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Molecular mechanisms of apoptosis in human lung diseases: a role for the DWNN and its protein products.
Hypothesis
DWNN, a novel apoptosis gene, controls the apoptosis cascade in pulmonary epithelial and inflammatory cells.
Specific Hypothesis
The expression pattern and intracellular distribution of the DWNN gene in:
i) airway epithelial and inflammatory cells,
ii) circulating eosinophils and neutrophils differs in asthma, chronic obstructive airway disease and lung cancer.
Objectives
1. The purpose of this study is to establish the expression levels of the RbBP6 gene in asthma subtypes, Chronic Obstructive Pulmonary Disease (COPD) and lung cancer. The objective is also to characterize the role of this gene and apoptosis in diverse lung diseases. An understanding of the role of this DWNN gene in the development of lung diseases may lead to insights into developing new therapeutic measures for those lung diseases in which apoptosis plays a prominent part.
2. Further to establish whether or not a correlation exists between the expression levels of RbBP6 and apoptosis and the development of asthma subtypes. COPD and lung cancer
Aims
To determine by Quantitative RT-PCR, immunolabelling (quantitative confocal microscopy and in situ hybridization), detection of apoptosis using the TUNEL method IN:
1. Cultured lung epithelial cells-A549 and NCI-H292
2. Circulating eosinophils and neutrophils.
3. Eosinophils and neutrophils in sputum
Background
Summary-Apoptosis
Apoptosis plays an important role in maintaining homeostatic balance between cell proliferation and cell death during development of an organism. In some instance, high or low levels of apoptosis might give rise to a variety of diseases including cancer and immune directed disorders (Mikes, 2002; Kiechle and Zhang, 1998; Rathmell and Thomson, 2002). There are three different mechanisms of apoptosis by which a cell commits suicide and these are, 1) generated by signals arising within the cell, 2) triggered by death activators binding to receptors at the cell surface and, 3) by dangerous reactive oxygen species (Knaapen et al., 2001 and Reed 2000).
DWNN Gene
DWNN (domain with no name) is a novel gene that is expressed as a 76-residue domain which is conserved throughout the plant and animal kingdoms. It was identified first by Inverse and Rapid Amplification of cDNA Ends Polymerase Chain Reaction (RACE PCR) on Chinese hamster ovary cell lines which had been mutagenised through a strategy called Promoter trap mutagenesis. CHO cells deficient in the DWNN gene showed resistance to cytotoxic T-lymphocyte killing and to apoptosis initiated by an inducer of apoptosis, staurosporine. In humans DWNN protein exists in two forms, as 13kD DWNN and 200kD DWNN. The 13kD DWNN protein has a single domain whereas the 200kD DWNN consist of the 13kD DWNN linked to other domains. The human domain shares high sequence similarity with the mouse homologue. These domains include C2H2 zinc finger [real interesting new gene (RING) finger], and p53-associated-domain. RING fingers are conserved cysteine rich domains of 40 to 60 residues that bind two atoms of zinc, and are probably involved in mediating protein-protein interactions The consensus sequence of the ring finger domains is arranged in the following manner: C-X2-C-X9-39-C-X1-3-H-X2-3-C-X2-C-X4-28-C-X2-C. (C for cysteines; X for any amino acid and H for Histidine).
Ubiquitin-ligases
Ubiquitin protein ligases are proteins that facilitate the ubiquitination of proteins for degradation. They are core components of the ubiquitin 26S proteosome pathway that degrades proteins, which are marked for degradation, by tagging them with polyubiquitin (Kevin L., et al, 1999; Martinez-Noel, 2001). Ubiquitin protein ligases are defined as proteins or protein complexes required for the recognition and ubiquitination of specific substrates, and in so doing, marking them for degradation. They catalyze the formation of an isopeptide bond between the carboxyl terminus of ubiquitin (an 8.5kd polypeptide) and the є-amino acid group of lysine residues on the target protein (Hochstrasser M., 1995; Scheffner M., et al., 1995). Among different classes of ubiquitin protein ligases, there are those that have a RING finger which have an important molecular sequence for enzymic function.
Recent studies have established a functional relationship between RING finger domain and ubiquitin ligase activity. Ubiquitination is a post-translational protein modification, which requires ATP and three different enzymes, a ubiquitin activating enzyme, a ubiquitin conjugating enzyme and a ubiquitin ligase. In short, free ubiquitin is attached to ubiquitin activating enzyme by thiol ester bond formation, and subsequently transferred to ubiquitin conjugating enzyme through a second thioester bond formation between ubiquitin activating enzyme and ubiquitin itself. Ubiquitin activating enzyme in conjunction with ubiquitin ligase transfers ubiquitin to target proteins. There are two major types or families of ubiquitin ligases. There are those that possess a Hect domain (Hect domain is a domain found in ubiquitin ligases and is homologous to the E6-COOH terminus), and there are those that possess a RING finger motif. Evidence has indicated that RING finger-containing proteins are ubiquitin ligases and are mostly involved in ubiquitination. The fact that DWNN domain is associated with both the RING Finger and p53-associated domains opens the possibility that DWNN is involved in p53 dependent apoptotic pathway as a ubiquitin-ligase enzyme. Ubiquitin protein ligases, of which DWNN may be one, are a very important group of enzymes that play a significant role in the pathogenesis of many human diseases through deregulation of targeted proteolysis.
Apoptosis
Apoptosis is widely defined as programmed cell death, which is a regulated process for the deletion of unwanted cells (Soto, 1997). In response to a variety of stimuli, cells die in a controlled, regulated manner through a series of sequential molecular steps known as apoptosis. Apoptosis is therefore characterized as a process or event that is distinct from other forms of cell death such as necrosis, in which uncontrolled cell death leads to cell lysis, and the release of potentially harmful inflammatory mediators. Apoptosis may be initiated by a variety of death triggering signals, and occurs in four distinct phases. The apoptosis stimuli generate signals that are either transmitted across the plasma membrane to the regulatory molecules or more directly at targets within the cell there is a signaling phase that initiates apoptosis, followed by an integration phase in which intracellular positive and negative regulatory molecules inhibit, stimulate or forestall apoptosis. These molecules determine the outcome of whether apoptosis occurs or not. In the final phase the killing of cells is directed by caspases, and dead cells are removed by phagocytosis. Two alternative pathways initiate apoptosis, either through death receptors on the cell surface (extrinsic pathway) or through mitochondria (intrinsic pathway) (Chandra et al, 2002).
What are caspases?
Caspaces are a family of cysteinyl aspartate specific proteases characterized by their absolute specificity for aspartic acid in the P1 position. Caspases contain a conserved amino acid sequence that forms a pentapeptide active site motif of QACXG (where X is either R or Q or G) (Cohen, 1997). Caspases are synthesized as inactive precursors known as procaspases, which are converted into mature enzymes by apoptotic signals. They comprise four distinct domains, which are activated following cleavage at specific aspartate cleavage sites (Cohen, 1997). These domains include an amino-terminal domain, a large subunit (20kDa), a small subunit (10 kDa) and a linker region between the two subunits. These cysteine proteases are activated by proteolytic cleavage between the domains, with the resultant removal of the prodomain and the linker regions, followed by assembly of the large and small subunits into an active enzyme complex. Upon cleavage and activation they exert their effect on different protein substrates that are involved in the elucidation of the apoptotic fate of the cell. They are implicated in the pathogenesis of many diseases caused because of faults in the regulation of apoptosis.
Caspases play a central role in apoptosis
The involvement of caspases in apoptosis was first discovered and described when it was found that they shared a significant sequence similarity with Interleukin-1 β-converting enzyme, which specifically cleaves at the c-terminus site of aspartic acid. Since it was the first cysteine protease to be discovered, it was called caspase 1. Over-expression of the wild type caspase 1 induces apoptosis while the mutant form inhibits apoptosis. Other caspases were numbered in the order of their discovery, for example, the next caspase identified, Nedd 2/ ICH1, was called caspase 2. It is uasually found in the nucleus, but procaspase 2 is also detected in mitochondrial and cytoplasmic fractions. In total there are over a dozen caspases that have been identified in humans; two thirds of them play a leading role in apoptosis (Hengartner, 2000).
Caspases are divided into initiators (which direct sequential molecular steps towards the death substrates and convert pro forms of caspases into active forms) and effectors (cleave death receptors) in the apoptotic pathways. Caspase 2 is an early effector in the apoptotic cascade and precedes the activation of caspase 3. Caspase 3 is synthesized as an inactive 32kd that is processed into an active form in order to act as a central control molecule of the programmed cell death in mammalian cells. Whereas Caspases 8 and 9 are initiators, caspase 2, 3 and 7 are effectors involved in the demise of the cell. Caspases are implicated in DNA laddering; nuclear shrinkage and budding that are observed in apoptotic cells. These effects occur because caspases are involved in the activation of a nuclease that cuts the genomic DNA between nucleosomes to generate DNA fragments of approximately 200 base pairs that are often used as apoptotic markers (Hengartner, 2000). Caspase-3 activates an inactive DNA ladder nuclease by cleaving off its inhibitory subunit which results in activation of the catalytic unit. Of the cysteine proteases, caspases-3 and -7 are able to remove the inhibitory unit of caspase-activated DNAase, but only caspase-3 promotes DNA fragmentation. This is the reason why caspases are known as executioners of apoptosis. In addition, caspases are involved also in the cleavage of cytoskeletal proteins such as fodrin and gelsolin that are involved in maintaining the integrity of cell membranes and shape, whereas cleavage of laminins by caspases leads to nuclear shrinkage and budding (Hengartner, 2000). Even blebbing of cells undergoing apoptosis has been shown to be caspase dependant. Cleavage of p21-activated kinase 2 (PAK2) is essential for the blebbing that is often observed in apoptotic cells (Walter et al, 1998). These findings clearly indicate that caspases play a central role in the manifestation of the morphological and biochemical characteristics of caspase-dependent apoptosis.
Several theories have been proposed for the activation of caspases. The key fact is that caspases are activated by proteolytic cleavage of the proenzyme, commonly called zymogen, between prodomain and the p20 domain at Asp-X-sites. This site is a candidate site for caspase cleavage (Hengartner, 2000); a site at which caspases either activate by self-cleavage (autocatalytic activation) or through another previously activated caspase. This particular manner of activation has been documented for the activation of the short prodomain caspases, namely 3, 6 and 7. Another mechanism involved in the activation of caspases is through what is called “induced proximity”. This is a process whereby death receptors such as CD95 (Apo-1/Fas) aggregate and form membrane-bound signaling complexes, which then recruit several molecules of procaspases through adaptor proteins that results in a high concentration of complexed aggregates.
Death receptor-extrinsic of apoptosis pathway
Signaling of apoptosis by the tumour necrosis factor family of death receptors (CD95/Fas/Apo1, TNFR1, DR3, 4 and 5) involves ligand binding as the first step. The signaling step that follows activation of the death receptor is initiated in a sequential manner but is often preceded by trimerization of the death receptors. The cytosolic domains of the death receptor form a death inducing signaling complex (DISC) that attracts and then links up with the adaptor molecule termed FADD (Fas-associated death domain containing protein). The DISC-FADD complex through their death effector domains bind to initiator caspases (caspase-8 and -10), thereby causing the autocatalytic cleavage of the procaspase-8. The activated caspase-8, in turn activates downstream executioner caspases. These proteases cleave cellular death substrates, resulting in morphological and biochemical changes observed in apoptotic cells (Krammer, 2000 and Hengartner, 1998).
Although death receptor domains direct cells to the apoptotic pathway, in some instances they may interact with molecules that have anti-apoptotic effects. This property is shown by the receptor interacting protein, which also has the death domain that stimulates the pathways that lead to activation of NF-κB, a inhibitor of apoptosis (Ashkenazi and Dixit, 1998). Downstream effector pro-caspases such as -3, -6, and –7 are enzymically converted into active forms by initiator caspases. These downstream effecter caspases next cleave and activate substrates that cause morphological and molecular changes observed in apoptotic cells (DNA fragmentation, nuclear shrinkage, plasma membrane blebbing and other characteristics. These proteins which include the inhibitory caspace activated DNase, acinus and laminins are associated with both biochemical and morphological changes that characterize apoptosis.
Death receptor pathway
Figure 1: Illustrates the sequential events that lead to apoptosis as signaled by death receptors
Mitochondrial: intrinsic pathway
Apoptotic signals result in the formation of pores within the inner membrane (that controls the membrane potential) and swelling of mitochondria.These signals cause an increase in the permeability of outer mitochondrial membrane which results in the translocation of cytochrome c from the mitochondrion into the cytosol, thereby initiating the death program, within which the Bcl-2 family proteins play a crucial role. The mitochondrion harbours a family of pro-apoptotic proteins that play a very significant role in the demise of the cell. These include cytochrome c, Smac and apoptosis-inducing factor-1, all of which are all believed to play a pivotal role in apoptosis even though the intrinsic pathway makes use of apoptotic caspases (executioners). The transmembrane apoptosis signal triggers the transllocation of the apoptotic Bcl-2 family members, such as Bax into the mitochondrion (Salvesen, 2002). Upon the release of cytochrome c, which is intiated by Bax on receipt of an apoptotic stimulus, cytochrome c binds to the apoptosis activating factor-1 (Apaf-1), so that it may to bind and promote the activation of procaspase-9 (Salvesen, 2002; Green, 1998; Yue et al, 1999). The bound proteins, cytochrome c, Apaf-1 and caspase-9, form a molecular complex known as an apoptosome. The active caspase-9 next activates downstream executioner caspases (-3, -6 and –7) which then promulgate the death of the cell.
Intrinsic apoptotic pathway: