Transcript Slide 1
A P O P toza Broj celija nije regulisan samo kontrolom celijske deobe nego je regulisan i celijskom smrti. Za svaku celiju postoji vreme kada treba da umre. Celije umiru na dva nacina: umiru usled povrede (nekroza) ili ukoliko vise nisu potrebne sprovode program smrti tj izvrse “samoubistvo” = APOPTOZA (programirana celijska smrt). Nekroza: • mehanicko ostecenje • hemijsko ostecenje Morfoloske karakteristike nekroze: • celija, kao i njene organele (posebno mitohondrije) “bubre”, zbog nesposobnosti njihove membrane da kontrolise prolaz vode i jona; • sadrzaj celije se izliva i dovodi do • imflamacije okolnog tkiva “Samoubistvo” celija se morfoloski manifestuje: • celja se “skuplja” zbog cepanja lamina i aktinskih filamenata (A) • degradacijom hromatina – nukleus apoptoticke celije ima izgled potkovice (B) • iz mitohondrija “izlazi” citohrom c • fosfatidilserini, koji su normalno sakriveni u plazma membrani, se okrecu ka spoljasnjoj povrsini membrane – “pojedi me” signali (C) • ovi signali se vezuju za receptore na fagocitima (makrofage ili dendritske celije) - - fagocitoza • fagociti sekretuju citokine koji inhibisu inflamaciju (npr. IL-10 i TGF-b) • celija se raspada na male membranom okruzene fragmente – apoptoticka tela (D) Apoptoticne celije trofoblasta (u toku 6h) Zasto celije izvrsavaju samoubistvo? 1. Programirana celijska smrt je neophodna za pravilan razvoj kao i mitoza Npr: resorpcija repa kod punoglavca u toku metamorfoze zabe; za formiranje prstiju kod fetusa neophodno je da se ukloni tkivo izmedju njih; eliminacija endometrijuma uterusa na pocetku menstruacije u toku razvoja CNS eliminise se veliki broj neurona 2. Programirana celijska smrt je neophodna za eliminaciju celija koje su pretnja integritetu organizma Npr: CTL pokrecu apoptozu u celijama inficrinam virusima eliminacija celija sa ostecenom DNA --- povecana produkcija p53 – potentan induktor apoptoze (celije kancera cesto imaju defektan p53) Sta “nagovori” celije da se ubiju? Smanjenje signala neophodnih za prezivljavanje Faktori rasta za neurone Interleukin-2 (IL-2), osnovni faktor mitoze za limfocite Bcl-2 proteini anti-apoptoticki vs. proapoptoticki proteini Bcl-2, bcl-XL Bad, Bax, Bid primanje negativnih signala Povecan nivo oksidanata u celiji (ultravioletno svetlo x-zraci, hemoterapeutski lekovi) Molekuli koji reaguju sa specificnim receptorima na pov. membrane – signali za apoptozu = signali smrti (TNF-α + TNF receptor; Lymphotoxin (TNF-β ) koji se takodje veze zaTNF receptor; Fas ligand (FasL), molekul koji se veze za Fas receptor (tzv. CD95). Apoptoza je posredovana intracelularnom proteolitickom kaskadom CASPASES: Cysteine Aspartate Specific ProteASEs Generalne karakteristike visoko specificne proteaze cepaju proteine iz ostatka aspartata 3 amino kiseline ispred aspartata odredjuju specificnost prema substratu funkcija: regulisu proteolizu tokom apoptoticke smrti celije obicno “sve ili nista” protezna kaskada je ireverzibilna = kad jednom dostigne kriticnu tacku na putu destrukcije, celija ne moze da se povrati Sve celije poseduju u sebi “seme” sopstvene destrukcije u obliku neaktivnih prokaspaza, koje cekaju signal za destrukciju celije. 2, 8, 9, 10 3, 6, 7 Class Activators Executioners Name Regulatory Adapter unit molecule Optimal tetrapeptide Caspase-2 CARD RAIDD DXXD Caspase-8 DED FADD (I/V/L)EXD Caspase-9 CARD Apaf-1 (I/V/L)EHD Caspase-10 DED FADD (I/V/L)EXD Caspase-3 DEXD Caspase-6 (I/V/L)EXD Caspase-7 DEXD Caspase-1 CARD Caspase-4 CARD Caspase-5 Cytokine processors mCaspase-11 mCaspase-12 Caspase-13 mCaspase-14 CARD: Caspase recruitment domain DED: Death effector domain CARDIAK (W/Y/F)EHD (W/Y/F)EHD (W/Y/F)EHD Kaskada kaspaza ukljucenih u apoptozu Aktivacija prokaspaza Aktivna kaspaza NH2 Mesto proteolize COOH Aktivna kaspaza prodomen Inaktivna prokaspaza Kaskada kaspaza Prokaspaze se aktiviraju vezivanjem za adapterne proteine koji povezuju veci broj inicijalnih prokaspaza u kompleks. Inicijalne prokaspaze imaju malu proteaznu aktivnost – agregacija – medjusobna aktivacija. 1 molekul aktivne kaspaze X Proteoliza citosolnih proteina Mnogo molekula aktivne kaspaze Y Proteoliza nuklearnih Lamina ili proteine koji cuvaju DNAse u neaktivnoj formi Jos vise molekula aktivne kaspaze Z Degradacija hromatina = je znak apoptoze 1. Inaktivacija enzima DNA obnove 2. Cepanje strukturnih nuklearnih proteina Caspasa 3 PARP (poly ADP-ribose polimerase) enzim obnove DNA 3. Fragmentacija DNA Caspasa 6 Caspasa 3 degradacija lamina CAD + ICAD (Caspase activated DNAse + inhibitor of CAD) CAD ICAD Brza fragmentacija DNA u nukleosomalne jedinice i. Spoljasnji put - aktivatori smrti koji se vezu za receptore na povrsini membrane: TNF-α Lymphotoxin Fas ligand (FasL) ii. Signali koji nastaju u celiji - unutrasnji ili mitohondrijalni put IAP proteins inhibit apoptosis by binding to activated caspases. They inhibit signals generated through both the two major pathways of apoptosis: the Extrinsic (death receptor mediated) and the Intrinsic (mitochondrial mediated) pathways i. Spoljasnji put In some cases the apoptotic stimuli comprise extrinsic signals such as the binding of death inducing ligands to cell surface receptors called death receptors. These ligands can either be soluble factors or can be expressed on the surface of cells such as cytotoxic T lymphocytes. Also, when T-cells recognise damaged or virus infected cells and initiate apoptosis in order to prevent damaged cells from becoming neoplastic (cancerous) or virus-infected cells from spreading the infection. Apoptosis can also be induced by cytotoxic T-lymphocytes using the enzyme granzyme. i. Receptori smrti – prenose apoptoticke signale inicirane ligandima kao sto su FasL, TNF i TRAIL (Tumour necrosis factor-related apoptosis-inducing ligand). Aktiviraju kaspaznu kaskadu u toku 1 sec od vezivanja liganda. Vezivanje za DR: produkcija ceramida na membrani – “lipid raft” – grupisanje velikog broja DR (“clustering”) = vazno za amplifikaciju signalnog puta neophodnog za aktivaciju apoptoze; Konformaciona promena intracelularnog domena R – “death domen” – regrutovanje adapternih i drugih proteina = proteinski kompleks DISC (Death Inducing Signaling Complex) – regrutovanje prokaspaza (obicno prokaspaza 8) i zapocinjanje apoptoze. i. Spoljasnji put Signaling preko TNFR1 TNF se produkuje u T limfocitima i aktiviranim makrofagama, kao odgovor na infekciju. Vezivanje za TNFR1 – nekoliko efekata: Trimerizacija receptora i “clustering” Vezivanje adapternog molekula TRADD (TNFR-associated death domains) za “death domene” Regrutacija razlicitih proteina nprTRAF-2 (TNF-associated factor 2) = aktivacija NFkB i JNK puta TRADD + FADD = indikcija apoptoze regrutacija – prokaspaze 8 Obicno vezivanje liganda za TNFR-1 nije dovoljno da inicira apoptozu (kao npr. vezivanje FasL ) Adapterni molekul aktivacija NFkB i JNK puta Aktivacija NF-kB i AP-1 – indukcija velikog broja gena i. Spoljasnji put Fas (CD95) signaling aktivira apoptozu i nema znacajnu ulogu u drugim aspektima signalinga Caspase activated DNAse • “clustering” receptora • FADD regrutuje direktno za death domen R (bez prethodne aktivacije TRADD-a) CAD = Caspase activated DNAse ICAD = inhibitor CAD i. Spoljasnji put TRAIL (TNF-related apoptosis inducing ligand) signaling Vezivanje za DR4 ili DR5 – brza apoptoza u mnogim celijama The death receptors DR4 and/or DR5 can be functionally engaged by recombinant soluble APO2L/TRAIL in its zinc-bound, trimeric form. Antibodies that bind to the extracellular portion of either receptor can also stimulate caspase-mediated apoptosis, with or without a requirement for receptor crosslinking. Nature Reviews Cancer 2, 420-430 (June 2002) i. Spoljasnji put The Ligand induced trimerisation model. The incoming trimeric ligand recruits three receptors into a complex. This induced juxtaposition of the intracellular domains triggers recruitment of the intracellular signalling components leading to either the caspase cascade and cell death, or activation of NF-B. Cell Research (2004); 14(5):359-372 Again With the Comics Sem DR4/5, TRAIL se vezu i za “decoy” receptore DcR1 I DcR2 (koji kompetituju sa DR4 i DR5 za TRAIL). “decoy” receptori DcR1 - nema citoplazmaticni domen; DcR2 - ima “death domen” kome nedostaje 4 od 6 AA = vaznih za regrutovanje adapternih proteina. Schematic of the five TRAIL Receptors. The extracellular cysteine-rich domains are represented by coloured ovals; yellow, for the first partial CRD1, which has one cystiene bond, and the other full cysteine rich domains in blue. Cell Research (2004); 14(5):359-372 TRAIL receptor signalling. Schematic showing the major signalling pathways initiated following TRAIL ligation of DR4 or DR5. The major pathway is the initiation of apoptosis. However, there is also some NF-kB and c-Jun activation. These transcription factors can have an anti-apoptotic effect via the upregulation of pro-survival genes, or can amplify the apoptotic signal through transcription of genes that can act via the mitochondria (the intrinsic pathway). Cell Research (2004); 14(5):359-372 ii. unutrasnji ili mitohondrijalni put Mitohondrije sadrze mnoge pro-apoptoticke proteine: AIF (Apoptosis Inducing Factor) Smac/DIABOLO Citohrom C Koji se oslobadjaju iz mitohondrija nakon formiranja PT pore (Permeability Transition pore), nastalih usled aktivacije pro-apoptotickih proteina iz familije bcl-2. Bcl-2 su aktivirani apoptotickim signalima – stres celije, slobodni radikali, ili nedostatak faktora rasta … Bcl-2 proteini mogu biti aktivirani I angazovanjem kaspaze 8 - amplifikuju apoptoticke signale od death receptora. ii. unutrasnji ili mitohondrijalni put Bcl-2 proteini – ukljuceni u apoptozu: pro- i anti-apoptoticki proteini. Prisustvo bcl-2 proteina na povrsini mitoh – formiranje PT pore PUMA p53-upregulated modulator of apoptosis Pro-apoptoticki bcl-2 proteini (npr Bad) – u citosolu – senzori celijskog stresa Mogu da se translociraju na povrsinu mitoh, gde su locirani anti-apoptoticki proteini. Interakcija pro- i antiapoptotickih proteina = narusavanje normalne antiapoptoticke funkcije bcl-2 proteina = formiranje PT pora i oslobadjanje cyt C i druhih proteina. cyt c u citosolu reaguje sa Apaf-1 proteinom (adapterni prot) = formiranje multiproteinskog kompleksa tzv apoptozom = angazovanje prokaspaze 9 u apoptozom ---- apoptoza. CTL Granzyme B Ili 7,8 i 10 SIGMA-ALDRICH Granzyme B and perforin, proteins released by an effector cell (cytotoxic T cell), can induce apoptosis in target cells by forming transmembrane pores and through cleavage of effector caspases such as caspase-3. In addition, caspase-independent mechanisms of granzyme Bmediated apoptosis have been suggested. Caspase-activated DNAse (CAD) is activated through the cleavage of its associated inhibitor ICAD by caspase-3. CAD is then able to interact with components such as topoisomerase II (Topo II) to condense chromatin, leading to DNA fragmentation and ultimately apoptosis. p53 (also known as protein 53 or tumor protein 53), is a transcription factor . p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. As such, p53 has been described as "the guardian of the genome," "the guardian angel gene," and the "master watchman," referring to its role in conserving stability by preventing genome mutation A model for p53-mediated apoptosis A model for p53mediated apoptosis. This model depicts the involvement of p53 in the extrinsic and intrinsic apoptotic pathways. p53 target genes are shown in red. The convergence of the two pathways through Bid is shown Haupt, S. et al. J Cell Sci 2003;116:4077-4085 Schematic representation of the p53-dependent apoptotic pathways by transcriptional activation of BAX, PUMA and Apaf-1 One important p53 effector is PUMA (p53-upregulated modulator of apoptosis) (Yee and Vousden, 2005). PUMA is a BH3-only member of the Bcl-2 family that is a potent inducer of apoptosis mediated by p53. It initiates the cell-death cascade by modulating Bax activity to facilitate cytochrome c release from the mitochondria. Role of PUMA and SLUG in Modulating Apoptosis Mediated by p53 Once activated by DNA damage, p53 induces various target genes, including Puma (which encodes a proapoptotic BH3-only protein) and Slug (which encodes a transcription factor that represses Puma transcription). In most cells, the amount of SLUG may not be sufficient to repress Puma and prevent apoptosis. Importantly, in hematopoietic progenitor cells, the endogenous amount of SLUG protein is sufficient to repress Puma and limit apoptosis induced by DNA damage. Also shown is the role of PUMA and p53 in coordinating cell death in the cytoplasm. Here, PUMA binds Bcl-xL and displaces p53, thereby allowing p53 to directly activate Bax and induce permeabilization of mitochondria and cell death Bcl-Xl = anti-apoptoticki bcl-2 protein Nitric oxide: NO apoptosis or turning it ON? Zavisno od koncentracije NO moze da ispolji anti- i pro-apoptoticki efekat Antiapoptoticki efekat NO: 1. Nitrosilacija i inaktivacija mnogih kaspasa (3,1,8) 2. Aktivacija p53 – heat shock proteina 70 = blokiranje regrutovanja prokaspase 9 za Apaf-1 i formiranje apoptosoma 3. “up”-regulacija Bcl-2 I Bcl-XL – inhibicija oslobadjanja cyt C iz mitoh 4. Aktiviranje cGMP signalinga – cGMP zavisnih kinaza = smanjenje aktivnosti kaspaza ili fosforilacije TAB-a i nemogucnosti aktivacije p38 … Model of GC-induced apoptosis in leukemic cells. Leukemia (2003) 17, 17–25. Glucocorticoids (GC) enter the cell by passive transport, and bind to the glucocorticoid receptor (GR). The unbound GR forms heterocomplexes consisting of heat-shock chaperone molecules hsp90 and 70, co-chaperone molecules hsp40, Hop (p60), p23 and immunophillins FKBp52 and CyP40, required for optimal configuration of the GR to be able to bind GC. As a homodimer the GC–GR complex translocates to the nucleus. There it interacts with either a GRE (consensus sequence (GGT ACA NNNTGT TCT) of a target gene (transactivation), or it interacts with other transcription factors such as AP-1 and NF-B (transrepression). Both processes may finally result in the induction of cell death (apoptosis). NF-B is kept in the cytoplasm in a complex with IB. Upon dissociation it can translocate to the nucleus. Simplified model of pro- and anti-apoptotic pathways in hen granulosa cells. Apoptosis Detection Kits 1. DNA Fragmentation Comet Assay™ Cells are immobilized on a slide in a bed of low melting point agarose and gently lysed and treated with alkali to unwind and denature the DNA. The samples are electrophoresed, stained, and visualized by fluorescence or light microscopy. Evidence of a "comet tail" shape and migration pattern indicates DNA Damage. DNA Laddering Kits Internucleosomal cleavage of DNA is a hallmark of apoptosis and can be assessed using Gel Electrophoresis with R&D Systems' colorimetric, ethidium bromide, luminescent, and isotope-based kits. Tissue supplement is also available. TUNEL Labeling Kits Apoptotic cells can be detected by terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL). . It may also label cells that have suffered severe DNA damage. 2. Caspase Activity anti-active caspase 3 antibody Caspase Colorimetric Activity Assays Caspase Fluorometric Activity Assays Microplate-based fluorometric and colorimetric assays for measuring Caspase activity in cell lysates. ApoStat (FITC-VD-FMK) ApoStat is designed to identify and quantitate caspase activity in apoptotic cells by flow cytometry. Cells undergoing apoptosis are labeled with a cell permeable, FITC-conjugated pan caspase inhibitor (ApoStat). Any unbound reagent diffuses out of the cell and is washed away. Cells are then analyzed by flow cytometry for the presence of bound reagent. Increased fluorescence is an indicator of caspase activity within individual cells. 3. Phosphatidylserine in the outer leaflet (Annexin V) Annexin V Kits Early in the apoptotic process, cell surface phospholipid asymmetry is disrupted leading to the exposure of phosphatidylserine (PS) on the outer leaflet of the cell membrane. Annexin V preferentially binds PS and can be used as an early indicator of apoptosis using either colorimetric or fluorescence-based detection. 4. PARP Activity/Inhibition PARP Universal Colorimetric Assay This assay allows for the measurment of Polymerase (PARP) activity can be used to determine whether DNA is damaged, or to test for PARP inhibition. Cleavage of PARP inactivates the enzyme and is an indicator of apoptosis. Format: Scintillation counting; [32P]-NAD 5. Mitochondrial Membrane Potential Disruption DePsipher™ Disruption of the mitochondrial transmembrane potential is one of the early intracellular events to occur following induction of apoptosis. DePsipher contains a lipophilic cation that can be used as a mitochondrial activity marker in live cells. The kit can be used to evaluate cellular viability, estimate the effect of drugs or other cytotoxic treatments, or detect early apoptosis in known models using fluorescence microscopy or flow cytometry. 6. Cell Viability and Proliferation MTT Cell Proliferation/Viability Assay Cells undergoing apoptosis demonstrate a decrease in their ability to reduce the tetrazolium salt, MTT. This microplate-based assay allows for the safe, sensitive, measurement of cell proliferation and viability. The rate of tetrazolium reduction is proportional to the rate of proliferation. XTT Cell Proliferation/Viability Assay Cells undergoing apoptosis demonstrate a decrease in their ability to reduce XTT, a yellow tetrazolium salt. XTT is cleaved to a soluble orange formazan dye which can be measured by absorbance on a microplate reader. This microplate-based assay allows for the safe, sensitive measurement of cell proliferation and viability and is ideal for high throughput platforms. The rate of tetrazolium reduction is proportional to the rate of proliferation. cellular damage signals and functions as a PKC - Apoptoza regulation of nuclear import and export of propose that in the absence of an apoptotic asm by a mechanism that is dependent on the such as etoposide induce post-translational rosine phosphorylation of the regulatory umulation of PKCδ. Active caspase 3 also se to etoposide, neration of δCF. In vely present in the s apoptosis through n cell damage as well ogether, our studies r import and export of hat caspase cleavage eversible commitment to Caspase-3 activation via tumor necrosis factor (TNF) family receptors (for example, Fas), FADD (Fas-activated death domain protein) and caspase-8 represents the extrinsic pathway (blue), whereas caspase-3 activation via the mitochondrial release of cytochrome c and Apaf1–mediated processing of caspase-9 represents the intrinsic pathway (red)3. For clarity, not all of the players are shown. Procaspase-3 is shown as a PAC-1–sensitive dormant single-chain precursor with an N-terminal prodomain (Pro). During apoptosis, caspase-3 assembles as an active p17-p12 heterotetramer after proteolytic processing between the p17 and p12 subunits (at Asp175) and removal of the prodomain2. PAC1 is proposed to regulate the Asp-Asp-Asp (DDD) safety catch at amino acids 179–181 in procaspase-3, consequently inducing a conformational change that leads to proteolytic processing into the active p17 and p12 subunits1. Cys163 is the catalytic cysteine in the active site of caspase-3; the sequence shown illustrates its proximity to the DDD safety catch and DDM motif. Although caspase-7 (not shown) is believed to be a downstream caspase, its position relative to caspase-3 in apoptosis pathways is unclear. Nature Chemical Biology 2, 509 - 510 (2006)