Dissecting the functional role of PTEN proteins from Physcomitrella patens

Tese de mestrado. Biologia (Biologia Celular e Biotecnologia). Universidade de Lisboa, Faculdade de Ciências, 2013PTEN (phosphate and tensin homolog) are dual specificity phosphatases with both protein and phosphoinositide phosphatase activity. In animal cells, they hydrolyze PtdIns(3,4,5)P3, a potent second messenger that modulates signaling pathways controlling cell division, cell size, angiogenesis and apoptosis. In addition, PTEN is a relevant gene in animal cells that is often lost in late-stage human tumors, especially those of the brain, prostate and endometrium. Interestingly, PtdIns(3,4,5)P3 is the only known phosphoinositide so far not detected in any plant system, and the enzymes that synthesizes PtdIns(3,4,5)P3 in animal cells do not exist in plants. In this thesis, I have studied PTEN genes in the moss Physcomitrella patens which has emerged as a model system in the last years due to its high frequency of homologous recombination allowing to study functional genomics by gene targeting. In additions, P. patens diverged from the lineage leading to flowering plants 450 million years ago, a feature that places mosses at an evolutionary position that is ideal for comparative studies of the evolution of biological processes in land plants. P. patens genome contains four PTEN genes, named PpPTENA, PpPTENB, PpPTENC and PpPTEND. PpPTEN proteins present the typical organization of PTENs with an N-terminal PTP domain (Protein Tyrosine Phosphatase) and a C-terminal C2-domain.Through protein modeling studies I show that PpPTENA and PpPTEND share high degree of structure similarity with CI_VSP (Ciona intestinales voltage sensor protein) a protein that harbors a transmembrane voltage sensor domain and a cytoplasmic PTEN domain. PpPTENs PTP domain presents the typical catalytic loops seen in the majority of PTENs but their C2 domain is markedly different. In terms of sequence comparison several differences are observed between PpPTENs and HsPTEN, being of main interest differences in the catalytic domain. I characterized a triple ptenb&c&d knockout line and results show that knockout moss plants produced more rhizoids and gametophytes, there is an earlier transition form the protonema juvenile stage to the gametophytic stage and interestingly chloronema and caulonemal cells have a higher growth rate compared to the WT. By using a knock in approach, I have obtained a PpTENB-GFP line to study its localization, revealing that PpPTENB is mostly localized in young growing cells such as chloronema, caulonemal but was well as in the tip of rhizoids.As proteínas PTEN (‘Protein Tensin Homologue deleted from chromosome 10), identificada primeiramente em Humano são fosfatases com capacidade de desfosforilar outras proteínas mas também fosfolípidos. Estas proteínas estão envolvidas na modulação de vias de sinal que regulam vários processos celulares como o crescimento, metabolismo ou processos de apoptose estando implicadas no aparecimento de várias doenças humanas, como por exemplo o desenvolvimento de tumores. Neste trabalho fiz a caracterização preliminar de quatro isoformas desta proteína, denominadas PpPTENA, PpPTENB, PpPTENC e PpPTEND, identificadas em Physcomitrlle patens. PpPTENs apresentam a organização estrutural típica das PTEN, com um domínio PTP (‘Protein Tyrosine Phosphatase’) N-terminal e um domínio C-terminal denominado C2. Recorrendo à modelação de proteínas foi-me possível demontrar que as PpPTENA a PpPTEND partilham um elevado grau de semelhança estrutural com a preteína CI_VSP (C. intestinales Voltage Sensor Protein) que apresenta um domínio transmembranar ‘Voltage Sensing’ e um domínio PTEN citoplasmático O mutante ‘knock out’ para PpPTENB,PpPTENC e PpPTEND (ptenb&c&d) revelou a produção de filamentos de caulonema mais longos, a produção de mais rizoides por gametófito e também uma taxa de crescimento das células do protonema marcadamente mais celerada, o que está de acordo com o demonstrado em células animais. Com a obtenção de um mutante ‘knock in’ para a proteína PpPTENB (PpPTENB_GFP) foi-me possível avaliar a sua localização ‘in vivo’ que revelou uma predominância desta proteína em células jovens do cloronema, caulonema e também dos rizoides