Suppression of the intrinsic apoptosis pathway by sinaptic activity

Synaptic activity promotes resistance to diverse apoptotic insults, the mechanism behind which is incompletely understood. We show here that a coordinated downregulation of core components of the intrinsic apoptosis pathway by neuronal activity forms a key part of the underlying mechanism. Activity-dependent protection against apoptotic insults is associated with inhibition of cytochrome c release in most but not all neurons, indicative of anti-apoptotic signaling both upstream and downstream of this step. We find that enhanced firing activity suppresses expression of the proapoptotic BH3-only member gene Puma in a NMDA receptor-dependent, p53-independent manner. Puma expression is sufficient to induce cytochrome c loss and neuronal apoptosis. Puma deficiency protects neurons against apoptosis and also occludes the protective effect of synaptic activity, while blockade of physiological NMDA receptor activity in the developing mouse brain induces neuronal apoptosis that is preceded by upregulation of Puma. However, enhanced activity can also confer resistance to Puma-induced apoptosis, acting downstream of cytochrome c release. This mechanism is mediated by transcriptional suppression of apoptosome components Apaf-1 and procaspase-9, and limiting caspase-9 activity, since overexpression of procaspase-9 accelerates the rate of apoptosis in active neurons back to control levels. Synaptic activity does not exert further significant anti-apoptotic effects downstream of caspase-9 activation, since an inducible form of caspase-9 overrides the protective effect of synaptic activity, despite activity-induced transcriptional suppression of caspase-3. Thus, suppression of apoptotic gene expression may synergize with other activity-dependent events such as enhancement of antioxidant defenses to promote neuronal survival

Dying a Thousand Deaths: Redundant Pathways From Different Organelles to Apoptosis and Necrosis

Cell death is an essential event in normal life and development, as well as in the pathophysiological processes that lead to disease. Although the literature on cell death has grown enormously in size and complexity, a pattern has emerged that each of several distinct organelles (plasma membrane, mitochondrion, nucleus, endoplasmic reticulum, lysosome) gives rise to signals that induce cell death. Most often these signals converge on mitochondria to initiate a common pathway to either caspase-dependent apoptosis or ATP depletion-dependent necrosis. This brief overview emphasizes the multiple and often redundant pathways between different organelles that lead ultimately to a cell's demise

CaDrA: A Computational Framework for Performing Candidate Driver Analyses Using Genomic Features

The identification of genetic alteration combinations as drivers of a given phenotypic outcome, such as drug sensitivity, gene or protein expression, and pathway activity, is a challenging task that is essential to gaining new biological insights and to discovering therapeutic targets. Existing methods designed to predict complementary drivers of such outcomes lack analytical flexibility, including the support for joint analyses of multiple genomic alteration types, such as somatic mutations and copy number alterations, multiple scoring functions, and rigorous significance and reproducibility testing procedures. To address these limitations, we developed Candidate Driver Analysis or CaDrA, an integrative framework that implements a step-wise heuristic search approach to identify functionally relevant subsets of genomic features that, together, are maximally associated with a specific outcome of interest. We show CaDrA’s overall high sensitivity and specificity for typically sized multi-omic datasets using simulated data, and demonstrate CaDrA’s ability to identify known mutations linked with sensitivity of cancer cells to drug treatment using data from the Cancer Cell Line Encyclopedia (CCLE). We further apply CaDrA to identify novel regulators of oncogenic activity mediated by Hippo signaling pathway effectors YAP and TAZ in primary breast cancer tumors using data from The Cancer Genome Atlas (TCGA), which we functionally validate in vitro. Finally, we use pan-cancer TCGA protein expression data to show the high reproducibility of CaDrA’s search procedure. Collectively, this work demonstrates the utility of our framework for supporting the fast querying of large, publicly available multi-omics datasets, including but not limited to TCGA and CCLE, for potential drivers of a given target profile of interest

The Antitumor Agent, Arglabin-DMA, Preferentially Induces Apoptosis in Human Colon Tumor Cells

Arglabin-DMA, an analog of farnesyl pyrophosphate (FPP), reportedly inhibits farnesyltransferase (FTase) directly by competitively blocking the binding of Ras protein and its posttranslational modification, as suggested in previous studies. But, the mechanisms by which Arglabin-DMA inhibits tumor growth in vivo and in vitro are still relatively poorly characterized. To determine the mechanism by which this drug inhibits tumor growth, the effects of Arglabin-DMA in two human colon tumor cell lines (mutant K-ras HCT 116 and wild-type ras HT-29) were explored on cell proliferation, apoptosis, and cell cycle kinetics in vitro. In cell viability studies, we showed that Arglabin-DMA had striking morphological and physiological effects on the two human colon tumor cell lines, possibly more so than those of other anticancer drugs. Also, Arglabin-DMA exhibited less harm to normal cells (Hs27) which retained their potential for cell growth. An add-back experiment showed that Arglabin-DMA had no effect on the isoprenoid biosynthetic pathway. The drug not only affects the mutant K-ras human colon tumor cell line, but also the wild-type ras human colon tumor cell line. It may therefore inhibit one or more non-Ras proteins to exert its antitumor effects. Gel electrophoresis, TUNEL assay, Annexin V assay, apoptosis dye-uptake assay, and morphological criteria were used to characterize apoptosis. Adherent cells and freely floating detached cells in Arglabin-DMA treatment were treated as two distinct populations We demonstrated that the detached cells caused by Arglabin-DMA exposure exhibited increased apoptosis in a p53-independent manner. Cell cycle effects were studied using flow cytometry. After Arglabin-DMA was added, the proportion of the two human colon tumor cells in G2/M phase increased, indicating a block in either G2 or M phase. We conclude that Arglabin-DMA has specific cytotoxic effects in two human colon tumor cell lines, and less cytotoxicity to normal cells. It induces arrest at the G2/M phase of the cell cycle. After treatment with Arglabin-DMA, rounded and detached cells enter apoptosis. This mechanism may be analogous to anoikis, which is the induction of apoptosis in response to loss of cell contact. The utility of this drug in combating cancer remains an attractive, though complex possibility

Multi-omic investigation of the mechanisms underlying the pathobiology of head and neck squamous cell carcinomas

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy associated with molecular heterogeneity, locoregional spread, resistance to therapy and relapse after initial treatment. Increasing evidence suggests that master developmental pathways with key roles in adult tissue homeostasis, including Hippo and Wnt/β-catenin signaling, are dysregulated in the initiation and progression of HNSCC. However, a comprehensive investigation into the crosstalk between these pathways is currently lacking, and may prove crucial to the discovery of novel targets for HNSCC therapy. More recent evidence points to the tumor microenvironment, mainly comprising cancer-associated fibroblasts (CAFs), as capable of influencing tumor cell behavior and promoting invasive HNSCC phenotypes. Nonetheless, current methods to screen for CAF markers in tumors are restricted to targeted immunostaining experiments with limited success and robustness across tissue types. The Cancer Genome Atlas network has generated multi-tiered molecular profiles for over 10,000 tumors spanning more than two dozen different cancer types, providing an unprecedented opportunity for the application and development of integrative methods aimed at the in silico interrogation of experimentally-derived signatures. These multi-omic profiles further enable one to link genomic anomalies, including somatic mutations and DNA copy number alterations, with phenotypic effects driven by pathogenic pathway activity. Effectively querying this vast amount of information to help elucidate subsets of functionally and clinically-relevant oncogenic drivers, however, remains an ongoing challenge. To address these issues, I first investigate the effects of oncogenic pathway perturbation in HNSCC using experimental models coupled with in vitro genome-wide transcriptional profiling. Next, I describe a new computational approach for the unbiased identification of CAF markers in HNSCC solely using bulk tumor RNA-sequencing information. Lastly, I have developed Candidate Driver Analysis or CaDrA - a statistical framework that allows one to query genetic and epigenetic alterations for candidate drivers of signature activity within a given disease context. Collectively, this work offers new perspectives on the molecular cues underlying HNSCC development, while simultaneously highlighting the power of integrative genomics methods capable of accelerating the discovery of novel targets for cancer diagnosis and therapy

Avaliação da ativação de mecanismos apoptóticos mediados pela via UPR (unfolded protein response) em células Jurkat estimulados com a proteína Tat do HIV-1

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2017.Indivíduos HIV-positivos geralmente apresentam uma elevada depleção de linfócitos T CD4. A morte de células infectadas ou não por HIV-1 é resultado, entre diversos outros fatores, da apoptose mediada por proteínas virais. Está apoptose pode ser ativada por mecanismos extrínsecos ou intrínsecos, respectivamente através de receptores celulares ou ativação de moléculas sinalizadoras de morte. Uma das vias celulares que regula a sobrevivência ou morte da célula é a via UPR (Unfolded Protein Response), que regula o estresse reticular causado pelo acúmulo de proteínas mal dobradas através do bloqueio da tradução proteica, aumento da expressão de chaperonas que auxiliam no dobramento e direcionam as proteínas mal dobradas para a via de degradação de proteínas associada ao retículo (ERAD). Quando o estresse reticular é prolongado, como no caso de infecções virais, a via UPR induz apoptose através da expressão da molécula pró-apoptótica CHOP. Neste trabalho, observou-se que após 72 horas de estímulo com 200 nM da proteína Tat houve um aumento significativo na taxa de apoptose das células Jurkat (12,05%, PAbstract : HIV-positive individuals usually have a high depletion of of CD4 lymphocytes. The death of cells infected or not by the HIV is a result, among many other factors, the apoptosis mediated by viral proteins. Extrinsic or intrinsic pathways, respectively through cell surface receptors or activation of death signaling molecules, can trigger apoptosis cell death. The Unfolded Protein Response (UPR) is one of the cellular pathways that regulate cell survival or cell death. UPR also regulates the Endoplasmic Reticulum (ER) stress caused by the accumulation of misfolded or unfolded protein, by blocking the cell protein translation, increased expression of chaperones that assist in protein folding and lead misfolded proteins for the degradation pathway associated with the ER. When the ER stress is prolonged, as in the case of viral infections, the UPR induces apoptosis through the expression of pro-apoptotic molecule CHOP. In the present work, it was observed that after 72 hours of stimulation with 200nM of Tat protein there was a significant increase in apoptosis rate in Jurkat cells (12,05%, p<0.0001), indicating that Tat protein most likely exerts a biologic effect which triggers the apoptosis pathway. Cells stimulated also showed significant changes in transcription profile of genes encoding proteins of the UPR pathway: PERK, ATF6, IRE1, BIP, eIF2a, XBP1-u, XBP1-s, CHOP; and the genes related to ER stress-mediated apoptosis: ATF4, CHOP, GADD34, BIM e BCL-2. The increase in CHOP transcription may indicate that the ER stress is involved in the mechanisms that induces apoptosis in Jurkat cells. Furthermore, Tat stimulation induces cell cycle arrest and loss of mitochondrial membrane potencial in Jurkat cells. Identification the role of Tat in ER stress-induced apoptosis may reinforce the suggestion of the UPR pathway as a possible therapeutic target

Identificación de moduladores del apoptosoma mediante química combinatoria.

RESUMEN La apoptosis es un proceso importante en una amplia variedad sistemas biológicos, incluyendo el recambio celular normal, el sistema inmunológico y el desarrollo embrionario. La apoptosis inadecuada está implicada en muchas enfermedades incluyendo enfermedades neurodegenerativas tales como las enfermedades de Alzheimer y Huntington, isquemia, desórdenes autoinmunes y varias formas de cáncer. La familia de proteínas Bcl-2 abarca una clase de estructuras homólogas que sirven para inhibir o para activar la apoptosis en un proceso intrincado y a su vez, bien orquestado. Los estímulos apoptósicos inducen la translocación de miembros pro-apoptósicos de la familia de Bcl-2 a la membrana mitochondrial externa donde forman canales iónicos que pueden contribuir a disipar el potencial transmembrana de la mitocondria y favorecer la liberación de citocromo c. En el citosol, esta proteína se une al factor activador de apoptosis (Apaf-1) para formar el complejo denominado apoptosoma que activa una familia de proteasas denominada caspasas. Las caspasas hidrolizan una serie de proteínas clave para la supervivencia celular y la célula muere de forma no necrótica. Estas moléculas moduladoras podrían ser consideradas como agentes "cabeza de serie" para el desarrollo de compuestos que puedan inhibir el crecimiento de células tumorales y/o paliar o aminorar los daños celulares asociados a las enfermedades neurodegenerativas En la tesis doctoral se utiliza la Química Combinatoria para la identificación de moléculas de interés biomédico moduladoras del apoptosoma. Para ello, en primer lugar es necesario poner a punto un ensayo de alto rendimiento que permita utilizar la diana terapéutica, el apoptosoma, y que permita el cribado funcional de un elevado número de moléculas con el objetivo de identificar efectores artificiales del sistema Apaf-1-caspasas. El trabajo presentado en esta tesis incorpora como novedad la utilización de los componentes básicos recombinantes purificados del apoptosoma y la reconstitución de su actividad in vitro para el cribado. Con este ensayo y formato de cribado, lo que se consigue una mejor definición de la diana molecular de búsqueda de moléculas moduladoras. Por otro lado, la posibilidad de manipulación de los distintos componentes del apoptosoma en las reacciones de reconstitución permite llevar a cabo la caracterización del mecanismo de acción de los compuestos actuvos identificados y su sitio de unión. Finalmente se llevo a cabo unestudio in vivo de los compuestos identificados como moduladores. __________________________________________________________________________________________________Protein-protein interactions represent points of chemical intervention for therapeutic gain in the biological processes associated with disease. Apoptosis is an interesting biological process because its importance in a wide variety of biological systems. Inappropriate apoptosis is involved in many human pathologies, including neurodegenerative diseases such as Alzheimer's and Huntington's, ischaemia, autoimmune disorders and several forms of cancer. Diverse apoptotic stimuli, including activation of cell surface death receptors, anticancer agents, irradiation, lack of survival factors, and ischemia induce signaling cascades that all activate a family of cysteine aspartyl proteases called caspases. It is these proteases that execute the apoptotic process. Effector caspases are responsible for the disassembly of cellular components while initiator caspases are responsible for activation of the effector caspases. Because of the critical consequences of apoptosis malfunctioning, the activation of caspases is scrupulously controlled. Some apoptotic signals activate the mitochondria-mediated or intrinsic pathway that utilizes caspase-9 as its initiator. Caspase-9 activation is triggered by the release to the cytosol of proapoptotic proteins from the mitochondrial inter-membrane space, in particular cytochrome c. The formation of the macromolecular complex named apoptosome is a key event in this pathway. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic protease-activating factor), dATP and procaspase-9. In this macromolecular complex apoptosome-associated caspase-9 is activated and then, in turn, activate effector caspases. To identify molecules that could ameliorate disease-associated apoptosis, drug discovery efforts have initially targeted caspase activity rather than activation. Nevertheless, protein-protein interactions upstream of caspase activation can be also relevant points of intervention for the development of modulators of apoptosis pathways. In particular, recent data propose the formation of the apoptosome as an interesting target for the development of apoptotic modulators. In the absence of detailed structural information, the conventional methods used for the identification of modulators of the apoptosome have been based in indirect measurements of the cytochrome c- and dATP-induced activation of caspase-3-like activity on defined cytosolic extracts. Using this methodology Lademann et al. have identified inhibitors of the apoptosome through the screening of small molecules using cytosolic extracts of selected cells. We have carried out a discovery program employing an in vitro reconstituted active apoptosome assembled from its recombinant constituent proteins. Here we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversity-oriented chemical library of N-alkylglycines. The active compounds rescued from the library were chemically optimized to obtain molecules that bind to both recombinant and human endogenous Apaf-1 and decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis

Σακχαρώδης διαβήτης κύησης και η επίδρασή του στο έμβρυο: ο ρόλος του οξειδωτικού στρες

Το οξυγόνο μπορεί να έχει επιβλαβή δράση σε παθολογικές καταστάσεις (όπως υποξία-επαναιμάτωση, υπεργλυκαιμία) μέσω υπερβολικής παραγωγής ελευθέρων ριζών οξυγόνου (ROS). Oι ROS είναι τοξικές για το κύτταρο γιατί προκαλούν των λιπών, των πρωτεϊνών και του DNA. Παραγωγή ROS που υπερβαίνει τους αντιοξειδωτικούς μηχανισμούς του κυττάρου οδηγεί σε οξειδωτικό στρες (OS). Το OS σε συνδυασμό με την υπεργλυκαιμία, οδηγούν σε προβληματική αναδίπλωση των πρωτεϊνών του κυττάρου, γνωστό ως στρες του ενδοπλασματικού δικτύου (ERS). OS, ERS, υπεργλυκαιμία και αντίσταση στην ινσουλίνη οδηγούν σε παθολογικό μεταβολισμό της γλυκόζης και ενεργοποίηση κυτταρικών σηματοδοτικών μονοπατιών που επάγουν τη φλεγμονή και την απόπτωση. Ο σακχαρώδης διαβήτης αποτελεί σοβαρό πρόβλημα δημόσιας υγείας στις δυτικές κοινωνίες και υπολογίζεται ότι επηρέασε περίπου το 14% του ελληνικού πληθυσμού το 2014. Φυσιολογικά ο πλακούντας προστατεύει το έμβρυο από το οξειδωτικό στρες, αλλά σε περιπτώσεις ΣΔ διαταράσσεται η εμφύτευση, η αγγειογένεση και μεταφορά ουσιών. ΣΔ στην αρχή της κύησης μπορεί να έχει οδηγήσει σε τερατογένεση μέσω υπερβολικής απόπτωσης και προβληματικής μετανάστευσης κυττάρων. Μονοπάτια που προάγουν την απόπτωση όπως ASK1, JNK, FOXO3,Bax/Bim διεγείρονται και αυτά που προάγουν την επιβίωση και τη μετανάστευση όπως PI3K, Akt, mTOR, MAPK-ERK καταστέλλονται. Μελέτες μεταβολομική και επιγενετικής έχουν δείξει αλλοιωμένα σηματοδοτικά μονοπάτια και διαφορική μεθυλίωση γονιδίων σε περιπτώσεις ΣΔΚ. Οι αλλαγές αυτές εξηγούν τη μεταβολική μνήμη που οδηγεί από την εμβρυική υπεργλυκαιμία στις νόσους της ενηλίκου ζωής των απογόνων. Περαιτέρω έρευνα είναι απαραίτητη ώστε να διαλευκανθούν οι μοριακές και κυτταρικές διαταραχές στο ΣΔΚ και να βρεθούν νεότερες θεραπείες και κλινικοί βιοχημικοί δείκτες.Oxygen can act as a deterrent in cases of cell pathology (ex hypoxia, hyperglycemia) by excess production of reactant oxygen species (ROS). ROS are toxic for the cell through lipid, protein and DNA peroxidation. Overproduction of ROS that overcomes the antioxidant mechanisms of the cell leads to oxidative stress OS. OS and hyperglycemia contribute to unfolded protein response, endoplasmic reticulum stress (ERS). OS, hyperglycemia, insulin resistance and ERS lead to alteration of glycose metabolism and activation of cellular signaling pathways (mainly protein and lipid kinase) that trigger inflammation and apoptosis. Diabetes mellitus is a major health burden in modern societies and is estimated to affect 14% of Greek population in 2014. Placenta protects the embryo from excess OS in cases of DM but has altered function caused by impaired implantation, angiogenesis/vasculogenesis and altered transfer of nutrients. Periconceptional diabetes mellitus can lead to dysmorphogenesis by excess apoptosis and impaired migration as a result of OS, ERS and inflammation. Signaling pathways such as ASK1, JNK, FOXO3,Bax/Bim that trigger apoptosis are upregulated, though prosurvival pathways, as PI3K, Akt, mTOR, MAPK-ERK are downregulated. Metabolomic and epigenetic studies have shown altered signaling pathways and diaphoric methylation in cases of GDM. These changes can explain the concept of metabolic memory leading from in uterus hyperglycemia to metabolic disease of adult life. Further research is needed to elucidate metabolic and cellular alteration that take place in DGM in order to find novel therapies and possible biomarkers

Physiological and pathological roles of Apaf1 and the apoptosome

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer

Physiological and pathological roles of Apaf1 and the apoptosome

Different cellular pathways can lead to apoptosis. Apaf1 is the molecular core of the apoptosome, a multiproteic complex mediating the so-called mitochondrial pathway of cell death. The importance of this pathway during development has been clearly demonstrated by knocking out key genes. Also, the relevance of Apaf1 dosage during development has been recently underlined. Moreover, a growing body of evidences seems to point out a possible role of the mitochondria-dependent apoptosis in different pathologies. In particular, we discuss here some recent evidences regarding the putative role of the apoptosome in neurodegeneration and cancer