A importância do mecanismo de “splicing” alternativo para a identificação de novos alvos terapêuticos

A motilidade progressiva dos espermatozóides é um factor essencial para a sua fertilidade. Ao nível molecular, este processo depende da fosforilação de proteínas ainda não identificadas, uma vez que o tratamento de espermatozóides imóveis com agentes moduladores da fosforilação proteica, incluindo inibidores específicos de proteínas fosfatases, induz a sua motilidade. Os nossos estudos anteriores indicam que a proteína fosfatase PP1 2, uma isoforma da proteína fosfatase 1 produzida por “splicing” alternativo e grandemente enriquecida nos espermatozóides, provavelmente tem um papel importante na regulação da sua motilidade. A PP1 actua ligando-se a proteínas reguladoras que a levam para locais específicos da célula e regulam a sua actividade. Através do Sistema Dois Híbrido de Levedura iniciámos um projecto de identificação das proteínas reguladoras da PP1 2 em testículo humano que poderão ser úteis para modular a motilidade dos espermatozóides e, assim, servir como alvos terapêuticos para o tratamento da infertilidade masculina e/ou para o desenvolvimento de novas estratégias de contracepção masculina. Várias das proteínas reguladoras identificadas são novas variantes, também produzidas por mecanismos de “splicing” alternativo, de proteínas previamente conhecidas. Neste artigo focamos a importância do “splicing” alternativo como mecanismo extraordinário de produção de complexidade proteica e para a identificação de alvos terapêuticos de enorme especificidade. Palavras-chave: Esperma, infertilidade, contracepção, Nek2, PP1.Spermatozoa leave the testis incapable of progressive motility, which is only acquired during transit through the epididymis. At the molecular level, this process is dependent on the phosphorylation of proteins not yet identified, since treatment of non-motile sperm with modulators of protein phosphorylation, including protein phosphatase-specific inhibitors, induces motility. Our previous studies showed that PP1gamma2, a protein phosphatase 1 isoform produced by alternative splicing, highly enriched in sperm, has a major role in the regulation of sperm motility. Since PP1 acts via binding to regulatory proteins that target it to specific cellular locations and regulate its activity, we initiated a project to identify putative regulators of PP1gamma2 in human testis that might serve as therapeutic targets to interfere with sperm motility and thus act on male infertility and contraception. Some of the protein regulators identified are also alternatively spliced variants of previously known proteins. In this paper we will address the importance of alternative splicing as an extraordinary mechanism to produce protein complexity and to identify therapeutic targets of great specificity.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

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

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

Descriptive Analysis of LAP1 Distribution and That of Associated Proteins throughout Spermatogenesis

Spermatogenesis comprises highly complex differentiation processes. Nuclear envelope (NE) proteins have been associated with these processes, including lamins, lamina-associated polypeptide (LAP) 2 and the lamin B-receptor. LAP1 is an important NE protein whose function has not been fully elucidated, but several binding partners allow predicting putative LAP1 functions. To date, LAP1 had not been associated with spermatogenesis. In this study, LAP1 expression and cellular/subcellular localization during spermatogenesis in human and mouse testes is established for the first time. The fact that LAP1 is expressed during nuclear elongation in spermiogenesis and is located at the spermatids' centriolar pole is singularly important. LAP1 binds to members of the protein phosphatase 1 (PP1) family. Similar localization of LAP1 and PP12, a testis-specific PP1 isoform, suggests a shared function for both proteins during spermiogenesis. Furthermore, this study suggests an involvement of LAP1 in manchette development and chromatin regulation possibly via interaction with acetylated-tubulin and lamins, respectively. Taken together, the present results indicate that, by moving to the posterior pole in spermatids, LAP1 can contribute to the achievement of non-random, sperm-specific chromatin distribution, as well as modulate cellular remodeling during spermiogenesis. In addition, LAP1 seems to be associated with dynamic microtubule changes related to manchette formation and flagella development.Institute for Biomedicine (Project UID/BIM/04501/2013), the Fundação para a Ciência e Tecnologia of the Ministério da Educação e Ciência (Project PTDC/BEX-BCM/0493/2012), the COMPETE program (Programa Operacional Factores de Competitividade), the QREN (Quadro de Referência Estratégica Nacional) and the European Union (Fundo Europeu de Desenvolvimento Regional). Filipa Martins was the recipient of an FCT fellowship. The authors would also like to thank William Dauer (Columbia University, USA) for providing the anti-LAP1 antibodyinfo:eu-repo/semantics/publishedVersio

CD81 promotes a migratory phenotype in neuronal-like cells

Tetraspanins, such as CD81, can form lateral associations with each other and with other transmembrane proteins. These interactions may underlie CD81 functions in multiple cellular processes, such as adhesion, morphology, migration, and differentiation. Since CD81's role in neuronal cells' migration has not been established, we here evaluated effects of CD81 on the migratory phenotype of SH-SY5Y neuroblastoma cells. CD81 was found enriched at SH-SY5Y cell's membrane, co-localizing with its interactor filamentous-actin (F-actin) in migratory relevant structures of the leading edge (filopodia, stress fibers, and adhesion sites). CD81 overexpression increased the number of cells with a migratory phenotype, in a potentially phosphatidylinositol 3 kinase (PI3K)-Ak strain transforming (AKT) mediated manner. Indeed, CD81 also co-localized with AKT, a CD81-interactor and actin remodeling agent, at the inner leaflet of the plasma membrane. Pharmacologic inhibition of PI3K, the canonical AKT activator, led both to a decrease in the acquisition of a migratory phenotype and to a redistribution of intracellular CD81 and F-actin into cytoplasmic agglomerates. These findings suggest that in neuronal-like cells CD81 bridges active AKT and actin, promoting the actin remodeling that leads to a motile cell morphology. Further studies on this CD81-mediated mechanism will improve our knowledge on important physiological and pathological processes such as cell migration and differentiation, and tumor metastasis.This work was supported by Fundação para a Ciência e Tecnologia (Portuguese Ministry of Science and Technology), Centro 2020 and Portugal2020, the COMPETE program, QREN, and the European Union (FEDER program) via the Institute for Biomedicine iBiMED UID/BIM/ 04501/2013, fellowship SFRH/BD/90996/2012, project PTDC/CVT-CVT/ 32261/2017, and the support of the LiM facility of iBiMED, a member of the Portuguese Platform of BioImaging (PPBI- POCI-01-0145-FEDER-022122).publishe

Identification and characterization of the BRI2 interactome in the brain

BRI family proteins are ubiquitous type II transmembrane proteins but BRI2 is highly expressed in some neuronal tissues. Possible BRI2 functions include neuronal maturation and differentiation. Protein complexes appear to be important in mediating its functions. Previously described BRI2 interactors include the Alzheimer's amyloid precursor protein and protein phosphatase 1, but clearly the identification of novel interactors provides an important tool to understand the role and function of BRI2. To this end three rat brain regions (cerebellum, hippocampus, and cerebral cortex) were processed by BRI2 immunoprecipitation;co-precipitating proteins were identified by Nano-HPLC-MS/MS. The pool of the brain regions resulted in 511 BRI2 interacting proteins (BRI2 brain interactome) of which 120 were brain specific and 49 involved in neuronal differentiation. Brain region-specific analyses were also carried out for cerebellum, hippocampus, and cerebral cortex. Several novel BRI2 interactors were identified among them DLG4/PSD-95, which is singularly important as it places BRI2 in the postsynaptic compartment. This interaction was validated as well as the interaction with GAP-43 and synaptophysin. In essence, the resulting BRI2 brain interactome, associates this protein with neurite outgrowth and neuronal differentiation, as well as synaptic signalling and plasticity. It follows that further studies should address BRI2 particularly given its relevance to neuropathological conditions

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

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

Exosomal aβ-binding proteins identified by “in silico” analysis represent putative blood-derived biomarker candidates for alzheimer´s disease

The potential of exosomes as biomarker resources for diagnostics and even for therapeutics has intensified research in the field, including in the context of Alzheimer´s disease (AD). The search for disease biomarkers in peripheral biofluids is advancing mainly due to the easy access it offers. In the study presented here, emphasis was given to the bioinformatic identification of putative exosomal candidates for AD. The exosomal proteomes of cerebrospinal fluid (CSF), serum and plasma, were obtained from three databases (ExoCarta, EVpedia and Vesiclepedia), and complemented with additional exosomal proteins already associated with AD but not found in the databases. The final biofluids’ proteomes were submitted to gene ontology (GO) enrichment analysis and the exosomal Aβ-binding proteins that can constitute putative candidates were identified. Among these candidates, gelsolin, a protein known to be involved in inhibiting Abeta fibril formation, was identified, and it was tested in human samples. The levels of this Aβ-binding protein, with anti-amyloidogenic properties, were assessed in serum-derived exosomes isolated from controls and individuals with dementia, including AD cases, and revealed altered expression patterns. Identification of potential peripheral biomarker candidates for AD may be useful, not only for early disease diagnosis but also in drug trials and to monitor disease progression, allowing for a timely therapeutic intervention, which will positively impact the patient’s quality of life.info:eu-repo/semantics/publishedVersio