Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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The nuclear pore complex (NPC) is a pivotal subcellular structure governing nucleocytoplasmic transport through a selectively permeable barrier. Comprising approximately 30 distinct proteins, it includes FG-Nups with phenylalanine-glycine (FG) motifs and non-FG Nups forming the pore's scaffold. The selectively permeable barrier formed by FG-Nups enables the passive diffusion of small molecules and facilitates the transport of larger ones recognized by nuclear transport receptors (NTRs). Their roles are critical in regulating mRNA and pre-ribosome nuclear export and the nuclear import of transcription factors, underscoring their significance in cellular processes. However, studying NPCs remains challenging due to their structural complexity, heterogeneity, dynamic interactions, and inaccessibility within live cells. In this dissertation, three core questions were investigated to elucidate the structure and function of the NPC. First, the nuclear export dynamics of pre-ribosomal subunits revealed significantly higher transport efficiency compared to other large cargos. Through inhibition of nuclear transport receptor (NTR), CRM1, by small-molecule inhibitor, leptomycin B, we found a dose-dependent inhibition of CRM1s played a crucial role in pre-ribosome export efficiency. We confirmed these results through a series of controlled environments with both import and export NTRs. Our results suggest that cooperative NTR mechanisms may enhance the nucleocytoplasmic transport of not only pre-ribosomal subunits but other protein complexes as well. Second, we investigated the dynamic properties of the NPC’s selectivity barrier by altering the concentration of O-linked β-N-acetylglucosamine (O-GlcNAc) sites on nuclear pore proteins. Using small-molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) to decrease or increase NPC O-GlcNAcylation, respectively, we found a significant change in the overall 3D spatial density of NPC O-GlcNAc sites. Then, by applying the same OGT- and OGA-inhibited conditions, we found that NPC O-GlcNAcylation significantly impacted the nuclear export of mRNA, suggesting that NPC O-GlcNAcylation regulates mRNA’s passage through the NPC’s selective permeability barrier. Third, we examined the nuclear transport mechanism for intrinsically disordered proteins (IDPs). Our findings revealed that IDPs, unlike large folded proteins, can passively diffuse through NPCs independent of size, and their diffusion behaviors are differentiated by the content ratio of charged (Ch) and hydrophobic (Hy) amino acids. Thus, we proposed a Ch/Hy-ratio mechanism for IDP nucleocytoplasmic transport. In summary, comprehending the dynamic behavior of the NPC selectivity barrier and its involvement in mediating large transiting complexes and IDPs has provided valuable insights into the fundamental nucleocytoplasmic transport mechanism, emphasizing the NPC's crucial role in cellular health and function. / Biology

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Physical and psychological stressors trigger activation of the hypothalamo-pituitary-adrenocortical (HPA) axis that leads to enhanced secretion of glucocorticoids e.g. cortisol. Moreover, chronic activation of this pathway may elevate oxidative stress that is linked to the onset of insulin resistance and cardiovascular diseases (CVD). Our laboratory previously found that oxidative stress increases flux through metabolic circuits such as the hexosamine biosynthetic pathway (HBP), in effect increasing its modification of target proteins post-transcriptionally with O-GlcNAc moeities. This in turn may alter protein function and contribute to the onset of myocardial insulin resistance and impaired contractile function. Since the underlying mechanisms linking chronic stress to cardiometabolic pathophysiology are poorly understood, we hypothesised that cortisol elicits myocardial oxidative stress, HBP activation, and decreased glucose uptake (due to attenuated glucose transport functionality) with detrimental outcomes, i.e. insulin resistance and apoptosis. To investigate this hypothesis we established an in vitro model using HL-1 cardiomyocytes, with which we evaluated the degree of O-GlcNAcylation and oxidative stress in response to a range of time-dose treatments with dexamethasone (synthetic glucocorticoid). Glucose transporter 4 (GLUT4) translocation to the sarcolemma was also assessed. In agreement with the literature, results suggest that GLUT4 translocation is significantly decreased subsequent to dexamethasone treatment. Although no significant differences were observed with regards to oxidative stress or O-GlcNAcylation, the data show that dexamethasone increased the latter with a maximal effect after two hours exposure to the 10-6 M dose. Although our results were not conclusive, the data suggest a potential novel link between dexamethasone exposure, HBP activation and decreased GLUT4 translocation. Based on our findings we propose that detrimental effects of chronic stress on the heart may be mediated by increased HBP flux. Given that glucocorticoid excess and GLUT4 dysregulation have been associated with insulin resistance (and related metabolic derangements and diseases), these results provide new targets for potential therapeutic agents. / AFRIKAANSE OPSOMMING: Fisiese sowel as psigologiese stressors veroorsaak die aktivering van die hipotalamiese-hipo seale-bynier (HHB) pad wat lei tot die verhoogde sekresie van glukokortikoïede soos kortisol. Kroniese aktivering van hierdie pad kan ook oksidatiewe stres verhoog wat weer tot insulienweerstandigheid en kardiovaskulêre siektes (KVS) kan lei. Navorsing uit ons laboratorium het voorheen bewys dat oksidatiewe stres 'n toename in vloei deur metaboliese paaie soos die heksoamine biosintetiese pad (HBP) kan veroorsaak deur die modi sering van teikenproteïene met O-GlcNAc motiewe. Dit kan weer proteïen funksie verander en bydra tot die ontstaan van miokardiale insulienweerstandigheid en verswakte kontraktiele funksie. Die onderliggende meganismes wat kroniese stres aan kardiometaboliese pato siologie verbind word nog nie goed verstaan nie, daarom is ons hipotese dat kortisol miokardiale oksidatiewe stres veroorsaak, die HBP pad aktiveer, en glukose opname verminder (deur die funksionele onderdrukking van glukose transport), wat nadelige uitkomste soos insulienweerstandigheid en apoptose tot gevolg kan hê. Om hierdie hipotese te ondersoek, is 'n in vitro model van HL-1 kardiomiosiete gebruik waarmee die graad van O-GlcNAsilering en oksidatiewe stres in reaksie op 'n reeks tyd-konsentrasie behandelings met deksametasoon (sintetiese glukokortikoïed), bepaal is. Glukose transporter 4 (GLUT4) translokasie na die sarkolemma is ook geasseseer. In ooreenstemming met die literatuur, is GLUT4 translokasie insiggewend onderdruk tydens deksometasoon behandeling. Alhoewel geen insiggewende verskille rakende oksidatiewe stres en O-GlcNAsilering gevind is nie, het ons data aangedui dat laasgenoemde deur deksametasoon vermeerder het na twee ure van blootstelling aan die 10-6 M konsentrasie. Alhoewel ons resultate geen afdoende bewys lewer nie, stel dit wel voor dat daar 'n potensiële verbintenis tussen deksametasoon behandeling en 'n afname in GLUT4 translokasie is. Gebasseer op ons bevindings, stel ons voor dat die nadelige e ekte van kroniese stres op die hart bemiddel kan word deur 'n toename in vloei deur die HBP. Gegewe dat 'n oormaat glukokortikoïede en GLUT4 wanregulering geassosieer is met insulien weerstandigheid (en verbandhoudende metaboliese veranderinge en siektes), verskaf hierdie resultate nuwe teikens vir potensiële terapeutiese ingrepe.

LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo. / LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.

LIMA, V.V. Papel da O-glicosilação com N-acetil-glucosamina (O-GlcNAc) nas alterações vasculares associadas a altos níveis de endotelina-1. 2012. 106 f. Tese (Doutorado) - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. A O-Glicosilação com N-acetilglucosamina (O-GlcNAc) é uma modificação pós-traducional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: UDP-NAc transferase (OGT) e O-GlcNAcase (OGA). A enzima OGT catalisa a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina ou treonina das proteínas alvo. Por outro lado, a OGA catalisa a remoção hidrolítica de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvos da O-GlcNAc, e recentemente demonstramos que a expressão de proteínas modificadas com O-GlcNAc está aumentada em artérias de ratos com hipertensão DOCA-sal. Considerando que a produção de endotelina-1 (ET-1) encontra-se aumentada na vasculatura de diferentes modelos de hipertensão sensível ao sal, nós investigamos a hipótese de que o aumento da resposta vascular contrátil induzida pela ET-1 é decorrente da hiperativação da via RhoA/Rho cinase, mediada pelo aumento dos níveis de proteínas O-GlcNAc. Durante a realização de nossos experimentos, demonstramos que a exposição de aortas ou células do músculo liso vascular (CMLV) à ET-1 (0,1 mol/L) aumenta a vasoconstrição para fenilefrina (PE) e serotonina, bem como os níveis de proteínas O-GlcNAc, além de modular a expressão das enzimas OGT e OGA. A infusão de ET-1 (2 pmol/Kg/min) por 14 dias também promoveu aumento dos níveis vasculares de proteínas O-GlcNAc e da resposta contrátil da aorta à PE. O tratamento de aortas ou CMLV com ST045849 (inibidor da OGT, 100 µMol/L) ou atrasentan (antagonista do receptor ETA, 1 mol/L), preveniu o aumento dos níveis de proteínas O-GlcNAc induzido pela ET-1. Além disso, o tratamento com atrasentan por cinco semanas (atrasentan - 5 mg/kg/dia, por via oral) normalizou os níveis vasculares de proteínas O-GlcNAc em ratos DOCA-sal e também diminuiu a resposta contrátil da aorta à PE. A transfecção de CMLV com siRNA para OGT aboliu o efeito da ET-1 sobre os níveis de proteínas O-GlcNAc. Considerando que o aumento nas contrações da aorta à PE, após o tratamento com PUGNAc (inibidor seletivo da OGA) ou ET-1, foi abolido pelo inibidor de Rho cinase (Y-27632, 1 mol/L) e que a ET-1 ativa a via de sinalização da RhoA/Rho cinase, decidimos investigar se aumento dos níveis de proteínas O-GlcNAc ativa/modula a via RhoA/Rho cinase. A incubação de CMLV com ET-1 não mudou a expressão protéica das formas totais de ROCK-, ROCK-, CPI-17, MYPT-1 ou MLC, porém aumentou a expressão das formas fosforiladas da MYPT-1 (Tre853), CPI-17 (Tre38) e MLC (Tre18/Ser19). Estes efeitos não foram observados quando CMLV foram tratadas com ST045849, atrasentan ou previamente transfectadas com o siRNA para OGT. Também observamos que a ET-1 aumentou a atividade e a expressão protéica da RhoA, assim como a expressão da PDZ-Rho GEF e p115-Rho GEF. Este efeito foi abolido, quando CMLV foram previamente transfectadas com siRNA para OGT, incubadas com o inibidor da OGT ou tratadas com o antagonista de receptores ETA. Em conclusão, nossos dados fornecem evidências de que a ET-1 aumenta os níveis vasculares de proteínas O-GlcNAc, resultando na ativação da via RhoA/Rho cinase e no aumento da reatividade vascular. É possível que o aumento de proteínas O-GlcNAc, induzido pela ET-1, possa representar um novo mecanismo para a disfunção vascular induzida por este potente peptídeo. / LIMA, V.V. O-GlcNAcylation contributes to the vascular effects of ET-1 via activation of RhoA/Rho-kinase pathway. 2012. 106 f. Ph.D. Thesis - Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2012. Glycosylation with O-linked -N-acetylglucosamine (O-GlcNAc) is a highly dynamic post-translational modification that plays a key role in signal transduction pathways. The cycling of O-GlcNAc is controlled by two enzymes: UDP-NAc transferase (OGT) and O-GlcNAcase (OGA). Whereas OGT catalyses the addition of O-GlcNAc to the hydroxyl group of serine and threonine residues of a target protein, OGA catalyses the hydrolytic cleavage of O-GlcNAc from post-translationally-modified target. Proteins with an important role in vascular function are targets for O-GlcNAcylation and we have recently shown that the vascular content of O-GlcNAc-proteins is augmented in arteries from DOCA-salt rats. Since endothelin-1 (ET-1) production is increased in the vasculature of salt-sensitive forms of hypertension, we tested the hypothesis that O-GlcNAc contributes to the vascular effects of ET-1, via activation of the RhoA/Rho-kinase pathway. Incubation of rat aortas or vascular smooth muscle cells (VSMCs) with ET-1 (0,1 mol/L) produced a time-dependent increase in O-GlcNAc levels, decreased expression of O-GlcNAc transferase (OGT) and -N-acetylglucosaminidase (OGA), key enzymes in the O-GlcNAcylation process. Overnight treatment of aortas with ET-1 increased phenylephrine (PE) vasoconstriction. ET-1 effects were not observed when vessels were previously instilled with anti-OGT antibody or after incubation with an OGT inhibitor (ST045849, 100 mol/L). Aortas from DOCA-salt rats, which exhibit increased pre-pro-ET-1 expression, displayed increased contractions to PE and augmented levels of O-GlcNAc proteins. Treatment of DOCA-salt rats with atrasentan (ETA antagonist) abrogated augmented vascular levels of O-GlcNAc and prevented increased PE vasoconstriction. Aortas from rats chronically infused with low rate of ET-1 (2 pmol/Kg/min, 14days) exhibited increased O-GlcNAc-proteins and enhanced PE responses. These changes are similar to those induced by PUGNAc (OGA inhibitor which increases O-GlcNAc levels). ET-1 as well as PUGNAc augmented contractions to PE in endothelium-denuded rat aortas, an effect that was abolished by the Rho kinase inhibitor Y-27632 (1 mol/L). Incubation of VSMCs with ET-1 did not change expression of ROCK-, ROCK-, CPI-17, MYPT-1 or MLC, but increased phosphorylation levels of MYPT-1 (Thr853), CPI-17 (Thr38) and MLC (Thr18/Ser19). The effects of ET-1 on MYPT-1, CPI-17 and MLC phosphorylation were prevented by the OGT inhibitor and OGT siRNA transfection, as well as by atrasentan. ET-1 increased RhoA expression and activity in VSMCs, and this effect was abolished by OGT siRNA transfection and OGT inhibition. ET-1 also augmented expression of PDZ-Rho GEF and p115-Rho GEF in VSMCs and this was prevented by OGT siRNA, OGT inhibition (ST045849) and ETA receptor blockade (atrasentan, 1 mol/L). In conclusion, our data strongly suggest that ET-1 augments O-GlcNAc levels and this modification contributes to increase vascular contractile responses, via activation of the RhoA/Rho-kinase pathway. We speculate that the modulatory effect of ET-1 on O-GlcNAcylation may represent a novel mechanism underlying the vascular effects of the peptide.

Self-assembling protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and many additional processes, which makes them appealing targets for therapeutics and biotechnological applications, including live vaccines, liposome drug delivery and biosensors. Unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of specialized S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Characterization of this interaction in the Gram-positive model organism Paenibacillus alvei CCM 2051T reveals that, remarkably, binding-site switches can occur between two distinct SLH-domain SCWP receptor-site grooves in the S-layer protein SpaA, possibly as part of a mechanism to alleviate strain in the S-layer. To date, however, analysis of this novel mechanism has been limited to the terminal SCWP monosaccharide and the internal SCWP repeat disaccharide ligand analogues, leaving open the role of subsequent SCWP sugar residues in binding, as well as whether the two receptor sites are also suited to accommodate longer SCWP ligands that better approximate the biological target at the surface of P. alvei. To address this, the objective of this work aims to uncover and characterize the details of the SpaA SLH-domain (SpaASLH¬) SCWP-interaction by determining the co-crystal structures of SpaASLH¬, and single (SpaASLH/G109A) and the corresponding double (SpaASLH/G46A/G109A) mutants in complex with synthetic terminal disaccharide and trisaccharide analogues of the P. alvei CCM 2051T SCWP target. These structural characterizations have been supplemented with disaccharide and trisaccharide binding data, which was obtained through thermodynamic ITC analyses carried out by collaborators. The co-crystal structures of P. alvei SpaASLH with synthetic, terminal SCWP disaccharide and trisaccharide analogues, together with previously published monosaccharide-bound SpaASLH structures, reveal that while the SLH trimer accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly-exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations for longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division. In addition, to further clarify the steps involved in SCWP biosynthesis, we present a crystal structure of the unliganded UDP-GlcNAc 2-epimerase enzyme MnaA, which catalyzes the interconversion of UDP-GlcNAc into UDP-ManNAc—an essential building block of the P. alvei SCWP target. The P. alvei MnaA epimerase adopts a GT-B fold that is consistent with the architecture of previously published structures of other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerase enzymes for which substrate binding is observed in the cleft located between the two domains. Characterization of this structure, coupled with an analysis of the sequence of the MnaA protein, reveals the presence of conserved residues that define the catalytic and allosteric sites in homologous enzymes from different organisms. These residues are positioned to accommodate substrate within the MnaA binding cleft in much the same manner as the published enzyme homologues, suggesting that allosteric regulation as a mechanism for enzyme regulation is conserved in P. alvei MnaA. These investigations are part of a greater effort toward understanding SLH domain-mediated SCWP-interactions in Gram-positive organisms, and provide insight into the structure and putative function of this SCWP biosynthetic enzyme. By understanding these processes, this knowledge may contribute to providing a platform for the rational design of Gram-positive inhibitors. Such inhibitors could selectively target, for example, the bacterial S-layer SCWP-binding interaction, or perhaps the essential biosynthetic enzymes involved in producing the exclusive targets that these S-layer proteins recognize and bind, and would thus represent a new class of antimicrobial therapeutics. / Graduate

Das Proteasomensystem der Zelle ist für die Degradation von Proteinen verantwortlich und spielt eine zentrale Rolle bei der Generierung von Epitopen, die auf MHC-Klasse-I Molekülen den cytotoxischen T-Lymphozyten (CTLs) präsentiert werden. Die Stimulation von Zellen mit Interferon-gamma (IFNgamma) führt zu der Bildung von Immunoproteasomen, die im Vergleich zu den konstitutiven Proteasomen eine verbesserte Generierung vieler MHC-Klasse-I Epitope aufweisen. In gesunden Mäusen werden Immunoproteasomen vorwiegend in den lymphatischen Geweben exprimiert, wohingegen nicht-lymphatische Gewebe hauptsächlich konstitutive Proteasomen enthalten. In der vorliegenden Arbeit wurde der Einfluss der Listeria monocytogenes Infektion auf die aus der Leber, der Milz, dem Dünndarm und dem Colon stammenden murinen 20S Proteasomen untersucht. Die Struktur der isolierten 20S Proteasomen wurde mittels zweidimensionaler Gelelektrophorese und Westernblot ermittelt, während die Funktion durch in vitro Prozessierung von drei oligomeren Peptidsubstraten analysiert wurde. Die Prozessierungsprodukte wurden mittels HPLC-ESI-Ionenfalle massenspektrometrisch identifiziert sowie quantifiziert. Die vorliegende Arbeit zeigt zum ersten Mal, dass nach einer Infektion die aus den nicht-lymphatischen Organen und Zellen isolierten 20S Proteasomen eine strukturelle und funktionelle Plastizität aufweisen: Nach der Infektion wurde die Bildung von Immunoproteasomen induziert, was mit der gesteigerten Generierung der immunrelevanten Fragmente korreliert werden konnte. Dies verlief unabhängig von der direkten Präsenz von Listeria monocytogenes in den Organen und wurde ausschließlich durch das Cytokin IFNgamma reguliert. Es konnte außerdem eine Zunahme der posttranslationalen Modifikation von Leberproteasomen mit dem Monosaccharid N-Acetylglucosamin nach der Infektion nachgewiesen werden. Des Weiteren wurde eine detaillierte Analyse der massenspektrometrischen Daten hinsichtlich des Schnittverhaltens der konstitutiven und Immunoproteasomen etabliert. Die Auswertung ergab, dass die Immunoproteasomen nach der Infektion durch schnellere und veränderte Nutzung bestehender Spaltstellen an der verbesserten Epitoppräsentation beteiligt sind. / The proteasome system of the cell is responsible for the degradation of proteins and plays a central role in the generation of epitopes which are presented to cytotoxic T-lymphocytes (CTLs) on MHC-class-I molecules. The stimulation of cells by interferon-gamma (IFNgamma) leads to the formation of immunoproteasomes that show an improved generation of many MHC-class-I epitopes compared to constitutive proteasomes. In healthy mice, immunoproteasomes are mainly expressed in the lymphatic tissues, whereas the non-lymphatic organs predominantly contain constitutive proteasomes. In this project the effect of Listeria monocytogenes infection on murine 20S proteasomes derived from the liver, spleen, small intestine and colon were investigated. The structure of the isolated proteasomes was analyzed by two-dimensional gel electrophoresis and western blots while the function was studied by in vitro processing of three oligomeric peptide substrates. Identification and quantification of the processing products was performed by HPLC-ESI-ion trap mass spectrometry. The project showed for the first time, that after infection 20S proteasomes isolated from non-lymphatic organs as well as from non-lymphatic cells displayed structural and functional plasticity: immunoproteasomes were induced post infection which could be correlated with the enhanced generation of immuno-relevant fragments. This was independent of the direct presence of Listeria monocytogenes in the organs and solely controlled by the cytokine IFNgamma. In addition, an increased posttranslational modification with the monosaccharide N-acetylglucosamine could be detected in liver-derived proteasomes after infection. Furthermore, a detailed analysis of the mass spectrometry data was established according to the cleavage site usage of constitutive and immunoproteasomes. The result was that immunoproteasomes are involved in improved generation of the immuno-relevant fragments by the faster cleavage and the changed usage of existing cleavage sites after infection.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.