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Margarida Fardilha - One of the best experts on this subject based on the ideXlab platform.
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Construction and analysis of a human testis/sperm-enriched interaction network: Unraveling the PPP1CC2 interactome
Biochimica et biophysica acta. General subjects, 2016Co-Authors: Joana Vieira Silva, S. Y. Yoon, Pieter-jan De Bock, Alexander V. Goltsev, Kris Gevaert, José F. F. Mendes, Margarida FardilhaAbstract:Abstract Background Phosphoprotein Phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a PPP1CC tissue-specific alternative splice restricted to testicular germ cells and spermatozoa, is essential for spermatogenesis and spermatozoa motility. The key to understand PPP1CC2 regulation lies on the characterization of its interacting partners. Methods We construct a testis/sperm-enriched protein interaction network and analyzed the topological properties and biological context of the network. Further the interaction of a potential target for pharmacological intervention was validated in human spermatozoa. Results A total of 1778 proteins and 32,187 interactions between them were identified in the testis/sperm-enriched network. The network analysis revealed the members of functional modules that interact more tightly with each other. In the network, PPP1CC was located in the fourth maximum core part (k = 41) and had 106 direct interactors. Sixteen PPP1CC interactors were involved in spermatogenesis-related categories. Also, PPP1CC had 50 direct interactors, highly interconnected and many of them part of the network maximum core (k = 44), associated with motility-related annotations, including several previously uncharacterized interactors, such as, LMNA, JAK2 and RIPK3. Conclusions In this study we integrated tissue-specific protein expression and protein-protein interaction data in order to identify key PPP1CC2 complexes for male reproductive functions. One of the most intriguing interactors was A-kinase anchor protein 4 (AKAP4), a testis-specific protein related to infertility phenotypes and involved in all major motility-related annotations. General significance We demonstrated for the first time the interaction between PPP1CC2 and AKAP4 in human spermatozoa and the potential of the complex as contraceptive target.
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characterisation of several ankyrin repeat protein variant 2 a Phosphoprotein Phosphatase 1 interacting protein in testis and spermatozoa
Reproduction Fertility and Development, 2016Co-Authors: Joana Vieira Silva, Edgar F. Da ,cruz E Silva, Luis Korrodigregorio, Georg H Luers, Maria Joao Cardoso, Antonio Patricio, Nuno Maia, Margarida FardilhaAbstract:Phosphoprotein Phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.
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The power of the yeast two-hybrid system in the identification of novel drug targets: building and modulating PPP1 interactomes.
Expert review of proteomics, 2015Co-Authors: Joana Vieira Silva, Juliana Felgueiras, Maria João Freitas, Margarida FardilhaAbstract:Since the description of the yeast two-hybrid (Y2H) method, it has become more and more evident that it is the most commonly used method to identify protein-protein interactions (PPIs). The improvements in the original Y2H methodology in parallel with the idea that PPIs are promising drug targets, offer an excellent opportunity to apply the principles of this molecular biology technique to the pharmaceutical field. Additionally, the theoretical developments in the networks field make PPI networks very useful frameworks that facilitate many discoveries in biomedicine. This review highlights the relevance of Y2H in the determination of PPIs, specifically Phosphoprotein Phosphatase 1 interactions, and its possible outcomes in pharmaceutical research.
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Phosphoprotein Phosphatase 1 complexes in spermatogenesis.
Current molecular pharmacology, 2015Co-Authors: Joana Vieira Silva, Maria João Freitas, Margarida FardilhaAbstract:The major post-translational modification in eukaryotes is protein phosphorylation which mediates responses to signals in a myriad of cellular processes. Not surprisingly, many steps in spermatogenesis involve the concerted action of the protein (de)phosphorylation key players, kinases and Phosphatases. Phosphoprotein Phosphatase 1 catalytic subunit (PPP1C), an evolutionarily conserved Ser/Thr-protein Phosphatase, catalyzes the majority of eukaryotic protein dephosphorylation reactions. Three genes, PPP1CA, PPP1CB and PPP1CC, encode four PPP1C isoforms, PPP1CA, PPP1CB, PPP1CC1, and PPP1CC2. After transcription, PPP1CC undergoes tissue-specific splicing, originating a ubiquitously expressed isoform, PPP1CC1 and a testis-enriched and sperm-specific isoform, PPP1CC2 which is essential for completion of spermatogenesis. Highly similar PPP1C isoforms - PPP1CA and PPP1CB - are capable of compensating the loss of Ppp1cc in every tissue except in testis. PPP1C cellular functions depend on the complexes it forms with PPP1C Interacting Proteins (PIPs), which together with the different catalytic subunits, account for PPP1C specificity. This review will focus on the role of the major serine/threonine Phosphatase - PPP1C and its holoenzymes in spermatogenesis. Furthermore, current challenges on the protein Phosphatases field as targets to male contraception will be addressed.
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Synphilin-1A is a Phosphoprotein Phosphatase 1-Interacting Protein and Affects PPP1 Sorting to Subcellular Compartments
Journal of Molecular Neuroscience, 2015Co-Authors: Emanuel Ferreira-fernandes, Luís Korrodi-gregório, Sara L. C. Esteves, Georg H Luers, Margarida Fardilha, Vera Afreixo, Odete A. B. Da ,cruz E SilvaAbstract:Lewy bodies (LBs) are synphilin-1 (Sph1)-containing aggregates and histological hallmarks of Parkinson’s disease. Therefore, understanding processes which modulate the aggregation of Sph1, or its isoform Sph1A, will contribute to our understanding of LBs formation. Protein phosphorylation promotes aggregation, but protein Phosphatases with activity towards Sph1 have not been described. The present study documents the identification of a novel Sph1A/Phosphoprotein Phosphatase 1 (PPP1) complex and unravels its regulatory effect on Sph1A aggregation. Using yeast co-transformation and overlay blot assay, the interaction between Sph1A and PPP1 was mapped to the Sph1A RVTF motif. Then, Sph1A overexpression in human embryonic kidney 293 cells demonstrated that Sph1A specifically targets endogenous PPP1 isoforms to inclusion bodies and that Sph1A/PPP1 complex disruption enhances inclusion bodies formation. Finally, as Sph1A interacted with PPP1CC2, a PPP1 sperm-specific isoform, Sph1 and Sph1A expression was addressed in male germ cells by qRT-PCR, revealing high expression levels in round spermatids. Together, these observations established Sph1A as a novel PPP1-interacting protein able to affect PPP1 sorting to subcellular compartments and Sph1A/PPP1 complex as a negative modulator of LBs formation. Contrarily, in physiological conditions, Sph1 isoforms are pointed as putative participants in vesicle dynamics with implications in neurotransmission and spermiogenesis.
Odete A. B. Da ,cruz E Silva - One of the best experts on this subject based on the ideXlab platform.
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An intriguing shift occurs in the novel protein Phosphatase 1 binding partner, TCTEX1D4: evidence of positive selection in a pika model
'Public Library of Science (PLoS)', 2017Co-Authors: Korrodi-gregório Luís, Lopes, Ana Margarida, Esteves, Sara L. C., Afonso Sandra, Lemos De Matos, Ana, Lissovsky, Andrey A., Odete A. B. Da ,cruz E Silva, Edgar F. Da ,cruz E Silva, Esteves, Pedro José, Fardilha MargaridaAbstract: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
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Phosphoprotein Phosphatase 1 isoforms alpha and gamma respond differently to prodigiosin treatment and present alternative kinase targets in melanoma cells
'Scientific Research Publishing Inc.', 2017Co-Authors: Fardilha Margarida, Korrodi-gregório Luís, Esteves, Sara L. C., Odete A. B. Da ,cruz E Silva, Edgar F. Da ,cruz E Silva, Figueiredo João, Espona Fiedler Margarita, Felgueiras Juliana, Rebelo Sandra, Pérez Tomás, Ricardo E.Abstract: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 well-known 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
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Synphilin-1A is a Phosphoprotein Phosphatase 1-Interacting Protein and Affects PPP1 Sorting to Subcellular Compartments
Journal of Molecular Neuroscience, 2015Co-Authors: Emanuel Ferreira-fernandes, Luís Korrodi-gregório, Sara L. C. Esteves, Georg H Luers, Margarida Fardilha, Vera Afreixo, Odete A. B. Da ,cruz E SilvaAbstract:Lewy bodies (LBs) are synphilin-1 (Sph1)-containing aggregates and histological hallmarks of Parkinson’s disease. Therefore, understanding processes which modulate the aggregation of Sph1, or its isoform Sph1A, will contribute to our understanding of LBs formation. Protein phosphorylation promotes aggregation, but protein Phosphatases with activity towards Sph1 have not been described. The present study documents the identification of a novel Sph1A/Phosphoprotein Phosphatase 1 (PPP1) complex and unravels its regulatory effect on Sph1A aggregation. Using yeast co-transformation and overlay blot assay, the interaction between Sph1A and PPP1 was mapped to the Sph1A RVTF motif. Then, Sph1A overexpression in human embryonic kidney 293 cells demonstrated that Sph1A specifically targets endogenous PPP1 isoforms to inclusion bodies and that Sph1A/PPP1 complex disruption enhances inclusion bodies formation. Finally, as Sph1A interacted with PPP1CC2, a PPP1 sperm-specific isoform, Sph1 and Sph1A expression was addressed in male germ cells by qRT-PCR, revealing high expression levels in round spermatids. Together, these observations established Sph1A as a novel PPP1-interacting protein able to affect PPP1 sorting to subcellular compartments and Sph1A/PPP1 complex as a negative modulator of LBs formation. Contrarily, in physiological conditions, Sph1 isoforms are pointed as putative participants in vesicle dynamics with implications in neurotransmission and spermiogenesis.
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Protein Phosphatase 1 and Its Complexes in Carcinogenesis
Current cancer drug targets, 2014Co-Authors: Joao Figueiredo, Odete A. B. Da ,cruz E Silva, Margarida FardilhaAbstract:Understanding the molecular mechanisms and the signaling pathways that underlie the pathology of cancer progression is crucial for the development of novel diagnostic and therapeutic tools. A major common mechanism used by cells to regulate intracellular signal transduction pathways is reversible protein phosphorylation which results in profound changes in cellular responses. This mechanism relies on the coordinated action of two families of proteins: protein kinases and protein Phosphatases. Interestingly, there are 3 to 5 times fewer Phosphatases than kinases, suggesting that the specificity of substrates is not only due to the variety of the catalytic subunits but also to the diversity of the regulatory subunits. This is particularly true for Phosphoprotein Phosphatase 1 (PPP1) for which more than 200 PPP1 Interacting Proteins (PIPs) have thus far been identified. PIPs can act as targeting subunits, substrates and activity regulators. Many PPP1/PIPs complexes are involved in signaling pathways that regulate cellular growth, cell cycle and apoptosis; processes known to be deregulated in cancer. This review will describe the cellular pathways, many of which involve PPP1/PIP complexes, that when deregulated lead to cancer. Furthermore, the possibility of PPP1/PIP complexes being considered novel targets to cancer diagnostic and therapy will be addressed.
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"Omics" of human sperm: profiling protein Phosphatases.
Omics : a journal of integrative biology, 2013Co-Authors: Margarida Fardilha, Luís Korrodi-gregório, Mónica Ferreira, Steven L. Pelech, Sandra I. Vieira, Sandra Rebelo, Mário Sousa, Alberto Barros, Vladimiro Silva, Odete A. B. Da ,cruz E SilvaAbstract:Abstract Phosphorylation is a major regulatory mechanism in eukaryotic cells performed by the concerted actions of kinases and Phosphatases (PPs). Protein phosphorylation has long been relevant to sperm physiology, from acquisition of motility in the epididymis to capacitation in the female reproductive tract. While the precise kinases involved in the regulation of sperm phosphorylation have been studied for decades, the PPs have only recently received research interest. Tyrosine phosphorylation was first implicated in the regulation of several sperm-related functions, from capacitation to oocyte binding. Only afterwards, in 1996, the inhibition of the serine/threonine-PP Phosphoprotein Phosphatase 1 (PPP1) by okadaic acid and calyculin-A was shown to initiate motility in caput epididymal sperm. Today, the current mechanisms of sperm motility acquisition based on PPP1 and its regulators are still far from being fully understood. PPP1CC2, specifically expressed in mammalian sperm, has been considered to be...
Thomas M. Chiang - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation–Dephosphorylation States at Different Sites Affect Phosphoprotein Phosphatase 1 Activity
Thrombosis Research, 1998Co-Authors: Thomas M. ChiangAbstract:Abstract We have previously reported that the binding of type I collagen to its receptor initiates platelet aggregation involving Phosphoprotein Phosphatase 1 (PP 1), which coprecipitates with the 65-kDa platelet type I collagen receptor. Phosphorylation of the anti-PP1 precipitation PP1 decreases its enzyme activity. In the present investigation, the mechanism of the decreased enzyme activity was studied by examining the phosphorylation of PP 1 on serine/threonine or tyrosine residues. Phosphoamino acid analysis of the PP 1 indicates that serine, threone, and tyrosine can all be phosphorylated. We find that the activity of PP 1 decreases with serine/threonine phosphorylation but that phosphorylation of tyrosine residue activates enzyme activity. These results indicate that the activity of platelet Phosphoprotein Phosphatase 1 is controlled by phosphorylation and dephosphorylation states at multiple, different site(s).
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phosphorylation dephosphorylation states at different sites affect Phosphoprotein Phosphatase 1 activity
Thrombosis Research, 1998Co-Authors: Thomas M. ChiangAbstract:We have previously reported that the binding of type I collagen to its receptor initiates platelet aggregation involving Phosphoprotein Phosphatase 1 (PP 1), which coprecipitates with the 65-kDa platelet type I collagen receptor. Phosphorylation of the anti-PP1 precipitation PP1 decreases its enzyme activity. In the present investigation, the mechanism of the decreased enzyme activity was studied by examining the phosphorylation of PP 1 on serine/threonine or tyrosine residues. Phosphoamino acid analysis of the PP 1 indicates that serine, threonine, and tyrosine can all be phosphorylated. We find that the activity of PP 1 decreases with serine/threonine phosphorylation but that phosphorylation of tyrosine residue activates enzyme activity. These results indicate that the activity of platelet Phosphoprotein Phosphatase 1 is controlled by phosphorylation and dephosphorylation states at multiple, different site(s).
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INVOLVEMENT OF Phosphoprotein Phosphatase 1 IN COLLAGEN-PLATELET INTERACTION
Thrombosis research, 1996Co-Authors: Thomas M. Chiang, Ellen S. Kang, Andrew H. KangAbstract:Abstract The binding of type I collagen to its receptor initiates platelet aggregation, but the relationship of the receptor to other signal transduction components is not yet established. Correlation of platelet aggregation and anti-type I collagen receptor antibody immunoprecipitation of type I collagen treated [ 32 PO4]-labeled platelets showed that there are two Phosphoproteins (M r 53 kDa and 21 kDa) that coprecipitated with the 65 kDa platelet type I collagen receptor. In the present investigation, we have identified one of the Phosphoproteins. A soluble component the 100,000×g supernatant fraction of 53 kDa protein is recognized by polyclonal anti-PP1 antibody. The activity of the precipitated Phosphatase is inhibited by okadaic acid and inhibitor 1, suggesting that it is protein Phosphatase 1 (PP 1). Phosphorylation decreases PP 1 activity as was found with [ 32 PO4]-phosphorylase b as the substrate. The immunocoprecipitation of the type-1 collagen receptor and PP 1 inot the result of cross reactivity of the anti-type I collagen receptor antibody with the PP I protein. These results indicate that the platelet type I collagen receptor, PP 1, and unidentified 21 kDa protein are in close association with the platelet type I collagen receptor upon the binding of type I collagen by the receptor. Copyright © 1996 Elsevier Science Ltd
Luís Korrodi-gregório - One of the best experts on this subject based on the ideXlab platform.
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An Intriguing Shift Occurs in the Novel Protein Phosphatase 1 Binding Partner, TCTEX1D4: Evidence of Positive Selection in a Pika Model
2016Co-Authors: Luís Korrodi-gregório, Andrey A. Lissovsky, Ana Margarida Lopes, Sara L. C. Esteves, Ra Afonso, Pedro JoséAbstract: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 positiv
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Synphilin-1A is a Phosphoprotein Phosphatase 1-Interacting Protein and Affects PPP1 Sorting to Subcellular Compartments
Journal of Molecular Neuroscience, 2015Co-Authors: Emanuel Ferreira-fernandes, Luís Korrodi-gregório, Sara L. C. Esteves, Georg H Luers, Margarida Fardilha, Vera Afreixo, Odete A. B. Da ,cruz E SilvaAbstract:Lewy bodies (LBs) are synphilin-1 (Sph1)-containing aggregates and histological hallmarks of Parkinson’s disease. Therefore, understanding processes which modulate the aggregation of Sph1, or its isoform Sph1A, will contribute to our understanding of LBs formation. Protein phosphorylation promotes aggregation, but protein Phosphatases with activity towards Sph1 have not been described. The present study documents the identification of a novel Sph1A/Phosphoprotein Phosphatase 1 (PPP1) complex and unravels its regulatory effect on Sph1A aggregation. Using yeast co-transformation and overlay blot assay, the interaction between Sph1A and PPP1 was mapped to the Sph1A RVTF motif. Then, Sph1A overexpression in human embryonic kidney 293 cells demonstrated that Sph1A specifically targets endogenous PPP1 isoforms to inclusion bodies and that Sph1A/PPP1 complex disruption enhances inclusion bodies formation. Finally, as Sph1A interacted with PPP1CC2, a PPP1 sperm-specific isoform, Sph1 and Sph1A expression was addressed in male germ cells by qRT-PCR, revealing high expression levels in round spermatids. Together, these observations established Sph1A as a novel PPP1-interacting protein able to affect PPP1 sorting to subcellular compartments and Sph1A/PPP1 complex as a negative modulator of LBs formation. Contrarily, in physiological conditions, Sph1 isoforms are pointed as putative participants in vesicle dynamics with implications in neurotransmission and spermiogenesis.
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Protein Phosphatase 1 catalytic isoforms: specificity toward interacting proteins
Translational research : the journal of laboratory and clinical medicine, 2014Co-Authors: Luís Korrodi-gregório, Sara L. C. Esteves, Margarida FardilhaAbstract:The coordinated and reciprocal action of serine-threonine protein kinases and protein Phosphatases produces transitory phosphorylation, a fundamental regulatory mechanism for many biological processes. Phosphoprotein Phosphatase 1 (PPP1), a major serine-threonine Phosphatase, in particular, is ubiquitously distributed and regulates a broad range of cellular functions, including glycogen metabolism, cell cycle progression, and muscle relaxation. PPP1 has evolved effective catalytic machinery but in vitro lacks substrate specificity. In vivo, its specificity is achieved not only by the existence of different PPP1 catalytic isoforms, but also by binding of the catalytic moiety to a large number of regulatory or targeting subunits. Here, we will address exhaustively the existence of diverse PPP1 catalytic isoforms and the relevance of their specific partners and consequent functions.
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TGF-β cascade regulation by PPP1 and its interactors -impact on prostate cancer development and therapy.
Journal of cellular and molecular medicine, 2014Co-Authors: Luís Korrodi-gregório, Joana Vieira Silva, Juliana Felgueiras, Maria João Freitas, Luís Santos-sousa, Margarida FardilhaAbstract:Protein phosphorylation is a key mechanism by which normal and cancer cells regulate their main transduction pathways. Protein kinases and Phosphatases are precisely orchestrated to achieve the (de)phosphorylation of candidate proteins. Indeed, cellular health is dependent on the fine-tune of phosphorylation systems, which when deregulated lead to cancer. Transforming growth factor beta (TGF-β) pathway involvement in the genesis of prostate cancer has long been established. Many of its members were shown to be hypo- or hyperphosphorylated during the process of malignancy. A major Phosphatase that is responsible for the vast majority of the serine/threonine dephosphorylation is the Phosphoprotein Phosphatase 1 (PPP1). PPP1 has been associated with the dephosphorylation of several proteins involved in the TGF-β cascade. This review will discuss the role of PPP1 in the regulation of several TGF-β signalling members and how the subversion of this pathway is related to prostate cancer development. Furthermore, current challenges on the protein Phosphatases field as new targets to cancer therapy will be addressed.
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"Omics" of human sperm: profiling protein Phosphatases.
Omics : a journal of integrative biology, 2013Co-Authors: Margarida Fardilha, Luís Korrodi-gregório, Mónica Ferreira, Steven L. Pelech, Sandra I. Vieira, Sandra Rebelo, Mário Sousa, Alberto Barros, Vladimiro Silva, Odete A. B. Da ,cruz E SilvaAbstract:Abstract Phosphorylation is a major regulatory mechanism in eukaryotic cells performed by the concerted actions of kinases and Phosphatases (PPs). Protein phosphorylation has long been relevant to sperm physiology, from acquisition of motility in the epididymis to capacitation in the female reproductive tract. While the precise kinases involved in the regulation of sperm phosphorylation have been studied for decades, the PPs have only recently received research interest. Tyrosine phosphorylation was first implicated in the regulation of several sperm-related functions, from capacitation to oocyte binding. Only afterwards, in 1996, the inhibition of the serine/threonine-PP Phosphoprotein Phosphatase 1 (PPP1) by okadaic acid and calyculin-A was shown to initiate motility in caput epididymal sperm. Today, the current mechanisms of sperm motility acquisition based on PPP1 and its regulators are still far from being fully understood. PPP1CC2, specifically expressed in mammalian sperm, has been considered to be...
Joana Vieira Silva - One of the best experts on this subject based on the ideXlab platform.
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Proteínas do espermatozoide como alvos para contraceção e biomarcadores de fertilidade
Universidade de Aveiro, 2018Co-Authors: Joana Vieira SilvaAbstract:Doutoramento em BiomedicinaO elevado número de gravidezes indesejadas a nível mundial (~41%) reflete a necessidade premente de novos métodos contracetivos. Para já, os contracetivos masculinos estão limitados ao preservativo, ao coito interrompido e à vasectomia dado que ainda não existem métodos farmacológicos disponíveis. Por outro lado, nos países desenvolvidos, 15% dos casais são inférteis e, em metade dos casos, as causas estão relacionadas com fatores masculinos, sendo a infertilidade idiopática o tipo mais comum de infertilidade masculina. Os principais objetivos deste trabalho consistiram em (1) identificar, caracterizar e modular alvos não-hormonais para a contraceção masculina; e (2) estabelecer um conjunto de biomarcadores para avaliar a fertilidade masculina. No que toca à identificação de alvos para a contraceção masculina, foi dada especial atenção a duas proteínas e aos seus interactors: a fosfoproteína fosfatase 1 (PPP1) e a proteína percursora amilóide (APP). Caracterizámos o interactoma da subunidade catalítica gama 2 da PPP1 (PPP1CC2), uma isoforma específica do testículo e espermatozoide. Demonstrando, pela primeira vez, a interação entre a PPP1CC2 e a A-kinase anchor protein 4 (AKAP4), uma proteína expressa especificamente no testículo essencial para a motilidade do espermatozoide, e o potencial desse complexo como alvo contracetivo. A motilidade do espermatozoide foi, então, modulada eficazmente recorrendo a cell penetrating peptides (CPPs) como sistemas de transporte intracelular de sequências peptídicas direcionadas para padrões únicos de interações proteicas. Um outro interactor da PPP1CC2, a several ankyrin repeat protein variant 2 (SARP2), foi também caracterizado em testículo e espermatozoide. Descrevemos ainda, o interactoma da APP em testículo e espermatozoide humano sendo que a nossa abordagem permitiu a identificação de novos interactores e o reconhecimento de interactores-chave na fertilidade masculina, particularmente na interação espermatozoide-oócito, o que representa um potencial mecanismo alvo para a contraceção masculina. Identificámos diversas proteínas sinalizadoras cuja atividade se correlaciona com parâmetros seminais distintos, tendo o potencial para integrar uma plataforma de diagnóstico que poderá ter várias aplicações: explicar situações de infertilidade idiopática; explicar o insucesso de técnicas de procriação medicamente assistida (PMA) ou abortos de repetição; auxiliar a escolha da técnica de PMA mais apropriada; avaliar a eficácia de intervenções médicas; e clarificar os mecanismos responsáveis pela deterioração da qualidade dos espermatozoides associada com a idade. Para além do potencial para integrar um painel de biomarcadores, essas proteínas permitiram o reconhecimento de vias de sinalização responsáveis por regular funções específicas do espermatozoide que podem ser utilizadas com fins terapêuticos.The large number of unintended pregnancies worldwide (~41%) highlights the need for new contraceptive methods. Still, male contraceptive methods are limited to condoms, withdrawal or vasectomy as, currently, no pharmaceutical contraceptive agents exist for men. In contrast, in developed countries, infertility affects 15% of couples attempting to conceive and in half of these cases the cause is related to male reproductive issues. Moreover, idiopathic infertility remains the most common type of male infertility. The main goals of this work were to (1) identify, characterize and modulate non-hormonal targets for male contraception; and (2) establish a biomarker “fingerprint” to assess male fertility. Concerning the identification of targets for male contraception, particular attention was given to two distinct proteins and its interacting partners: Phosphoprotein Phosphatase 1 (PPP1) and amyloid precursor protein (APP). We characterized the interactome of PPP1 catalytic subunit gamma 2 (PPP1CC2), a testis-enriched/sperm-specific PPP1 isoform, in human testis/spermatozoa. We demonstrated for the first time the interaction between PPP1CC2 and A-kinase anchor protein 4 (AKAP4), a testis-specific protein essential for sperm motility, in human spermatozoa and the potential of the complex as a contraceptive target. Sperm motility was then successfully modulated by specific protein complex disruption using cell-penetrating peptides (CPPs) as a drug intracellular delivery system. Herein, we demonstrated for the first time the potential of CPPs to deliver peptide sequences that target unique protein-protein interactions in spermatozoa. Another PPP1CC2 interacting protein – several ankyrin repeat protein variant 2 (SARP2) – was also characterized in testis and spermatozoa. Moreover, we provided the first report on APP interactome in human testis/spermatozoa and our approach allowed the identification of novel interactions and the recognition of key APP interacting proteins for male reproduction, particularly in spermoocyte interaction, which represent a potential mechanism for male contraception modulation. We identified several signaling proteins that showed a high degree of differential activity in spermatozoa samples with distinct seminal parameters and have the potential to integrate a diagnostic array, which may have several applications: explain idiopathic infertility, failure in assisted reproductive techniques (ART) or repeated abortion; choice of the appropriate ART; assess the efficacy of medical interventions; and clarify the mechanisms responsible for age-dependent declines in spermatozoa quality. Besides the potential to integrate a biomarker "fingerprint" to assess sperm quality, those proteins allowed the recognition of the signaling pathways accountable for regulating specific spermatozoa functions that by future modulation could serve for therapeutic proposes
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Construction and analysis of a human testis/sperm-enriched interaction network: Unraveling the PPP1CC2 interactome
Biochimica et biophysica acta. General subjects, 2016Co-Authors: Joana Vieira Silva, S. Y. Yoon, Pieter-jan De Bock, Alexander V. Goltsev, Kris Gevaert, José F. F. Mendes, Margarida FardilhaAbstract:Abstract Background Phosphoprotein Phosphatase 1 catalytic subunit gamma 2 (PPP1CC2), a PPP1CC tissue-specific alternative splice restricted to testicular germ cells and spermatozoa, is essential for spermatogenesis and spermatozoa motility. The key to understand PPP1CC2 regulation lies on the characterization of its interacting partners. Methods We construct a testis/sperm-enriched protein interaction network and analyzed the topological properties and biological context of the network. Further the interaction of a potential target for pharmacological intervention was validated in human spermatozoa. Results A total of 1778 proteins and 32,187 interactions between them were identified in the testis/sperm-enriched network. The network analysis revealed the members of functional modules that interact more tightly with each other. In the network, PPP1CC was located in the fourth maximum core part (k = 41) and had 106 direct interactors. Sixteen PPP1CC interactors were involved in spermatogenesis-related categories. Also, PPP1CC had 50 direct interactors, highly interconnected and many of them part of the network maximum core (k = 44), associated with motility-related annotations, including several previously uncharacterized interactors, such as, LMNA, JAK2 and RIPK3. Conclusions In this study we integrated tissue-specific protein expression and protein-protein interaction data in order to identify key PPP1CC2 complexes for male reproductive functions. One of the most intriguing interactors was A-kinase anchor protein 4 (AKAP4), a testis-specific protein related to infertility phenotypes and involved in all major motility-related annotations. General significance We demonstrated for the first time the interaction between PPP1CC2 and AKAP4 in human spermatozoa and the potential of the complex as contraceptive target.
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characterisation of several ankyrin repeat protein variant 2 a Phosphoprotein Phosphatase 1 interacting protein in testis and spermatozoa
Reproduction Fertility and Development, 2016Co-Authors: Joana Vieira Silva, Edgar F. Da ,cruz E Silva, Luis Korrodigregorio, Georg H Luers, Maria Joao Cardoso, Antonio Patricio, Nuno Maia, Margarida FardilhaAbstract:Phosphoprotein Phosphatase 1 (PPP1) catalytic subunit gamma 2 (PPP1CC2), a PPP1 isoform, is largely restricted to testicular germ cells and spermatozoa. The key to understanding PPP1 regulation in male germ cells lies in the identification and characterisation of its interacting partners. This study was undertaken to determine the expression patterns of the several ankyrin repeat protein variant 2 (SARP2), a PPP1-interacting protein, in testis and spermatozoa. SARP2 was found to be highly expressed in testis and spermatozoa, and its interaction with human spermatozoa endogenous PPP1CC2 was confirmed by immunoprecipitation. Expression analysis by RT-qPCR revealed that SARP2 and PPP1CC2 mRNA levels were significantly higher in the spermatocyte fraction. However, microscopy revealed that SARP2 protein was only present in the nucleus of elongating and mature spermatids and in spermatozoa. In spermatozoa, SARP2 was prominently expressed in the connecting piece and flagellum, as well as, to a lesser extent, in the acrosome. A yeast two-hybrid approach was used to detect SARP2-interacting proteins and a relevant interaction with a novel sperm-associated antigen 9 (SPAG9) variant, a testis and spermatozoa-specific c-Jun N-terminal kinase-binding protein, was validated in human spermatozoa. Given the expression pattern of SARP2 and its association with PPP1CC2 and SPAG9, it may play a role in spermiogenesis and sperm function, namely in sperm motility and the acrosome reaction.
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The power of the yeast two-hybrid system in the identification of novel drug targets: building and modulating PPP1 interactomes.
Expert review of proteomics, 2015Co-Authors: Joana Vieira Silva, Juliana Felgueiras, Maria João Freitas, Margarida FardilhaAbstract:Since the description of the yeast two-hybrid (Y2H) method, it has become more and more evident that it is the most commonly used method to identify protein-protein interactions (PPIs). The improvements in the original Y2H methodology in parallel with the idea that PPIs are promising drug targets, offer an excellent opportunity to apply the principles of this molecular biology technique to the pharmaceutical field. Additionally, the theoretical developments in the networks field make PPI networks very useful frameworks that facilitate many discoveries in biomedicine. This review highlights the relevance of Y2H in the determination of PPIs, specifically Phosphoprotein Phosphatase 1 interactions, and its possible outcomes in pharmaceutical research.
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Phosphoprotein Phosphatase 1 complexes in spermatogenesis.
Current molecular pharmacology, 2015Co-Authors: Joana Vieira Silva, Maria João Freitas, Margarida FardilhaAbstract:The major post-translational modification in eukaryotes is protein phosphorylation which mediates responses to signals in a myriad of cellular processes. Not surprisingly, many steps in spermatogenesis involve the concerted action of the protein (de)phosphorylation key players, kinases and Phosphatases. Phosphoprotein Phosphatase 1 catalytic subunit (PPP1C), an evolutionarily conserved Ser/Thr-protein Phosphatase, catalyzes the majority of eukaryotic protein dephosphorylation reactions. Three genes, PPP1CA, PPP1CB and PPP1CC, encode four PPP1C isoforms, PPP1CA, PPP1CB, PPP1CC1, and PPP1CC2. After transcription, PPP1CC undergoes tissue-specific splicing, originating a ubiquitously expressed isoform, PPP1CC1 and a testis-enriched and sperm-specific isoform, PPP1CC2 which is essential for completion of spermatogenesis. Highly similar PPP1C isoforms - PPP1CA and PPP1CB - are capable of compensating the loss of Ppp1cc in every tissue except in testis. PPP1C cellular functions depend on the complexes it forms with PPP1C Interacting Proteins (PIPs), which together with the different catalytic subunits, account for PPP1C specificity. This review will focus on the role of the major serine/threonine Phosphatase - PPP1C and its holoenzymes in spermatogenesis. Furthermore, current challenges on the protein Phosphatases field as targets to male contraception will be addressed.