₂-O-3′ (5′CP3′) type. Simulations were performed by using the AMBER 5.0 software package with the force field completed by a set of parameters needed to model the modified segments. Both modifications were found to lead to double helical complexes, in which the thymidine strand as well as deoxyriboses and unmodified linkages in the adenosine strand adopted conformations typical for the B-type structure. For each of the two conformational richer modified linkages two stable conformations were found at 300 K: the -ggt and ggt for the 5′PC3′ and ggg, tgg for the 5′CP3′, respectively. Both modified chains adopted helical conformations with heightened values of the inclination parameter but without affecting the Watson-Crick hydrogen bonds.