Apoptosis

BY- VISHAKHA UPADHYAY B.tech 4th year

Apoptosis is a biological mechanism which is one type of programmed cell death. Apoptosis is used by multicellular organisms to remove cells that are not needed by the body.

Apoptosis has distinctive morphological characteristics such as plasma membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation, and begins with the enzymes of the caspase proteases and form a complex group of Cysteine protease activation of multi sub-unit called apoptosome.

Morphological hallmarks of apoptosis:

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Apoptosis Necrosis

Natural: Yes caused by factors external to the cell or tissue, such as infection, toxins, or trauma

Effects: Beneficial as well as detrimental

Detrimental

Introduction: Apoptosis programmed cell death (PCD) in humans & multicellular organisms. PCD involves a series of biochemical events leading to a cell destruction and death.

Necrosis is the premature death of cells and living tissue. 

result: Can prevent tumor formation (homeostasis between cell death rate and mitosis rate)

Necrosis results in inflammation, which could become chronic.

definition: programmed cell death the cell or tissue damage due to external factors.

process: membrane blebbing, shrinkage of cell, nuclear collapse, apoptopic body formation. Then, engulf by white blood cells

membrane disruption, respiratory poisons and hypoxia which cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation.

On the basis of their working

ROLE OF APOPTOSIS IN PHYSIOLOGICAL CONDITIONS

Programmed cell destruction in embryonic development for the purpose of sculpting of tissue.

Mouse paws, for example, are sculpted by cell death during embryonic development.

They start out as spadelike structures and the individual digits separate only as the cells between them die.

• In other cases, cells die when the structure they form is no longer needed.

• When a tadpole changes into a frog, the cells in the tail die, and the tail, which is not needed in the frog disappears .

Sculpting the digits in the developing mouse paw by apoptosis.

Apoptosis during the metamorphosis of a tadpole into a frog. As a tadpole changes into a frog, the cells in the tadpole tail are induced to undergo apoptosis; as a consequence, the tail is lost. All the changes that occur during metamorphosis, including the induction of apoptosis in the tail, are stimulated by an increase in thyroid hormone in the blood.

In adult tissues, cell death exactly balances cell division. If this were not so, the tissue would grow or shrink. If part of liver is removed in an adult rat for ex liver cell proliferation increase to make up the loss.

Conversely, if a rat is treated with the drug phenobarbital which stimulates liver cell division (and thereby liver enlargement) and then the phenobarbital treatment is stopped.

Apoptosis in the liver greatly increases until the liver has returned to its original size, usually within a week or so.

Thus, the liver is kept at a constant size through the regulation of both the cell death rate and the cell birth rate.

Hormonal regulation of physiological cell turnover and apoptosis

Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues.

Cell turnover is precisely regulated by the interplay of various endocrine and paracrine factors.

They modulate tissue and cell-specific responses on proliferation and apoptosis, either directly, or by altering expression and function of key cell proliferative and/or death genes.

Several similarities exist among the various tissues with regard to the intermediates that regulate tissue homeostasis, enabling a better understanding of the general mechanisms involved in the process.

Age‐related thymic involution

The thymus continues to grow between birth and puberty and then begins to atrophy. This thymic involution is directed by the high levels of circulating hormones. Proportional to thymic size, thymic activity (T-cell output) is most active before puberty. Upon atrophy, the size and activity are dramatically reduced, and the organ is primarily replaced with fat (a phenomenon known as "organ involution“). The atrophy is due to the increased circulating level of sex hormones, and chemical or physical castration of an adult results in the thymus increasing in size and activity.Patients with the autoimmune disease Myasthenia gravis commonly (70%) are found to have thymic hyperplasia or malignancy.

Age related thymic involution

Age Mass

Birth about 15 grams;

puberty about 35 grams

twenty-five years 25 grams

sixty years less than 15 grams

seventy years as low as 5 grams

Cell death by apoptosis during involution of the lactating breast in mice and rats.

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy.

Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species.

In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens.

This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium.

Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats.

Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats.

Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes.

Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands.

It characteristically results in shrinkage of a tissue without disruption of its basic architecture.

Apoptosis in human endometrium and endometriosis

Apoptosis plays a critical role in maintaining tissue homeostasis and represents a normal function to eliminate excess or dysfunctional cells.

Apoptosis helps to maintain cellular homeostasis during the menstrual cycle by eliminating senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phase of the cycle.

The BCL‐2 family and Fas/FasL system have been extensively studied in human endometrium and endometriotic tissues.

CASPASESThe machinery responsible for apoptosis depends on the family of proteases that have a cysteine at their active site and cleave their target proteins at specific aspartic acids.The activation of a group of enzymes belonging to the cysteine protease family named caspases. The “c” of “caspase” refers to a cysteine protease, while the “aspase” refers to the enzyme’s unique property to cleave after aspartic acid residues.

Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in apoptosis (programmed cell death), necrosis, and inflammation.

Caspases are essential in cells for apoptosis, or programmed cell death, in development and most other stages of adult life, and have been termed "executioner" proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of lymphocytes. Failure of apoptosis is one of the main contributions to tumour development and autoimmune diseases; this, coupled with the unwanted apoptosis that occurs with ischemia or Alzheimer's disease, has stimulated interest in caspases as potential therapeutic targets since they were discovered in the mid-1990s.

TYPES OF CASPASES As of November 2009, twelve caspases have been identified in humans. There are two types of apoptotic caspases: initiator (apical)

caspases and effector (executioner) caspases. (1) Initiator caspases  CASP2, CASP8, CASP9, and CASP10 They cleave inactive pro-forms of effector caspases, thereby activating

them.

(2)Effector caspases CASP3, CASP6, CASP7 These in turn cleave other protein substrates within the cell, to trigger

the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors.

CASP 1,CASP4 ,CASP 5, CASP 11, CASP 12 , which are overexpressed in some cases of vitiligo and associated autoimmune diseases caused by NALP1 variants, are not currently classified as initiator or effector , because they are inflammatory enzymes that are involved in T-cell maturation.

PROTEINS AND GENES INVOLVED IN APOPTOSIS

p53Location: short arm of chromosome 17Subcellular location: Nucleus (kinetochore). These are essential for cell growth regulation and

apoptosis induced by genotoxic and non-genotoxic stresses .

In normal unstressed cells, the level of p53 protein is downregulated via the binding of proteins such as MDM2, COP1, JNK that promote p53 degradation via the ubiquitin/proteasome pathway. As most of these genes are up regulated by p53, this lead to a regulation loop that will keep p53 level very low in a normal cells.

After genotoxic or non-genotoxic stresses, activation of p53 is a two steps process.

Downstream signalling includes a large series of genes that are activated by the transactivating properties of p53. This occurs via specific DNA binding of the p53 protein to a p53 response element (p53 RE) that isfound either in the promoter or in the intron of target genes .

Regardless of the type of stress, the final outcome of p53 activation is either cell cycle arrest and DNA repair or apoptosis, but the mechanism leading to the choice between these fates has not yet been elucidated .

The p53 pathways has been conveniently divided into five parts :

p21p21 is a potent cyclin dependent kinase inhibitor (CKI). The p21 protein binds to and inhibits the

activity of cyclin- CDK2, -CDK1, and -CDK4/6complexes, and thus functions as a regulator of cell cycle progression at G1and S phase. 

Location: Chromsome no. 6(in humans).Subcellular location: Cytoplasm , nucleus.P21 is transcriptionally activated by p53.Sometimes p21 is expressed without being

induced by p53. This kind of induction plays a big role in p53

independent differentiation which is promoted by p21

. Expression of p21 is mainly dependent on two factors:

1) stimulus provided 2) type of the cell. Growth arrest by p21 can

promote cellular differentiation. p21 therefore prevents cell

proliferation.

mdm2Mouse double minute 2 homolog (MDM2)

also known as E3 ubiquitin-protein ligase.Mdm2 is a protein that in humans is encoded

by the MDM2 gene. Mdm2 is an important negative regulator of

the p53  tumor suppressor. Mdm2 protein functions both as an E3

ubiquitin ligase that recognizes the N-terminal trans-activation domain (TAD) of the p53 tumor suppressor and

an inhibitor of p53 transcriptional activation.

The key target of Mdm2 is the p53 tumor suppressor. Mdm2 has been identified as a p53 interacting protein that represses p53 transcriptional activity.

Mdm2 is a p53 responsive gene—that is, its transcription can be activated by p53. Thus when p53 is stabilized, the transcription of Mdm2 is also induced, resulting in higher Mdm2 protein levels.

Location: chrmosome no. 12Subcellular location: Nucleus ,

Nucleoplasm

Akt1

The serine-threonine protein kinase AKT1.Location: chromsome no. 14.Subcellular location: Cytoplasm, Cell membrane,

Nucleus AKT1 and the related AKT2 are activated

by platelet-derived growth factor. The activation is rapid and specific, and it is abrogated by mutations in the pleckstrin homology domain of AKT1.

It was shown that the activation occurs through phosphatidylinositol 3-kinase.

In the developing nervous system AKT is a critical mediator of growth factor-induced neuronal survival.

Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase AKT1, which then phosphorylates (pAkt) and inactivates components of the apoptotic machinery.

Mice lacking Akt1 display a 25% reduction in body mass, indicating that Akt1 is critical for transmitting growth promoting signals, most likely via the igf1 receptor.

Mice lacking Akt1 are also resistant to cancer: they experience considerable delay in tumor growth initiated by the large T antigen or the Neu oncogene . A single-nucleotide polymorphism in this gene causes Proteus syndrome.

Apoptosis Triggered via Two Pathways-

1.Intrinsic pathway

2. Extrinsic pathway

Bcl-2bcl-2 (B-cell lymphoma 2), encoded by

the BCL2 gene, is the founding member of the Bcl-2 family of regulator proteins that regulate cell death .

Damage to the Bcl-2 gene has been identified as a cause of a number of cancers including -

*melanoma,* breast and prostate cancer , * chronic lymphocytic leukaemia, * lung cancer.a possible cause of  autoimmunity. It is also a cause

of resistance to cancer treatments.Antibodies to Bcl-2 can be used with immuno

histochemistry to identify cells containing the antigen.

Caspase cascadeCaspases are regulated at a post-translational

level, ensuring that they can be rapidly activatedThey are first synthesized as inactive pro-caspases, that consist of a prodomain, a small subunit and a large subunit.

initiator caspases possess a longer prodomain than the effector caspases, whose prodomain is very small

The prodomain of the initiator caspases contain domains such as a CARD domain (e.g., caspases-2 and -9) or a death effector domain (DED) (caspases-8 and -10) that enables the caspases to interact with other molecules that regulate their activation.

These molecules respond to stimuli that cause the clustering of the initiator caspases. Such clustering allows them to activate automatically, so that they can proceed to activate the effector caspases.

The caspase cascade can be activated by: granzyme B (released by cytotoxic T

lymphocytes and NK cells), which is known to activate caspase-3 and -7

death receptors (like Fas , TRAIL receptors and TNF receptor ), which can activate caspase-8 and -10

the apoptosome  (regulated by cytochrome c and the Bcl-2 family), which activates caspase-9

Intrinsic apoptosis

Apoptosis involves the degradation of cellular components by a group of cysteine proteases called caspases.

The first mechanism through which the caspases get activated is –intrinsic pathway . (which means mitochondrial mediated).

Pathway is characterized by permeabilization of the mitochondria

then the release of cytochrome c into the cytoplasm Cytochrome c forms a multi protein complex called

apoptosome. Initiates the activation of the caspases cascade

through caspase 9.

Intrinsic pathway (damage):

Mitochondria

Cytochrome c release

Pro-caspase 9 cleavage

Pro-execution caspase (3) cleavage

BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3Caspase (3) cleavage of cellular proteins,

nuclease activation, etc.

Tumors arises more from intrinsic pathwayMitochondrial proteins are known as SMAC’s and

they are released into the cytosol following an increase in permeability.

Cytochrome c is released from mitochondria due to formation of a channel, ie MAC , in the outer mitochondrial membrane .

Once the cytochrome is released , it binds to APAF-1 and ATP , then it binds to pro caspase 9 to create a protein complex called as apoptosome.

the apoptosome cleaves the pro-caspase to its active form of caspase9 , which in turn activates the effector caspase3.

The intrinsic signalling pathway involves non-receptor–mediated intracellular signals.

inducing activities in the mitochondria that initiate apoptosis.

Stimuli for the intrinsic pathway include viral infections or damage to the cell by toxins, free radicals, or radiation.

Damage to the cellular DNA can also induce the activation of the intrinsic pathway for programmed cell death

These stimuli induce changes in the inner mitochondrial membrane that result in the loss of transmembrane potential, causing the release of pro-apoptotic proteins into the cytosol

Pro-apoptotic proteins activate caspases that mediate the destruction of the cell through many pathways.

These proteins also translocate into the cellular nucleus, inducing DNA fragmentation, a hallmark of apoptosis

The regulation of pro-apoptotic events in the mitochondria occurs through activity of members of the Bcl-2 family of proteins and the tumor suppressor protein p53.

 Members of the Bcl-2 family of proteins may be pro- or anti-apoptotic..

The anti-apoptotic proteins are Bcl-2, Bcl-x, Bcl-xL, Bcl-XS, Bcl-w, and BAG. Some of these proteins are currently under investigation as potential targets for anti-cancer therapy.

 Pro-apoptotic proteins include Bcl-10, Bax, Bak, Bid, Bad, Bim, Bik, and Blk.

It has been suggested that up regulation of these proteins or their increased activation may offer an approach for cancer therapy.

Cellular pathways that modulate the activities of the p53 protein are also currently being evaluated as targets for potential anticancer therapies

Extrinsic pathway The extrinsic pathway is activated by death receptors on

the plasma membrane such as TNF receptor1 and CD95. As ligands bind to these receptors , the death inducing

signalling complex (DISC) is formed leading to initiation of the caspase cascade through caspase 8.

Molecules that stimulate the activity of these pro-apoptotic proteins or activate these receptors are currently under evaluation for their therapeutic potential in the treatment of cancer.

The signal transduction of the extrinsic pathway involves several caspases which are proteases with specific cellular targets.

 Once activated, the caspases affect several cellular functions as part of a process that results in the death of the cells