No description available.

No description available.

No description available.

<p><em>Chlamydia pneumoniae</em> is a Gram-negative intracellular pathogen that uses type III secretion to invade and survive within eukaryotic cells. The T3SS secretes specific effector proteins during the infection process to facilitate immune evasion and nutrient acquisition. Unfortunately, the genetic intractability and difficult culturing conditions of Chlamydiae has inhibited progress in the chlamydial T3S field. This thesis characterizes fundamental aspects of the <em>C. pneumoniae </em>injectisome such as the ATPase, the inner-membrane export apparatus, and a specific effector protein Cpn0803. Initially, we explored whether <em>C. pneumoniae</em> encodes a functional T3S ATPase and if it associates with other T3S components. We found that CdsN has enzymatic activity consistent with other Gram-negative T3S ATPases, and that CdsN associates with inner-membrane and soluble components such as CdsD, CdsQ, CopN and CdsL. We also found that CdsN has binding surfaces for either structural or putative effector / chaperone T3S proteins. Next, we explored the putative flagellar genes, which were of interest since <em>Chlamydia</em> is a non-motile bacteria that lacks flagellum. We found that the flagellar proteins associate with the T3S apparatus, suggesting that they play a role in T3S during the life-cycle. We extended this observation to show that CdsL, a T3S component, down-regulates both CdsN and FliI enzymatic activity, suggesting that the flagellar proteins are involved in T3S. Furthermore, we characterized Cpn0803 as an exemplary effector, which associates with both CdsN and FliI. We found that Cpn0803 is secreted into host cells upon<em> Chlamydia</em> infection. Cpn0803 was thought to be the T3S needle-tip protein; however, the crystal structure does not support this hypothesis. Presently, the actual role of Cpn0803 in the T3S apparatus remains unknown. Overall, our data suggests that CdsN and FliI both function during the chlamydial life-cycle in the T3S process, possibly coordinating effector proteins (such as Cpn0803) for secretion into host cells.</p> / Doctor of Philosophy (PhD)

The establishment of non-resolving inflammation underlies the pathogenesis of chronic inflammatory diseases in humans. Super low dose (SLD) endotoxin has been associated with exacerbating inflammation and the pathogenesis of chronic inflammatory diseases. However, the underlying molecular mechanisms are not well studied. In this study, I tested the hypothesis that SLD endotoxin may potentiate non-resolving innate immune cell inflammation through disrupting cellular endoplasmic reticulum (ER) homeostasis. We chose to study the dynamics of ER homeostasis in macrophages stimulated with SLD endotoxin. In naïve cells, ER stressor such as tunicamycin (TM) not only will induce cellular stress and inflammation through JNK and NFkβ activation, but also will cause subsequent compensatory homeostasis through inducing homeostatic molecules such as XBP1 and GRP78/BiP. We observed that cells challenged with SLD endotoxin have significantly reduced expression of homeostatic molecules XBP1 and BiP. Mechanistically, we observed that SLD-LPS increases phosphorylated HCK expression in TM treated cells. Phosphorylated HCK activation resulted in the phosphorylation of Golgi protein GRASP, leading to unstacking of Golgi cisterna and overall dysfunction of the Golgi apparatus. Dysfunctional Golgi apparatus and its effect on protein transport and secretion, may account for decreased levels of Site 2 Protease, reduced generation of ATF6 and its transcriptional target BiP. Taken together, our study reveal that super low dose endotoxin exacerbates low grade inflammation through increasing phosphorylation of HCK, inducing Golgi dysfunction, and decreasing BiP /homeostatic protein expression in innate immune cells. / Ph. D.

Molecular chaperones have emerged in recent years as major players in many aspects of cell biology. Molecular chaperones are also known as heat shock proteins (HSPs) since many were originally discovered due to their increased synthesis in response to heat shock. They were initially identified when Drosophila salivary gland cells were exposed to a heat shock at 37°C for 30 min and then returned to their normal temperature of 25°C for recovery. A “puffing” of genes was found to have occurred in the chromosome of recovering cells, which was later shown to be accompanied by an increase in the synthesis of proteins with molecular masses of 70 and 26 kDa. These proteins were hence named “heat shock proteins”. The first identification of a function for HSPs was the discovery in Escherichia coli that five proteins synthesized in response to heat shock were involved in λ phage growth. The products of the groEL and groES genes were found to be essential for phage head assembly while the dnaK, dnaJ and grpE gene products were essential for λ phage replication. It was later shown that GroEL and GroES are part of a chaperonin system for protein folding in the prokaryotic cytosol while DnaK is a member of the Hsp70 family that works in conjunction with the DnaJ (Hsp40) co-chaperone and the nucleotide exchange factor GrpE to promote phage replication by dissociating the DnaB helicase from the phage-encoded P protein. Since then, a large number of other proteins collectively referred to as HSPs have been discovered. However, heat shock is not the only signal that induces synthesis of heat shock proteins. Stress of any kind, such as nutrient deprivation, chemical treatment and oxidative stress among others causes increased production of HSPs and therefore, they are also known as stress proteins. The term “molecular chaperone” was originally used to describe the function of nucleoplasmin, a Xenopus oocyte protein that promotes nucleosome assembly by binding tightly to histones and donating the bound histone to chromatin. However, since then, chaperones have been defined as “a family of unrelated classes of proteins that mediate the correct assembly of other proteins, but are not themselves components of the final functional structure”. This view of molecular chaperones, though undoubtedly correct, doesn’t capture the multifaceted roles they have since been discovered to play in cellular processes. In recent years, molecular chaperones have been shown to perform other functions in addition to the maintenance of protein homeostasis: translocation of proteins across organelle membranes, quality control in the endoplasmic reticulum, turnover of misfolded proteins as well as signal transduction. As a result, many chaperones are also essential under non-stress conditions and play crucial roles in cell growth and development, cell-cell communication and regulation of gene expression. Heat shock protein 90 (Hsp90) is one of the most abundant and highly conserved molecular chaperones in organisms ranging from bacteria to all branches of eukarya. It has been shown to be essential for cell viability in Saccharomyces cerevisiae, Schizosaccharomyces pombe and Drosophila melanogaster. Although the bacterial homolog HtpG is dispensable under normal conditions, it is important for cell survival during heat shock. In addition to its role as general chaperone in protein folding following stress, Hsp90 has a more specialized role as a chaperone for several protein kinases and transcription factors. Many Hsp90 client proteins are signaling proteins involved in regulation of cell growth and survival. These proteins are critically dependent on Hsp90 for their maturation and conformational maintenance resulting in a key role for Hsp90 in these processes. Recent reports have also highlighted a role for Hsp90 in linking the expression of genetic and epigenetic variation in response to environmental stress with morphological development in Drosophila melanogaster and Arabidopsis thaliana. In Candida albicans, Hsp90 augments the development of drug resistance, implicating a role for Hsp90 in the evolution of infectious diseases. The malarial parasite, Plasmodium falciparum, is the causative agent of the most lethal form of human malaria. The parasite life cycle involves two hosts: an invertebrate mosquito vector and a vertebrate human host. As the parasite moves from the mosquito to the human body, it experiences an increase in temperature resulting in a severe heat shock. The mechanisms by which the parasite adapts to changes in temperature have not been deciphered. Our laboratory has been interested in investigating the role of heat shock proteins during acclimatization of the parasite to such temperature fluctuations. Heat shock proteins of the Hsp40, Hsp60, Hsp70 and Hsp90 families have been characterized in the parasite and are being examined in our laboratory. This thesis pertains to understanding the functional role of Plasmodium falciparum Hsp90 (PfHsp90) during adaptation of the parasite to fluctuations in environmental temperature. The parasite expresses a single gene for cytosolic Hsp90 on chromosome 7 (PlasmoDB accession no.: PF07_0029) coding for a protein of 745 amino acids with a pI of 4.94 and Mw of 86 kDa. Eukaryotic Hsp90 regulates several protein kinases and transcription factors involved in cell growth and differentiation pathways resulting in a crucial role for Hsp90 in developmental processes. A role for PfHsp90 in parasite development, therefore, seems likely. Indeed, PfHsp90 has previously been implicated in parasite development from the ring stage to the trophozoite stage during the intra-erythrocytic cycle. Pharmacological inhibition of PfHsp90 function using geldanamycin (GA), a specific inhibitor of Hsp90 activity, abrogates stage progression. These experiments suggest that PfHsp90 may play a critical role in parasite development. This is further substantiated by the fact that several pathogenic protozoan parasites such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii and Eimeria tenella depend on Hsp90 function during different stages of their life cycles. It appears, therefore, that a principal role of Hsp90 in protozoan parasites may be the regulation of their developmental cycles. However, the precise functions of PfHsp90 during the intra-erythrocytic cycle of the malarial parasite are not clear. In this study we have carried out a functional analysis of PfHsp90 in the malarial parasite. We have examined the role of PfHsp90 in parasite development during repeated exposure to febrile temperatures. We have investigated its involvement in parasite development during a commonly used synchronization protocol involving cyclical changes in temperature. We have examined the interaction of GA with the Hsp90 multi-chaperone complex from P. falciparum as well as the human host. Finally, we have carried out a systems level analysis of chaperone networks in the malarial parasite as well as its human host using an in silico approach. We have analyzed the protein-protein interactions of PfHsp90 in the chaperone network and predicted putative cellular processes likely to be regulated by parasite chaperones, particularly PfHsp90.

No description available.

HIV-NC is a multifunctional protein which plays an important role in almost every step of the retroviral life cycle. NC is essential in catalyzing stand transfers of HIV-1 reverse transcription, including the annealing of the transactivation response element (TAR) of the viral genome to the complementary TAR DNA in minus-strong-stop DNA. In this dissertation, the research starts with focus on elucidating the reaction mechanism of NC-facilitated TAR DNA/RNA annealing using single molecule spectroscopy (SMS) approaches. The results indicate that nucleation of TAR DNA/RNA annealing occurs in an encounter complex form in which one or two DNA/RNA strands in the partially open “Y” form associated with multiple NC molecules. This encounter complex leads to annealing through the 3’/5’ termini, namely “zipper” pathway and the annealing through the hairpin loop region, namely “kissing” pathway. By employing target oligonucleotides for specific TAR regions, we directly probed kinetic reversibility and the chaperone role of NC. Concentration-dependence of NC chaperoned melting and annealing of TAR hairpins was investigated and the results further support the proposed reaction mechanism. Additionally, we used a single-stranded DNA (ssDNA) as model to study ssDNA conformational change upon NC binding. Here we present observation of NC binding to d(TG)n and d(T)n, including NC effect on flexibility and conformation of these oligonucleotides chains. Our results reveal that the rigidity of ssDNA chain is dramatically reduced through interaction with NC. Meanwhile the results of NC dissociation experiments indicate the interaction of NC/ssDNA is complex and heterogeneous. Finally, we used SMS in vitro to systematically compare and contrast the RNA/protein interactions for the zinc-finger-binding-motif protein (NC) and the arginine-rich-binding-motif (ARM) protein (Tat) encoded by HIV-1. Tat and NC use different RNA binding motifs to recognize and interact with RNA hairpin, giving rise to very different changes in the RNA secondary structure upon protein binding. Competition experiments show that the presence of Tat can effectively inhibit the NC binding-induced local melting of TAR RNA hairpins. These results indicate that Tat specifically binds and stabilizes the TAR RNA hairpin structure, which likely inhibits the local melting of the hairpin induced by NC. / text

Cyanobacteria are photosynthetic organisms that efficiently capture light by utilizing the light-harvesting complexes called phycobilisomes. In many cyanobacteria, phycobilisomes are composed of an allophycocyanin core with phycocyanin and phycoerythrin (PE) rods radiating from the core. These phycobiliproteins have multiple bilin chromophores, such as phycoerythrobilin (PEB), covalently attached to specific cysteine (Cys) residues for efficient photosynthetic light capture. Chromophore ligation on phycobiliprotein subunits occurs through bilin lyase catalyzed reactions. This study mainly focuses on characterizing the roles of enzymes that are involved in the biosynthetic pathway of the phycobiliproteins within two cyanobacteria Thermosynechococcus elongatus and Fremyella diplosiphon. A combination of molecular and biochemical techniques were used to better understand the roles of these proteins in the post-translational modification and/or stability of phycobiliproteins. Using a heterologous plasmid coexpression system in E. coli, recombinant CpcS-III from T. elongatus was shown to ligate three different bilins to both subunits of allophycocyanin and to the beta subunit of phycocyanin, thus, acting as a bilin lyase. The crystal structure of CpcS-III was also solved, the first bilin lyase structure. Next, the roles of three proteins from F. diplosiphon CpeY, CpeZ, and CpeF were analyzed using a combination of gene knock-out mutants and recombinant protein expression techniques. In the absence of cpeY, chromophorylation to the alpha subunit of PE at Cys-82 was reduced, coinciding with the recombinant data that CpeY is the lyase that attaches PEB to this site. Removing cpeZ from the genome resulted in the destabilization and reduced accumulation of PE, especially the beta subunit CpeB. Recombinant CpeZ was shown to act like a chaperone-like protein and increased the solubility and fluorescence of both recombinant and native CpeB by increasing the stability of the phycobiliprotein and/or by increasing the activities of other lyases. The deletion of cpeF resulted in a reduced-PE phenotype with the doubly attached PEB missing from CpeB at Cys-48/Cys-59. Recombinant CpeF was shown to ligate PEB to CpeB-Cys-48/Cys-59 in the presence of recombinant CpeS (lyase attaches PEB to CpeB-Cys-80) and CpeZ. CpeF also showed a chaperone-like function by stabilizing CpeB, but its main role appears to be as a bilin lyase.

Hsp27 appartient à la famille des protéines dites de survie comme Bcl2 ou la survivine. C’est une protéine anti-apoptotique qui subit une dérégulation de son expression dans de nombreux types tumoraux. Elle est caractérisée comme étant une cible thérapeutique majeure. Au cours de ma thèse, j’ai isolé des peptides stabilisés, dit aptamères, capables d’inhiber fonctionnellement les activités anti-apoptotiques et tumorigènes d’Hsp27. Ces aptamères perturbent la biochimie structurale de Hsp27 et induisent le blocage du cycle cellulaire in vivo. Parallèlement à cette étude, j’ai caractérisé les effets de la déplétion de Hsp27 sur la formation de métastases et de tumeurs osseuses. J’ai aussi montré que la modification du taux de Hsp27 induisait la dégradation de différentes protéines, dites clientes, comme la caspase3, HDAC6 et STAT2. / Hsp27 belongs to the class of survival proteins like Bcl2 or survivin. This protein was well categorized as a major anti apoptotic protein as displaying a high level of expression in lot of tumor types. Moreover, Hsp27 is referenced as a major therapeutic target in cancer. During my PhD, I characterized stable peptides called aptamers, which functionally blocked Hsp27 antiapoptotic and tumorigenic properties. These aptamers disrupted biochemical and structural states of Hsp27 and promoted cell cycle arrest in xenografts. In the same time, I have characterized the effect of Hsp27 depletion in metastasis establishment and bone marrow tumor growth. I have shown that targeting level of Hsp27 induced degradation of several of its client proteins like caspase3, HDAC6 and STAT2