Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.

Human papillomaviruses (HPV) are among the most common sexually transmitted infections and are responsible for 5% of all human cancers. HPV type 16 is the most prevalent of the high-risk HPVs (a subgroup of HPVs with potential to cause cancer), accounting for ~55% of HPV-associated cancers. HPV16 is a nonenveloped virus, composed of the major capsid protein L1, the minor capsid protein L2, and a circular double-stranded DNA genome (vDNA) condensed with human histones. HPV initially infects undifferentiated basal keratinocytes and viral replication is dependent on epithelial differentiation. Like many other DNA viruses, HPV must deliver its vDNA to the host cell nucleus to successfully replicate. Initial binding of HPV16 to host cells is through L1 interactions with cell surface heparan sulfate receptors. Shortly after virus binding, L2 is believed to undergo furin cleavage-dependent conformational changes, resulting in spanning of the protein across the local membrane and exposure of the central and C-terminal regions of L2 (which was lumenal and and inaccessible before furin cleavage) to the host cell cytosol. L2 is critical for transport of the L2/vDNA from endosomes to the trans-Golgi network (TGN). We hypothesize that furin-dependent early L2 spanning, through the direct binding and recruitment of cytosolic sorting factors, may contribute to viral endocytosis and subcellular retrograde trafficking (trafficking from endosomes to Golgi) of vDNA. We have developed a Tac receptor (CD25 or IL2 receptor, a transmembrane cell surface protein) chimera system to study L2-dependent endocytosis and trafficking. In this system the Tac ecto- and transmembrane domains are fused to the ~400 amino acid portion of L2 that is likely cytosolic upon L2 spanning. Through transient expression of Tac-L2 chimera we use anti-Tac ectodomain antibodies to label and track cell surface populations by immunofluorescence and confocal microscopy. We have also adopted this system to study endocytosis through a cell surface biotinylation approach. Both approaches suggest that L2 may enhance endocytosis and preliminary evidence suggests that the Tac-L2 chimera may recruit the cytosolic retromer complex (the host cytosolic factors help protein retrograde trafficking) to preferentially traffic to the TGN. Retromer-dependent trafficking of cargo from early endosomes to the TGN is known to involve certain members of the sorting nexin family, specifically the SNX-BAR proteins. We performed a small siRNA screen and identify SNX6 and SNX32 (aka SNX6b) as SNX-BAR proteins that may be specifically involved in retrograde trafficking of HPV16 L2/vDNA during infection. Future work will focus on the mechanisms through which L2 and SNX6 influence HPV16 entry and trafficking.

Ein sehr wichtiger Tumorsuppressor ist der Transkriptionsfaktor p53, der Zellschicksals-Entscheidungen wie Zellzyklus-Arrest und programmierten Zelltod (Apoptose) kontrolliert. Die Wirkung von p53 und von seinen Familienmitgliedern p63 und p73 beruht überwiegend auf der Fähigkeit, als Transkriptionsfaktoren die Genexpression zu regulieren. Die DNA-Bindung an Promotoren von Zielgenen ist dabei von grundlegender Bedeutung und wird durch die hoch konservierte zentrale DNA-Bindungs-Domäne und den Carboxy-Terminus bestimmt. In dieser Arbeit wurden die DNA-Bindungseigenschaften von p53 und verschiedener Carboxy-terminalen p73 Isoformen untersucht. In „electrophoretic mobility shift assay” (EMSA) Experimenten bildeten p53 und p73gamma nur schwache Sequenz-spezifische DNA-Komplexe, wohingegen p73alpha, beta und delta die DNA deutlich stärker banden. Die schwache DNA-Bindung von p53 und p73gamma kann durch mehrfach positiv geladene Carboxy-Termini erklärt werden, die über eine Sequenz-unabhängige DNA-Bindung ein Gleiten entlang der DNA ermöglichen. Die Deletion der Carboxy-terminalen Domäne (CTD) von p53 („p53delta30“) verstärkte dementsprechend die Sequenz-spezifische DNA-Bindung in vitro und seine Übertragung auf p73alpha („p73alpha+30“) schwächte sie ab. Mittels „fluorescence recovery after photobleaching“ (FRAP) Experimenten konnte in lebenden Zellen eine Verminderung der intra-nukleären Mobilität von p53 und p73alpha+30 durch die CTD gezeigt werden, die aus der Sequenz-unabhängigen DNA-Bindung resultiert. Zusätzlich reduzierte die CTD die Sequenz-spezifische DNA-Bindung von p53 an den p21 (CDKN1A) Promotor. Das Spektrum der regulierten Zielgene wurde in einer Genom-weiten Genexpressions-Analyse nicht durch die CTD verändert, sondern maßgeblich durch das Protein-Rückgrat von p53 beziehungsweise p73 bestimmt. Allerdings verminderte die CTD das Ausmaß der Transkriptions-Regulation und hemmte die Induktion von Zellzyklus-Arrest und Apoptose. Die mehrfach positiv geladene CTD in p53 besitzt demzufolge eine negativ regulatorische Wirkung, die in den wichtigsten p73 Isoformen alpha, beta und delta fehlt. Die zentrale DNA-Bindungs-Domäne trägt durch elektrostatische Wechselwirkungen zwischen H1-Helices (Aminosäurereste 177 bis 182) unterschiedlicher p53 Monomere zu kooperativer DNA-Bindung und zu Zellschicksals-Entscheidungen bei. Anhand von Mutanten, die unterschiedlich starke H1-Helix-Interaktionen ermöglichen, konnte gezeigt werden, dass starke Interaktionen die Bindung an Promotoren von pro-apoptotischen Genen verstärkte, wohingegen die Bindung an anti-apoptotische und Zellzyklus-blockierende Gene unabhängig von der Interaktions-Stärke war. Diese Unterschiede in der Promotor-Bindung ließen sich nicht auf eine veränderte zelluläre Lokalisation der Mutanten zurückführen, da alle Mutanten überwiegend nukleär lokalisiert waren. Eine an Serin 183 Phosphorylierungs-defekte Mutante von p53 bildete stabile DNA-Komplexe, entsprechend einer Mutante mit starker H1-Helix-Interaktion, und trans-aktivierte pro-apoptotische Promotoren stärker als Mutanten, die Phosphorylierung von p53 an Serin 183 simulieren. Da zusätzlich bekannt ist, dass Serin 183 mit der H1-Helix wechselwirkt, könnte diese Phosphorylierung einen physiologischen Mechanismus zur Regulation der H1-Helix-Interaktion und damit des Zellschicksals darstellen. Zusammenfassend ließ sich zeigen, dass sowohl die Interaktions-Stärke zweier DNA-Bindungs-Domänen als auch die elektrische Ladung des Carboxy-Terminus die DNA-Bindungseigenschaften von p53 Familienmitgliedern bestimmen und so Zellschicksals-Entscheidungen der p53 Familie beeinflussen. / A very important tumour suppressor is the transcription factor p53 that controls cell fate decisions like cell cycle arrest and programmed cell death (apoptosis). The effects of p53 and its family members p63 and p73 are mainly based on their transcription factor activities to regulate gene expression. The DNA binding to promoters of target genes is of fundamental importance for their functionality and is determined by the highly conserved core DNA binding domain and the carboxy-terminus. In this thesis the DNA binding properties of p53 and different carboxy-terminal p73 isoforms were examined. In electrophoretic mobility shift assays (EMSA) p53 and p73gamma formed only weak sequence-specific protein-DNA-complexes while p73alpha, beta and delta bound considerably stronger to DNA. A highly positively charged carboxy-terminus can explain the weak DNA binding of p53 and p73gamma by enabling sequence-nonspecific DNA binding leading to sliding on DNA. According to this the deletion of the carboxy-terminal domain (CTD) of p53 („p53delta30“) reinforced DNA binding in vitro, and its fusion to p73alpha („p73alpha+30“) attenuated it. In living cells the CTD reduced intranuclear mobility of p53 and p73alpha+30 in fluorescence recovery after photobleaching (FRAP) experiments by mediating sequence-nonspecific binding to DNA. In addition, the CTD reduced sequence-specific occupancy of the p21 (CDKN1A) promoter by p53 in vivo. In an unbiased genome-wide gene expression analysis the spectrum of target genes was not changed by the presence of the CTD, but mainly determined by the p53 and p73 protein backbone, respectively. However, the CTD diminished the level of target gene activation and inhibited the induction of cell cycle arrest and apoptosis. As a result, the highly positively charged carboxy-terminus of p53 exhibits a negative regulatory effect that is missing in the most important p73 isoforms alpha, beta and delta. The core DNA binding domain adds to cooperative DNA binding and cell fate decisions by electrostatic interactions between H1 helices (residues 177 to 182) of different p53 monomers. Strong H1 helix interactions increased binding to promoters of pro-apoptotic genes, whereas binding to anti-apoptotic and proliferation inhibiting genes was independent of the interaction strength as shown by mutants with different strengths of the H1 helix interactions. These differences in promoter binding were not caused by different cellular localizations of the mutants as they were all predominantly localized to the nucleus. A serine 183 phosphorylation-defective mutant of p53 formed stable protein-DNA-complexes, comparable to a mutant with strong H1 helix interactions, and trans-activated pro-apoptotic promoters stronger than mutants that mimicked p53 phosphorylated on serine 183. Due to the fact that serine 183 interacts with the H1 helix, these data suggest that phosphorylation of serine 183 is a physiological mechanism to regulate H1 helix interactions and thereby cell fate decisions. In summary, it was shown that both the interaction strength of two DNA binding domains and the electrostatic charge of the CTD define the DNA binding properties of p53 family members and thereby influence cell fate decisions of the p53 family.

Não consta resumo na publicação. / Abstract not available.

Protein aggregation, oligomer and fibril formation is one of the dominant characteristics in the pathogenesis of a number of neurodegenerative diseases, such as Alzheimer’s disease (AD). Inhibition of toxic oligomer and fibril formation is one of the approaches to find potential drug candidates for AD. Additionally, early diagnosis of these amyloid species can provide mechanistic understanding of protein aggregation and thus can pave the way for preventing the onset of AD. The aim of this dissertation was 1) to explore the effects of charged cholesterol derivatives on the aggregation kinetic behavior of Amyloid-β40 (Aβ40), 2) to probe Aβ40 oligomer and amyloid formation in vitro using gold nanoparticles (AuNPs), and 3) to monitor the kinetic effect of various natural product molecules on Aβ40 aggregation in vitro. In the first chapter, a general introduction about AD as an amyloidogenic disease, amyloid cascade hypothesis, and the manipulation of Aβ peptides aggregation kinetics using different approaches was presented. In the second chapter, we studied the effects of oppositely charged cholesterol derivatives on the aggregation kinetics of Aβ. In the third chapter, we developed a gold nanoparticles (AuNPs) assay to probe Aβ40 oligomers and amyloid formation. In chapter IV, we monitored the effects of various small molecules on the aggregation kinetics of Aβ40. In chapter V, we discussed the methods and experimental details. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Protozoários do gênero Trypanosoma infectam milhões de pessoas todo ano e coletivamente contribuem muito para as misérias humanas, pois são causa de muitas das doenças negligenciadas tropicais. Várias vias metabólicas essenciais são encontradas nesses parasitas tornando-os particularmente atrativos para investigações moleculares. Mecanismos de controle pós-transcricional tem sido alvo de estudo por sua peculiaridade nesses organismos. Nesse cenário, proteínas da classe das poli-A-binding proteins (PABP) possuem função no início da tradução, turnover do mRNA e interação com o 5´-CAP. Nesse trabalho foi identificada a homóloga poli-A binding protein 1 (PABP1) de Trypanosoma brucei. O silenciamento do gene pabp1 revelou que a ausência da proteína é letal ao parasita, comprovando sua essencialidade nesse organismo. Da mesma maneira, na ausência da proteína observou-se erro no processamento do mRNA sugerindo possível função nos eventos de cis e trans splicing. Sua localização subcelular foi avaliada indicando localização citoplasmática, bem como o são suas homólogas. No citoplasma, a proteína apresenta-se em estrutura reticulada, co-localizada com proteínas de retículo endoplasmático. Porém, sob estresse induzido a proteína relocaliza para o compartimento nuclear, indicando ser uma proteína com trânsito núcleo-citoplasma ainda não demonstrada na literatura. As funções identificadas sugerem a existência de um sub-complexo a 3´ do mRNA que acopla poliadenilação e splicing. Além disso, a relocalização nuclear parece ocorrer em resposta a estímulo externo, sugerindo que a relocalização do mRNA para o núcleo pode ser uma estratégia da célula para modular sua resposta gênica frente a variações do ambiente. / Protozoa of the genus Trypanosoma infect millions of people every year and collectively contribute to the human misery by causing several neglected tropical diseases. Several intriguing molecular pathways are found in these parasites also, rendering them particularly attractive for biochemical investigation. This unique eukaryotic cells lack mechanisms to control gene expression at the transcriptional level, they mostly control protein synthesis by posttranscriptional regulation process. Several RNAs and proteins are involved in this process, including poly(A) binding proteins. The poly(A)- binding protein of eukaryotes plays a role in polyadenylation, translation initiation and metabolism of mRNA. In this work the poly(A) binding protein 1 (PABP1) was identified in Trypanosoma brucei. Depletion of TbPABP1 showed its essential role in the procyclic form of the parasite. Immunofluorescence assays showed localization in the cytosolic compartment despite of its functions in cis and trans splicing as shown by RNA analysis of cells free from PABP1. As it was shown in the homologs, PABP1 it´s not only a cytosolic protein but it shuttles between the nucleus and the cytoplasm. Together with the literature, these results suggest an active complex in the 3´ end of the mRNA which works in synchrony with the splicing and capping machinery implying PABP1 as possible link between these processes.

L'objectif de cette thèse est de développer des outils de prédiction d'interactions entre protéines qui puissent être appliqués en particulier sur le réseau d’interaction autour de la protéine CFTR, qui est impliquée dans la mucoviscidose. Le développement de méthodes de prédiction in silico peut s'avérer utile pour suggérer aux biologistes de nouvelles cibles d'interaction. Nous proposons une nouvelle méthode pour la prédiction de liens dans un réseau. Afin de bénéficier de l'information des données non étiquetées, nous nous plaçons dans le cadre de l'apprentissage semi-supervisé. Nous abordons ce problème de prédiction comme une tâche d'apprentissage d'un noyau de sortie. Un noyau de sortie est supposé coder les proximités existantes entres les nœuds du graphe et l'objectif est d'approcher ce noyau à partir de descriptions appropriées en entrée. L'utilisation de l'astuce du noyau dans l'ensemble de sortie permet de réduire le problème d'apprentissage à celui d'une fonction d'une seule variable à valeurs dans un espace de Hilbert. En choisissant les fonctions candidates pour la régression dans un espace de Hilbert à noyau reproduisant à valeur opérateur, nous développons, comme dans le cas de fonctions à valeurs scalaires, des outils de régularisation. Nous établissons en particulier des théorèmes de représentation, qui permettent de définir de nouveaux modèles de régression. Nous avons testé l'approche développée sur des données artificielles, des problèmes test ainsi que sur un réseau d'interaction chez la levure et obtenu de très bons résultats. Puis nous l'avons appliquée à la prédiction d'interactions entre protéines dans le cas d'un réseau construit autour de CFTR. / The aim of this thesis is to develop tools for predicting interactions between proteins that can be applied to the human proteins forming a network with the CFTR protein. This protein, when defective, is involved in cystic fibrosis. The development of in silico prediction methods can be useful for biologists to suggest new interaction targets. We propose a new method to solve the link prediction problem. To benefit from the information of unlabeled data, we place ourselves in the semi-supervised learning framework. Link prediction is addressed as an output kernel learning task, referred as Output Kernel Regression. An output kernel is assumed to encode the proximities of nodes in the target graph and the goal is to approximate this kernel by using appropriate input features. Using the kernel trick in the output space allows one to reduce the problem of learning from pairs to learning a single variable function with output values in a Hilbert space. By choosing candidates for regression functions in a reproducing kernel Hilbert space with operator valued kernels, we develop tools for regularization as for scalar-valued functions. We establish representer theorems in the supervised and semi-supervised cases and use them to define new regression models for different cost functions. We first tested the developed approach on transductive link prediction using artificial data, benchmark data as well as a protein-protein interaction network of the yeast and we obtained very good results. Then we applied it to the prediction of protein interactions in a network built around the CFTR protein.

"November 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 131-152). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

A defining characteristic of neurodegenerative disease is the accumulation of mutant or misfolded proteins within neurons. Selective macroautophagy of aggregates, or aggrephagy, is a lysosome-mediated protein degradation pathway implicated in the turnover of disease-relevant accumulated proteins, but its specific function in vivo in the mammalian nervous system is poorly understood. The large PI3P-binding protein Alfy (Autophagy-linked FYVE protein) is an adaptor required for selective macroautophagy of aggregated proteins in cellular model systems. We sought to address Alfy-mediated aggrephagy in the mammalian brain in mouse models of Huntington’s disease (HD). HD is a neurodegenerative disorder caused by autosomal dominant inheritance of an expanded CAG repeat within the IT15, or huntingtin (htt) gene. The mutation causes an expansion of a polyglutamine (polyQ) tract in the protein Huntingtin (Htt), which results in psychiatric, cognitive, and motor symptomology. A pathological hallmark of HD is the accumulation of intracellular deposits of mutant Htt and ubiquitin. The exact relevance of these deposits remains unclear, but their elimination, hypothesized to occur via macroautophagy, correlates with behavioral improvements in mouse models of HD. The selective mechanisms of this phenomenon are largely unexplored in vivo. We have created two mouse models to address the role of Alfy-mediated selective macroautophagy in mammalian HD brain. First, we created tamoxifen-inducible Alfy knockout mice (Alfy iKO) and crossed them with a redesigned inducible HD mouse (HD103Q) that uses a tetracycline-regulated system to control reversible expression of mutant exon-1 Htt. Western blot, in situ, and PCR analysis confirm that Alfy can be eliminated from brain in adult Alfy iKO mice. A timecourse of Htt aggregation and clearance reveals that HD103Q mice accumulate huntingtin deposits, which clear in a linear manner upon transgene suppression over the course of four months. The loss of Alfy significantly impedes the removal of these deposits. Second, an Alfy knockout mouse was created using gene-trap technology, and mice hemizygous for Alfy knockout were crossed with BACHD mice expressing full-length human mutant Htt. We find that 50% Alfy depletion in the BACHD leads to increased insoluble Htt aggregate deposition along with accelerated decline in motor behavioral performance. Furthermore, inducible knockout of Alfy alone has a severe and age-dependent motor behavioral phenotype. This work reveals an in vivo role for Alfy in turnover of mutant Htt deposits, suggests that the accumulation of detergent-insoluble mutant Htt species contributes to behavioral pathogenesis, and supports an important function for Alfy at the intersection of HD and aging.