Evolutionary History of Tissue Kallikreins

The gene family of human kallikrein-related peptidases (KLKs) encodes proteins with diverse and pleiotropic functions in normal physiology as well as in disease states. Currently, the most widely known KLK is KLK3 or prostate-specific antigen (PSA) that has applications in clinical diagnosis and monitoring of prostate cancer. The KLK gene family encompasses the largest contiguous cluster of serine proteases in humans which is not interrupted by non-KLK genes. This exceptional and unique characteristic of KLKs makes them ideal for evolutionary studies aiming to infer the direction and timing of gene duplication events. Previous studies on the evolution of KLKs were restricted to mammals and the emergence of KLKs was suggested about 150 million years ago (mya). In order to elucidate the evolutionary history of KLKs, we performed comprehensive phylogenetic analyses of KLK homologous proteins in multiple genomes including those that have been completed recently. Interestingly, we were able to identify novel reptilian, avian and amphibian KLK members which allowed us to trace the emergence of KLKs 330 mya. We suggest that a series of duplication and mutation events gave rise to the KLK gene family. The prominent feature of the KLK family is that it consists of tandemly and uninterruptedly arrayed genes in all species under investigation. The chromosomal co-localization in a single cluster distinguishes KLKs from trypsin and other trypsin-like proteases which are spread in different genetic loci. All the defining features of the KLKs were further found to be conserved in the novel KLK protein sequences. The study of this unique family will further assist in selecting new model organisms for functional studies of proteolytic pathways involving KLKs

Vzájemné interakce mezi nádorovým mikroprostředím a kalikreinovými proteázami v myším modelu karcinomu mléčné žlázy

Breast cancer is the most common cancer type with a high annual death rate. Finding meaningful tissue-related or body-fluid-accessible biomarkers is necessary to characterize cancer subtype, predict tumor behavior, choose the most effective therapy, predict severe treatment-related toxicities, and also the opportunity to personalize treatments for each patient. There is increasing evidence that various kallikrein-related peptidases (Klk) gene family members can modulate the immune response and are differentially regulated in breast cancer, and therefore are proposed to be potential prognostic biomarkers. This work established and validated an experimental setup to study the roles of selected kallikrein-related peptidases (KLK5, KLK7, KLK14) in breast cancer in vivo using gene-deficient mouse models previously generated in our laboratory. We used the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) editing system to generate several E0771 cell line-based reporter and gene-deficient cell lines. These allowed enhanced monitoring of cancer progression in vivo and studying KLKs roles in tumor immune microenvironment of C57Bl/6N mice. Finally, we present the analysis of the initial in vivo experiments using these tools combined with established Klk-deficient mouse models. Our...(CZ) Celosvětově je nejběžnějším typem rakoviny je rakovina prsu, jež je současně spojena s vysokou smrtností. Nalezení vhodných prognostických ukazatelů je důležitým aspektem pro bližší určení, o jaký typ rakoviny prsu se jedná a jaký průběh onemocnění lze u pacienta očekávat. Je známo, že kalikreinové proteázy jsou při rakovině prsu dysregulovány, a proto se spekuluje o jejich využití jako prognostických ukazatelů. Jejich schopnost ovlivňovat imunitní odpovědi včetně té protinádorové je též diskutována. Tento diplomový projekt využívá technologie CRISPR-Cas (clustered regularly interspaced short palindromic repeats) ke genetické editaci myší buněčné linie rakoviny prsu E0771. Geneticky modifikované linie E0771 jsou v projektu využívány ke studiu vzájemných interakcí mezi nádorovým mikroprostředím a kalikreinovými proteázami (KLK5, KLK7, KLK14) v C57Bl/6 myším modelu karcinomu mléčné žlázy. Diplomová práce přináší výsledky analýzy první části "in vivo" experimentů využívajících vytvořené geneticky modifikované buněčné linie v kombinaci se zavedenými myšími modely nesoucími mutace v genech pro zmíněné kalikreinové proteázy. Z předběžných výsledků tohoto diplomového projektu vyplývá, že zmíněné kalikreinové proteázy hrají roli při vývoji rakoviny prsu v použitém myším modelu a výsledky poukazují na...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Identificação de complexos proteicos na doença de Alzheimer

Doutoramento em Ciências BiomédicasA Doença de Alzheimer (AD) é a maior doença neurodegenerativa a nível mundial, e a principal causa de demência na população idosa. O processamento da proteína precursora de amilóide (APP) pelas β- e g- secretases origina o peptídeo Aβ, que agrega em oligómeros neurotóxicos e em placas senis. Estes são eventos-chave na patogénese da DA que levam à rutura da neurotransmissão sináptica, morte neuronal e inflamação neuronal do hipocampo e córtex cerebral, causando perda de memória disfunção cognitiva geral. Apesar dos grandes avanços no conhecimento do papel do processamento da APP na DA, a sua função fisiológica ainda não foi totalmente elucidada. Os mapas de interações proteína-proteína (PPI) humanos têm desempenhado um papel importante na investigação biomédica, em particular no estudo de vias de sinalização e de doenças humanas. O método dois-híbrido em levedura (YTH) consiste numa plataforma para a produção rápida de redes de PPI em larga-escala. Neste trabalho foram realizados vários rastreios YTH com o objetivo de identificar proteínas específicas de cérebro humano que interagissem com a APP, ou com o seu domínio intracelular (AICD), tanto o tipo selvagem como com os mutantes Y687F, que mimetizam o estado desfosforilado do resíduo Tyr-687. De facto, a endocitose da APP e a produção de Aβ estão dependentes do estado de fosforilação da Tyr-687. Os rastreios YTH permitiram assim obter de redes proteínas que interagem com a APP, utilizando como “isco” a APP, APPY687F e AICDY687F. Os clones positivos foram isolados e identificados através de sequenciação do cDNA. A maior parte dos clones identificados, 118, correspondia a sequências que codificam para proteínas conhecidas, resultando em 31 proteínas distintas. A análise de proteómica funcional das proteínas identificadas neste estudo e em dois projetos anteriores (AICDY687E, que mimetiza a fosforilação, e AICD tipo selvagem), permitiram avaliar a relevância da fosforilação da Tyr-687. Três clones provenientes do rastreio YTH com a APPY687F foram identificados como um novo transcrito da proteína Fe65, resultante de splicing alternativo, a Fe65E3a (GenBank Accession: EF103274), que codifica para a isoforma p60Fe65. A p60Fe65 está enriquecida no cérebro e os seus níveis aumentam durante a diferenciação neuronal de células PC12, evidenciando o potencial papel que poderá desempenhar na patologia da DA. A RanBP9 é uma proteína nuclear e citoplasmática envolvida em diversas vias de sinalização celulares. Neste trabalho caracterizou-se a nova interação entre a RanBP9 e o AICD, que pode ser regulada pela fosforilação da Tyr-687. Adicionalmente, foi identificada uma nova interação entre a RanBP9 e a acetiltransferase de histonas Tip60. Demonstrou-se ainda que a RanBP9 tem um efeito de regulação inibitório na transcrição mediada por AICD, através da interação com a Tip60, afastando o AICD dos locais de transcrição ativos. O estudo do interactoma da APP/AICD, modelado pela fosforilação da Tyr-687, revela que a APP poderá estar envolvida em novas vias celulares, contribuindo não só para o conhecimento do papel fisiológico da APP, como também auxilia a revelar as vias que levam à agregação de Aβ e neurodegeneração. A potencial relevância deste trabalho relaciona-se com a descoberta de algumas interações proteicas/vias de sinalização que podem que podem ser relevantes para o desenvolvimento de novas estratégias terapêuticas na DA.Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder worldwide and the leading cause of dementia in the elderly. Processing of amyloid-β precursor protein (APP) by β- and g-secretases produces Aβ, which aggregates into neurotoxic oligomers and senile plaques. These are key events in the pathogenesis of AD that lead to the disruption of synaptic neurotransmission, neuronal cell death, and inflammation in the hippocampus and cerebral cortex, thus causing memory loss and global cognitive dysfunction. Despite advances in understanding the role of APP processing in AD, the normal physiological function of this protein has proven more difficult to elucidate. Human protein-protein interaction (PPI) maps play an increasingly important role in biomedical research and have been shown to be highly valuable in the study of a variety of human diseases and signaling pathways. The yeast twohybrid (YTH) system provides a platform for the rapid generation of large scale PPI networks. Several YTH screens were performed to identify human brainspecific proteins interacting with APP, or with its intracellular domain (AICD), either the wild-type or the Y687F mutant, which mimics the dephosphorylated residue. In fact, APP endocytosis and Aβ generation are dependent upon Tyr- 687 phosphorylation. A human APP network comprised of the protein interactions was assembled through YTH screening, using as baits APP, APPY687F and AICDY687F. Positive clones were isolated and identified by DNA sequencing and database searching. The majority of these clones, 118, matched to a protein coding sequence, yielding 31 different proteins. Functional proteomics analysis of the proteins identified in this study, and two additional screens from previous projects (phospho-mutant AICDY687E and wild-type AICD), allowed to infer the relevance of Tyr-687 phosphorylation. Three clones from YTH with APPY687F were identified as a new splice variant of the APP binding protein Fe65, Fe65E3a (GenBank Accession EF103274), encoding the p60Fe65 isoform. Fe65E3a is expressed preferentially in the brain and the p60Fe65 protein levels increased during PC12 cell differentiation. This novel Fe65 isoform and the regulation of the splicing events leading to its production, may contribute to elucidating neuronal specific roles of Fe65 and its contribution to AD pathology. RanBP9 is an evolutionarily conserved nucleocytoplasmic protein implicated as a scaffolding protein in several signaling pathways. In this work a novel interaction between RanBP9 and AICD, which can be regulated by Tyr-687 phosphorylation, was characterized. Moreover, a novel interaction between RanBP9 and the histone acetyltransferase Tip60 was identified. RanBP9 was demonstrated to have an inhibitory regulatory effect on AICD-mediated transcription, through physical interaction with Tip60, relocating AICD away from transcription factories. Overall, the APP/AICD interactome shaped by the phosphorylation state of Tyr- 687 provided clues to elucidate APP pathways leading to amyloid deposition and neurodegeneration. As such the work here described brings us nearer to unravelling the physiological functions of APP. This in turn is of potential significant relevance in the pathology of AD, and for the design of effective novel therapeutic strategies

Copy number variation and Huntington’s disease

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the expansion of an unstable triplet repeat within the huntingtin gene. The length of this repeat is inversely correlated with the age of onset (AO) of the disease, which ranges from 1 to 80 years of age. The length of this repeat explains 50-70% of the variance of AO with the remaining variation attributable to environmental and other genetic factors. Copy number variation (CNV) is a structural variation of the human genome wherein a genomic sequence is duplicated or deleted compared to a reference genome. As CNVs have the potential to affect gene expression either directly by dosage effects or indirectly by affecting gene product interactions and pleiotropic effects, they are found to be associated with phenotypic variance, disease susceptibility and Mendelian disorders. The aim of the study was to investigate CNVs as candidate genetic modifiers of the AO in HD. Specifically we investigated CNV of the human β-defensin region (including DEFB4) and CNV involving SLC2A3, with potential impacts, respectively, in the neuroinflammatory response and in neuronal glucose uptake. CNVs were analysed within a large HD sample cohort (provided by EHDN) using the paralogue ratio test (PRT) to test their potential impact on a variance of the AO in HD. In 490 HD individuals analysed the frequency distribution of β-defensin copy number was shown to be equivalent to the general European population. Furthermore, no significant association was shown between β-defensin CNV and a variance of the AO in HD. 987 HD patients were genotyped for SLC2A3 CNV and a modest but significant association with a variance of AO in HD was found (p value = 0.028). Individuals with three copies showed a delay in the AO of up to nearly 6 years compared to individuals with one or two copies. In order to test if SLC2A3 CNV affects gene expression 15 cell lines from patients with different SLC2A3 copy number were immunoblotted for GLUT3 (encoded by SLC2A3) and it was found that the protein level was significantly correlated with the genomic copy number (p value = 0.020). Therefore, we concluded that SLC2A3 CNV is a genetic modifier of the AO in HD, associated to a variance of the disease onset and affecting the gene expression. To investigate the functional basis of this effect, we analysed lines showing over- or under-expression of the functional homolog of SLC2A3 (Glut1) in a Drosophila model of HD. After analysing several disease-relevant metrics, including neurodegeneration of the photoreceptors, eclosion rate and longevity, we found that gain and loss of Glut1 expression can delay and worsen, respectively, neurodegeneration in HD flies. In conclusion, β-defensin genomic copy number is not associated with modulation of HD AO. On the other hand, SLC2A3 CNV is a genetic modifier of the AO in HD, and likely has functional consequences, based on our findings in patient cells and in a Drosophila HD model

Oncogene and Cancer

This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

Hippo and Sonic Hedgehog signalling pathway modulation of human urothelial tissue homeostasis

The urinary tract is lined by a barrier-forming, mitotically-quiescent urothelium, which retains the ability to regenerate following injury. Regulation of tissue homeostasis by Hippo and Sonic Hedgehog signalling has previously been implicated in various mammalian epithelia, but limited evidence exists as to their role in adult human urothelial physiology. Focussing on the Hippo pathway, the aims of this thesis were to characterise expression of said pathways in urothelium, determine what role the pathways have in regulating urothelial phenotype, and investigate whether the pathways are implicated in muscle-invasive bladder cancer (MIBC). These aims were assessed using a cell culture paradigm of Normal Human Urothelial (NHU) cells that can be manipulated in vitro to represent different differentiated phenotypes, alongside MIBC cell lines and The Cancer Genome Atlas resource. Transcriptomic analysis of NHU cells identified a significant induction of VGLL1, a poorly understood regulator of Hippo signalling, in differentiated cells. Activation of upstream transcription factors PPARγ and GATA3 and/or blockade of active EGFR/RAS/RAF/MEK/ERK signalling were identified as mechanisms which induce VGLL1 expression in NHU cells. Ectopic overexpression of VGLL1 in undifferentiated NHU cells and MIBC cell line T24 resulted in significantly reduced proliferation. Conversely, knockdown of VGLL1 in differentiated NHU cells significantly reduced barrier tightness in an unwounded state, while inhibiting regeneration and increasing cell cycle activation in scratch-wounded cultures. A signalling pathway previously observed to be inhibited by VGLL1 function, YAP/TAZ, was unaffected by VGLL1 manipulation. In MIBC, overexpression of VGLL1 was observed in a subset of differentiated tumours associated with significantly reduced survival, indicative of dysregulated VGLL1 function. This study reveals a novel Hippo pathway-independent function of VGLL1, with the protein observed to play an important role in regulating urothelial tissue homeostasis. Consequently, these findings contribute to the current understanding of how dysregulation of tissue homeostasis can facilitate MIBC progression

Investigating the mechanism of human beta defensin-2-mediated protection of skin barrier in vitro

The human skin barrier is a biological imperative. Chronic inflammatory skin diseases, such as Atopic Dermatitis (AD), are characterised by a reduction in skin barrier function and an increased number of secondary infections. Staphyloccocus aureus (S. aureus) has an increased presence on AD lesional skin and contributes significantly to AD pathology. It was previously demonstrated that the damage induced by a virulence factor of S. aureus, V8 protease, which causes further breakdown in skin barrier function, can be reduced by induction of human β- defensin (HBD)2 (by IL-1β) or exogenous HBD2 application. Induction of this defensin is impaired in AD skin. This thesis examines the mechanism of HBD2-mediated barrier protection in vitro; demonstrating that in this system, HBD2 was not providing protection through direct protease inhibition, nor was it altering keratinocyte proliferation or migration, or exhibiting specific localisation within the monolayer. Proteomics data demonstrated that HBD2 did not induce expression of known antiproteases but suggested that HBD2 stimulation may function by modulating expression of extracellular matrix proteins, specifically collagen- IVα2 and Laminin-β-1. Alternative pathways of protection initiated by IL-1β and TNFα stimulation were also investigated, as well as their influence over generalised wound healing. Finally, novel 3D human skin epidermal models were used to better recapitulate the structure of human epidermis and examine alterations to skin barrier function in a more physiological system. These data validate the barrier-protective properties of HBD2 and extended our knowledge of the consequences of exposure to this peptide in this context