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

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 dithienylcyclopentene-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 1H 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

Identification and characterization of two distinct PPP1R2 isoforms in human spermatozoa

Background: Protein Ser/Thr Phosphatase PPP1CC2 is an alternatively spliced isoform of PPP1C that is highly enriched in testis and selectively expressed in sperm. Addition of the phosphatase inhibitor toxins okadaic acid or calyculin A to caput and caudal sperm triggers and stimulates motility, respectively. Thus, the endogenous mechanisms of phosphatase inhibition are fundamental for controlling sperm function and should be characterized. Preliminary results have shown a protein phosphatase inhibitor activity resembling PPP1R2 in bovine and primate spermatozoa. Results: Here we show conclusively, for the first time, that PPP1R2 is present in sperm. In addition, we have also identified a novel protein, PPP1R2P3. The latter was previously thought to be an intron-less pseudogene. We show that the protein corresponding to the pseudogene is expressed. It has PPP1 inhibitory potency similar to PPP1R2. The potential phosphosites in PPP1R2 are substituted by non-phosphorylable residues, T73P and S87R, in PPP1R2P3. We also confirm that PPP1R2/PPP1R2P3 are phosphorylated at Ser121 and Ser122, and report a novel phosphorylation site, Ser127. Subfractionation of sperm structures show that PPP1CC2, PPP1R2/PPP1R2P3 are located in the head and tail structures. Conclusions: The conclusive identification and localization of sperm PPP1R2 and PPP1R2P3 lays the basis for future studies on their roles in acrosome reaction, sperm motility and hyperactivation. An intriguing possibility is that a switch in PPP1CC2 inhibitory subunits could be the trigger for sperm motility in the epididymis and/or sperm hyperactivation in the female reproductive tract.publishe

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

An intriguing shift occurs in the novel protein phosphatase 1 binding partner, TCTEX1D4: evidence of positive selection in a pika model

T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms

TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood-testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood-testis barrier

An intriguing shift occurs in the novel protein phosphatase 1 binding partner, TCTEX1D4: evidence of positive selection in a pika model

T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif 90RVSF93 is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif 96NLS98, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The dN/dS for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms.publishe

From proteomic analysis to potential therapeutic targets: functional profile of two lung cancer cell lines, A549 and SW900, widely studied in pre-clinical research

Lung cancer is a serious health problem and the leading cause of cancer death worldwide. The standard use of cell lines as in vitro pre-clinical models to study the molecular mechanisms that drive tumorigenesis and access drug sensitivity/effectiveness is of undisputable importance. Label-free mass spectrometry and bioinformatics were employed to study the proteomic profiles of two representative lung cancer cell lines and to unravel the specific biological processes. Adenocarcinoma A549 cells were enriched in proteins related to cellular respiration, ubiquitination, apoptosis and response to drug/hypoxia/oxidative stress. In turn, squamous carcinoma SW900 cells were enriched in proteins related to translation, apoptosis, response to inorganic/organic substances and cytoskeleton organization. Several proteins with differential expression were related to cancer transformation, tumor resistance, proliferation, migration, invasion and metastasis. Combined analysis of proteome and interactome data highlighted key proteins and suggested that adenocarcinoma might be more prone to PI3K/Akt/mTOR and topoisomerase IIα inhibitors, and squamous carcinoma to Ck2 inhibitors. Moreover, ILF3 overexpression in adenocarcinoma, and PCNA and NEDD8 in squamous carcinoma shows them as promising candidates for therapeutic purposes. This study highlights the functional proteomic differences of two main subtypes of lung cancer models and hints several targeted therapies that might assist in this type of cancer.publishe

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

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 dithienylcyclopentene-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 1H 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.Financial support from the Spanish Ministerio de Economía y Competitividad/FEDER (Project Nos. CTQ2015-70371-REDT, CTQ2014-55293-P, and CTQ2017-88446-R) is acknowledged. P.G. thanks the Institució Catalana de Recerca i Estudis Avançats (ICREA). This work was partially supported by a grant from the Spanish government and the EU (FIS PI13/00089), a grant from La Marató de TV3 Foundation (20132730). Also, an individual contract to L.K.G. supported by a program from the Barcelona Univ. in collaboration with Obra Social de la Fundació Bancaria “La Caixa” is acknowledged. This research used resources of the Advanced Light Source, which is a Department of Energy Office of Science User Facility under Contract No. DE-AC02-05CH11231.Peer reviewe