Facilitation of Nanoscale Thermal Transport by Hydrogen Bonds

Thermal transport performance at the nanoscale and/or of biomaterials is essential to the success of many new technologies including nanoelectronics, biomedical devices, and various nanocomposites. Due to complicated microstructures and chemical bonding, thermal transport process in these materials has not been well understood yet. In terms of chemical bonding, it is well known that the strength of atomic bonding can significantly affect thermal transport across materials or across interfaces between materials. Given the intrinsic high strength of hydrogen bonds, this dissertation explores the role of hydrogen bonds in nanoscale thermal transport in various materials, and investigates novel material designs incorporating hydrogen bonds for drastically enhanced thermal conduction. Molecular dynamics simulation is employed to study thermal transport processes in three representative hydrogen-bonded materials: (1) crystalline motifs of the spider silk, silkworm silk and synthetic silk, (2) crystalline polymer nanofibers, and (3) polymer nanocomposites incorporating graphene or functionalized graphene. Computational and theoretical investigations demonstrate that hydrogen bonds significantly facilitate thermal transport in all three material systems. The underlying molecular mechanisms are systematically investigated. The results will not only contribute new physical insights, but also provide novel concepts of materials design to improve thermal properties towards a wide range of applications

Identification and functional characterization of an ABC transporter of Haemonchus contortus, the P-glycoprotein 13

Les lactones macrocycliques (LM) sont des anthelminthiques (AH) à effet paralysant très utilisés chez les animaux et les humains contre les nématodes parasites. Cependant, leur succès thérapeutique est compromis par la résistance croissante aux LM, qui pourrait être en partie dû aux ABC transporteurs P-glycoprotéines (Pgps) sélectionnés et surexprimés chez les nématodes résistants aux LM. Dans ce travail, nous avons étudié plus précisément la P-glycoprotéine 13 du parasite de petits ruminants, Haemonchus contortus. Son orthologue chez le modèle nématode C. elegans, Cel-Pgp-13, est exprimé dans les amphides, structures qui ont été associées à la sensibilité aux AH chez C. elegans et H. contortus. Pour prédire la capacité des Pgps de nematode à transporter des drogues, incluant des LM et autres AH, nous avons développé un modèle de docking in silico. Nous avons utilisé la structure cristallographique de C. elegans Pgp-1 (Cel-Pgp-1), et nous avons montré la liaison avec une forte affinité de plusieurs ligands décrits comme activateurs de sa fonction ATPasique. Nous avons aussi décrit une forte affinité des LM, et un site spécifique de liaison de ces composés à Cel-Pgp-1. Cette approche représente un outil important pour prédire les interactions entre AH, et pour concevoir rationnellement de nouveaux inhibiteurs compétitifs des Pgps de nématode, dans le but d'améliorer les stratégies thérapeutiques. Sur la base de cette approche, nous avons prédit la structure 3D de Hco-Pgp-13 à partir du cristal de Cel-Pgp-1 afin d'étudier son intéraction avec des substrats potentiels, en particulier les LM. Nous avons trouvé des affinités similaires pour différents composés précédemment testés sur Cel-Pgp-1. In vitro, la mesure de l'activité ATPasique montre que l'actinomycine D est un substrat de Hco-Pgp-13. Nos données démontrent la présence possible d'un domaine de reconnaissance multispécifique sur ce transporteur de parasite. La détermination par immunofluorescence de l'expression de Hco-Pgp-13 a montré une distribution tissulaire large indiquant que Hco-Pgp-13 pourrait jouer un role important dans le transport de substrats endogènes et/ou exogènes. En conclusion, ce travail permet de mieux comprendre le rôle des Pgps de nématodes dans le transport de médicaments AH, tant au niveau de l'organisme modèle C. elegans que du nématode parasite H. contortus. Cette étude suggère la conservation de la fonction de tranporteur ABC multidrogue dans ces espèces. La localisation de Hco-Pgp-13 sur les structures amphidiales, et son éventuelle implication dans la résistance aux médicaments et à la survie de H. contortus à l'exposition à des composés AH, restent à préciser.Macrocyclic lactones (ML) are paralyzing anthelmintics used in animals and humans against parasite nematodes. However, their therapeutic success is compromised by the spread of ML resistance. This might be at least partly due to P-glycoproteins (Pgps) ABC transporters that are selected and overexpressed in ML-resistant nematodes. Deciphering the role of the 10 Pgps expressed in the parasite of small ruminants Haemonchus contortus is thus of major importance to guaranty anthelmintic (AH) efficacy of various drugs. Here we focused on Hco-Pgp-13 due to the expression in the amphids of its closest ortholog in the model nematode C. elegans. Indeed, the amphids represent a putative entry route of drugs to reach AH targets in the nervous system and have been linked to AH susceptibility in C. elegans and H. contortus. In order to predict the capacity of nematode Pgps to transport drugs, including ML and otherAH, we have developed an in silico drug docking model. We have used C. elegans Pgp-1 (Cel-Pgp-1) crystal structure and have showed a high affinity binding of several ligands that have been shown to be activators of its ATPase function. ML were also found to bind with high affinity to Cel-Pgp-1, on a specific binding site. This approach provides a valuable tool to predict drug-drug interactions and to rationally design new competitive inhibitors of nematode Pgps, in order to improve anthelmintic therapeutics. We then predicted a putative 3D structure of Hco-Pgp-13 based on the recently released crystal of Cel-Pgp-1, with which it presented a high homology. This allowed the study of the interaction of Hco-Pgp-13 with potential substrates, in particular ML. We found similar affinities for various drugs previously tested on Cel-Pgp-1, supporting the good homology of these two proteins. Together with in vitro ATPase assay experiments that confirmed the substrate status of actinomycin D, this indicates a possible multispecifc recognition capacity of this parasitic transporter. The determination of Hco-Pgp-13 localization using immunohistochemistry showed a wide tissue expression consistent with a critical role for Hco-Pgp-13 in endogenous and/or exogenous substrate transport. In conclusion, this work provides insights into the role of nematode Pgps in transporting AH drugs, both at the level of the model organism C. elegans and of the parasitic nematode H. contortus. This suggests a high homology of function conserved between ABC tranporters in these species. The localization of such protein on amphidial structures and its possible involvement in drug resistance and survival of H. contortus to exposure to AH compounds remain to be precised

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Mechanisms of ER Protein Import

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome. Most of these proteins fulfill their functions either in the membrane or lumen of the ER plus the nuclear envelope, in one of the organelles of the pathways for endo- and exocytosis (ERGIC, Golgi apparatus, endosome, lysosome, and trafficking vesicles), or at the cell surface as plasma membrane or secreted proteins. An increasing number of membrane proteins destined to lipid droplets, peroxisomes or mitochondria are first targeted to and inserted into the ER membrane prior to their integration into budding lipid droplets or peroxisomes or prior to their delivery to mitochondria via the ER-SURF pathway. ER protein import involves two stages, ER targeting, which guarantees membrane specificity, and the insertion of nascent membrane proteins into or translocation of soluble precursor polypeptides across the ER membrane. In most cases, both processes depend on amino-terminal signal peptides or transmembrane helices, which serve as signal peptide equivalents. However, the targeting reaction can also involve the ER targeting of specific mRNAs or ribosome–nascent chain complexes. Both processes may occur co- or post-translationally and are facilitated by various sophisticated machineries, which reside in the cytosol and the ER membrane, respectively. Except for resident ER and mitochondrial membrane proteins, the mature proteins are delivered to their functional locations by vesicular transport

Funktionelle Charakterisierung der C-terminalen-Domänen des Korepressors N-CoR

Although in general cells are genetically identical in multicellular organisms, the differential expression of genomic information enables cell type definition and specific organ function. In eukaryotic cells, the DNA is associated with histone and non-histones proteins into a restrictive structure called chromatin. Assembly into chromatin does not only protect and package the linear double stranded DNA into the nucleus but is fundamental for the execution of diverse genetic programs. Posttranslational modifications of histones regulate the accessibility of the DNA to transcription factors and serve as scaffold for binding of regulatory proteins. Nuclear receptors are transcription factors that bind specific target sequences on the DNA and recruit transcriptional coregulators at the promoter. These are able to modify the chromatin structure in an activating or repressing manner. The contribution of corepressors to the biological actions of nuclear receptors has turned out to be essential. Impaired corepressor function can be the cause of endocrine malfunctions, neoplastic diseases or severe developmental abnormalities. To better understand the role of the nuclear receptor corepressor N-CoR the unknown function of the extreme C-terminus was investigated. In this thesis the interaction of N-CoR with the non-POU-domain containing octamer-binding protein Non0/p54nrb, that was found tobe a potential interaction partner in a yeast-two-hybrid screen, was confirmed. This protein contains two RNA recognition motifs (RRM) and is described as a multifunctional protein since it is involved in transcription Initiation as well as in pre-mRNA processing. The RRM1 motif was determined to be essential and sufficient for the interaction with N-CoR. Obtaining dominant negative effect with the Non0/p54nrb RRM1 deletion mutant in functional reporter assays, data support that NonO modulates the capacity of N-CoR to repress and alters the recruitment of N-CoR by nuclear receptors to targeted Promoters. Additional analyses suggest that the N- and C- terminus of N-CoR are involved in intramolecular interactions and that they regulate each other. Taken results together a functional model is proposed that supports the biological relevance of the interaction of N-CoR with NonO and the function of N-CoR C-terminus acting as asensor that evaluates the ratio of corepressors and coactivators in the nuclear receptor environment. N-CoR repressive capacity would be altered by modulating factors like NonO that interacts with N-CoR C-terminus. The mechanism support that splicing and transcription regulation are physically and functionallylinked to ensure the appropriate amount of messager RNA to be transcript and process in response to stimulation intensity and cell context.Die fehlerhafte Rekrutierung von Korepressoren wie dem Protein N-CoR (nuclear receptor co-repressor) ist die Ursache von genetischer Krankheiten und verschiedener Leukämien. Darüber hinaus ist N-CoR vermittelte Repression für die Entwicklung von Säugern entscheidend. N-CoR Knockoutmäuse sterben in der mittleren Phase der Embryonalentwicklung und zeigen Defekte in der Reifung von Erythrozyten, Thymozyten und in der Entwicklung verschiedener neuronaler Strukturen. Dies ist zum Teil auf die Störung der Repression zurückzuführen, die von nuklearen Hormonrezeptoren wie Retinsäure- und Thyroidhormonrezeptoren vermitteltet wird. N-CoR ist in der Zelle mit Histondeacetylasen (HDACs) komplexiert. Diese Enzyme bewirken im Zusammenspiel mit den Histonacetyltransferasen durch Deacetylierung beziehungsweise Acetylierung von Histonen eine dynamische Modifikation des Chromatins und beeinflussen so die Transkription von Genen, Obwohl N-CoR ein seit längerer Zeit bekanntes Protein ist, enthält es bislang uncharakterisierte Domänen wie die äußerste carboxyterminale Region. Diese Domäne wurde als Köder in einem Hefe-Zwei-Hybrid Screen zur Identifizierung von Interaktionspartnern des N-CoR C-Terminus eingesetzt. In der vorliegenden Arbeit wurde die Interaktion von N-CoR mit dem multifunktionellen Protein Non0/p54nrb (non POU domain containing octamer binding protein) bestätigt und die biologische Bedeutung dieser Interaktion wurde untersucht. Non0/p54nrb ist an verschiedenen Prozessen wie der RNA Polymerase II Komplexbildung und -aktivierung, Splicesosom Komplexbildung und der Bildung des RNA Polyadenylierungskomplexes beteiligt. Dieses RNA-Erkennungsmotive (RRM) enthaltende Protein spielt eine regulatorische Rolle in der Hormonrezeptor- abhängigen Transkriptionsregulation. Es wurde festgestellt, dass das RRM1 Motiv des Proteins Non0/p54nr für die Interaktion mit N-CoR entscheidend ist und, dass NonO sowohl die Interaktion zwischen nuklearen Rezeptoren und N-CoR als auch die N-CoR Repressionsaktivität beeinflussen kann. Ferner wurde gezeigt, dass der extreme N-CoR C- Terminus möglicherweise eine regulierende Domäne ist, welche die N-CoR Repressionsaktivität des Amino-Terminus modulieren kann. Das vorgeschlagene funktionelle Modell stellt ein bislang nicht bekanntes Regulationselement in der Kontrolle der Genexpression dar. N-CoR repressive Kapazität und die Rekrutierung des Korepressors zu Promotorenregionen wäre nicht nur von dem Aktivierungsstandder Nuklearen Rezeptoren abhängig aber auch von dem Verhältnis von Koaktivator und Korepressor in der Umgebung der Nuklearen Rezeptoren und von den Interaktionen mit modulierende Protein wie Non0/p54nrb welche die physische Verbindung zwischen transkriptionsakivierenden und -reprimierenden Prozessen sein könnte

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Single-Molecule Measurements of Complex Molecular Interactions in Membrane Proteins using Atomic Force Microscopy

Single-molecule force spectroscopy (SMFS) with atomic force microscope (AFM) has advanced our knowledge of the mechanical aspects of biological processes, and helped us take big strides in the hitherto unexplored areas of protein (un)folding. One such virgin land is that of membrane proteins, where the advent of AFM has not only helped to visualize the difficult to crystallize membrane proteins at the single-molecule level, but also given a new perspective in the understanding of the interplay of molecular interactions involved in the construction of these molecules. My PhD work was tightly focused on exploiting this sensitive technique to decipher the intra- and intermolecular interactions in membrane proteins, using bacteriorhodopsin and bovine rhodopsin as model systems. Using single-molecule unfolding measurements on different bacteriorhodopsin oligomeric assemblies - trimeric, dimeric and monomeric - it was possible to elucidate the contribution of intra- and interhelical interactions in single bacteriorhodopsin molecules. Besides, intriguing insights were obtained into the organization of bacteriorhodopsin as trimers, as deduced from the unfolding pathways of the proteins from different assemblies. Though the unfolding pathways of bacteriorhodopsin from all the assemblies remained the same, the different occurrence probability of these pathways suggested a kinetic stabilization of bacteriorhodopsin from a trimer compared to that existing as a monomer. Unraveling the knot of a complex G-protein coupled receptor, rhodopsin, showed the existence of two structural states, a native, functional state, and a non-native, non-functional state, corresponding to the presence or absence of a highly conserved disulfide bridge, respectively. The molecular interactions in absence of the native disulfide bridge mapped onto the three-dimensional structure of native rhodopsin gave insights into the molecular origin of the neurodegenerative disease retinitis pigmentosa. This presents a novel technique to decipher molecular interactions of a different conformational state of the same molecule in the absence of a high-resolution X-ray crystal structure. Interestingly, the presence of ZnCl2 maintained the integrity of the disulfide bridge and the nature of unfolding intermediates. Moreover, the increased mechanical and thermodynamic stability of rhodopsin with bound zinc ions suggested a plausible role for the bivalent ion in rhodopsin dimerization and consequently signal transduction. Last but not the least, I decided to dig into the mysteries of the real mechanisms of mechanical unfolding with the help of well-chosen single point mutations in bacteriorhodopsin. The monumental work has helped me to solve some key questions regarding the nature of mechanical barriers that constitute the intermediates in the unfolding process. Of particular interest is the determination of altered occurrence probabilities of unfolding pathways in an energy landscape and their correlation to the intramolecular interactions with the help of bioinformatics tools. The kind of work presented here, in my opinion, will not only help us to understand the basic principles of membrane protein (un)folding, but also to manipulate and tune energy landscapes with the help of small molecules, proteins, or mutations, thus opening up new vistas in medicine and pharmacology. It is just a matter of a lot of hard work, some time, and a little bit of luck till we understand the key elements of membrane protein (un)folding and use it to our advantage

Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 2.0

This book, "Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity", presents five papers on human cytochrome P450 (CYP) and P450 reductase, three reviews on the role of CYPs in humans and their use as biomarkers, six papers on CYPs in microorganisms, and one study on CYP in insects. The first paper reports the in silico modeling of human CYP3A4 access channels. The second uses structural methods to explain the mechanism-based inactivation of CYP3A4 by mibefradil, 6,7-dihydroxy-bergamottin, and azamulin. The third article compares electron transfer in CYP2C9 and CYP2C19 using structural and biochemical methods, and the fourth uses kinetic methods to study electron transfer to CYP2C8 allelic mutants. The fifth article characterizes electron transfer between the reductase and CYP using in silico and in vitro methods, focusing on the conformations of the reductase. Then, two reviews describe clinical implications in cardiology and oncology and the role of fatty acid metabolism in cardiology and skin diseases. The second review is on the potential use of circulating extracellular vesicles as biomarkers. Five papers analyze the CYPomes of diverse microorganisms: the Bacillus genus, Mycobacteria, the fungi Tremellomycetes, Cyanobacteria, and Streptomyces. The sixth focuses on a specific Mycobacterium CYP, CYP128, and its importance in M. tuberculosis. The subject of the last paper is CYP in Sogatella furcifera, a plant pest, and its resistance to the insecticide sulfoxaflor

Dissecting Dopamine D2 Receptor Signaling

Dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that activates G protein and arrestin signaling molecules. D2R antagonism has been a hallmark of antipsychotic medications for more than half a century. However, this drug-class is associated with substantial side effects that decrease quality of life and medication compliance. The development of novel antipsychotic medications with superior therapeutic and side effect profiles has been hampered in part due to a poor understanding of the specific D2R populations and downstream signaling molecules that must be blocked to confer therapeutic efficacy. It has been proposed that antipsychotic medications confer their effects through the blockade of arrestin but not G protein signaling downstream of D2R, and thus substantial efforts have gone towards the development of ligands that selectively block arrestin signaling. However, this approach suffers from several major limitations, namely that blockade of G protein signaling may also be important in conferring antipsychotic effects. Moreover, currently available pharmacological and genetic tools that have been used to probe G protein and arrestin signaling downstream of D2R in vivo suffer from on- and off-target effects that add substantial confounds to our understanding of these processes. Herein, we describe the development of several tools that can be used to probe G protein and arrestin-mediated processes in vivo with high specificity, as well as mechanisms by which these processes are activated