Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Dual modulation of cell survival and cell death by beta(2)-adrenergic signaling in adult mouse cardiac myocytes.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

Spatiotemporal Dynamics of CaMKI During Structural Plasticity of Single Dendritic Spines

Multifunctional calcium/calmodulin dependent protein kinases (CaMKs) are key regulators of spine structural plasticity and long-term potentiation (LTP) in neurons. CaMKs have promiscuous and overlapping substrate recognition motifs, and are distinguished in their regulatory role based on differences in the spatiotemporal dynamics of activity. While the function and activity of CaMKII in synaptic plasticity has been extensively studied, that of CaMKI, another major class of CaMK required for LTP, still remain elusive.

Here, we develop a Förster’s Resonance Energy Transfer (FRET) based sensor to measure the spatiotemporal activity dynamics of CaMK1. We monitored CaMKI activity using 2-photon fluorescence lifetime imaging, while inducing LTP in single dendritic spines of rat (Rattus Norvegicus, strain Sprague Dawley) hippocampal CA1 pyramidal neurons using 2-photon glutamate uncaging. Using RNA-interference and pharmacological means, we also characterize the role of CaMKI during spine structural plasticity.

We found that CaMKI was rapidly and transiently activated with a rise time of ~0.3 s and decay time of ~1 s in response to each uncaging pulse. Activity of CaMKI spread out of the spine. Phosphorylation of CaMKI by CaMKK was required for this spreading and for the initial phase of structural LTP. Combined with previous data showing that CaMKII is restricted to the stimulated spine and required for long-term maintenance of structural LTP, these results suggest that CaMK diversity allows the same incoming signal – calcium – to independently regulate distinct phases of LTP by activating different CaMKs with distinct spatiotemporal dynamics.

Interaction between Candida albicans and Pseudomonas aeruginosa

Fungal pathogen Candida albicans causes serious nosocomial infections in patients, in part, due to formation of drug-resistant biofilms. Protein kinases (PK) and transcription factors (TF) mediate signal transduction and transcription of proteins involved in biofilm development. To discover biofilm-related PKs, a collection of 63 C. albicans PK mutants was screened twice independently with microtiter plate-based biofilm assay (XTT). Thirty-eight (60%) mutants showed different degrees of biofilm impairment with the poor biofilm formers additionally possessing filamentation defects. Most of these genes were already known to encode proteins associated with Candida morphology and biofilms but VPS15, PKH3, PGA43, IME2 and CEX1, were firstly associated with both processes in this study. Previous studies of Holcombe et al. (2010) had shown that bacterial pathogen, Pseudomonas aeruginosa can impair C. albicans filamentation and biofilm development. To investigate their interaction, the good biofilm former PK mutants of C. albicans were assessed for their response to P. aeruginosa supernatants derived from two strains, wildtype PAO1 and homoserine lactone (HSL)-free mutant ΔQS, without finding any nonresponsive mutants. This suggested that none of the PKs in this study was implicated in Candida-Pseudomonas signaling. To screen promoter sequences for overrepresented TFs across C. albicans gene sets significantly up/downregulated in presence of bacterial supernatants from Holcombe et al. (2010) study, TFbsST database was created online. The TFbsST database integrates experimentally verified TFs of Candida to analyse promoter sequences for TF binding sites. In silico studies predicted that Efg1p was overrepresented in C. albicans and C. parapsilosis RBT family genes.

Structural and Functional Characterization of the Human Tribbles Homologue 2 Pseudokinase

The Tribbles Homologues are a family of three eukaryotic pseudokinases (Trb1, Trb2, Trb3) that act as allosteric inhibitors and regulatory scaffold sites in pathways governing adipogenesis, cell proliferation and insulin signaling. The Tribbles Homologues have the same overall tertiary structure of the eukaryotic protein kinase domain, but lack multiple residues necessary to catalysis in the nucleotide-binding P-loop and the Mg2+-coordinating DFG motif. Trb1 has been shown conclusively to be incapable of binding ATP, whereas a recent study presents evidence that Trb2 autophosphorylates independently of Mg2+ in vitro. This finding is surprising given the high degree of sequence similarity between the two proteins (71%), and suggests unique nucleotide binding and phosphotransfer mechanisms. The goal of this project was to investigate whether Trb2 possesses kinase activity or not and determine its structural basis. A method for the high-yield recombinant expression and purification of stable Trb2 was developed. Trb2 nucleotide binding and autophosphorylation could not be detected across multiple experimental approaches, including thermal shift assays, MANT-ATP fluorescence, radiolabeled phosphate incorporation, and nonspecific ATPase activity assays. Further characterization also revealed that Trb2 forms homomultimers with possible functional consequences, and extensive crystallization screening has yielded multiple promising conditions that could produce diffraction-quality crystals with further optimization. This project explores the difficulties in functionally characterizing putatively active pseudokinases, and proposes a structural basis for the conserved pseudokinase features of the Tribbles homologues.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Tumor-related splicing variant RAC1b in colorectal cancer: regulation and role in tumorigenesis

Purpose: Alternative splicing of the small GTPase RAC1 generates RAC1b, a hyperactivated variant that is overexpressed in a subtype of colorectal tumors. The objective of our studies is to understand the molecular regulation of this alternative splicing event and how it contributes to tumorigenesis. Experimental description: The regulation of the RAC1b splicing event in human colon cell lines was dissected using a transfected RAC1 minigene and the role of upstream regulating protein kinases through an RNA interference approach. The functional properties of the RAC1b protein were characterized by experimental modulation of Rac1b levels in colon cell lines. Results: The RAC1b protein results from an in-frame inclusion of an additional alternative exon encoding 19 amino acids that change the regulation and signaling properties of the protein. RAC1b is a hyperactive variant that exists predominantly in the GTP-bound active conformation in vivo and promotes cell cycle progression and cell survival through activation of the transcription factor NF-κB. RAC1b overexpression functionally cooperates with the oncogenic mutation in BRAF-V600E to sustain colorectal tumor cell survival. The splicing factor SRSF1 was identified to bind an exonic splice enhancer element in the alternative exon and acts as a prime regulator of Rac1b alternative splicing in colorectal cells. SRSF1 is controlled by upstream protein kinase SRPK1, the inhibition or depletion of which led to reduced SRSF1 phosphorylation and nuclear translocation with a concomitant reduction in RAC1b levels. As further SRSF1-regulating pathways we discovered kinase GSK3 and a cyclooxygenase independent effect of the non-steroidal anti-inflammatory drug ibuprofen. Conclusions: Expression of tumor-related RAC1b in colorectal cancer depends critically on SRSF1 for the observed deregulation of alternative splicing during tumorigenesis and is controlled by upstream protein kinases that can be pharmacologically targeted.

Transcription of TIR1-Controlled Genes Can be Regulated within 10 Min by an Auxin-Induced Process. Can TIR1 be the Receptor?

ABP1 and TIR1/AFBs are known as auxin receptors. ABP1 is linked to auxin responses several of which are faster than 10 min. TIR1 regulates auxin-induced transcription of early auxin genes also within minutes. We use transcription of such TIR1-dependent genes as indicator of TIR1 activity to show the rapid regulation of TIR1 by exogenous auxin. To this end, we used quantification of transcription of a set of fifteen early auxin-induced reporter genes at t = 10 and t = 30 min to measure this as a TIR1-dependent auxin response. We conducted this study in 22 mutants of auxin transporters (pin5, abcb1, abcb19, and aux1/lax3), protein kinases and phosphatases (ibr5, npr1, cpk3, CPK3-OX, d6pk1, d6pkl1-1, d6pkl3-2, d6pkl1-1/d6pkl2-2, and d6pkl1-1/d6pkl3-2), of fatty acid metabolism (fad2-1, fad6-1, ssi2, lacs4, lacs9, and lacs4/lacs9) and receptors (tir1, tir1/afb2, and tir1/afb3) and compared them to the wild type. After 10 min auxin application, in 18 out of 22 mutants mis-regulated expression of at least one reporter was found, and in 15 mutants transcription of two-to-three out of five selected auxin reporter genes was mis-regulated. After 30 min of auxin application to mutant plants, mis-regulation of reporter genes ranged from one to 13 out of 15 tested reporter genes. Those genes chosen as mutants were themselves not regulated in their expression by auxin for at least 1 h, excluding an influence of TIR1/AFBs on their transcription. The expression of TIR1/AFB genes was also not modulated by auxin for up to 3 h. Together, this excludes a feedback or feedforward of these mutant genes/proteins on TIR1/AFBs output of transcription in this auxin-induced response. However, an auxin-induced response needed an as yet unknown auxin receptor. We suggest that the auxin receptor necessary for the fast auxin-induced transcription modulation could be, instead, ABP1. The alternative hypothesis would be that auxin-induced expression of a protein, initiated by TIR1/AFBs receptors, could initiate these responses and that this unknown protein regulated TIR1/AFB activities within 10 min.

The Kinome of Pacific Oyster Crassostrea gigas, Its Expression during Development and in Response to Environmental Factors

Oysters play an important role in estuarine and coastal marine habitats, where the majority of humans live. In these ecosystems, environmental degradation is substantial, and oysters must cope with highly dynamic and stressful environmental constraints during their lives in the intertidal zone. The availability of the genome sequence of the Pacific oyster Crassostrea gigas represents a unique opportunity for a comprehensive assessment of the signal transduction pathways that the species has developed to deal with this unique habitat. We performed an in silico analysis to identify, annotate and classify protein kinases in C. gigas, according to their kinase domain taxonomy classification, and compared with kinome already described in other animal species. The C. gigas kinome consists of 371 protein kinases, making it closely related to the sea urchin kinome, which has 353 protein kinases. The absence of gene redundancy in some groups of the C. gigas kinome may simplify functional studies of protein kinases. Through data mining of transcriptomes in C. gigas, we identified part of the kinome which may be central during development and may play a role in response to various environmental factors. Overall, this work contributes to a better understanding of key sensing pathways that may be central for adaptation to a highly dynamic marine environment.

Investigação do papel modulador do Haloperidol sobre os níveis de proteínas relacionadas à plasticidade e preferência por objetos novos em camudongos

Dopamine (DA) is known to regulate both sleep and memory formations, while sleep plays a critical role in the consolidation of different types of memories. We believe that pharmacological manipulation of dopaminergic pathways might disrupt the sleep-wake cycle, leading to mnemonic deficits, which can be observed in both behavioral and molecular levels. Therefore, here we investigated how systemic injections of haloperidol (0.3 mg/kg), immediately after training in dark and light periods, affects learning assessed in the novel object preference test (NOPT) in mice. We also investigated the hippocampal levels of the plasticity-related proteins Zif-268, brain-derived neurotrophic factor (BDNF) and phosphorylated Ca2+/calmodulin-dependent protein kinases II (CaMKII-P) in non-exposed (naïve), vehicle-injected controls and haloperidol-treated mice at 3, 6 and 12 hours after training in the light period. Haloperidol administration during the light period led to a subsequent impairment in the NOPT. In contrast, preference was not observed during the dark period neither in mice injected with haloperidol, nor in vehicle-injected animals. A partial increase of CaMKII-P in the hippocampal field CA3 of vehicle-injected mice was detected at 3h. Haloperidol-treated mice showed a significant decrease in the dentate gyrus of CaMKII-P levels at 3, 6 and 12h; of Zif-268 levels at 6h, and of BDNF levels at 12h after training. Since the mnemonic effects of haloperidol were only observed in the light period when animals tend to sleep, we suggest that these effects are related to REM sleep disruption after haloperidol injection

Design and Synthesis of Novel Kinase Inhibitors for the Treatment of Chronic Respiratory Diseases

In 2017, Chronic Respiratory Diseases accounted for almost four million deaths worldwide. Unfortunately, current treatments are not definitive for such diseases. This unmet medical need forces the scientific community to increase efforts in the identification of new therapeutic solutions. PI3K delta plays a key role in mechanisms that promote airway chronic inflammation underlying Asthma and COPD. The first part of this project was dedicated to the identification of novel PI3K delta inhibitors. A first SAR expansion of a Hit, previously identified by a HTS campaign, was carried out. A library of 43 analogues was synthesised taking advantage of an efficient synthetic approach. This allowed the identification of an improved Hit of nanomolar enzymatic potency and moderate selectivity for PI3K delta over other PI3K isoforms. However, this compound exhibited low potency in cell-based assays. Low cellular potency was related to sub optimal phys-chem and ADME properties. The analysis of the X-ray crystal structure of this compound in human PI3K delta guided a second tailored SAR expansion that led to improved cellular potency and solubility. The second part of the thesis was focused on the rational design and synthesis of new macrocyclic Rho-associated protein kinases (ROCKs) inhibitors. Inhibition of these kinases has been associated with vasodilating effects. Therefore, ROCKs could represent attractive targets for the treatment of pulmonary arterial hypertension (PAH). Known ROCK inhibitors suffer from low selectivity across the kinome. The design of macrocyclic inhibitors was considered a promising strategy to obtain improved selectivity. Known inhibitors from literature were evaluated for opportunities of macrocyclization using a knowledge-based approach supported by Computer Aided Drug Design (CADD). The identification of a macrocyclic ROCK inhibitor with enzymatic activity in the low micro molar range against ROCK II represented a promising result that validated this innovative approach in the design of new ROCKs inhibitors.

Protein tyrosine kinases in Schistosoma mansoni

The identification and description of signal transduction molecules and mechanisms are essential to elucidate Schistosoma mansoni host-parasite interactions and parasite biology. This mini review focuses on recent advancements in the study of signalling molecules and transduction mechanisms in S. mansoni, drawing special attention to the recently identified and characterised protein tyrosine kinases of S. mansoni.

Phosphorylation of phosphatidylinositol-3-kinase-protein kinase B and extracellular signal-regulated kinases 1/2 mediate reoxygenation-induced cardioprotection during hypoxia.

In vivo exposure to chronic hypoxia (CH) depresses myocardial performance and tolerance to ischemia, but daily reoxyenation during CH (CHR) confers cardioprotection. To elucidate the underlying mechanism, we tested the role of phosphatidylinositol-3-kinase-protein kinase B (Akt) and p42/p44 extracellular signal-regulated kinases (ERK1/2), which are known to be associated with protection against ischemia/reperfusion (I/R). Male Sprague-Dawley rats were maintained for two weeks under CH (10% O(2)) or CHR (as CH but with one-hour daily exposure to room air). Then, hearts were either frozen for biochemical analyses or Langendorff-perfused to determine performance (intraventricular balloon) and tolerance to 30-min global ischemia and 45-min reperfusion, assessed as recovery of performance after I/R and infarct size (tetrazolium staining). Additional hearts were perfused in the presence of 15 micromol/L LY-294002 (inhibitor of Akt), 10 micromol/L UO-126 (inhibitor of ERK1/2) or 10 micromol/L PD-98059 (less-specific inhibitor of ERK1/2) given 15 min before ischemia and throughout the first 20 min of reperfusion. Whereas total Akt and ERK1/2 were unaffected by CH and CHR in vivo, in CHR hearts the phosphorylation of both proteins was higher than in CH hearts. This was accompanied by better performance after I/R (heart rate x developed pressure), lower end-diastolic pressure and reduced infarct size. Whereas the treatment with LY-294002 decreased the phosphorylation of Akt only, the treatment with UO-126 decreased ERK1/2, and that with PD-98059 decreased both Akt and ERK1/2. In all cases, the cardioprotective effect led by CHR was lost. In conclusion, in vivo daily reoxygenation during CH enhances Akt and ERK1/2 signaling. This response was accompanied by a complex phenotype consisting in improved resistance to stress, better myocardial performance and lower infarct size after I/R. Selective inhibition of Akt and ERK1/2 phosphorylation abolishes the beneficial effects of the reoxygenation. Therefore, Akt and ERK1/2 have an important role to mediate cardioprotection by reoxygenation during CH in vivo.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.