Caracterização de proteínas que interagem com a PPP1 na reprodução masculina

Doutoramento em BioquímicaA fosforilação reversível de proteínas é um importante mecanismo de controlo em eucariotas. A fosfoproteína fosfatase 1 (PPP1) é uma fosfatase de serina/treonina envolvida em vários processos celulares. Existem três isoformas da subunidade catalítica (α/CA, δ/β/CB e γ/CC) com pequenas diferenças nos terminais amino e carboxílico. O gene PPP1CC sofre ainda splicing alternativo para produzir duas isoformas, a PPP1CC1 ubíqua e a PPP1CC2 enriquecida em testículo e específica de esperma. A localização e especificidade de substratos da PPP1 está dependente da formação de complexos oligoméricos com proteínas que interagem com a PPP1 (PIPs). O objetivo principal desta tese foi estudar novas PIPs, específicas de testículo e esperma, a fim de melhor caracterizar o papel desta fosfatase e dos respetivos complexos na reprodução em mamíferos. Com este fim, estudou-se a presença, localização e possíveis funções de uma PIP previamente conhecida, PPP1R2, e de duas novas PIPs, PPP1R2P3 e Tctex1d4. PPP1R2 e PPP1R2P3 estão presentes em esperma humano colocalizando com a PPP1CC2, na cabeça e na cauda. A hipótese é que as holoenzimas localizadas na cabeça terão um papel na reação acrossómica, enquanto que as holoenzimas presentes no axonema são relevantes para o controlo da motilidade flagelar. De seguida foram estudados os pseudogenes da PPP1R2, em termos de história evolutiva e de possíveis funções. Na espécie humana, a PPP1R2 tem 10 pseudogenes, 7 deles específicos de primatas. Estudos de bioinformática e dados de expressão mostram que os PPP1R2P1/P3/P9 são os pseudogenes com maior probabilidade de serem transcritos e traduzidos. Também identificámos o PPP1R2P9 em esperma humano e mostrámos que alguns pseudogenes poderão estar associados a estados fisiopatológicos. Isto indica que o processo de evolução poderá estar ligado á formação de novos genes ou ao controlo do mRNA da PPP1R2. A sobre-expressão da PPP1R2 ou PPP1R2P3 em testículo de ratinho também foi realizada, para caracterizar os mecanismos envolvidas na função dos complexos PPP1R2/PPP1R2P3-PPP1CC2 na espermatogénese e fisiologia dos espermatozoides. A dineína de cadeia leve, Tctex1d4, foi encontrada como interagindo com a PPP1C e como estando presente em testículo de ratinho e em esperma humano. Demonstrámos que a Tctex1d4 e a PPP1 colocalizam no centro organizador de microtúbulos e nos microtúbulos e que o motivo de ligação à PPP1 presente na Tctex1d4 parece ser importante para manter a PPP1 no centro organizador de microtúbulos e/ou para disromper ou atrasar o seu movimento ao longo dos microtúbulos emergentes. Estes resultados abrem novos caminhos para os possíveis papéis do complexo Tctex1d4-PPP1 na dinâmica dos microtúbulos, motilidade do esperma, reação acrossómica e na regulação da barreira hemato-testicular, provavelmente, através da via de sinalização do TGFß. A análise do motivo de ligação à PPP1 mostra que este é altamente conservado entre os mamíferos, com exceção das Pikas, sugerindo que esta perda aconteceu antes da radiação das Pikas, há 6-20 milhões de anos atrás. Através de um rastreio por mutações demonstrámos que a capacidade da Tctex1d4 se ligar à PPP1 é mantida nas Pikas, embora o motivo de ligação à PPP1 esteja disrompido. Este estudo abre portas para novas descobertas na área da reprodução mostrando o papel da PPP1CC2 na espermatogénese e fisiologia do esperma.Reversible phosphorylation of proteins is an important intracellular control mechanism in eukaryotes. Phosphoprotein Phosphatase 1 (PPP1) is a major serine/threonine protein phosphatase involved in a wide range of cellular processes. Three closely related catalytic subunit isoforms (/CA, δ//CB and /CC) exist with only minor differences at their N- and C-terminus. PPP1CC gene can also undergo tissue-specific processing to yield a ubiquitously expressed PPP1CC1 and the testis-enriched and sperm-specific PPP1CC2 isoforms. PPP1C exists in the cell as an oligomeric complex binding to a spectrum of PPP1 interacting proteins (PIPs), which modulate both its intracellular localization and substrate specificity. The main goal of this thesis was to study novel PIPs in testis and sperm, in order to further characterize the role of PPP1CC2 and the respective complexes in mammalian reproduction. To this end we addressed the presence, localization and putative roles of a previously known PIP, PPP1R2, in testis and sperm, and two novel PPP1CC2 testis/sperm specific PIPs, PPP1R2P3 and Tctex1d4. PPP1R2/PPP1R2P3 were shown to be present in human sperm co-localizing with PPP1CC2, in the head and tail. It was shown that PPP1R2P3 is a heat stable inhibitor of PPP1CC that cannot be phosphorylated by GSK3. We hypothesize that the holoenzymes localized in the head may have a role in the acrosome reaction while the axoneme bound holoenzymes are relevant for the control of flagellar motility. To further address the PPP1R2 significance, its pseudogenes were described in terms of evolutionary history and putative functions. In human specie, PPP1R2 has ten pseudogenes most of them primate-specific. Besides PPP1R2P3, bioinformatic studies and expression data show that PPP1R2P1, PPP1R2P2 and PPP1R2P9 are the pseudogenes with more probability of being transcribed and eventually translated. Moreover, we identified PPP1R2P9 in human sperm and showed that several pseudogenes appear to be associated with physiological and pathological states. This indicates that evolution processes might be in part related with the formation of new genes or in the control of the parental PPP1R2 message. Overexpression of human PPP1R2 or PPP1R2P3 in mouse testis was also pursued to provide the molecular tools to initiate the characterization of the mechanisms behind PPP1R2/PPP1CC2 and PPP1R2P3/PPP1CC2 role in spermatogenesis and sperm physiology. Dynein light chain, Tctex1d4, was found to bind to PPP1C and to be present in mouse testis and human sperm. Tctex1d4-PPP1CC complex was shown to co-localize in the microtubule organizing centre and in microtubules. Moreover, the Tctex1d4 PPP1 binding motif seems to be important to retain PPP1CC in the microtubule organizing centre, and also to disrupt or delay its movement along microtubules. These results open new avenues to the possible roles of Tctex1d4-PPP1 complex in microtubule dynamics, sperm motility, acrosome reaction and in regulation of the blood testis barrier possibly via TGFß signaling. Moreover, PPP1 binding motif is highly conserved among mammals, except in Pikas, suggesting that this event happened before the Pikas radiation, 6-20 Million years ago. Mutational screening shows that the ability of Tctex1d4 to bind to PPP1 is maintained in Pikas, although the PPP1 binding motif is disrupted. This work opens doors to new discoveries in male reproduction and unravels the roles of PPP1CC2 and its PIPs in spermatogenesis and sperm physiology

Protein phosphorylation: deregulation and oncogenesis

A fosforilação reversível proteica é o principal mecanismo regulador das cascatas de sinalização celular em eucariotas sendo este catalisado por cinases e revertido por fosfatases. A desregulação da fosforilação de proteínas integrantes das vias de sinalização (ex. PI3K, TGFβ e apoptóticas) têm sido associadas ao desenvolvimento de diversos tipos de cancro(ex. carcinomas da mama e próstata, o melanoma e o retinoblastoma). O processo oncogénico é dependente de alterações na maquinaria de fosforilação que permitem à célula a manipulação das vias de sinalização resultando numa promoção da proliferação celular. O intuito desta revisão é apresentar como alterações no mecanismo de fosforilação desempenham um papel fulcral no desenvolvimento do processo oncogénico e como pode este ser manipulado com intuitos terapêuticos

Identification of therapeutic and diagnostic targets through yeast two hybrid system: molecular biology in medicine

Nas últimas décadas, o desenvolvimento da biologia molecular tem impulsionado a medicina, principalmente na identificação de novos alvos terapêuticos e de diagnóstico. Nas células são as proteínas as principais intervenientes responsáveis pelo funcionamento de todos os processos celulares, desde a síntese de novas moléculas de DNA, à formação de RNA e de novas proteínas, ao transporte de todos os componentes celulares bem como da composição estrutural da própria célula. Também são as proteínas um dos componentes mais relevantes das vias de sinalização entre as células. Estudos apontam para que, normalmente, as proteínas não funcionem sozinhas mas em complexos. Daí a importância de estudar as interações entre proteínas e, por outro lado, encontrar compostos que interfiram com esses complexos para tratamentos farmacológicos. Já existem alguns fármacos com estas características. A tricostatina A, um inibidor duma diacetilase de histonas (DH), atua no complexo Proteína Fosfatase 1-DH, sendo um bom alvo na terapia anti-cancerígena. Em 1989, de um modo revolucionário para a época, Fields e Songs desenvolveram o Yeast Two Hybrid system (YTH). Este método baseia-se na genética da levedura Saccharomyces cerevisiae para detetar interações entre proteínas. Desde a sua descoberta sofreu várias modificações que permitiram a sua aplicação à investigação translacional. Por exemplo, esta técnica permite fazer um rastreio em larga escala para determinar que droga pode interferir com uma determinada interação proteica. Por outro lado, pode também utilizar-se para se determinar que proteínas num determinado tecido (por exemplo, cérebro ou testículo) interagem com a nossa proteína de interesse. Deste modo é possível desvendar funções de novas proteínas, vias de sinalização e funcionamento de tecidos. A grande quantidade de informação produzida por esta abordagem é de eleição para a identificação e validação de alvos de diagnóstico, terapêuticos e mesmo desenvolvimento de novos fármacos. Esta revisão tem como intuito elucidar o funcionamento do Yeast Two Hybrid system e a sua contribuição para a identificação de novos tratamentos farmacológicos

Identificação de alvos terapêuticos e de diagnóstico através do Yeast Two Hybrid System: a biologia molecular na medicina

Nas últimas décadas, o desenvolvimento da biologia molecular tem impulsionado a medicina, principalmente na identificação de novos alvos terapêuticos e de diagnóstico. Nas células são as proteínas as principais intervenientes responsáveis pelo funcionamento de todos os processos celulares, desde a síntese de novas moléculas de DNA, à formação de RNA e de novas proteínas, ao transporte de todos os componentes celulares bem como da composição estrutural da própria célula. Também são as proteínas um dos componentes mais relevantes das vias de sinalização entre as células. Estudos apontam para que, normalmente, as proteínas não funcionem sozinhas mas em complexos. Daí a importância de estudar as interações entre proteínas e, por outro lado, encontrar compostos que interfiram com esses complexos para tratamentos farmacológicos. Já existem alguns fármacos com estas características. A tricostatina A, um inibidor duma diacetilase de histonas (DH), atua no complexo Proteína Fosfatase 1-DH, sendo um bom alvo na terapia anti-cancerígena. Em 1989, de um modo revolucionário para a época, Fields e Songs desenvolveram o Yeast Two Hybrid system (YTH). Este método baseia-se na genética da levedura Saccharomyces cerevisiae para detetar interações entre proteínas. Desde a sua descoberta sofreu várias modificações que permitiram a sua aplicação à investigação translacional. Por exemplo, esta técnica permite fazer um rastreio em larga escala para determinar que droga pode interferir com uma determinada interação proteica. Por outro lado, pode também utilizar-se para se determinar que proteínas num determinado tecido (por exemplo, cérebro ou testículo) interagem com a nossa proteína de interesse. Deste modo é possível desvendar funções de novas proteínas, vias de sinalização e funcionamento de tecidos. A grande quantidade de informação produzida por esta abordagem é de eleição para a identificação e validação de alvos de diagnóstico, terapêuticos e mesmo desenvolvimento de novos fármacos. Esta revisão tem como intuito elucidar o funcionamento do Yeast Two Hybrid system e a sua contribuição para a identificação de novos tratamentos farmacológicos.In the last decades, molecular biology development was driven medicine, mainly in identification of novel therapeutic and diagnostics targets. In cells, proteins are the main responsible for the functioning of all cellular processes, from DNA synthesis to RNA and protein production, transport of cellular components and structural composition of the cell. Proteins are also an important component of signaling pathways between cells. Studies show that proteins normally do not function as singular units but as protein complexes. Understand protein interactions and discover compounds that interfere with such protein complexes are important to develop new pharmacologic treatments. There are already some drugs with such characteristics. Trichostatin A, a histone diacetilase, acts in Phosphatase protein 1 - Histone diacetilase complex, being a good target for anti-cancer therapy. In 1989, in a revolutionary way, Fields and Songs developed the Yeast Two Hybrid system (YTH). This method is based in the genetic properties of Saccharomyces cerevisiae and allows the detection of protein interactions in vivo. Since its development it suffered a few modifications that allowed its application in translational medicine. For example, this technique allows a high throughput screening to assess if a drug can interfere with a protein interaction. In the other hand, YTH can be used to ascertain which proteins interact with a protein of interest in a specific tissue (for example, brain or testis). Thus it is possible to unveil protein functions, signaling pathways and tissue functions. The great amount of data produced with YTH allows the identification and validation of diagnostic and therapeutic targets and also the development of new drugs. This review has the purpose to clarify the YTH system function and its contribution in identification of new pharmacologic treatments.publishe

Therapeutic strategies involving survivin inhibition in cancer

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.Consejería de Educación, Junta de Castilla y León. Grant Number: BU092U16 Instituto de Salud Carlos III. Grant Number: FIS PI18/0044

The natural-based antitumor compound T21 decreases survivin levels through potent STAT3 inhibition in lung cancer models

Lung cancer is the leading cause of cancer-related deaths worldwide; hence novel treatments for this malignancy are eagerly needed. Since natural-based compounds represent a rich source of novel chemical entities in drug discovery, we have focused our attention on tambjamines, natural compounds isolated from marine invertebrates that have shown diverse pharmacological activities. Based on these structures, we have recently identified the novel indole-based tambjamine analog 21 (T21) as a promising antitumor agent, which modulates the expression of apoptotic proteins such as survivin. This antiapoptotic protein plays an important role in carcinogenesis and chemoresistance. In this work, we have elucidated the molecular mechanism by which the anticancer compound T21 exerts survivin inhibition and have validated this protein as a therapeutic target in di erent lung cancer models. T21 was able to reduce survivin protein levels in vitro by repressing its gene expression through the blockade of Janus kinase/Signal Transducer and Activator of Transcription-3 (JAK/STAT3)/survivin signaling pathway. Interestingly, this occurred even when the pathway was overstimulated with its ligand interleukin 6 (IL-6), which is frequently overexpressed in lung cancer patients who show poor clinical outcomes. Altogether, these results show T21 as a potent anticancer compound that e ectively decreases survivin levels through STAT3 inhibition in lung cancer, appearing as a promising therapeutic drug for cancer treatment

Not so pseudo: the evolutionary history of protein phosphatase 1 regulatory subunit 2 and related pseudogenes

Background: Pseudogenes are traditionally considered “dead” genes, therefore lacking biological functions. This view has however been challenged during the last decade. This is the case of the Protein phosphatase 1 regulatory subunit 2 (PPP1R2) or inhibitor-2 gene family, for which several incomplete copies exist scattered throughout the genome. Results: In this study, the pseudogenization process of PPP1R2 was analyzed. Ten PPP1R2-related pseudogenes (PPP1R2P1-P10), highly similar to PPP1R2, were retrieved from the human genome assembly present in the databases. The phylogenetic analysis of mammalian PPP1R2 and related pseudogenes suggested that PPP1R2P7 and PPP1R2P9 retroposons appeared before the great mammalian radiation, while the remaining pseudogenes are primate-specific and retroposed at different times during Primate evolution. Although considered inactive, four of these pseudogenes seem to be transcribed and possibly possess biological functions. Given the role of PPP1R2 in sperm motility, the presence of these proteins was assessed in human sperm, and two PPP1R2-related proteins were detected, PPP1R2P3 and PPP1R2P9. Signatures of negative and positive selection were also detected in PPP1R2P9, further suggesting a role as a functional protein. Conclusions: The results show that contrary to initial observations PPP1R2-related pseudogenes are not simple bystanders of the evolutionary process but may rather be at the origin of genes with novel functions.publishe

The effect of potential supramolecular-bond promoters on the DNA-interacting abilities of copper-terpyridine compounds.

Three copper(II) coordination compounds have been prepared from three different 2,2′:6′,2′′-terpyridine-based ligands, which have been selected to investigate the potential role of supramolecular interactions on the DNA-interacting and cytotoxicity properties of the corresponding metal complexes. Hence, the ligands 4′-((naphthalen-2-yl)methoxy)-2,2′:6′,2′′-terpyridine (Naphtpy) and 4′-((1H-benzo[d]imidazol-2-yl)methoxy)-2,2′:6′,2′′-terpyridine (Bimztpy) have been synthesized from commercially-available 4′-chloro-2,2′:6′,2′′-terpyridine (Cltpy), and their copper(II) complexes have been obtained by reaction with copper(II) nitrate. The DNA-interacting abilities of the corresponding compounds [Cu(Cltpy)(H2O)(NO3)2] (1), [Cu(naphtpy)(NO3)(H2O)](NO3)(MeOH) (2) and [Cu(bimztpy)(NO3)(H2O)](NO3) (3) have been investigated using different techniques, and cytotoxicity assays with several cancer cell lines have revealed interesting features, viz. the more efficient complex is 2, which although it does not act as a DNA cleaver, displays the most effective DNA-interacting and cytotoxic properties, compared to 1 and 3

Phosphoprotein Phosphatase 1 isoforms alpha and gamma respond differently to prodigiosin treatment and present alternative kinase targets in melanoma cells

Reversible protein phosphorylation is a central regulatory mechanism of cell function. Deregulation of the balanced actions of protein kinases and phosphatases has been frequently associated with several pathological conditions, including cancer. Many studies have already addressed the role of protein kinases misregulation in cancer. However, much less is known about protein phosphatases influence. Phosphoprotein Phosphatase 1 (PPP1) is one of the major serine/threonine protein phosphatases who has three catalytic isoforms: PPP1CA, PPP1CB, and PPP1CC. Its function is achieved by binding to regulatory subunits, known as PPP1-interacting proteins (PIPs), which may prefer a catalytic isoform. Also, some inhibitors/enhancers may exhibit isoform specificity. Here we show that, prodigiosin (PG), a molecule with anticancer properties, promotes the formation of PPP1CA-AKT complex and not of PPP1CC-MAPK complex. Both, AKT and MAPK, are wellknown PIPs from two pathways that crosstalk and regulate melanoma cells survival. In addition, the analysis performed using surface plasmon resonance (SPR) technology indicates that PPP1 interacts with obatoclax (OBX), a drug that belongs to the same family of PG. Overall, these results suggest that PG might, at least in part, act through PPP1C/PIPs. Also, this study is pioneer in demonstrating PPP1 isoform-specific modulation by small molecules.publishe

Photoactivation of the cytotoxic properties of platinum(II) complexes through ligand photoswitching

The development of photoactivatable metal complexes with potential anticancer properties is a topical area of current investigation. Photoactivated chemotherapy using coordination compounds is typically based on photochemical processes occurring at the metal center. In the present study, an innovative approach is applied that takes advantage of the remarkable photochemical properties of diarylethenes. Following a proof-of-concept study with two complexes, namely, C1 and C2, a series of additional platinum(II) complexes from dithienylcydopentene-based ligands was designed and prepared. Like C1 and C2, these new coordination compounds exhibit two thermally stable, interconvertible photoisomers that display distinct properties. The photochemical behavior of ligands L3-L7 has been analyzed by H-1 NMR and UV-vis spectroscopies. Subsequently, the corresponding platinum(II) complexes C3-C7 were synthesized and fully characterized, including by single-crystal X-ray diffraction for some of them. Next, the interaction of each photoisomer (i.e., containing the open or closed ligand) of the metal complexes with DNA was examined thoroughly using various techniques, revealing their distinct DNA-binding modes and affinities, as observed for the earlier compounds C1 and C2. The antiproliferative activity of the two forms of the complexes was then assessed with five cancer cell lines and compared with that of C1 and C2, which supported the use of such diarylethene-based systems for the generation of a new class of potential photochemotherapeutic metallodrugs