APOPtoza

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-)• 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 mitozaNpr: 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

primanje negativnih signala

Faktori rasta za neuroneInterleukin-2 (IL-2), osnovni

faktor mitoze za limfocite

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).

Bcl-2 proteinianti-apoptoticki vs. proapoptoticki proteini

Bcl-2, bcl-XL Bad, Bax, Bid

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

Apoptoza je posredovana intracelularnomproteolitickom kaskadom

2, 8, 9, 10

3, 6, 7

Sve celije poseduju u sebi “seme” sopstvene destrukcije u obliku neaktivnih prokaspaza, koje cekaju signal za

destrukciju celije.

Class NameRegulatoryunit

Adaptermolecule

Optimaltetrapeptide

Activators

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

Executioners

Caspase-3 DEXD

Caspase-6 (I/V/L)EXD

Caspase-7 DEXD

Cytokine processors

Caspase-1 CARD CARDIAK (W/Y/F)EHD

Caspase-4 CARD (W/Y/F)EHD

Caspase-5 (W/Y/F)EHD

mCaspase-11

mCaspase-12

Caspase-13

mCaspase-14

CARD: Caspase recruitment domainDED: Death effector domain

Kaskada kaspaza ukljucenih u apoptozu

COOH

NH2

Mestoproteolize

prodomen

Aktivna kaspaza

Aktivna kaspaza

Inaktivna prokaspaza

Proteolizacitosolnih proteina

Proteoliza nuklearnih Lamina ili proteine koji cuvaju DNAse u neaktivnoj formi

Aktivacija prokaspaza

Kaskada kaspaza1 molekulaktivne kaspaze X

Mnogo molekula aktivne kaspaze Y

Jos vise molekula aktivne kaspaze Z

Prokaspaze se aktiviraju vezivanjem za adapterne proteine koji povezuju veci broj inicijalnih prokaspaza u kompleks. Inicijalne prokaspaze imaju malu proteaznu aktivnost – agregacija – medjusobna aktivacija.

Brza fragmentacija DNA u nukleosomalne jedinice

Caspasa 3

CAD + ICAD

(Caspase activated DNAse + inhibitor of CAD)

CAD ICAD

1. Inaktivacija enzima DNA obnove

Caspasa 3

PARP(poly ADP-ribose polimerase)enzim obnove DNA

3. Fragmentacija DNA2. Cepanje strukturnih nuklearnih proteina

Caspasa 6

degradacijalamina

Degradacija hromatina = je znak apoptoze

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

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. Spoljasnji put

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.

TNF se produkuje u T limfocitima i aktiviranim makrofagama, kao odgovor na infekciju. Vezivanje za TNFR1 – nekoliko efekata:

Aktivacija NF-kB i AP-1 – indukcija velikog broja gena

Obicno vezivanje liganda za TNFR-1 nije dovoljno da inicira apoptozu (kao npr. vezivanje FasL )

Adapterni molekul

Trimerizacija receptora i “clustering” Vezivanje adapternog molekula TRADD (TNFR-associated death domains) za “death domene”

Regrutacija razlicitih proteina npr- TRAF-2 (TNF-associated factor 2) = aktivacija NFkB i JNK puta

TRADD + FADD = indikcija apoptoze regrutacija – prokaspaze 8

aktivacija NFkB i JNK puta

Signaling preko TNFR1

i. Spoljasnji put

CAD = Caspase activated DNAseICAD = inhibitor CAD

Fas (CD95) signaling

• “clustering” receptora• FADD regrutuje direktno za death domen R(bez prethodne aktivacije TRADD-a)

aktivira apoptozu i nema znacajnu ulogu u drugim aspektima signalinga

i. Spoljasnji put

Caspase activated DNAse

Nature Reviews Cancer 2, 420-430 (June 2002)

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.

i. Spoljasnji put

TRAIL (TNF-related apoptosis inducing ligand) signalingVezivanje za DR4 ili DR5 – brza apoptoza u mnogim celijama

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

i. Spoljasnji put

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

Again With the Comics

“decoy” receptori

Sem DR4/5, TRAIL se vezu i za “decoy” receptore DcR1 I DcR2 (koji kompetituju sa DR4 i DR5 za TRAIL).

DcR1 - nema citoplazmaticni domen;

DcR2 - ima “death domen” kome nedostaje 4 od 6 AA = vaznih za regrutovanje adapternih proteina.

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 up-regulation 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

PUMAp53-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 anti-apoptotickih proteina = narusavanje normalne anti- apoptoticke 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.

Granzyme B

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 B-mediated 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.

CTL

Ili 7,8 i 10

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

Haupt, S. et al. J Cell Sci 2003;116:4077-4085

A model for p53-mediated apoptosis

A model for p53-mediated 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

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.

Schematic representation of the p53-dependent apoptotic pathways by transcriptional activation of BAX, PUMA and Apaf-1

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 pro-kaspase 9 za Apaf-1 i formiranje apoptosoma3.“up”-regulacija Bcl-2 I Bcl-XL – inhibicija oslobadjanja cyt C iz mitoh4.Aktiviranje cGMP signalinga – cGMP zavisnih kinaza = smanjenje aktivnosti kaspaza ili fosforilacije TAB-a i nemogucnosti aktivacije p38 …

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.

Model of GC-induced apoptosis in leukemic cells. Leukemia (2003) 17, 17–25.

Simplified model of pro- and anti-apoptotic pathways in hen granulosa cells.

Apoptosis Detection Kits1.DNA FragmentationComet 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 KitsInternucleosomal 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 KitsApoptotic 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 ActivityCaspase 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 KitsEarly 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 beused as an early indicator of apoptosis using either colorimetric or fluorescence-based detection.

anti-active caspase 3 antibody

4. PARP Activity/InhibitionPARP 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 DisruptionDePsipher™

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 ProliferationMTT 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 AssayCells 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.

PKC - Apoptoza

The PKC (protein kinase C) family of serine/threonine kinases regulates many cellular functions including proliferation and survivalPKCα, β, ε and ζ have been identified as isoforms that can suppress apoptosis, while PKCδ is a critical pro-apoptotic signal in many cell typesPKCδ is required for execution of the apoptotic programmePKCδ is activated downstream of cellular damage signals and functions as a proximal regulator of apoptosis.Under normal growth conditions, tight regulation of nuclear import and export of PKCδ is required for cell survival.We propose that in the absence of an apoptotic signal, PKCδ is retained in the cytoplasm by a mechanism that is dependent on the regulatory domain. Apoptotic signals such as etoposide induce post-translational modifications in the PKCδ, such as tyrosine phosphorylation of the regulatory domain, which may allow nuclear accumulation of PKCδ. Active caspase 3 also accumulates in the nucleus in response to etoposide,resulting in cleavage of PKCδ and generation of δCF. Incontrast with PKCδ, δCF is constitutively present in thenucleus where it presumably regulates apoptosis throughphosphorylation of proteins involved in cell damage as wellas other apoptotic mediators. Taken together, our studiessuggest that tight regulation of nuclear import and export ofPKCδ is critical for cell survival and that caspase cleavageof PKCδ in the nucleus signals an irreversible 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 Apaf-1–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. PAC-1 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)