Kazuhisa Nishizawa

Molecular Dynamics Simulation of Kv Channel Voltage Sensor Helix in a Lipid Membrane with Applied Electric Field

In this article, we present the results of the molecular dynamics simulations of amphiphilic helix peptides of 13 amino-acid residues, placed at the lipid-water interface of dipalmitoylphosphatidylcholine bilayers. The peptides are identical with, or are derivatives of, the N-terminal segment of the S4 helix of voltage-dependent K channel KvAP, containing four voltage-sensing arginine residues (R1-R4). Upon changing the direction of the externally applied electric field, the tilt angle of the wild-type peptide changes relative to the lipid-water interface, with the N-terminus heading up with an outward electric field. These movements were not observed using an octane membrane in place of the dipalmitoylphosphatidylcholine membrane, and were markedly suppressed by 1), substituting Phe located one residue before the first arginine (R1) with a hydrophilic residue (Ser, Thr); or 2), changing the periodicity rule of Rs from at-every-third to at-every-fourth position; or 3), replacing R1 with a lysine residue (K). These and other findings suggest that the voltage-dependent movement requires deep positioning of Rs when the resting (inward) electric field is present. Later, we performed simulations of the voltage sensor domain (S1-S4) of Kv1.2 channel. In simulations with a strong electric field (0.1 V/nm or above) and positional restraints on the S1 and S2 helices, S4 movement was observed consisting of displacement along the S4 helix axis and a screwlike axial rotation. Gating-charge-carrying Rs were observed to make serial interactions with E183 in S1 and E226 in S2, in the outer water crevice. A 30-ns-backward simulation started from the open-state model gave rise to a structure similar to the recent resting-state model, with S4 moving vertically approximately 6.7 A. The energy landscape around the movement of S4 appears very ragged due to salt bridges formed between gating-charge-carrying residues and negatively charged residues of S1, S2, and S3 helices. Overall, features of S3 and S4 movements are consistent with the recent helical-screw model. Both forward and backward simulations show the presence of at least two stable intermediate structures in which R2 and R3 form salt bridges with E183 or E226, respectively. These structures are the candidates for the states postulated in previous gating kinetic models, such as the Zagotta-Hoshi-Aldrich model, to account for more than one transition step per subunit for activation.

A DNA sequence evolution analysis generalized by simulation and the markov chain monte carlo method implicates strand slippage in a majority of insertions and deletions.

To study the mechanisms for local evolutionary changes in DNA sequences involving slippage-type insertions and deletions, an alignment approach is explored that can consider the posterior probabilities of alignment models. Various patterns of insertion and deletion that can link the ancestor and descendant sequences are proposed and evaluated by simulation and compared by the Markov chain Monte Carlo (MCMC) method. Analyses of pseudogenes reveal that the introduction of the parameters that control the probability of slippage-type events markedly augments the probability of the observed sequence evolution, arguing that a cryptic involvement of slippage occurrences is manifested as insertions and deletions of short nucleotide segments. Strikingly, ?80% of insertions in human pseudogenes and ?50% of insertions in murids pseudogenes are likely to be caused by the slippage-mediated process, as represented by BC in ABCD ? ABCBCD. We suggest that, in both human and murids, even very short repetitive motifs, such as CAGCAG, CACACA, and CCCC, have ?10- to 15-fold susceptibility to insertions and deletions, compared to nonrepetitive sequences. Our protocol, namely, indel-MCMC, thus seems to be a reasonable approach for statistical analyses of the early phase of microsatellite evolution.

Effects of Lys to Glu mutations in GsMTx4 on membrane binding, peptide orientation, and self-association propensity, as analyzed by molecular dynamics simulations.

GsMTx4, a gating modifier peptide acting on cationic mechanosensitive channels, has a positive charge (+5e) due to six Lys residues. The peptide does not have a stereospecific binding site on the channel but acts from the boundary lipids within a Debye length of the pore probably by changing local stress. To gain insight into how these Lys residues interact with membranes, we performed molecular dynamics simulations of Lys to Glu mutants in parallel with our experimental work. In silico, K15E had higher affinity for 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine bilayers than wild-type (WT) peptide or any other mutant tested, and showed deeper penetration than WT, a finding consistent with the experimental data. Experimentally, the inhibitory activities of K15E and K25E were most compromised, whereas K8E and K28E inhibitory activities remained similar to WT peptide. Binding of WT in an interfacial mode did not influence membrane thickness. With interfacial binding, the direction of the dipole moments of K15E and K25E was predicted to differ from WT, whereas those of K8E and K28E oriented similarly to that of WT. These results support a model in which binding of GsMTx4 to the membrane acts like an immersible wedge that serves as a membrane expansion buffer reducing local stress and thus inhibiting channel activity. In simulations, membrane-bound WT attracted other WT peptides to form aggregates. This may account for the positive cooperativity observed in the ion channel experiments. The Lys residues seem to fine-tune the depth of membrane binding, the tilt angle, and the dipole moments.

Potential of mean force analysis of the self-association of leucine-rich transmembrane α-helices: Difference between atomistic and coarse-grained simulations

Interaction of transmembrane (TM) proteins is important in many biological processes. Large-scale computational studies using coarse-grained (CG) simulations are becoming popular. However, most CG model parameters have not fully been calibrated with respect to lateral interactions of TM peptide segments. Here, we compare the potential of mean forces (PMFs) of dimerization of TM helices obtained using a MARTINI CG model and an atomistic (AT) Berger lipids-OPLS/AA model (AT(OPLS)). For helical, tryptophan-flanked, leucine-rich peptides (WL15 and WALP15) embedded in a parallel configuration in an octane slab, the AT(OPLS) PMF profiles showed a shallow minimum (with a depth of approximately 3 kJ/mol; i.e., a weak tendency to dimerize). A similar analysis using the CHARMM36 all-atom model (AT(CHARMM)) showed comparable results. In contrast, the CG analysis generally showed steep PMF curves with depths of approximately 16-22 kJ/mol, suggesting a stronger tendency to dimerize compared to the AT model. This CG > AT discrepancy in the propensity for dimerization was also seen for dilauroylphosphatidylcholine (DLPC)-embedded peptides. For a WL15 (and WALP15)/DLPC bilayer system, AT(OPLS) PMF showed a repulsive mean force for a wide range of interhelical distances, in contrast to the attractive forces observed in the octane system. The change from the octane slab to the DLPC bilayer also mitigated the dimerization propensity in the CG system. The dimerization energies of CG (AALALAA)3 peptides in DLPC and dioleoylphosphatidylcholine bilayers were in good agreement with previous experimental data. The lipid headgroup, but not the length of the lipid tails, was a key causative factor contributing to the differences between octane and DLPC. Furthermore, the CG model, but not the AT model, showed high sensitivity to changes in amino acid residues located near the lipid-water interface and hydrophobic mismatch between the peptides and membrane. These findings may help interpret CG and AT simulation results on membrane proteins.

Atomistic Molecular Simulation of Gating Modifier Venom Peptides – Two Binding Modes and Effects of Lipid Structure

GsMTx4, a gating-modifier peptide obtained from tarantula venom has been a valuable tool for investigating the gating mechanisms of mechanosensitive channels. GsMTx4 is thought to act at the channel/lipid interface by modifying the structure of the surrounding lipid molecules. However, the atomistic details of these actions are poorly understood. Here, the studies of GsMTx4 and related peptide toxins that inhibit the voltage activation of various ion channels are reviewed, with emphasis on the results of molecular dynamic (MD) simulation analyses. Free energy profile analyses suggest that these toxins exhibit two modes of binding to lipid membrane, namely, the shallow mode and the deep mode. These toxins favor the deep mode, especially in membranes rich in saturated lipid acyl chains, which make the headgroup layer tight. It is hypothesized that in the case of HaTx the deep mode is the action mode, while for GsMTx4 the two modes can explain the concentration-dependent (biphasic) effect of GsMTx4 that has recently been reported. The possibility that such toxins seek out specific types of lipid molecules is discussed. Simulation results support the view that the channel/GsMTx4 (or HaTx)/lipids make a tertiary complex crucial to the effectiveness of the toxin and therefore binding of the toxin to channels occurs only in the presence of lipid molecules with appropriate structures.

Mechanisms of immunosuppression by mesenchymal stromal cells: a review with a focus on molecules

Immunosuppressive treatment with mesenchymal stromal (stem) cells (MSCs) has been performed in many human transplantation settings with the goal to prevent rejection. The therapeutic potential of MSCs has also been explored in a broad spectrum of applications including the treatment of autoimmune diseases. As the immunomodulatory function of MSCs is a multifactorial process, it is important to occasionally review the precise molecular mechanisms/modalities and insights into how they are orchestrated and deployed in clinical settings. This article aimed to review the mechanisms of the immunosuppressive activities of MSCs from a molecular (modality) perspective, with an emphasis on recent reports published between 2014 and mid 2016. The article highlights the subtle differences due to cell type, timing, and types of priming that could lead to better quality control and pre-enhancement of MSCs to optimize their therapeutic potential. Correspondence to: Kazuhisa Nishizawa, Department of Clinical Laboratory Science, Teikyo University, Japan, Tel: +81-3-3964-1211, ext: 46136, E-mail: kazunet@med.teikyo-u.ac.jp Received: May 24, 2016; Accepted: July 20, 2016; Published: July 23, 2016 MSCs and their immunomodulatory capabilities Adult MSCs have been proposed to be a potential therapeutic alternative for inflammatory diseases and tissue transplantation, based on their capacity to modulate the function of most types of immune cells [1]. MSCs are a heterogeneous population of cells that were originally isolated from bone marrow as progenitor cells of the osteogenic lineage by Friedenstein [2]. Although first isolated from bone marrow, now MSCs can be obtained from other adult and fetal sources, including adipose [3], dental pulp [4], and the umbilical cord [5] tissues. The ability of MSCs to differentiate into adipocytes, chondrocytes, and osteoblasts is also well documented [6]. MSCs are characterized by their proliferation on plastic-adherents in a culture, their fibroblast-like shape, and their expression of stromal markers such as CD105, CD73, and CD90, and not hematopoietic markers including CD45, CD34, CD14 (or CD11b), CD79alpha (or CD19), or human leukocyte antigen (HLA)-DR molecules. Among the mammals, the expression of CD105 is variable, but CD45 was found to be absent across species tested by Su et al. [7]. A more formal definition of MSCs could be the one proposed by the International Society for Cellular Therapy [8-10]. A population of MSCs has the following features: 1) They are plastic-adherent when cultured using standard protocols. 2) Phenotypically, ex vivo generated MSCs express a number of nonspecific markers including CD105 (SH2 or endoglin), CD73 (SH3 or SH4), CD90, CD166, CD44, and CD29. MSCs do not express hematopoietic and endothelial markers such as CD11b, CD14, CD31, and CD45 (of note, CD106 may be included). 3) MSCs can be differentiated into bone, fat, and cartilage tissue with the appropriate stimulation. Significantly, MSCs have multilineage potential and immunomodulatory capacity [11]. Bartholomew et al. [12] showed that intravenous administration of donor MSCs to MHC-mismatched recipient baboons caused prolonged survival of third-party skin grafts. In an analysis of MSC immunogenicity, Tse et al. [13] reported that MSCs do not elicit an allogeneic proliferative response, and furthermore that MSCs are capable of suppressing allogeneic T cell proliferation through third party peripheral blood mononuclear cells (PBMCs). Studies on MSCs led to the consensus that the beneficial effects of MSCs on immune cells include not only the inhibition of proinflammatory polarization [14], and effector functions and pathways [15-17], but also the generation of regulatory cells [18,19]. Leukocyte subpopulations including regulatory T cells, type-2 macrophages, immature dendritic cells (DCs) are now well known to exert immunosuppressive effects; however, MSCs can induce and collaborate with these cells. Thus, MSCs are able to induce peripheral tolerance, which shows their potential as therapeutic tools for immunemediated disorders including graft-versus-host disease (GVHD). Furthermore, MSCs are known to exhibit low-level expression of MHC-I (major histocompatibility complex-1) proteins and lack the expression of MHC-II and co-stimulatory molecules CD80, CD86, and CD40. Owing to this feature, MSCs are not able to trigger T cell activation [12]. In addition, MSCs can inhibit the proliferation of allostimulated T cells and both CD4+ and CD8+ phytohemagglutininstimulated human T cells [20] and induce the proliferation of Treg cells [21,22]. In vitro co-culture of T cells with MSCs results in a shift towards a CD4+ CD25+ Foxp3+ Treg phenotype. Other than T cells [23], MSCs have been shown to exert strong inhibitory effects on a variety of immune cells including B cells [24,25], NK cells [26], and dendritic cells (DCs) [11,27,28]. Reflecting recent preclinical and clinical research activities using Nishizawa K (2016) Mechanisms of immunosuppression by mesenchymal stromal cells: a review with a focus on molecules Volume 1(3): 82-96 Biomed Res Clin Prac, 2016 doi: 10.15761/BRCP.1000116 MSCs, a substantial number of recent review articles appear to have focused on challenges associated with the quality control of MSCs for therapeutic use. As Galipeau and Krampera [29] suggest, robust markers and assays that result in the approval and release of MSCs as commercial products need to be established. The current research is focused on a better understanding of the immunoregulatory properties of MSCs with the ultimate goal of establishing clinically useful methods for quality control. Recent review articles covering preclinical and clinical challenges include that of Gao et al. [1]. The molecular mechanisms mediating the anti-inflammatory function of MSCs have been the focus of intense study. Soluble and cell-bound factors identified to have a role in mediating this function include transforming growth factor (TGF-β), nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), IL-6, Notch-receptors, human leukocyte antigen (HLA-G), and programmed cell-death ligand 1 (PD-1). However, MSCs generally exhibit context-dependency, which suggests that caution needs to be exercised when extrapolating In vitro or in vivo phenomena, including data regarding different diseases as well as diverse host and donor backgrounds. Given the preclinical and clinical challenges, such molecular mechanisms might be better reviewed by focusing not only on positive but also negative findings. This article focuses on recent (in particular 2014 to mid-2016) findings about the immunosuppressive molecules expressed by MSCs. Recent review articles focusing on these various molecules include English [30], Shi et al. [31], and Uccelli et al. [11]. We acknowledge that our coverage is limited to a few selected papers allowing us to focus on details for each molecule.

On Slippage-Like Mutation Dynamics Within Genes: A Study of Pseudogenes and 3′UTRs

Recent analyses show that even very short repetitive sequences are prone to slippage-like mutations. Characterization of the dynamics of such mutations should increase our knowledge about the background frequencies of extension and contractions commonly occurring within genes, allowing the effect of the selections on particular repetitive motifs to be assessed. Consideration of the slippage-like changes may also help the reconstruction of the phylogenetic tree of a gene. In our previous report (Nishizawa and Nishizawa 2002), we described a method which finds the best alignment between two sequences by performing simulations of various types of changes including slippage-like ones. The method can be used to optimize the ‘‘slippage-controlling parameters’’ so that they explain the observed evolution of sequences well. While we believe that the presented framework was reasonable, we acknowledge that the accuracy of the parameter estimation in the paper was limited due to a dearth of informative pseudogenes at the time. The purpose of this letter is to present (1) the result of the pseudogene analysis that supplements our previous report and, in addition, (2) the results of 3¢UTR analyses, which have implications for evolutionary changes of 3¢UTR sequences. We extended the analyses to 160 human and 120 mouse pseudogenes, which were randomly chosen from the recent database (Echols et al. 2002; Zhang et al. 2004). The total numbers of insertions and deletions (‘‘the indel-total’’) that we estimated to have occurred for human and mouse pseudogene/functional-gene pairs were 627 and 931, respectively. Based on the simple binominal model, these numbers can be considered to produce only the negligible sampling errors, and therefore the p-values in the following primarily reflect the potential error in our likelihood estimations. Our method simulates the changes of the sequences in a manner such that the various patterns of insertions and deletions can be examined (Nishizawa and Nishizawa 2002). We have categorized the slippage-like changes into the three types of changes that are accounted for by the parameters f1, f2, and f3, respectively. The f1 parameter indicates the probability, for the insertion events scheduled during the simulation, that each insertion becomes a duplication (instead of an insertion of random nucleotides), as represented by the example ATCAGC fi ATCA CAGC or ATCAGCGC, where the introduced 2-nt segment is underlined. (If, for example, f1 is set to 0.6, the simulation is performed such that 60% of insertions [of any lengths] are duplicative, where 30% are the duplications of the upstream nucleotides and the remaining 30% are those of the downstream nucleotides.) Our previous results suggested that human pseudogenes are more likely to undergo the f1-type slippages than those of murids. In fact, further analyses show no difference between human and murids. For both the human and the mouse pseudogenes, the likelihood profile for f1 shows peak at f1 = 0.5, or 50% are duplicative, indicating that pseudogenes of human and mouse do not have an appreciable difference in f1 -mutations. The f2 and f3 parameters specify the ‘‘extra’’ probability (compared with nonrepetitive sequences) that the very short repetitive sequences, such as GGGG and CACACA, are subjected to the extension and contraction of the repeat, respectively. (For Correspondence to: Manami Nishizawa; email: nmanami@aol.com J Mol Evol (2005) 60:274–275 DOI: 10.1007/s00239-004-0109-5

Free energy of helical transmembrane peptide dimerization in OPLS-AA/Berger force field simulations: inaccuracy and implications for partner-specific Lennard-Jones parameters between peptides and lipids

Abstract Interactions between transmembrane (TM) peptides are important in biophysical chemistry, but there are few studies assessing atomistic simulation parameters concerning the energetics of interactions of TM helical peptides. Our potential of mean force analysis using OPLS-AA protein/Berger lipid force fields (FFs) shows that the dimerisation energy of (AALALAA)3 helical peptides in the dioleoylphosphatidylcholine bilayer is −4.4 kJ/mol, which was much smaller than the reported experimental value (−12.7 kJ/mol), thus calling for improvement of parameters of the combined FFs. As each of the FFs has been independently developed, we then tested the effects of downscaling the Lennard-Jones (LJ) energy terms between the OPLS-AA atoms and Berger lipid atoms, preserving the parameters within each FF. A 0.9-fold rescaling of the LJ energies was found to render the dimerisation energy close to the experimental value. Solvation of backbone atoms as well as side chain atoms in lipids is crucial for the TM helix interaction. In similar analyses, GROMOS 53A6 FF exhibited as weak dimerisation propensity (~−5.2 kJ/mol) as OPLS-AA/Berger, but CHARMM36 showed relatively accurate propensity (~−9.9 kJ/mol). Challenges and strategies in rendering the TM interaction energy realistic within the framework of current FFs are discussed.