Sara C. Domingues

Identification of the human testis protein phosphatase 1 interactome

Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins-PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

S655 phosphorylation enhances APP secretory traffic.

Cellular protein phosphorylation regulates proteolytic processing of the Alzheimer’s Amyloid Precursor Protein (APP). This appears to occur both indirectly and directly via APP phosphorylation at residues within cytoplasmic motifs related to targeting and protein–protein interactions. The sorting signal 653YTSI656 comprises the S655 residue that can be phosphorylated by PKC, particularly in mature APP molecules. The YTSI domain has been associated with APP internalization and Golgi polarized sorting, but no functional significance has been attributed to S655 phosphorylation thus far. Using APP695-GFP S655 phosphomutants we show that S655 phosphorylation is a signal that positively modulates APP secretory traffic. The phosphomimicking and dephosphomimicking S655 mutants exhibited contrasting Golgi dynamics, which correlated with differential Golgi vesicular exit and secretory cleavage to sAPP. The role of S655 phosphorylation in APP trafficking at sorting stations, such as the Golgi, its contribution toward cytoprotective alpha sAPP production, and implications for Alzheimer’s disease are discussed.

Protein Phosphatase 1α Interacting Proteins in the Human Brain

Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved.

Protein Phosphatase 1γ Isoforms Linked Interactions in the Brain

Posttranslational protein modifications, in particular reversible protein phosphorylation, are important regulatory mechanisms involved in cellular signaling transduction pathways. Thousands of human proteins are phosphorylatable and the tight regulation of phosphorylation states is crucial for cell maintenance and development. Protein phosphorylation occurs primarily on serine, threonine, and tyrosine residues, through the antagonistic actions of protein kinases and phosphatases. The catalytic subunit of protein phosphatase 1 (PP1), a major Ser/Thr-phosphatase, associates with a large variety of regulatory subunits that define substrate specificity and determine specific cellular pathway responses. PP1 has been shown to bind to different proteins in the brain in order to execute key and differential functions. This work reports the identification of proteins expressed in the human brain that interact with PP1γ1 and PP1γ2 isoforms by the yeast two-hybrid method. An extensive search of PP1-binding motifs was performed for the proteins identified, revealing already known PP1 regulators but also novel interactors. Moreover, our results were integrated with the data of PP1γ interacting proteins from several public web databases, permitting the development of physical maps of the novel interactions. The PP1γ interactome thus obtained allowed for the identification of novel PP1 interacting proteins, supporting novel functions of PP1γ isoforms in the human brain.

Isoform Specific Amyloid- β Protein Precursor Metabolism

Alzheimers amyloid-beta protein precursor (AbetaPP) can occur in different isoforms, among them AbetaPP(751), which is the most abundant isoform in non-neuronal tissues, and AbetaPP(695), often referred to as the neuronal isoform. However, few isoform-specific roles have been addressed. In the work here described, AbetaPP isoforms, both endogenous and as cDNA fusions with green fluorescent protein (GFP), were used to permit isoform-specific monitoring in terms of intracellular processing and targeting. Differences were particularly marked in the turnover rates of the immature isoforms, with AbetaPP(751) having a faster turnover rate than AbetaPP(695) (0.8 h and 1.2 h respectively for endogenous proteins and 1.1 h and 2.3 h for transfected proteins). Hence, AbetaPP(751) matures faster. Additionally, AbetaPP(751) responded to both okadaic acid (OA) and phorbol 12-myristate 13-acetate (PMA), as determined by sAbetaPP production, with PMA inducing a more robust response. For the AbetaPP(695) isoform, however, although PMA produced a strong response, OA failed to elicit such an induction in sAbetaPP production, implicating isoform specificity in phosphorylation regulated events. In conclusion, it seems that the AbetaPP(695) isoform is processed/metabolized at a slower rate and responds differently to OA when compared to the AbetaPP(751) isoform. The relevance of isoform-specific processing in relation to Alzheimers disease needs to be further investigated, given the predominance of the AbetaPP(695) isoform in neuronal tissues and isoform-specific alterations in expression levels associated with the pathology.

Altered Subcellular Distribution of the Alzheimer's Amyloid Precursor Protein Under Stress Conditions

Abstract:  Altered metabolism of the Alzheimers amyloid precursor protein (APP) appears to be a key event in the pathogenesis of Alzheimers disease (AD), and both altered phosphorylation and oxidative stress appear to affect the production of the toxic Abeta fragment. Our results show that altered processing of APP was observed under conditions of stress induced by sodium azide in the presence of 2‐deoxy‐d‐glucose (2DG). As previously reported, the production of the secreted fragment of APP (sAPP) was inhibited. Using APP‐GFP fusion proteins, we show that 2DG causes the accumulation/delay of APP in the endoplasmic reticulum (ER)/Golgi (G). The 751 isoform accumulated preferentially in the G, whereas the 695 isoform was blocked preferentially at the ER. This effect was augmented in the presence of sodium azide. APP subcellular distribution was also affected at the plasma membrane. The involvement of protein phosphorylation in APP subcellular localization was reinforced by the effect of drugs, such as phorbol 12‐myristate 13‐acetate (PMA), since APP was completely depleted from the membrane in the presence of 2DG and PMA. Thus, the hypothesis that APP is processed in a phosphorylation‐dependent manner and that this may be of clinical relevance appears to hold true even under stress conditions. Our results provide evidence for a role of protein phosphorylation in APP sorting under stress conditions and contribute to the understanding of the molecular basis of AD.

Identification of a novel human LAP1 isoform that is regulated by protein phosphorylation.

Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation.

Wnt signalling is a relevant pathway contributing to amyloid beta- peptide-mediated neuropathology in Alzheimer's disease.

One of the most important contributions to our understanding of neurodegenerative diseases in the last decade has been the demonstration that several disorders have a common biochemical cause, involving aggregation and deposition of abnormal proteins. Abnormal protein deposition leads to neuronal degeneration with consequences to impaired brain function. Protein deposition can be extracellular (beta-amyloid peptide (A beta), prion protein) or intracellular (Tau, alpha-synuclein, huntingtin). Individuals with Alzheimers disease (AD) exhibit extracellular senile plaques (SPs) of aggregated A beta and intracellular neurofibrillary tangles that contain hyperphosphorylated Tau protein (NFTs), and also an extensive loss in basal forebrain cholinergic neurons that innervate the hippocampus and neocortex. The SPs and NFTs contribute to neurodegeneration, although the mechanisms inducing basal forebrain cholinergic cell loss and cognitive impairment remain unclear. Furthermore, the pathophysiological relationship between NFTs and SPs remains undefined, and controversy still rages over which of the two hallmark pathologies of AD is the primary cause of neurodegeneration in the brain. However, consensus is beginning to develop that the two pathologies are not separate processes, and the Wnt signalling pathway may provide a pathological link between both. In fact, work in transgenic mice showed that A beta or the amyloid precursor protein can influence the formation of Tau tangles in areas of the brain known to be affected in AD. Furthermore, A beta can contribute to synaptic dysfunction. Thus, A beta appears to be a recurring player affecting protein phosphorylation, signal transduction mechanisms, cytoskeletal organization, multiprotein complex formation, synaptotoxicity and ultimately culminating in protein aggregation. Consequently this peptide and the downstream signalling cascades are presently considered as potential therapeutic targets.

Sodium azide and 2-deoxy-D-glucose-induced cellular stress affects phosphorylation-dependent AβPP processing

Production of the amyloid beta (Abeta) peptide via altered metabolism of the amyloid beta-Protein Precursor (AbetaPP) appears to be a key event in the pathology of Alzheimer Disease (AD). Accordingly, altered processing of AbetaPP was observed under conditions of abnormal cellular stress induced by sodium azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of sAbetaPP (the secreted fragment of AbetaPP) was inhibited. However, our data further suggests that 2DG alone can account for most of the observed effects on AbetaPP processing in COS-1 cells and PC12 cells. It appears that 2DG interferes with the normal glycosylation of AbetaPP and its maturation process, having direct consequences on sAbetaPP production. Interestingly, PMA (phorbol 12-myristate 13-acetate)-induced sAbetaPP production was maintained under the stress conditions used, suggesting that potential non-amyloidogenic AbetaPP processing can still be favoured. This is of potential therapeutic interest, since it indicates that even under adverse stress conditions drugs such as PMA can affect AbetaPP processing, leading to increased sAbetaPP production and a concomitant reduction in Abeta production. However, the induction of sAbetaPP production was not identical when the phosphatase inhibitor OA (okadaic acid) was used. In fact, the typical OA-induced increase in sAbetaPP production could be abolished under specific conditions. This constitutes an interesting precedent for the possible dissociation of the PMA and OA responses in terms of sAbetaPP production. The involvement of protein phosphatases, which are inhibited by OA, inbetaPP processing, was reinforced by the increased co-localization of AbetaPP and PP1alpha (protein phosphatase 1alpha) at the cell surface upon exposure to OA and PMA. Overall, our results support the notion that signal transduction processes may be of particular relevance for our understanding of the molecular basis of AD, and for the design of rational signal transduction therapeutics.

RanBP9 Modulates AICD Localization and Transcriptional Activity via Direct Interaction with Tip60

Proteolytic processing of the amyloid-β protein precursor (AβPP) occurs via alternative pathways, culminating with the production of the AβPP intracellular domain (AICD). AICD can translocate to the nucleus and regulate transcription, but its activity is modulated by interactions with other proteins. In the nucleus, AICD, FE65, and Tip60 associate into AFT complexes, which are targeted to nuclear spots which correspond to transcription factories. Here we report that RanBP9 interacts with the cytoplasmic domain of AβPP, through the NPXY internalization motif. Moreover, RanBP9 interaction with Tip60 is also described. The RanBP9-Tip60 interaction dramatically relocated RanBP9 from a widespread cellular distribution to nuclear speckles. AβPP processing is a central aspect in determining the proteins function and that of its resulting proteolytic fragments, among them AICD. The latter results from the amyloidogenic pathway and is the peptidic species predominantly involved in nuclear signaling. Of note RanBP9 transfection was previously demonstrated to increase amyloid-β generation. Here we show that RanBP9 relocates AICD to the Tip60-enriched nuclear speckles, and prevented the formation of nuclear spots formation, having therefore a negative effect on AICD mediated nuclear signaling and consequently AFT complex formation. Furthermore, by transfecting cells with increasing amounts of RanBP9, the expression of AICD-regulated genes, including AβPP itself, was reduced. Given the data presented, one can deduce that RanBP9 has an inhibitory regulatory effect on AICD-mediated transcription and the effect is mediated by relocating AICD away from transcription factories.