Thèses sur le sujet « HU Protein »

La spermatogenèse est un processus élaboré permettant d'une part le maintien de cellules souches par divisions mitotiques et d'autre part la production de spermatozoïdes par différenciation. Au cours des dernières étapes de la différenciation, la chromatine se compacte, ne laissant plus à la cellule la possibilité de transcrire ses gènes. Du fait de l'arrêt brutal de la transcription, bien avant la fin du processus de différenciation, la cellule germinale utilise le stock d'ARN messagers (ARNm) préexistants pour finaliser sa différenciation. Ce phénomène repose sur la régulation fine du stockage et de la traduction des ARNm au cours du temps, deux régulations post-transcriptionnelles encore très peu documentées dans les cellules germinales. Au cours de ma thèse je me suis intéressée au rôle potentiel de deux protéines de liaison à l'ARN exprimées dans le testicule de souris: HuR/ELAVL1 et AUF1/hnRNP D. Dans les cellules somatiques, ces protéines lient les séquences riches en adénines et uridines (AU-rich element ou ARE) localisées dans la région 3' non codante de certains ARNm (ARN à ARE). HuR protége de la dégradation ses ARN à ARE cibles et favorise leur traduction, alors qu'AUF1 induit leur dégradation. Afin d'étudier la contribution d'HuR et d'AUF1 aux mécanismes post-transcriptionnels indispensables au bon déroulement de la spermatogénèse, nous avons dans un premier temps examiné leur patron d'expression. Nous avons montré que l'expression d'HuR est étroitement régulée au cours de la spermatogénèse, alors que celle d'AUF1 est ubiquitaire. Dans un second temps, nous avons utilisé des lignées de souris transgéniques surexprimant HuR (HuRtg) ou AUF1 (AUF1tg) établies au laboratoire et montré que la surexpression d'HuR et non celle d'AUF1 altère la spermatogenèse, entraînant leur stérilité dans 25% des cas (Sertoli Cells Only syndrome). Par la suite, nous avons mis évidence que de nombreux ARN à ARE, naturellement abondamment exprimés dans le testicule, sont dérégulés dans les cellules germinales HuRtg et AUF1tg. Une étude approfondie des ARN cibles d'HuR et d'AUF1, a révélé que ces deux protéines ont une activité différente car elles s'associent à des ARN différents dans les cellules germinales. .<br>Spermatogenesis, the elaborate process by which sperm are produced, is marked by dramatic proliferation and differentiation. During the late steps of spermatogenesis, transcription suddenly ceases prior the end of differentiation, because of drastic epigenetic modifications that result in chromatin compaction. Thus, haploid germ cells make use of extensive temporal mRNA storage and translation regulation to ensure stage-specific protein synthesis. Factors and cellular compartments involved in these post-transcriptional controls are still poorly understood. During my PhD, I hypothesized that the two RNA binding proteins HuR/ELAVL1 and AUF1/hnRNP D, might play a role in these controls. They bind AU-rich element-containing mRNAs (ARE-mRNAs) in somatic cells and regulate their stability and translation: HuR protects ARE-mRNAs from degradation and favours their translation, whereas AUF1 usually induces their degradation. First, to investigate the contribution of HuR and AUF1 to the post-transcriptional mechanisms occurring in germ cells, I used transgenic mice derived in our laboratory overexpressing HuR (HuRtg) and AUF1 (AUF1tg) in their testes. Strikingly, whereas spermatogenesis proceeded normally in AUF1tg mice, HuR overexpression impaired spermatogenesis, revealing the importance of a regulated expression of HuR to fulfill male germ cell differentiation. The comparative analysis of AU-transcriptome of pre-pubertal wild type testes with that of HuRtg and AUF1tg testes, combined with computational analyses and RNA/Protein immunoprecipitation experiments, revealed that these two proteins regulate different targets mRNAs and thus exhibit different activities. .

Proteins that bind to both DNA and RNA embody the ability to perform multiple functions by a single gene product. These nucleic acid binding proteins in prokaryotes can play a vital role in many cellular processes, including replication, transcription, gene expression, recombination, and repair, to name a few. Nucleic acid binding proteins have unique functional characteristics that stem from their structural attributes that have evolved in a widely-conserved manner. In Escherichia coli (E. coli), the highly-conserved histone-like protein, HU, which predominates as a heterodimer of HUα and HUβ, has been found to bind to both dsDNA and ssDNA. Likewise, RNA-binding proteins contain various structural motifs, many of which are also conserved amongst many bacterial species like the RNA recognition motif. However, in Porphyromonas gingivalis, a periodontal pathogen, the histone-like, HU proteins and the RNA-binding protein (RBP) are not well characterized compared to their respective structures in E. coli. In our study, we sought to characterize and compare the HU proteins and RBP in order to gain a better understanding of their structure and function in the cell. Our data showed the HU proteins predominate as homo-tetramers and RBP as a monomer. We demonstrated single-stranded DNA binding with all three proteins. We found both P. gingivalis HU subunits bind non-specifically to ssDNA but show preferential binding to poly(dG) content, while binding to poly(dA) the weakest. These results show that HUα, HUβ and RBP are novel ssDNA binding proteins in P. gingivalis, indicating an expanded role and function within the cell.

Le but de cette thèse était de comprendre le rôle des protéines de la famille ELAV/Hu dans le développement, le maintien et la fonction des neurones par l’analyse phénotypique de mutants de dérégulation d'elav/Hu sur deux organismes modèles. Chez le xénope, nous avons montré que 3 homologues de la famille elav/Hu (elrB, elrC et elrD) sont exprimés de façon différentielle au cours du développement du système nerveux, suggérant un rôle dans différentes phases de la maturation des neurones et la définition de différents sous-domaines neuraux. La dérégulation de l’expression d'elrB dans les embryons de xénope réduit l'expression de marqueurs neuraux précoces, stoppe la prolifération cellulaire et induit l’apoptose, suggérant un rôle dans la régulation post-transcriptionnelle de gènes impliqués dans le contrôle du cycle cellulaire et/ou l'apoptose. Chez la drosophile, l’expression ubiquitaire d’elav obtenue grâce au système UAS/Gal4 produit un phénotype de dépigmentation de la cuticule et des soies similaire à celui de mutants de la voie de biosynthèse de la dopamine (DA ; neuromodulateur et intermédiaire de synthèse de la cuticule chez la drosophile). Ce phénotype de dépigmentation suggère qu'elav régulerait l'obtention de la tyrosine hydroxylase (TH) neurale qui contrôle la 1ère étape de la biosynthèse de DA. Ce travail suggère que les protéines ELAV/Hu participent au contrôle du cycle cellulaire et/ou de l'apoptose en plus de leur rôle dans la différenciation des neurones. L'identification de la TH comme cible potentielle d'ELAV suggère un rôle dans le contrôle de la synthèse de la DA, établissant ainsi un lien entre la présence d'ELAV et la fonction du système nerveux<br>The goal of this thesis was to understand the role of ELAV/Hu family proteins in the development, maintenance and function of neurons, using phenotypic analyses of deregulation mutants in two models. In Xenopus, we showed that 3 elav/Hu family homologs (elrB, elrC, and elrD) are differentially expressed during nervous system development, suggesting a role in different phases of neuron maturation and the definition of different neural sub-domains. Deregulating elrB expression in Xenopus embryos reduced early neuronal markers expression, stopped cell proliferation, and induced apoptosis, suggesting a role in post-transcriptional regulation of genes controlling cell cycle and/or apoptosis. In Drosophila, ubiquitous expression of elav, obtained with the UAS/Gal4 system, produced a phenotype of cuticle and bristles depigmentation. This phenotype, similar to that of mutants of dopamine (DA; neuromodulator and cuticle biosynthesis intermediate in Drosophila) biosynthesis. This depigmentation phenotype suggests that elav could regulate production of neural tyrosine hydroxylase (TH), which control the first step of DA biosynthesis. This work suggests that ELAV/Hu proteins participate in the control of cell cycle and/or apoptosis in addition to their role in neuron differentiation. Identification of TH as a potential target of ELAV suggests a role in controlling the biosynthesis of DA, thus establishing a link between ELAV presence and its function in the nervous system

HU est une protéine bactérienne de type histone impliquée dans de nombreuses fonctions biologiques telles que la compaction, la transcription, la réplication et la réparation de l'ADN. Chez E. coli, il existe trois espèces dimériques de HU (HUα2, HUβ2 et HUαβ) ayant des rôles biologiques distincts. La formation de l'hétérodimère repose sur un échange de chaines peptidiques entre les homodimères. Un mécanisme modèle a été proposé par Ramstein et collaborateurs (J.M.B. 331, 101-121 2003) et a servi de point de départ a ce travail. Dans ce modèle, les homodimères transitent d'une conformation native (N2) vers une conformation intermédiaire (I2). Les homodimères sous forme I2 s'associent ensuite dans un hétérotetramère transitoire qui se redissocie en formant des hétérodimères. L'objectif principal de ce travail de thèse a été de caractériser chaque étape du mécanisme d'échange du point de vue structural et cinétique. Parmi les principaux résultats de ce travail, deux structures originales, HUβ2 de E. coli et HU de L. lactis, ont été obtenues par diffraction des rayons X et complètent la caractérisation structurale des protéines HU sous leur forme N2. Un modèle de la conformation I2, partiellement désordonnée, a été élaboré à partir des résultats obtenus en RMN et en simulation de dynamique moléculaire. De plus, l'existence de la conformation tétramérique a été mise en évidence en faible concentration par spectrométrie de masse en conditions natives. Des protocoles de production/purification/oxydation ont été mis au point pour l'introduction de ponts disulfure afin de stabiliser la conformation tétramérique en vue de sa caractérisation structurale. L'ensemble des données acquises par ces différentes méthodes biophysiques affine la compréhension du mécanisme d'échange de chaines d'un point de vue structural et cinétique et met en lumière le rôle clef de la conformation I2 dans le contrôle de la composition des dimères de HU<br>HU is a histone like protein of bacteria involved in numerous biological functions such as DNA compaction, transcription, replication and repair. In E. coli, three HU dimers types are present (HUα2, HUβ2 et HUαβ) and show distinct biological roles. The heterodimer is formed from homodimers through a peptidic chain exchange. A model of this mechanism has been proposed by Ramstein and coworkers (J.M.B. 331, 101-121 2003) and was used as a starting point for this study. In this model, homodimers undergo a conformationnal change from a native state (N2) toward an intermediate state (I2). Then, I2 homodimers associate to form a transient heterotetramer which then dissociate into heterodimers. The main aim of this work was to characterize the structure and kinetic of each step of this mechanism. Major results of this work include the elucidation of two original crystal structures : HUβ2 from E. coli and HU from L. lactis in N2 states. A model of the partially disordered I2 state has also been proposed for HUβ2 and HUα2, and is consistent with results obtained from both NMR and molecular dynamics experiments. In addition, the existence of a low concentration tetrameric conformation has been evidenced by native mass spectrometry experiments. A protocol of production/purification/oxydation as been developped for the introduction of disulfide bridges in order to stabilize this conformation and characterize its structure. Together, results obtained from these different biophysical means refine our understanding of the chain exchange mechanism at the molecular level and highlight the role of the I2 conformation in controlling HU dimers composition

Protein (mis)folding into highly ordered, fibrillar structures, amyloid fibrils, is a hallmark of several, mainly neurodegenerative, disorders. The mechanism of this supra-molecular self-assembly reaction, as well as its relationship to protein folding are not well understood. In particular, the molecular origin of the metastability of the soluble state of proteins with respect to the aggregated states has not been clearly established. In this dissertation, it is demonstrated, that highly accurate kinetic experiments, using a novel biosensing method, can yield fundamental insight into the dynamics of proteins in the region of the free energy landscape corresponding to protein aggregation. First, a section on Method development describes the extension and elaboration of the previously established kinetic assay relying on quartz crystal microbalance measurements for the study of amyloid fibril elongation (Chapter 3). This methodology is then applied in order to study in great detail the origin of the various contributions to the free energy barriers separating the soluble state of a protein from its aggregated state. In particular, the relative importance of residual structure, hydrophobicity (Chapter 4) and electrostatic interactions (Chapter 5) for the total free energy of activation are discussed. In the last part of this thesis (Chapter 6), it is demonstrated that this biosensing method can also be used to study the binding of small molecules to amyloid fibrils, a very useful feature in the framework of the quest for potential inhibitors of amyloid formation. In addition, it is shown that Thioflavin T, to-date the most frequently employed fluorescent label molecule for bulk solution kinetic studies, can in the presence of potential amyloid inhibitor candidates be highly unreliable as a means to quantify the effect of the inhibitor on amyloid formation kinetics. In summary, the work in this thesis contributes to both the fundamental and the applied aspects of the field of protein aggregation.

Hu est parmi les proteines affines a l'adn la plus abondante d'e. Coli. C'est un dimere constitue de deux sous-unites, qui existe sous trois formes: alpha beta, alpha 2 et beta 2. Hu ne reconnait pas de sequence particuliere. Il a ete montre recemment qu'hu reconnait des structures cruciformes de l'adn. Par ailleurs, l'effet d'hu sur la conformation de l'adn a ete bien etabli. Nous avons etudie le role d'hu dans trois mecanismes: la replication, la recombinaison et la reparation ; ainsi que la compensation d'hu par ihf. Nos resultats ont montre qu'ihf ne compense pas l'absence d'hu dans la cellule. Nous montrons qu'hu joue un role dans la reparation de l'adn in vivo et in vitro. En effet, le double mutant d'hu presente une grande sensibilite aux rayonnements ionisants hu intervient egalement dans la recombinaison et nous montrons qu'hu peut moduler la repartition et l'activite de la proteine reca. En ce qui concerne le role d'hu dans la replication, nous montrons que la surproduction de beta 2 compense la thermosensibilite de la proteine dnaa dans un triple mutant dnaa46ts hupab

La régulation post-transcriptionnelle joue un rôle essentiel dans le système nerveux, de l’assemblage à la fonction. Chez les métazoaires, la famille multigénique ELAV / Hu code pour des protéines de liaison à l'ARN produites dans les neurones et impliqués dans cette régulation. Le gène elav (embryonic lethal abnormal visual system) code pour une protéine essentielle produite dans tous les neurones et est présent seulement chez les diptères. L’élucidation des fonctions des plus anciens paralogues de drosophile, fne (found in neurons) et rbp9 (RNA binding protein 9), a fait l'objet de ce travail, à cause de (1) l’étroite relation de ces gènes avec les orthologues de vertébrés, (2) la disponibilité de mutants nuls viables.En collaboration avec le laboratoire de Brian Oliver, NIH, États-Unis, nous avons utilisé le RNA-Seq pour analyser le transcriptome de tête de mutants fne-. Les données ont été analysées ) avec les programmes Cuffdiff et DESeq pour identifier des différences dans les niveaux de transcripts mutants et ceux d’une souche sauvage de référence (WT). L'épissage alternatif a été examiné avec Spanki (SPlicing ANalysis Kit), un suite de programmes que nous avons contribué à développer. Spanki utilise les données de séquence des jonctions exon-exon et compare l’abondance de formes alternatives et mutuellement exclusives de transcrits issus d’un événement d'épissage. Les premières analyses se sont concentrées sur les différences de transcriptomes entre males et femelles sauvages. La détermination sexuelle somatique et le comportement chez la drosophile sont sous le contrôle d'une cascade génétique bien caractérisée et contrôlée par les gènes Sxl, tra, msl2, dsx et fru, mais les cibles en aval de cette voie canonique demeurent largement inconnues. Notre approche nous a permis d’identifier (et de valider par qPCR) de nouveaux gènes exprimés différemment dans les deux sexes. J’ai montré que l’un d’entre eux est le gène fne, que sa régulation est indépendante de tra et tra2, mais dépend de Sxl. Ces propriétés font de fne la première cible identifiée dans une voie alternative de détermination sexuelle prédite par le laboratoire de T. Cline sur la base d'autres études. J'ai aussi montré l’existence d'un rôle précédemment non documenté pour tra et tra2 dans la lignée somatique mâle. Afin d'identifier des réseaux fonctionnels contrôlés par fne, les données du transcriptome de mutants ont été comparées à celles de la souche sauvage de référence. Plusieurs gènes dont le niveau de transcrits ou l’épissage alternatif est altéré en absence du gène fne ont été identifiés et validés par qPCR. Par exemple, l'épissage alternatif de unc-13, essentiel pour l'exocytose des vésicules, est affecté dans les mutants fne. D'autres gènes impliqués dans les fonctions synaptiques ont été identifiés, y compris n-syb, tomosyn, brp et Clc. En outre, il existe des interactions génétiques entre la mutation fne et des mutations affectant les fonctions synaptiques. Ces interactions peuvent causer une létalité synthétique dans les doubles mutants. Finalement, les males mutants fne- ont un comportement de « chaining» également compatible avec une fonction à la synapse. Ainsi, le gène fne établit un lien entre la régulation post-transcriptionnelle, la fonction synaptique et le comportement.Dans la dernière partie de mon travail, j'ai utilisé une approche évolutive pour tenter de distinguer les fonctions spécifiques de fne ou de rbp9, des fonctions ancestrales. Des approches moléculaires, anatomiques et comportementales ont été employées. Les résultats distinguent différentes catégories de gènes, spécifiquement ceux dont l'expression est affectée (1) uniquement par fne (2) uniquement par rbp9 (3) à la fois par fne et rbp9 de manière redondante ou synergique. J’ai mis en évidence une redondance fonctionnelle partielle des deux gènes causant une létalité synthétique à l'âge adulte dans les doubles mutants, mais j’ai aussi identifié des fonctions spécifiques<br>Post-transcriptional regulation plays pivotal roles from assembly to function of the nervous system. In metazoans, the ELAV/Hu genes constitute a conserved multigene family of pan-neuronal RNA-binding protein involved in post-transcriptional regulation. The elav (embryonic lethal abnormal visual system) gene is the first described family member, it encodes a vital protein and is restricted to dipterans. Elucidating the functions of the more ancient Drosophila paralogs, fne (found in neurons) and rbp9 (RNA-binding-protein-9), has been the focus of this work, because of (1) the close relationship of these genes to the vertebrate orthologs, (2) the availability of viable null mutants.In collaboration with the laboratory of Brian Oliver, NIH, USA, we used RNA-Seq to analyze the head transcriptomes of fne- mutants and a wild type reference strain (WT). The RNA-Seq data was searched for differences in transcript levels using the programs Cuffdiff and DESeq. Alternative splicing was examined using a suite of programs called Spanki (SPlicing ANalysis Kit), whose development we participated in. Spanki utilizes only sequence reads spanning exon-junctions to compare pairs of mutually exclusive alternative splicing events.Initial analyses included sex-specific comparisons in WT transcriptomes. Somatic sexual determination and behavior in Drosophila are under the control of a well characterized genetic cascade initiated by Sxl (Sex lethal), but the targets downstream of this canonical pathway remain largely unknown. As expected, the genes Sxl, tra (transformer), msl2 (male-specific lethal gene), dsx (doublesex) and fru (fruitless), which belong to the canonical sex-determination/dosage compensation pathways were identified by our analyses. In addition, new genes whose transcript expression levels differ between the sexes were identified and validated by qPCR. Further, I obtained evidence for previously undocumented roles of tra and tra2 (transformer 2) in the male soma. Finally, a sex-biased alternative splicing event was identified in fne, whose regulation is independent of tra or tra2, but dependent upon Sxl. This makes of fne the first Sxl-dependent, tra/tra2-independent target identified in a sex determination/differentiation pathway, previously been predicted to exist based upon other studies.The data was analyzed to identify functional networks controlled by fne. I found that it affects the expression of several genes at the level of transcript expression and alternative splicing involved in synaptic functions. They include of unc-13, n-syb (neuronal Synaptobrevin), tomosyn, brp (bruchpilot) and Clc (Clathrin light chain). Further, genetic interactions between fne and shi (shibire) or nrg (neuroglian), which also possess synaptic functions, reveal synthetic lethality in the double mutants. In addition, fne mutant males display a fruitless-like chaining behavior, also consistent with a function at the synapse. Thus the fne gene links post-transcriptional regulation to synaptic function and behavior.Finally, I used an evolutionary approach to distinguish newly evolved functions, i.e. specific to fne or rbp9, from those that are shared and thus more likely to be ancestral. Molecular, anatomical and behavioral approaches in parallel analyses of fne and rbp9, show that they possess both gene-specific and overlapping functions. The latter is evident from synthetic lethality in early adulthood of double mutants. My results distinguish different gene categories with respect to their regulation: genes whose expression is affected (1) only by fne (2) only by rbp9 (3) both by fne and rbp9 in redundant or synergistic ways. Finally, I showed that male-male interactions dramatically differ between fne versus rbp9 mutants, revealing the emergence of a new (or loss of an ancestral) function in behavior

Si hu contribue a la formation des structures de type nucleosomique dans la bacterie, son absence devrait modifier l'equilibre des topoisomerases responsable du maintien de la superhelicite non contrainte. Les differents resultats obtenus montrent effectivement une augmentation de l'activite relaxante de la topoisomerase i inversement proportionnelle a la quantite d'hu presente dans la cellule bacterienne. Nous avons egalement observe une diminution de la synthese de la sous-unite gyra dans les doubles mutants d'hu. Ces variations constatees contribuent a eliminer l'exces de la superhelicite negative generee par l'absence d'hu et retablissent une superhelicite de l'adn compatible aux besoins essentiels de la cellule. Par ailleurs, nous avons mis en evidence, aussi bien in vivo qu'in vitro, un mecanisme d'inhibition de la topoisomerase i en presence de fortes quantites de la proteine hu. Afin de mieux comprendre la relation entre la proteine hu et la topoisomerase i nous avons essaye de construire, par transduction, des doubles mutants topa10 hupa. Il s'est avere qu'une souche defectueuse au niveau de l'activite relaxante ne peut accepter une mutation au niveau du gene hupa. Cette incompatibilite diminue lorsque l'adn chromosomique du mutant topa est relache par un inhibiteur de la gyrase. Ces resultats suggerent que la relation existant entre la topoisomerase i et la proteine hu pourrait s'effectuer par le biais de la superhelicite de l'adn chromosomique. Par ailleurs l'etude des pseudo-revertants des doubles mutants hupab a montre l'existence de mutations compensatoires localisees au niveau des deux genes codant pour la gyrase. Ces mutations qui n'affectent pas la superhelicite de l'adn bacterien semblent permettre a la gyrase d'accomplir ses autres activites (decatenation, relaxation des supertours positifs lors de la transcription etc) en absence de la proteine hu

Hu est la proteine affine a l'adn la plus abondante chez e. Coli qui reconnait une structure particuliere de l'adn. En se liant a l'adn elle modifie la conformation de l'adn, ce faisant elle intervient dans de nombreux mecanismes moleculaires essentiels pour la survie de la bacterie. Hu est tres conservee et existe sous trois formes dimeriques (, 2 et 2) associees a une phase de croissance bien precise. Nous avons developpe un systeme pour optimiser la surproduction et la purification des dimeres sous leur forme native depourvue de contamination nucleasique. Nous avons determine les caracteristiques de liaison de 2 et 2 pour differentes structures d'adn et nous les avons comparees a celles obtenues pour. Si 2 presente les memes caracteristiques de liaison que , 2 se differencie dans ces interactions avec l'adn par rapport a 2 et 2. Les dimeres 2 et presentent une grande affinite pour l'adn cruciforme et les adns contenant un nick et un gap et tres peu d'affinite pour un adn double-brin. 2 quant a lui, se fixe preferentiellement a l'adn cruciforme. Cette etude comparative des proprietes de liaison des dimeres nous a permis aussi de montrer que 2 et 2 reconnaissent preferentiellement, comme , une conformation particuliere dans l'adn plutot qu'une sequence. Nous avons ainsi mis en evidence chez les trois dimeres deux modes de fixation : une interaction non-specifique se transformant en une interaction hautement specifique quand hu rencontre une structure d'adn. Nous avons mis en evidence les modalites de ces deux modes de fixation. Les experiences de mutagenese nous ont permis de cibler le domaine de la proteine responsable de la fixation structure-specifique et l'amino-acide responsable de la reconnaissance par hu d'une structure d'adn. D'autre part, en generant et purifiant deux proteines hybrides, nous avons mis en evidence que l'interaction non-specifique a l'adn se fait uniquement par l'intermediaire des bras de la proteine.

La proteine hu d'escherichia coli appartient a un groupe de proteines abondantes et affines a l'adn que l'on qualifie de type-histone, en raison des proprietes qu'elle partage avec les histones des eucaryotes. Cette proteine hu participe au maintien du niveau de superhelicite du chromosome bacterien au sein du nucleoide. D'autre part hu intervient dans la replication, la transcription, la transposition, la recombinaison et la reparation de l'adn. Ses roles dans la bacterie sont donc consideres comme relativement divers. L'etude de la regulation de la synthese d'hu nous a amene a aborder la regulation de la transcription des deux genes hupa et hupb, codant respectivement pour les deux sous-unites a et b de la proteine. Nous avons ainsi pu montrer que les proteines regulatrices de la transcription crp (camp receptor protein) et fis (factor inversion stimulation) interviennent dans l'expression des deux genes d'hu. Nous avons egalement pu mettre en evidence un controle par la phase de croissance sur l'accumulation des arn messagers des genes hupa et hupb. L'ensemble de ces regulations au niveau transcriptionnel permet de moduler la synthese des deux sous-unites a et b de la proteine. Ainsi nous avons montre que la quantite d'hu accumulee dans la cellule ainsi que la composition en formes dimeriques de la proteine varient au cours de la phase de croissance des bacteries. L'approche physiologique du role des differents dimeres a2, ab et b2 nous a conduit a designer un lien entre hu et certains stress associes a la phase stationnaire des bacteries. En effet, il semble que la presence des differentes formes dimeriques de la proteine influe sur la survie des cellules face a des conditions de carences en nutriments et face au stress oxydatif. Il existe egalement une relation entre hu et le facteur ss de l'arn polymerase. Ce facteur contribue, via le controle de l'expression de nombreux genes, a la mise en oeuvre de diverse reponses a de nombreux stress. Ces resultats nous amenent donc a envisager un role important d'hu pour la survie des bacteries dans des conditions de stress.

La proteine hu d'escherichia coli est le prototype d'une nouvelle classe de proteines bacterienne de faible poids moleculaire, affines a l'adn comprenant hu mais aussi ihf, fis et h-ns. La proteine hu joue un role pleiotropique dans la cellule puisqu'elle intervient au niveau de la replication, de la transposition, de l'inversion genique ou encore de la reparation. Nous avons mis en evidence deux mecanismes reliant hu, proteine de type histone, a des modifications de la membrane bacterienne. Dans un premier temps, la deregulation de la synthese d'hu par transformation d'une souche sauvage par un plasmide portant un des deux genes d'hu (hupa ou hupb) rend la bacterie mucoide. Cette mucosite correspond a la synthese massive d'un polysaccharide de capsule, l'acide colanique. Nous avons montre que dans ce mecanisme, les genes cps pour capsular polysaccharide synthesis sont actives via l'activation transcriptionnelle d'un gene codant pour un des regulateurs positifs, la proteine rcsa. Parallelement a ce mecanisme de production de polysaccharide de capsule, la cellule declenche l'induction du systeme sos qui est un mecanisme de defense developpe lors de l'exposition face a des conditions qui endommagent l'adn. Dans un second temps nous avons montre que l'absence d'hu produit un changement drastique dans la composition de la membrane externe e. Coli avec surproduction de la porine ompf. Une des regulations de ompf s'exerce a un niveau post-transcriptionnel par une hybridation grace a une complementarite partielle entre l'arnm issu du gene ompf et micf un petit arn. Nous avons montre qu'en absence d'hu la production de l'arn micf est affectee ce qui entraine physiologiquement une augmentation de la permeabilite membranaire avec comme consequence une hypersensibilite envers certains antibiotiques, detergents et sels biliaires.

L'adn est une molecule polymorphe dont les differentes conformations sont liees a des fonctions cellulaires precises. Elle est sensible a certains agents physiques et chimiques. Cette propriete est exploitee dans le cas de la chimiotherapie ou un agent chimique comme le cis-dichlorodiamminoplatine (ii) (cis-ddp) modifie, en se fixant a l'adn, la conformation de la double helice ce qui le rend particulierement toxique pour les cellules en division rapide. Etant donne que la cytotoxicite du cis-ddp n'a pas encore pu etre correlee au type d'adduit forme sur l'adn, il est important d'avoir un apercu des differentes deformations de l'adn induites par la drogue. La resolution de la structure cristallographique d'un decamere double-brin ponte par un adduit interbrin du cis-ddp, nous a permis d'analyser les deformations de la double helice induites par un tel adduit ainsi que le positionnement particulier de certaines molecules de solvant autour de celui-ci. La reconnaissance proteique des lesions induites par le cis-ddp sur l'adn semble jouer un role important pour l'activite de la drogue mais egalement pour la resistance des cellules face a celle-ci. La proteine procaryote hu reconnait specifiquement les deformations induites par certains adduits intrabrins du cis-ddp. En plus de son role architecturale, elle semble impliquee dans de nombreux mecanismes cellulaires. Dans le but d'etudier, par cristallographie, la structure d'un complexe entre la proteine hu et un oligonucleotide porteur d'un adduit intrabrin du cis-ddp, nous avons prepare et caracterise les differents partenaires impliques dans ce complexe. Chez escherichia coli, la proteine hu existe sous trois formes dimeriques (2, 2 et ) dont les proportions varient au cours du cycle cellulaire et qui ont des affinites differentes pour des formes transitoires de l'adn. La structure cristalline de la proteine homodimerique hu2 a ete resolue, analysee et comparee aux structures des proteines homologues.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.<br>The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

碩士<br>國立陽明大學<br>生醫光電工程研究所<br>95<br>We setup an optical manipulation system to trap a polystyrene particle via optical tweezers and measured the transverse optical spring constant by analyzing the Brownian motion of the trapped particle driven by thermal fluctuation. In our experiments, the transverse optical spring constant was measured to be approximately 115±1pN/mm when a polystyrene particle (diameter =2.88μm) was trapped with a 19mW IR laser beam (wavelength = 1064nm) focused by an oil-immersion objective lens with a numerical aperture (NA) of 1.24. In biological application, we focused on the study of the interaction of Lon protease and HU protein by coating Lon on one polystyrene particle and HU on the other and measured their binding force by optical tweezers. From biochemical experiment data, we know Lon protease can degrade HUβ, but not HUα. To investigate the origin of this difference, we measure the binding force of Lon protease interacting with HUα、HUβ and the mutant protein, HUβ-A20D, in which. In experiments without energy supplement of ATP, the binding forces of Lon protease interacting with BSA, HUβ,HUα and HUβ-A20D were determined to be approximately 0(pN), 24±6(pN), 28±6(pN) and 31±4(pN), respectively. The lack of interaction of BSA with Lon protease indicates the specificity of the enzyme and the substrates, whereas the very small differences in the binding force of Lon-HUβ and Lon-HUβ-A20D indicate that the binding site and the cleavage site of Lon protease on HU protein may not be the same. However, with ATP supplement, the binding force of Lon protease interacting with HUα,HUβ and HUβ-A20D were measured to be approximately 14±2(pN), 11±4(pN) and 15±2(pN), respectively. The significant decrease in the binding force in all cases (in the presence of ATP) is speculated to be due to the conformation change of the Lon protease in the presence of ATP.

Of the four Hu genes found in most vertebrates (HuA, HuB, HuC and HuD), all except HuA exhibit mRNA and protein expression that is essentially restricted to post-mitotic neurons of the developing and adult nervous systems. Spatial and temporal examination of individual “neuronal Hu” (nHu = HuB, HuC and HuD) proteins in brain tissue suggests nHu proteins may play a functional role during neuronal differentiation; as RNA-binding proteins, the nHu proteins may participate in gene regulatory events that are essential for acquisition of the neuronal phenotype. We have identified a number of candidate mRNA targets of the nHu proteins. Our data suggest that the majority of these mRNAs interact with nHu proteins through sequences present in their 3´ untranslated regions (UTRs). From this 3´UTR target subset, several mRNAs were selected for further examination based on reported roles for their encoded proteins during axonogenesis, a critical developmental process during which nascent neurons grow and extend axons that eventually connect to and form synaptic connections with other neurons. The mRNAs chosen encode for cytoskeleton-modifying proteins; Cofilin, Vasodilator-Stimulated Phosphoprotein (VASP) and the Rho GTPase Cdc42. The primary aim of the work reported in this thesis was to characterise the effect of interactions between the neuronal Hu protein HuC, and the CLIP-identified 3´UTRs listed above. To do this, the 3´UTR sequences were cloned into reporter vectors (both fluorescent and luciferase reporter-based) to produce reporter protein-encoding messages that included a putative target 3´UTR. These vectors were then used in co-transfection experiments with or without HuC and measurements of reporter protein and mRNA abundance obtained. Interestingly, despite initial speculation that HuC might be involved in directly regulating protein expression from target mRNAs, no significant effect of HuC on protein production from any of the 3´UTR-reporter mRNAs tested was observed. However and quite unexpectedly, measurement of 3´UTR-reporter mRNA abundance from co-transfection assays revealed a potential role for HuC in modulating alternative polyadenylation site choice for one of the CLIP-identified 3´UTR sequences. Regulation of mRNA polyadenylation site choice may be a novel mechanism by which nHu proteins post-transcriptionally control gene expression during neuronal development.<br>Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2011

碩士<br>國立臺灣大學<br>生化科學研究所<br>92<br>HU, a prokaryotic histone-like protein, associates with bacterial nucleoids and plays architectural roles in replication initiation, transcription regulation and site-specific recombination. To gain an insight into the role of Lon protease in regulation of HU, the purified Bt-Lon was incubated with HU of Bacillus subtilis (Bs-HU). Lon selectively degraded Bs-HU in an ATP-dependent manner, thus confirming the ability of Lon to directly recognize and degrade specific substrates. Cleavage occurred at Leu44-Ile45 and Ala57-Arg58 peptide bonds in the early period of degradation, which are known to be in the DNA-binding motif of Bs-HU. Plasmid DNA and ds-DNA can both enhance proteolysis, with plasmid being better enhancer than ds-DNA . In addition, the degradation of Bs-HU by Bt-Lon in the early period does not influence its ability to bind single-stranded DNA even removing the DNA-binding motif; however, the degradation causes the loss of the DNA-binding ability after incubation with Bt-Lon for 2 h. Furthermore, the DNA conformation altered by Hu can be reformed from the degradation of HU by Bt-Lon. This is the first example to show that HU, a DNA binding protein, is a physiological substrate of Lon protease and Lon protease is probably involved in the regulation of gene expression by degradation of HU.

Bacteria face the challenging task of compacting their chromosomes to accommodate them in a small cytoplasmic volume and at the same time maintaining the nucleoids in a highly organized and dynamic state for transcription, DNA replication, and chromosome partitioning to take place with accuracy and speed. DNA also has to be protected from damage to preserve the genetic information. The structure and organization of the bacterial chromatin is shaped by compacting forces, such as DNA supercoiling, macromolecular crowding, and by nucleoid-associated proteins (NAPs). The NAPs are often referred to as histone-like proteins due to their functional similarity with eukaryotic histones. In Escherichia coli, a dozen proteins have been identified as NAPs. The well characterized members of this family are HU, H-NS, IHF, Lrp, and Fis. NAPs carry out wrapping, bridging, and bending of the bacterial DNA, resulting in its compaction and topological rearrangements. Most NAPs display broad specificity toward DNA and can thus have a global effect on gene expression. NAPs are known to influence the virulence and pathogenesis of different pathogenic bacteria. In spite of their critical role in bacterial physiology and virulence, very limited information is available on NAPs of Mycobacterium tuberculosis (Mtb). The work presented in this thesis describes the functional characterization of two important NAPs of Mtb namely, Rv3852 and Rv2986c (HU) and understanding their role in genome organization and topology modulation. Chapter 1 of the thesis provides introduction on bacterial nucleoid and its architectural organization. The importance of nucleoid-associated proteins in maintenance of genome architecture and supercoiling have been discussed. Further, the functions of some of the well characterized NAPs have been described with specific examples. Finally, a brief overview of Mtb genome, disease epidemiology, and pathogenesis is presented along with the description of the initial studies on mycobacterial NAPs. In Chapter 2 studies have been directed to functionally characterize Rv3852, a NAP of Mtb, conserved among the pathogenic and slow growing species of mycobacteria. Data presented in this part show that the NAP binds DNA in a sequence independent manner and ectopic expression of the protein in Mycobacterium smegmatis cells causes spreading of the nucleoid. The protein has both DNA binding and membrane anchoring properties and is predominantly localizes in the cell membrane. The carboxyl terminal region of the protein has the propensity to form transmembrane helix which is shown to be necessary for its membrane localization. The protein is involved in genome organization and its ectopic expression in M. smegmatis results in defects in biofilm formation, sliding motility and change in a polar lipid profile. The study demonstrates the crucial role of Rv3852 in regulating the expression of KasA, KasB and GroEL1 proteins which are in turn involved in controlling the surface phenotypes in mycobacteria. Chapter 3 describes the studies on an essential NAP, Rv2986c, the homologue of histone-like protein HU in Mtb (MtHU). HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. The work described in this chapter reports the functional characterization of HU from Mtb. Although HU is essential for growth of Mtb, there have been no reported attempts to perturb MtHU function with small molecules. Based on the crystal structure, a core region within the MtHU-DNA interface has been identified that can be targeted using stilbene derivatives. These small molecules specifically inhibit MtHU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. The results indicate that HU is a potential target for development of therapeutics against tuberculosis. The work presented in Chapter 4 focuses on understanding the role of MtHU in maintenance of DNA topology. The topological homeostasis of bacterial chromosomes is achieved by the balance between compaction and topological organization of genomes. Two classes of proteins play major roles in chromosome organization: The NAPs and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. The data presented here demonstrates that MtHU specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between MtHU and TopoI is necessary for enhancing the enzyme activity both in vitro and in vivo. The interaction is between amino terminal domain of MtHU and carboxyl terminal domain of TopoI. Binding of MtHU does not affect the two catalytic trans-esterification steps but enhances the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism of regulation of topoisomerase activity. An interaction deficient mutant of MtHU is compromised in enhancing the strand passage activity. The species specific physical and functional cooperation between MtHU and TopoI may be the key to achieve DNA relaxation levels needed to maintain optimal superhelical density of mycobacterial genomes.

Thesis (Ph.D.)--Jacobs University Bremen, 2007.<br>School of Engineering and Science. "Doctor of Philosophy in Biochemistry." Includes bibliographical references (leaves 100-105). Also available online.

In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen. MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens. The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome. Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work. The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected. The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser. Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.

In bacteria, nucleoid associated proteins (NAPs) represent a prominent group of global regulators that perform the tasks of genome compaction, establishing chromosomal architecture and regulation of various DNA transactions like replication, transcription, recombination and repair. HU, a basic histone like protein, is one of the most important NAPs in Eubacteria. Mycobacterium tuberculosis produces a homodimeric HU (MtbHU), which interacts with DNA non-specifically through minor groove binding. Exploration for essential genes in Mtb (H37Rv) through transposon insertion has identified HU coding gene [Rv2986c, hupB; Gene Id: 15610123; Swiss-Prot ID: P95109)] to be vital for the survival and growth of this pathogen. MtbHU contains two domains, the N-terminal domain which is considerably conserved among the HU proteins of the prokaryotic world, and a C–terminal domain consisting of Lys-Ala rich multiple repeat degenerate motifs. Sequence analysis carried out by the thesis candidate showed that MtbHU exhibits 86 to 100 percent identity within the N-term region among all the mycobacterium species and some of the members of actinobacteria, including important pathogens like M. tuberculosis, M. leprae, M. ulcerans, M. bovis, Nocardia; while C term repeat region varies relatively more. This strikingly high cross species identity establishes the MtbHU N-terminal domain (MtbHUN) as an important representative structural model for the above mentioned group of pathogens. The thesis candidate has solved the X-ray crystal structure of MtbHUN, crystallized in two different forms, P2 and P21. The crystal structures in combination with computational analyses elucidate the structural details of MtbHU interaction with DNA. Moreover, the similar mode of self assembly of MtbHUN observed in two different crystal forms reveals that the same DNA binding interface of the protein can also be utilized to form higher order oligomers, that HU is known to form at higher concentrations. Though the bifunctional interface involved in both DNA binding and self assembly is not akin to a typical enzyme active site, the structural analysis identified key interacting residues involved in macromolecular interactions, allowing us to develop a rationale for inhibitor design. Further, the candidate has performed virtual screening against a vast library of compounds, and design of small molecules to target MtbHU and disrupt its binding to DNA. Various biochemical, mutational and biological studies were performed in the laboratory of our collaborator Prof. V. Nagaraja, MCBL, IISc., to investigate these aspects. After a series of iterations including design, synthesis and validation, we have identified novel candidate molecules, which bind to MtbHU, disrupt chromosomal architecture and arrest M. tuberculosis growth. Thus, the study suggests that, these molecules can serve as leads for a new class of DNA-interaction inhibitors and HU as a druggable target, more so because HU is essential to Mtb, but absent in human. Our study proposes that, targeting the nucleoid associated protein HU in Mtb can strategize design of new anti-mycobacterial therapeutics. Perturbation of MtbHU-DNA binding through the identified compounds provides the first instance of medium to small molecular inhibitors of NAP, and augurs well for the development of chemical probe(s) to perturb HU functions, and can be used as a fundamental chemical tool for the system level studies of HU-interactome. Section I: “Crystal structure of Mycobacterium tuberculosis histone like protein HU and structure based design of molecules to inhibit MtbHU-DNA interaction: Leads for a new target.” of this thesis presents an elaborate elucidation of the above mentioned work. The candidate has additionally carried out structure based computational and theoretical work to elucidate the interaction of amino acid based metal complexes which efficiently bind to DNA via minor-groove, major-groove or base intercalation interaction and display DNA cleavage activity on photo-irradiation. This understanding is crucial for the design of molecules towards Photodynamic Therapy (PDT). PDT is an emerging method of non-invasive treatment of cancer in which drugs like Photofrin show localized toxicity on photoactivation at the tumor cells leaving the healthy cells unaffected. The work carried out in our group in close collaboration with Prof. A.R. Chakravarty of Inorganic and Physical Chemistry Department elaborates the structure based design of Amino acid complexes containing single Cu (II), such as [Cu(L-trp)(dpq)(H2O)]+ , [Cu (L-arg) 2](NO3)2 , Amino acid complexes containing oxobridged diiron Fe(III), such as [{Fe(L-his)(bpy)}2(μ-O)](ClO4)2 , [{Fe(L-his)(phen)}2(μ-O)](ClO4)2 , and Complexes containing Binuclear Cu(II) coordinated organic moiety, such as [{(dpq) CuII}2(μ-dtdp)2], which bind to DNA through minor groove/major groove/base intercalation interactions. Docking analysis was performed with the X-ray crystallographic structure of DNA as receptor and the metal complexes as ligands, to study the mode of binding to DNA and to understand the possible mode of DNA cleavage (single/double strand) when activated with laser. Section II: “Structure based computational and theoretical analysis of metal coordinated complexes containing amino acids and organic moieties designed for photo induced DNA cleavage” of this thesis presents a detailed presentation of the above mentioned work.

DNA- and RNA-binding proteins play a central role in gene regulation, which includes transcriptional control, alternative splicing, post-translational and transcriptional modifications like methylation and acetylation among other roles. In this way, they control most of the working machinery of the cell in direct or indirect manner. Although more than 60 years ago the structure of DNA was proposed by Watson and Crick, our understanding of how RNA- and DNA-binding proteins interact with the genome and transcriptome remains scarce. One of the most important questions in biology is how a large number of DNA- and RNA-binding proteins find their target, interact and later disassociate. These nucleic acid binding proteins either recognizes the unique structural and chemical signatures of the bases (base readout) which give the specificity or it recognizes a sequence-dependent shape (shape readout). Methyltransferases are enzymes with diverse folds, which perform methyltransfer to various substrates using mainly S-adenosyl-L-methionine (AdoMet) as a methyl donor. RNA methylation is one of the most crucial post-transcriptional modifications which influences a wide variety of cellular processes like metabolic stabilization of RNA, quality control in protein synthesis, resistance to antibiotics, mRNA reading frame maintenance, splicing, viral nucleoprotein stabilization among others. Specificity in recognition and methylation in ribosomal RNA (rRNA) methyltransferases is very crucial, as rRNA is highly conserved and lack of specificity would influence the stabilization of RNA and thus, will affect the ribosome. In recent years, rRNA modifications which confer resistance to ribosomal antibiotics have also been observed. The mechanism of recognition to their unique rRNA target site with high selectivity and their evolution still remains an enigma. Thus, the evolution of antibiotic resistance-conferring methyltransferases in pathogenic organisms needs to be investigated from the structural and evolutionary perspective. In the last two decades, many global regulators in both eukaryotes and prokaryotes have been discovered, which promiscuously bind to a large number of DNA sequences. In prokaryotes, they are called as ‘Nucleoid-associated proteins’ (NAPs), which influence the transcriptional process and exhibit multi-specificity or promiscuity. They also take part in the formation of many multi-protein complexes. HU and Integration Host Factor (IHF) are NAPs which belong to prokaryotic DNA-bending protein family (DNABII family). HU and IHF play crucial architectural roles in bacterial DNA condensation and additionally play a regulatory role in many cellular processes. Although sharing structural similarity, the DNA binding and bending features of HU and IHF are strikingly different, allowing them to selectively regulate genes from different genomic locations. HU binds to DNA in a sequence promiscuous manner while IHF is moderately sequence specific. The molecular mechanism of DNA binding multi-specificity (differential specificity with varied binding affinity) of HU/IHF proteins remains unexplored, as little attention has been paid to the determinants at the sequence level. Now, the fundamental question which the author attempted to understand is the structural and evolutionary determinants of specificity in DNA- and RNA-binding proteins. The candidate has taken nucleoid-associated protein HU and SPOUT superfamily RNA methyltransferase as model systems. As the very limited number of structural folds makes up the DNA- and RNA-binding proteins, it is intriguing to examine closely related nucleic acid binding domains or folds carrying out specific functions. Also, we observed that some proteins having a particular structural fold (or homologous ancestry) bind to DNA or RNA with high specificity, while its other homolog binds promiscuously. These observations tempted us to find the sequence and structural determinants which guide this phenomenon, not just specific to only a single protein family, but, determinants are of more general nature, where results can possibly be applied to other nucleic acid binding proteins too. The first part of the thesis reports the crystal structures of native and AdoMet bound ribosomal RNA Methyltransferase from Sinorhizobium meliloti (smMtase), by single anomalous dispersion (SAD) phasing on seleno-methionine substituted crystal, which diffracted to 2.28Å and 2.9 Å resolutions respectively in space group P212121. smMtase belong to an rRNA binding SPOUT superfamily protein, which is fused with an RNA binding L30e domain at the N-terminus. We focused our study on these types of proteins among the large superfamily (henceforth termed as SPOUTL30). The author also has conducted a phylogenetic study, which revealed 11 major clades, out of which we focused our present study in understanding the sequence conservation and variations of 5 (A-E) clades, for which structural, biochemical and functional data is available. These proteins share homology to antibiotic resistance conferring methyltransferases. The availability of experimentally determined structures of native and AdoMet bound smMtase along with an analysis of other homologous crystal structures has enabled a critical examination of factors influencing RNA binding specificity. Also, the thesis reports for the first time an evolutionary and structural inter-connectivity of the three conserved motifs (I-III) in SPOUT superfamily, which is responsible for AdoMet binding and catalysis. The results highlight that both the location of conserved positive and negatively charged residues influence the RNA binding, specificity, and affinity. The conservation of these residues could be at superfamily, family or at clade level, and the position of these charged residues at specific sites, alters their salt-bridge geometry, which ultimately fixes the conformation of RNA-binding residues, thus defining a particular binding site specific to its cognate RNA. The study conducted by the author reveals that the dynamics of salt-bridge and other directional interactions like hydrogen bonding and aromatic interactions essentially determines the specificity of SPOUTL30. The second part of the thesis reports evolutionary, structural and functional studies on nucleoid-associated proteins HU and IHF. To understand the sequence determinants, which influence the degree of DNA binding specificity, we undertook a phylogenetic study in conjunction with analysis of three-dimensional structures. The phylogenetic analysis revealed three major clades, belonging to HU, IHFα, and IHFβ like proteins with reference to E. coli. The author observed statistically significant amino acid compositional bias in the DNA binding sites of HU and IHF clade proteins. The author proposes that the molecular mechanisms giving rise to specificity or multi-specificity depend on a combination effect of the amino acid composition of the binding site, its flexibility, ionic and steric constraints. In continuation of this part of the thesis, the candidate examined the role of protein interacting interface of HU-IHF family proteins, understanding its evolutionary history and utilizing it in designing inhibitors for Mycobacterium tuberculosis HU (MtbHU). The present results give a model example of an evolutionary study of a protein interface of nucleoid-associated protein, which is used to understand the interface and computationally design inhibitors targeting it. The author was a part of the study (Bhowmick et al. 2014, Nature communications) which has determined the crystal structure of Mycobacterium tuberculosis HU, inhibited it using stilbene derivatives (SD1 and SD4) which curtailed the Mtb cell growth. In the present thesis, the candidate observed from microarray analysis that the SD1 stimulon consists of genes involved majorly in lipid biosynthesis pathway, ribosomal genes which affect the overall translation, aerobic respiration pathways, antigenic membrane proteins involved in pathogenicity. Nearly half of the genes in affected by SD1 are essential in nature, thus could explain the curtailing of cellular growth. The whole study provides a system inspired view of probing as well, inhibiting global regulator HU using novel chemical molecules.

Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Vor 2 Jahren wurden erstmals die wahrscheinlich wichtigsten Tight Junction Proteine, Claudin-1 und -2 in der Maus beschrieben. An Hand von Sequenzhomologien konnten bis heute dieser 4 Transmembrandomänen Proteinfamilie 18 Mitglieder mit unterschiedlichen Gewebeverteilungen zugeordnet werden. Parallel zum murinen Claudin-1 wurde von (Swisshelm et al. 1999) das humane Claudin-1 mit einer 91 prozentigen Sequenzhomologie zum murinen Protein isoliert und molekulargenetisch beschrieben. In der überwiegenden Mehrzahl von Brusttumorzellinien ist die Expression von hu-CLDN1 verloren gegangen. In der vorliegenden Arbeit sollte deshalb die biologische Funktion des humanen Claudin-1 (hu-CLDN1) und dessen Relevanz während der Tumorgenese untersucht werden. Für diese Aufgabenstellung mußten monoklonale Antikörper mittels DNA Vakzinierung und hu-CLDN1 Retroviren zur effektiven Transduktion von Tumorzellen entwickelt werden. Die Antikörper wurden in ELISA, kompetitiven hu-CLDN1 Peptid ELISA und Western Blot auf ihre hu-CLDN1 Spezifität und Epitoperkennungsstelle überprüft. Für biologische Untersuchungen wurden für die neuen Antikörper optimale immunzytochemische und immunhistochemische Methoden etabliert. Mit Hilfe der monoklonalen Antikörper wurde die zelluläre Proteinexpression und die Lokalisation, als auch die Expression von hu-CLDN1 in Gewebeschnitten von Tumoren- und Normalgewebe untersucht. Für die Reexpression von hu-CLDN1 in Brusttumorzellen wurden retrovirale Genshuttlesysteme hergestellt. Die retroviralen Genshuttlesysteme basieren auf dem MoMuLV Grundgerüst und als Besonderheit wurde der l-NGFR Rezeptor zur schnellen und sicheren Identifkation transduzierter Zellen einkloniert. Hergestellt wurde ein Mock Kontrollvektor (nur l-NGFR) und der hu-CLDN1 Vektor mit l-NGFR. Die hu-CLDN1 retroviralen Überstände wurden verwendet, um verschiedene Brusttumorzellinien zu transduzieren. Die Expression von hu-CLDN1 wurde mittels eigens entwickelter quantitativer gekoppelter Reverser Transkription Polymerase Ketten Reaktion (qRT PCR) in verschiedenen Brusttumorzellinien untersucht. Wie aus der vorliegenden Arbeit ersichtlich, konnten erstmalig monoklonale Antikörper gegen hu-CLDN1 entwickelt werden. Diese sind spezifisch und haben eine hohe Sensitivität gegenüber hu-CLDN1. Desweiteren gelang es erstmals Antikörper gegen alle 4 extra- und intrazellulären Domänen zu gewinnen. Mit diesen Antikörpern gelang es nachzuweisen, daß es sich bei hu-CLDN1 um ein ausschließlich membranständiges Protein handelt. Die Expression des Proteins findet sich nur in konfluenten Zellkulturen von natürlicherweise hu-CLDN1 exprimierenden Brusttumorzellen (T47D, MCF7), wobei die Lokalisation ausschließlich auf die Zell-Zell Kontaktstelle beschränkt ist. Bei der hu-CLDN1 Transduktion in hu-CLDN1 negativen Brusttumorzellinen zeigte sich eine konstitutive mRNA Expression in subkonfluenten und konfluenten Zellkulturen. Allerdings ist fluoreszenzmikroskopisch hu-CLDN1 Protein nur in konfluenten Zellkulturen nachweisbar, wobei diese Tumorzellen das Protein korrekt nur an der Zell-Zell Kontaktstelle einbauen. Offensichtlich haben die hu-CLDN1 negativen Brusttumorzellen noch den intakten Signalweg zur korrekten Expression mit einer noch unbekannten posttranskriptionellen Kontrolle. Bemerkenswert ist in diesem Zusammenhang, daß in Brusttumorzellen, die weder Occludin noch ZO-1 exprimieren (MDA-MB-435) die physiologisch korrekte Expression von hu-CLDN1 existiert. Offensichtlich ist die Lokalisation von hu-CLDN1 von beiden Proteinen unabhängig. Die Analyse von unterschiedlichen hu-CLDN1 positiven und negativen Brusttumorzellen zeigte, daß der Verlust der hu-CLDN1 Expression besser zur in vitro Invasivität von Brusttumorzellen korreliert als der Expressionsverlust von Occludin und ZO-1. Die klinische Relevanz des Verlustes von hu-CLDN1 während der Tumorgenese wurde bei den Expressionsanalysen auf normalen Brust- und Darmgewebe gegenüber transformierten Brust- und Darmgewebe untersucht. Bei der Analyse von normalem Brustgewebe konnte festgestellt werden, daß hu-CLDN1 ein rein epthelial/endotheliales Protein mit eindeutiger Membranlokalisation darstellt. In den untersuchten transformierten Geweben zeigte keines der transformierten Gewebe mehr die membranständige Färbung, sondern nur noch zytoplasmatische Färbung. Desweiteren war eine signifikant reduzierte oder nicht vorhandene hu-CLDN1 Färbung in einer größeren Anzahl von Tumoren zu beobachten. Diese Ergebnisse zeigen, daß der Verlust der Expression zumindest bei der Brust- und Darmtumorentwicklung offensichtlich mit der in vivo Tumorprogression korreliert. Um die Bedeutung der hu-CLDN1 Relokalisation bzw. verringerten Expression während der Tumorgenese zellphysiologisch zu verstehen, wurden in vitro Zellkulturstudien mit hu-CLDN1 negativen Zellinien und ihren hu-CLDN1 transduzierten Tochterpopulationen durchgeführt. In den adhärenten Zellkulturen hat die hu-CLDN1 Expression keinen Einfluß auf Zellwachstum und Apoptose. Allerdings zeigte sich in drei dimensional wachsenden Tumorzellaggregaten, daß die Reexpression von hu-CLDN1 zu einer drastischen Erhöhung der Apoptose führt. Die parazellulären Fluxstudien ergaben, daß bei der Reexpression von hu-CLDN1 in Brusttumorzellen der parazelluläre Flux zwischen den Zellen deutlich zurückgeht. Somit könnte die reduzierte Wachstumskapazität bzw. erhöhte Apoptose in 3 D Kulturen mit einer reduzierten Zugänglichkeit von Nährstoffen und Wachstumsfaktoren in hu-CLDN1 transduzierten Zellen verursacht sein. Dies würde auch erklären, warum bei den untersuchten transformierten noch hu-CLDN1 positiven Brust- und Darmtumorgeweben sich ausschließlich eine zytoplasmatische Lokalisation in den Tumorzellen findet. Während in Organen und Drüsengeweben Epithelien einschichtig vorkommen und die Versorgung der Zellen mit Wachstumsfaktoren basolateral oder apikal durch Diffusion und Mikro-/Makropinozytose erfolgen kann, wachsen Tumorepithelzellen mehrschichtig. Wären die Tight Junctions im Tumor noch intakt, so könnte es zu einer Mangelversorgung und somit zur Apoptose kommen. Für das in vivo Wachstum eines Tumors ist es also notwendig, Membranproteine, die den parazellulären Flux inhibieren, zu verringern. Wie die zellphysiologischen in vitro Studien der vorliegenden Arbeit zeigen, ist es aber möglich, einen tumorinhibierenden Effekt allein durch die Reexpression von hu-CLDN1 unabhängig von Occludin und ZO-1 zu erreichen. In diesem Sinne kann zumindest zellphysiologisch hu-CLDN1 als Tumorsuppressorprotein betrachtet werden<br>Two years ago probably the most important tight junction proteins in mice were identified: claudin-1 and -2 . By sequence homology up to 18 members could be assigned to this 4 transmembrane protein family. The human claudin-1 with a 91 per cent sequence similarity to the murine claudin-1 was isolated in parallel (Swisshelm et al. 1999) and it has been shown that most breast cancer cell lines lost the expression of hu-CLDN1. The goal of this Ph. D. thesis was the evaluation of the cell physiological function of the human Claudin-1 (hu-CLDN1) and its relevance during tumorigenesis. To investigate the protein expression and cellular homing monoclonal antibodies using DNA vaccination were generated. The antibodies were analyzed for hu-CLDN1 specifity by Western blot and the epitope binding sites were identified by a competetive hu-CLDN1 peptide ELISA. Using the monoclonal antibodies optimal immunocytochemical and immunohistochemical methods for biological methods were established. With these antibodies the cellular expression and localization, and the expression of hu-CLDN1 in tissue slices of tumor versus normal tissues was analyzed. For the reexpression of hu-CLDN1 in breast tumor cells, retroviral shuttle systems were generated, based on a MoMuLV backbone using the l-NGFR receptor for efficient and rapid transduction of breast cancer cells with hu-CLDN1. For this purpose a mock vector (containing l-NGFR only ) and a hu-CLDN1 vector with l-NGFR were cloned. The expression of hu-CLDN1 in various breast cancer cell lines was analysed using quantitative reverse transcription polymerase chain reaction (qRT PCR). The monoclonal antibodies against hu-CLDN1 are specific against hu-CLDN1. In addition antibodies for all 4 extra- and cytosolic domains were generated. Using these antibodies it was shown by immunofluorescence microscopic analysis that hu-CLDN1 is an exclusively membrane located protein. The expression of the protein is detectable only in confluent cell cultures of naturally hu-CLDN1 expressing cell lines (T47-D, MCF7) and only in the areas of direct cell cell contact. The transduction of hu-CLDN1 negative cell lines analyzed by qRT PCR displayed a mRNA expression in subconfluent and confluent cell cultures, while the protein was only detectable in confluent cell cultures. This indicates that the hu-CLDN1 negative breast tumor cell lines still have the intact signal transduction pathway for the correct expression with an up to now unknown posttranscriptional control mechanism of protein expression. In this context it is noteworthy that even occludin and ZO-1 negative breast tumor cell lines (e. g. MDA-MB-435) still have the physiological homing mechanism of hu-CLDN1. Therefore it can be suggested that expression and homing at hu-CLDN1 might be independent of occludin and ZO-1. The analysis of different hu-CLDN1 positive and negative breast tumor cells showed that the loss of expression of hu-CLDN1 correlates more significantly to the tumorigenesis of cells in comparison to occludin and ZO-1. The clinical relevance of the downregulation and loss of hu-CLDN1 was analyzed using expression analysis of breast and colon tissue versus tumor tissue on tissue microarrays. Normal breast tissue displayed a epithelial/endothelial hu-CLDN1 membrane localization only. None of the breast tumor tissue displayed a membrane localization of the hu-CLDN1 however a relocalization to the cytoplasm was evident. Additionally a significant reduction or loss of expression could be detected in the majority of tumor tissues. These results show that the loss of expression of hu-CLDN1 in breast and colon tumors correlates with tumor progression. In order to analyze the relevance of hu-CLDN1 relocalization and reduction of expression during tumorigenesis on a cellphysiological level, in vitro cell culture studies were performed using hu-CLDN1 negative cell lines (MDA-MB-361) and their hu-CLDN1 transduced counterparts. Hu-CLDN1 transduced cells growing in 2 D cell cultures displayed no altered growth or increased levels of apoptosis. However in three dimensional growing tumor cell aggregates the reexpression of hu-CLDN1 results in a significantly increased induction of apoptosis. In addition paracellular flux analysis revealed that reexpression of hu-CLDN1 decreased the paracellular flux significantly. This data suggests that the reduced growth and increased apoptosis in hu-CLDN1 positive tumor cell aggregates could be due to reduced accessibility of growth factors in hu-CLDN1 transduced cell aggregates. This would explain the necessity for the cytoplasmic homing and loss of expression of hu-CLDN1 during tumorprogression in breast and colon tumors. Most epithelia exist as single layers in organs and lobular structures resulting in a good accessibility of growth factors via diffusion or micro-/macropinocytosis. However, carcinoma tumor cells grow in multilayers. Intact tight junction complexes in carcinoma multi layers would result in a reduced accessibility of growth factors and nutrients. Therefore it is suggested that it is essential for the in vivo growth of a tumor to decrease the expression of tight junctions regulating the paracellular flux. According to the molecular and cell physiological studies presented it might be possible to achieve a tumor inhibiting effect by re-expression of hu-CLDN1. Therefore at least from a cell physiological point of view hu-CLDN1 can be considered to be a tumor suppressor protein