蛋白質分泌及胞吐作用是指蛋白質在內質網(ER)中合成後前往質膜(PM) ， 隨後， 被分泌到達細胞外的過程。 而細胞分泌路徑是指蛋白質途經數個含有膜包被的細胞器後運送到细胞之外。 這些細胞器，包括內質網， 高爾基體， 反式高爾基網絡(TGN) 及質膜。 分泌蛋白分泌出细胞之外後， 在细胞外基質中進行其功能。 / 為達到這項研究的目的，我們結合了細胞、分子和生物化學上的方法， 來對蛋白質運輸路徑及參與蛋白質分泌的細胞器進行研究。首先，通過MALDI-MS/MS對煙草懸浮BY-2細胞中的原態分泌性蛋白質進行分析。第二，把已識別的分泌蛋白包括陽離子過氧化物酶同工酶40K（40K）和N1過氧化物酶（N1），透過轉基因細胞的GFP融合表達方式、及應用特異性抗體於免疫螢光和膠體金免疫電鏡上的測定來對其特性作進一步分析。 第三，總合以上的研究，煙草懸浮BY-2細胞的典型蛋白質分泌路徑次序為質網 - 高爾基體 - 反式高爾基網絡 - 質膜。 / Protein secretion or exocytosis is the process by which proteins synthesized in the endoplasmic reticulum (ER) travel to the plasma membrane (PM) for their subsequent secretion outside of the cell. The secretory pathway responsible for protein secretion contains several membrane-bounded organelles such as the ER, Golgi apparatus, trans-Golgi Network (TGN), and PM. The secreted proteins move outside of the cell and perform their functions in the extracellular matrix. / The general objective of this study was to examine the transport pathways and organelles involved in protein secretion in plant cells using a combination of cellular, molecular and biochemical approaches. First, major native secreted proteins in suspension cultures of tobacco BY-2 culture cells were identified via MALDI-MS/MS analysis. Second, the identified secreted proteins, cationic peroxidase Isozyme 40K (40K) and peroxidase N1 (N1), were further characterized by examining the GFP fusion expression of transgenic cell lines and by generating specific antibodies in immunofluorescent and immunogold electron microscope (EM) studies. Third, throughout all of these studies, a typical ER-Golgi-TGN-PM pathway was mapped for protein secretion in tobacco BY-2 cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lam, Chun Kok. / "December 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-84). / Abstracts also in Chinese. / Thesis /Assessment Committee --- p.i / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Secreted protein --- p.1 / Chapter 1.2. --- Secretory Pathway --- p.1 / Chapter 1.3. --- Protein secretion --- p.2 / Chapter 1.4. --- Plant Peroxidases --- p.3 / Chapter 1.5. --- Project Objective --- p.4 / Chapter 1.6. --- Significance --- p.4 / Chapter Chapter 2 --- Materials and Methods --- p.6 / Chapter 2.1. --- Mass spectrometry analysis --- p.6 / Chapter 2.2. --- Generation of 40K/N1-GFP construct --- p.7 / Chapter 2.2.1. --- For transient expression --- p.7 / Chapter 2.2.2. --- For stable expressing constructs --- p.7 / Chapter 2.3. --- Transient expression of 40K/N1-GFP --- p.7 / Chapter 2.4. --- Generation of transgenic cell lines --- p.8 / Chapter 2.5. --- Fluorescence microscopic screening --- p.9 / Chapter 2.6. --- Generation and characterization of antibodies specific for 40K/N1 peroxidase --- p.9 / Chapter 2.7. --- Confocal immunofluorescence studies --- p.10 / Chapter 2.8. --- (TIRF) Total internal reflection fluorescence microscopy --- p.11 / Chapter 2.9. --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.11 / Chapter 2.10. --- Drug Treatment --- p.12 / Chapter 2.10.1. --- Dexamethasone (dex) --- p.12 / Chapter 2.10.2. --- Brefeldin A (BFA)/Concanamycin A (ConcA) --- p.12 / Chapter 2.11. --- Salt treatment (plasmolysis) --- p.13 / Chapter 2.12. --- EM (electron microscopy) study --- p.13 / Chapter Chapter 3 --- Results --- p.14 / Chapter 3.1. --- Protein secretion from tobacco BY2 cells --- p.14 / Chapter 3.2. --- Western blot analysis --- p.15 / Chapter 3.3. --- Protein expression in tobacco plant tissues --- p.15 / Chapter 3.4. --- EM labeling on the wild type BY-2 cells --- p.16 / Chapter 3.5. --- Localization in the tobacco root tip apoplast --- p.17 / Chapter 3.6. --- 40K/N1 peroxidase transient/stable cell line expression --- p.18 / Chapter 3.7. --- Time course study of 40K/N1 peroxidase-GFP cell line expression after induction --- p.18 / Chapter 3.8. --- Plasmolysis (salt treatment analysis) --- p.20 / Chapter 3.9. --- Brefeldin A (BFA) and concanamycin A (ConcA): Trafficking through the Golgi and TGN --- p.20 / Chapter 3.10. --- Examining exocytosis by total internal reflectance fluorescence (TIRF) --- p.22 / Chapter 3.11. --- Immunolabeling study --- p.23 / Chapter 3.12. --- EM study on transgenic 40K & N1 peroxidase-GFP cell lines --- p.24 / Chapter Chapter 4 --- Discussion --- p.26 / Chapter 4.1. --- Trafficking from the ER to the extracellular matrix --- p.26 / Chapter 4.2. --- Secretion through PM by exocytosis --- p.28 / Chapter 4.3. --- Time required for the secretory pathway --- p.29 / Chapter 4.4. --- Similarities of 40K and N1 --- p.30 / Chapter 4.5. --- Future perspectives --- p.30 / References --- p.79

R1162 is a broad-host range, mobilizable plasmid conferring resistance to streptomycin and sulfonamides. Efficient conjugative mobilization of R1162 requires three plasmid-encoded proteins: MobA, MobB and MobC. MobA binds plasmid DNA at the origin of transfer (oriT), nicks the subsequently transferred strand and ligates the ends of the strand after transfer into the recipient. The N-terminal region of this protein carries out this DNA processing. The C-terminal half is a primase required to initiate DNA synthesis at two single-stranded priming sites sites, oriL and oriR, during vegetative plasmid replication. The primase region of MobA is not necessary for DNA processing by the N-terminal part of the protein, however its role in strand replacement during conjugation is not clearly defined. This study demonstrates that R1162 can undergo multiple rounds of transfer from a single plasmid molecule. The presence of oriL increases the frequency of second-round transfer, presumably due to initiation of replacement strand synthesis at this site by R1162 primase in the donor. Priming at oriR by the primase region of MobA is required for efficient replacement strand synthesis in the recipient when the plasmid is transferred to Salmonella. When the plasmid is transferred into E. coli, the plasmid-encoded priming system is not required for strand replacement in the recipient, presumably due to a host-encoded mechanism capable of priming the transferred strand. Transport of MobA through the R751 conjugative pore was also investigated. The two domains of MobA can be transported to recipient cells independently of each other. However, MobB is required for the transport of either fragment. Two sites, named the R-site and the P-site, are located in the relaxase and primase domains of MobA, respectively, and make up part of the signals required for MobA transport. Unlike previously described type IV transport signals, domain structure is required for the MobA transport signals to be active. / text

The work presented in this thesis centres on the development of a work-flow in which coarse-grained molecular dynamics (MD) simulations of a planar phospholipid bilayer, containing membrane proteins, is used to parameterize a larger-scale simplified bilayer model. Using this work-flow, repeat simulations and simulations of larger systems are possible, better enabling the calculation of bulk statistics for the system. The larger-scale simulations can be run on commercial hardware, once the initial parameterization has been performed. In the simplified representation, each protein was initially only represented by the position of its centre of mass and later with the inclusion of its orientation. The membrane protein used throughout most of this work was the bacterial outer membrane protein NanC, a member of the KdgM family of proteins. To parameterize the motion and interaction of proteins using MD, the potential of mean force (PMF) for the pairwise association of two proteins in a bilayer was calculated for a variety of orientational combinations, using a modified umbrella sampling procedure. The relative orientations chosen represented extreme examples of the contact regimes between the two proteins: they approximately corresponded to maxima and minima of the solvent inaccessible surface area, calculated when the proteins were in contact. These PMFs showed that there was a correlation between the buried surface area and the depth of the potential well in the PMF; this is something that, to date, has only been observed in these relatively-'featureless' membrane proteins (but is seen in globular proteins), where the effect of the interactions with lipids in the bilayer plays a larger role. Features in the PMF were observed that resulted from the preferential organization of lipids in the region between the two proteins. These features were small wells in the PMF, which occurred at protein separations that corresponded to the intervening lipids being optimally packed between the proteins. This result further highlighted the role that the lipids in the bilayer played in the interaction between the NanC proteins. The simplified bilayer model was parameterized using the PMFs and the relationship between buried surface area and potential well depth. The initial model included only the proteins' positions. A series of Monte Carlo simulations were performed in order to compare the system behaviour to that of an equivalent MD simulation. Initially, the MD simulation and our parameterized model did not show a good agreement, so a Monte Carlo scheme that incorporated cluster-based movements was implemented. The agreement between the MD simulation and the simulations of our model using the cluster-based scheme, when comparing diffusive and clustering behaviour, was good. Including the orientation-dependent features of the parameterization resulted in the emergence of behaviour that was not clearly detectable in the MD simulation. Finally, attempts were made to parameterize the model using PMFs for the association of rhodopsin from the literature. Rhodopsin was a much more complicated protein to represent: there was not a clear correlation between surface area and the features of the PMF, and the geometry of the interaction between two rhodopsins was more complicated. Simulations of the 'rows-of-dimers' system of rhodopsin, observed in disc membranes, was not entirely well represented by the model; for such a closely packed system, where the number of lipids is much closer to the number of proteins, the use of an implicit-lipid model meant that the effect of the reduced lipid mobility was not adequately captured. However, the model accurately captures the orientational composition of the system. Future work should be focussed on incorporating explicit representations of the lipid in the system so that the behaviour of close-packed systems are better represented.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are dynamic membrane spanning proteins which mediate the rapid influx of Na+ during the upstroke of the action potential (AP). In addition to the large inward Na+ currents responsible for the upstroke of the AP, some VGSC isoforms produce smaller, subthreshold Na+ currents, which can influence the excitable properties of neurons. An example of such a subthreshold current is resurgent Na+ current (INaR). These unusual currents are active during repolarization of the membrane potential, where the channel is normally refractory to activity. INaR exhibit slow gating kinetics and unusual voltage-dependence derived from a novel mechanism of channel inactivation which allows the channel to recover through an open configuration resulting in membrane depolarization early in the falling phase of the AP, ultra-fast re-priming of channels, and multiple AP spikes. Although originally identified in fast spiking central nervous system (CNS) neurons, INaR has recently been observed in a subpopulation of peripheral dorsal root ganglion (DRG) neurons. Because INaR is believed to contribute to spontaneous and high frequency firing of APs, I have hypothesized that increased INaR may contribute to ectopic AP firing associated with inherited and acquired disorders of excitability. Specifically, this dissertation explores the mechanisms which underlie the electrogenesis of INaR in DRG neurons and determines whether the biophysical properties of these unique currents were altered by mutations that cause inherited muscle and neuronal channelopathies or in an experimental model of nerve injury. The results demonstrate that (1) multiple Na+ channel isoforms are capable of producing INaR in DRG neurons, including NaV1.3, NaV1.6, and NaV1.7, (2) inherited muscle and neuronal channelopathIy mutations that slow the rate of channel inactivation increase INaR amplitude, (3) temperature sensitive INaR produced by select skeletal muscle channelopthy mutations may contribute to the triggering of cold-induced myotonia, and (4) INaR amplitude and distribution is significantly increased two weeks post contusive spinal cord injury (SCI). Taken together, results from this dissertation provide foundational knowledge of the properties and mechanism of INaR in DRG neurons and indicates that increased INaR likely contributes to the enhanced membrane excitability associated with multiple inherited and acquired disorders of excitability.

Chan, Wai Hon. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 134-141). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / 摘要 --- p.5 / Content --- p.6 / List of Abbreviations and symbols --- p.10 / Chapter Chapter 1 --- Introduction --- p.13 / Chapter 1.1 --- The Severity of Influenza A Virus --- p.14 / Chapter 1.2 --- Introduction to Influenza A Virus --- p.15 / Chapter 1.3 --- What is Nucleoprotein? --- p.17 / Chapter 1.4 --- Multifunctional role of Nucleoprotein --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Other Viral Components --- p.19 / Chapter 1.4.1 --- Interaction of Nucleoprotein with Cellular Components --- p.22 / Chapter 1.5 --- Aims of study --- p.23 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.25 / Chapter 2.1.1 --- Chemical reagents --- p.25 / Chapter 2.1.2 --- Buffers --- p.28 / Chapter 2.1.2.1 --- Preparation of Buffers --- p.28 / Chapter 2.1.2.2 --- Buffer for Common Use --- p.28 / Chapter 2.1.3 --- Plasmids and Strains --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Molecular Cloning --- p.32 / Chapter 2.2.2 --- "Expression of the Recombinant NP-WT/ mutants and importin α1, 3and 5 in E.coli" --- p.46 / Chapter 2.2.3 --- Purification of the NP WT/ variants --- p.50 / Chapter 2.2.4 --- "Purification of importin αl, 3 and 5" --- p.53 / Chapter 2.2.5 --- In vitro interaction study between NP and importin α --- p.56 / Chapter 2.2.6 --- In vivo interaction study between NP and importin α --- p.59 / Chapter 2.2.7 --- In vivo analysis to study NP-NP homo-oligomerization --- p.64 / Chapter 2.2.8 --- In vitro static light scattering analysis to determine NP oligomeric state --- p.68 / Chapter 2.2.9 --- Control experiments to verify NP-polymerases and NP-RNA interaction --- p.69 / Chapter Chapter 3 --- Functional analysis of influenza H5N1 nucleoprotein tail loop for oligomerization and ribonucleoprotein activities / Chapter 3.1 --- Introduction --- p.72 / Chapter 3.2 --- Results --- p.75 / Chapter 3.2.1 --- Tail loop insertion is maintained by intra- and inter-molecular interactions --- p.75 / Chapter 3.2.2 --- NP mutants display defective transcription-replication activity --- p.77 / Chapter 3.2.3 --- Expression and purification of defective NP variants --- p.80 / Chapter 3.2.4 --- Defective NP variants interact with RNA and the polymerase complex --- p.85 / Chapter 3.2.5 --- Defective NP variants possess abnormal oligomeric states in vitro --- p.91 / Chapter 3.2.6 --- NP variants with impaired RNP activity cannot form homo-oligomers in vivo --- p.95 / Chapter 3.3 --- Discussion --- p.98 / Chapter Chapter 4 --- Biophysical characterization of the interaction between influenza nucleoprotein and importin α / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Results --- p.108 / Chapter 4.2.1 --- Expression and Purification of NP-WT/ NLS variants and Importin a --- p.108 / Chapter 4.2.2 --- Pull down Assay --- p.113 / Chapter 4.2.3 --- Light scattering analysis (NP-lmportin α5) --- p.114 / Chapter 4.2.4 --- Binding assay of NP NLS variants with importin α5 --- p.115 / Chapter 4.2.5 --- BIAcore 3000 Surface Plasmon Resonance (NP-lmportin α5) --- p.118 / Chapter 4.2.6 --- QRT-PCR (NP-lmportin a5) --- p.123 / Chapter 4.3 --- Discussion --- p.125 / References --- p.134

Many biomolecular processes involve mechanical force-induced reactions in the cell, such as translocation, and mechanical stretching of biopolymers. Recent advances in single molecule manipulation techniques make it possible to apply mechanical force to individual biomolecules and study their dynamics. To gain molecular level understanding of these processes and to interpret the single-molecule experiments, we used Langevin dynamics simulations of coarse-grained biopolymer models. Our result show that the mechanism of translocation of proteins through pores depends on the pore diameter, on the magnitude of the pulling force and on whether the force is applied at the N- or the C-terminus of the chain. In addition, the translocation kinetics of peptides varies with their stability. The mechanism of protein translocation is found to be different from that of a structureless polypeptide of the same length. We further showed that unfolding mechanism of translocation process is different from when the same protein is stretched between its C- and N-termini. We also studied the mechanical and chemical/thermal denaturation of proteins. We observed that the free energy profile along the mechanical reaction coordinate and the chemical reaction coordinate are different. In our protein model, the mechanical and chemical/thermal denaturation cannot be simply explained in terms of a simple onedimensional free energy landscape. We further analyzed the spontaneous folding and refolding under a constant force and found that refolding generally occurs via different mechanisms. Similarly, we investigated the protein unfolding/refolding under the applied force that varies with a constant loading rate. This study shows that unfolding/refolding pathways are generally similar for low loading/unloading rates while they become different for high loading/unloading rates. Finally, we studied the dynamics of molecular friction knots formed by a pair of polymer strands. We examined different knot types, and different polymer sequences. Depending on the knot type and the nature of the polymer, we observed two different behaviors when the force F is exerted to separate the polymer strands. The knot between polymer strands can be strong (the time [tau] the knot stays tied increases with the force F applied to separate the strands) or weak ([tau]decreases with increasing F).

Indiana University-Purdue University Indianapolis (IUPUI) / Disruptions of the endoplasmic reticulum (ER) cause perturbations in protein folding and result in a cellular condition known as ER stress. ER stress and the accumulation of unfolded protein activate the unfolded protein response (UPR) which is a cellular attempt to remedy the toxic accumulation of unfolded proteins. The UPR is implemented through three ER stress sensors PERK, ATF6, and IRE1. Phosphorylation of the α-subunit of eIF2 by PERK during ER stress represses protein synthesis and also induces preferential translation of ATF4, a transcriptional activator of stress response genes. Early UPR signaling involves translational and transcriptional changes in gene expression that is geared toward stress remedy. However, prolonged ER stress that is not alleviated can trigger apoptosis. This dual signaling nature of the UPR is proposed to mimic a 'binary switch' and the regulation of this switch is a key topic of this thesis. Adaptive gene expression aimed at balancing protein homeostasis encompasses the first phase of the UPR. In this study we show that the PERK/eIF2~P/ATF4 pathway facilitates both the synthesis of ATF6 and trafficking of ATF6 from the ER to the Golgi where ATF6 is activated. Liver-specific depletion of PERK significantly lowers expression of survival genes, leading to reduced expression of protein chaperones. As a consequence, loss of PERK in the liver sensitizes cells to stress which ultimately leads to apoptosis. Despite important roles in survival, PERK signaling is often extended to the vii activation of other downstream transcription factors such as CHOP, a direct target of ATF4-mediated transcription. Accumulation of CHOP is a hallmark of the second phase in the binary switch model where CHOP is shown to be required for full activation of apoptosis. Here the transcription factor ATF5 is found to be induced by CHOP and that loss of ATF5 improves the survival of cells following changes in protein homeostasis. Taken together this study highlights the importance of UPR signaling in determining the balance between cell survival and cell death. A topic that is important for understanding the more complex pathological conditions of diseases such as diabetes, cancer, and neurodegeneration.

The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. <p>Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.<p>The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.<p>In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses. <p>In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand.<p>/<p>Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.<p>Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée. <p>Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11. <p>Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.

Indiana University-Purdue University Indianapolis (IUPUI) / Intrinsically disordered proteins are proteins characterized by lack of stable tertiary structures under physiological conditions. Evidence shows that disordered proteins are not only highly involved in protein interactions, but also have the capability to associate with more than one partner. Short disordered protein fragments, called “molecular recognition features” (MoRFs), were hypothesized to facilitate the binding diversity of highly-connected proteins termed “hubs”. MoRFs often couple folding with binding while forming interaction complexes. Two protein disorder mechanisms were proposed to facilitate multiple partner binding and enable hub proteins to bind to multiple partners: 1. One region of disorder could bind to many different partners (one-to-many binding), so the hub protein itself uses disorder for multiple partner binding; and 2. Many different regions of disorder could bind to a single partner (many-to-one binding), so the hub protein is structured but binds to many disordered partners via interaction with disorder. Thousands of MoRF-partner protein complexes were collected from Protein Data Bank in this study, including 321 one-to-many binding examples and 514 many-to-one binding examples. The conformational flexibility of MoRFs was observed at atomic resolution to help the MoRFs to adapt themselves to various binding surfaces of partners or to enable different MoRFs with non-identical sequences to associate with one specific binding pocket. Strikingly, in one-to-many binding, post-translational modification, alternative splicing and partner topology were revealed to play key roles for partner selection of these fuzzy complexes. On the other hand, three distinct binding profiles were identified in the collected many-to-one dataset: similar, intersecting and independent. For the similar binding profile, the distinct MoRFs interact with almost identical binding sites on the same partner. The MoRFs can also interact with a partially the same but partially different binding site, giving the intersecting binding profile. Finally, the MoRFs can interact with completely different binding sites, thus giving the independent binding profile. In conclusion, we suggest that protein disorder with post-translational modifications and alternative splicing are all working together to rewire the protein interaction networks.