INTRODUCTION

Leukemia is a malignant hematological disorder characterized by proliferation of abnormal white cells that infiltrate the bone marrow, peripheral blood and other important organs. Leukemia arising from myeloid cells is known as Myeloid Leukemia which may either be chronic myeloid leukemia (CML) and/or acute myeloid leukemia (AML). AML is a complex disease caused by mutations, deregulated gene expression and epigenetic modifications leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Several important molecular markers have been discovered in AML to better characterize patients. C/EBPα is an important regulator of Granulopoiesis. Several groups have reported mutations in the C/EBPα gene in a subset of patients with AML presenting with normal karyotypes. A significant percentage of AML patients without chromosomal translocations have demonstrated abnormalities in C/EBPα protein or function, suggesting that loss of transcriptional control is a common mechanism of leukemogenesis. Even in the setting of other proleukemogenic genetic abnormalities, such as the (8; 21) translocation, C/EBPα has been demonstrated to be aberrantly regulated, in this case by down regulation of expression. Functional alterations of C/EBPα in AML include mutations of the C/EBPα gene and deregulated expression of C/EBPα by chromosomal translocations. Further, post-transcriptional or post-translational suppression of C/EBPα has been demonstrated to be involved in hematopoietic malignancies. AML is also characterized as, a malignant disease of hematopoietic system in which cells accumulate in an undifferentiated state due to mutations that prevent their normal differentiation and allow undifferentiated cells to survive and proliferate. The molecular changes that occur in AML usually lead to either abnormal cell proliferation (FLT3 and Ras mutations) or block in differentiation (AML1/ETO, PML/RAR alpha, C/EBPalpha mutations) or suppression of apoptosis (bcl2 overexpression). Despite of block in differentiation, native AML cells often show some morphological signs of differentiation that allow a classification into different subsets, and further differentiation may be induced by exposure to various soluble mediators, e.g., all trans-retinoic acid (ATRA) and several cytokines in t(15;17).

All-trans retinoic acid (ATRA) is a derivative of vitamin A and it affects cellular development including haematopoiesis, in particular granulocytic differentiation. ATRA could induce a dose-dependent differentiation of HL-60 promyeloblasts to mature, functioning neutrophils. ATRA induces growth inhibition, differentiation, and apoptosis in cancer cells, including acute promyelocytic leukemia (APL). In APL, expression of promyelocytic leukemia protein retinoic acid receptor (PMLRARα) fusion protein, owing to the t (15; 17) reciprocal translocation, leads to a block in the promyelocytic stage of differentiation.

E3 Ubiquitin ligases are a large family of proteins engaged in the regulation of protein turnover and activity through a multistep proteolytic cascade, called ubiquitination. Ubiquitination of a target protein involves 2 distinct steps: covalent attachment of multiple ubiquitin molecules to the protein substrates and degradation of the polyubiquitylated proteins by the 26S proteasome system. The first step is mediated by a cascade of 3 enzymes: ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) [1, 2]. Ubiquitin is a 76-amino acid polypeptide that is highly conserved among eukaryotic organisms. It is first activated in an ATP-dependent manner via binding to E1 through a thioester bond between a cysteine residue at the active site of E1 and the C-terminal glycine (G76) of ubiquitin. Activated ubiquitin in an E1-ubiquitin complex is then transferred to E2, which also participates in the formation of a thioester bond between its active site cysteine residue and the G76 of ubiquitin. Finally, ubiquitin is covalently attached to the target protein through an is opeptide bond between the G76 of ubiquitin and the ε-amino group of an internal lysine residue of the target protein, in a reaction catalyzed by E3 ligase. Subsequent to the linkage of ubiquitin to the target protein, a polyubiquitin chain is formed in which the C-terminus of each ubiquitin moiety is linked to a specific lysine residue (most commonly Lys48) of the previous ubiquitin to form K48-linked polyubiquitylated conjugates which are rapidly recognized by the 19S regulatory subunit of the 26S proteasome and degraded by the 20S core particle [1-3].

There are approximately 600 E3 ligases in the human genome that can be classified into 3 major types the N-end rule ubiquitin ligases; HECT-type; and the RING-type, on the basis of domain structure and substrate recognition[1].

The N-end rule ubiquitin E3 ligases target protein substrates bearing specific destabilizing N-terminal residues, including Arg, Lys, His (type I) and Phe, Trp, Leu, Tyr, Ile (type II)[1].

The second type HECT (homology to E6AP C-Terminus) E3-ubiquitin protein ligases, found from yeast to humans range in size from 80kDa to more than 500kDa. They are characterised by the HECT domain, a C-terminal region of approximately 350 amino acids in length with significant similarity to C-terminus of E6AP. Unlike RING E3s which act as scaffolds facilitating interaction between E2s and substrates, HECT E3 ligases form an intermediate thioester bond with the ubiquitin C-terminus through an evolutionarily conserved cysteine residue before catalyzing substrate ubiquitination. Hence, HECT E3s play a direct catalytic role in the final attachment of ubiquitin moieties to target proteins. The N-terminus is highly variable and may be involved in substrate recognition. On the basis of distinct amino acid sequence motifs within the N-terminus, human HECT E3s can be classified into 3 sub-families: HECT E3s with RLDs (RCC1-like domains, termed as HERC (HECT and RCC-1like domain E3s), HECT E3s with WW domains (called Nedd4/Nedd4- like E3s), and HECT E3s that neither contain RLDs nor WW domains (called SI(ngle)- HECT E3s). E6AP, the prototype of HECT E3 family belongs to the third sub-family of HECT E3 ligases [3-5].

The third and the largest type of E3 ligase is the RING (Really Interesting New Gene) family. RING-based E3 ligases are specified by over 600 human genes surpassing 518 protein kinase genes. These are characterised by a classic C₃H₂C₃ or C₃HC₄ RING finger domain with a characteristic linear sequence Cys-X2-Cys-X9-39-Cys-X1-3-His-X2-3-Cys/His-X2-Cys-X4-48-Cys-X2-Cys, where X can be any amino acid. The RING domain provides a docking site for the E2 enzyme, which mediate transfer of ubiquitin to the substrate, facilitating assembly of mono- or polyubiquitylated conjugates via different lysine residues of ubiquitin. The resulting modifications have a diverse range of biological functions, from proteasome-dependent proteolysis (Lys48- and Lys 11-linked polyubiquitin) to post-translational regulation of protein function, structure, assembly, and/or localization (Lys 63 and other linkages)[1, 6].

E3 ligases can also be classified into single subunit E3s (e.g. Mdm2, Cbl) and multi-subunit complexes (APC, SCF). E3 enzymes bind their target substrates through various protein-protein interaction domains (e.g. WD 40 repeats). However, for substrate recognition post-translational modifications such as phosphorylation or proteolytic cleavage are required[7]. The modified motif in the substrate is called degron. There are many different types of degrons (e.g. phosphodegron, PEST). Once modified, a degron in a substrate might be recognized by a specific E3 ligase, which forms the basis for its subsequent ubiquitination[8].

Through ubiquitin-mediated covalent modification of diverse range of cellular proteins, E3 ubiquitin ligases regulate several cellular functions or biological processes such as cell cycle progression, Oncogenesis, signal transduction, transcription regulation, DNA repair, endocytois, transport and development via proteolytic or non-proteolytic mechanisms [2, 9].

A direct molecular link between cell-cycle control, Oncogenesis and E3 ubiquitin ligase activity was supported by several clinical findings and wealth of experimental data on E6AP, SKP2 and FBW7, Nedd 4-1, Pirh2, CDC20, CDH1 and also on other E3 ligases [3, 10, 11].

Understanding the physiological role of E6-AP, the founding member of the HECT E3 family, is of interest because inactivation of UBE3A gene encoding E6AP has been associated with Angelman Syndrome, a hereditary neurological disorder. Moreover, in the case of cervical cancer, the E6/E6-AP complex not only targets p53 for ubiquitin-mediated degradation, but also targets other proteins, which is necessary for HPV-induced cervical carcinogenesis[12]. E6-AP forms a stable complex with the adaptor protein E6. The dimeric complex binds to and targets p53 for ubiquitin-mediated proteolysis, thus eventually interfering with the negative growth regulating activities of this tumor suppressor protein [13-15]. In addition, the expression of E6-AP protein is decreased in human invasive breast and prostate cancers compared with their adjacent normal tissues. This down-regulation of E6-AP is accompanied by the elevation of ER in breast and AR in prostate carcinomas[16]. Furthermore, in vivo data from E6-AP-knockout animals indicated that the expression levels of ER and AR are increased in E6-AP-null mammary and prostate glands, respectively, when compared with that of normal control animals, suggesting that E6-AP modulates the protein levels of ER in breast and AR in prostate glands [17].

E6AP, a 100-kDa cellular protein is a member of functionally related E3-ubiquitin-protein ligases defined by the domain homologous to the carboxy terminus hect domain.²⁰ E3 ligases ubiquitinate and degrade several regulatory proteins including p53, p27, promyelocytic leukemia retinoic acid receptor α and others, which serve as tumor suppressors and cell-cycle inhibitors.

E6AP promotes C/EBPα ubiquitination leading to its proteasome-mediated degradation and thus functional inactivation. E6AP negatively regulate Granulopoiesis by targeting C/EBPα for degradation via ubiquitin proteasome pathway.

Promyelocytic leukemia tumor suppressor (PML) has been recently identified as a target of catalytically active form of E6AP. PML tumor suppressor is essential for the formation of PML nuclear bodies. Recent studies have implicated role of PML and PML nuclear bodies in the regulation of growth inhibition, senescence and apoptosis. PML is down regulated in multiple human cancers and experimental data has correlated reduced PML activity and expression to E3 ubiquitin ligase activity of E6AP, regulating protein turnover and activity[18].

Recently, Annexin I involved in the inhibition of cell proliferation, regulation of cell differentiation, anti-inflammatory effects, cell death signalling, carcinogenesis has been identified as a novel target of E6AP in addition to classical substrates, including p53 tumor suppressor, PDZ domain-containing protein scribble, a transcriptional repressor of the gene encoding hTERT[19]. In addition, studies have also implicated the role of E6AP ubiquitin ligase activity in ubiquitin-dependent degradation of Peroxiredoxin1 and presumably open avenues to investigate the functional link between lack of E6AP expression and stability of Peroxiredoxin 1with regard to the pathogenesis of Angelman syndrome[20].

p53 is targeted for proteasomal degradation by mdm2 which is a p53 target gene containing E3 ubiquitin ligase activity[21]. While mdm2 targets p53 for degradation, mdm2 is self -ubiquitinated and degraded. Cyclin dependent kinase inhibitor p21waf/cip, another p53 target gene, is degraded by proteasome and GSK3 (glycogen synthase kinase 3) mediated phosphorylation [22]. Rb (Retinoblastoma) protein is a tumor suppressor and negatively regulates G1/S transition by interacting with E2F transcription factor. Rb protein is degraded in an ubiquitin dependent manner [23]. In addition, free E2F is also degraded in ubiquitin dependent manner by the 26S proteasome. Thus, collectively HECT domain containing E3 ligases are important for homeostasis of protein levels and defects in their function may lead to various diseases including cancer.

Thus, wealth of experimental data and clinical findings identifying many substrates targeted by E3 ubiquitin ligases, indicate that the deregulation of Ubiquitin proteasome system in cell cycle control is tightly linked to malignancies and tumorigenesis.

Due to the above relevance and role of E6AP in malignancies and tumorigenesis

• The project is based on the expression, purification and validation of GST tagged protein that is GST- E6AP.
• The current study includes
• Cell culture: HL-60 cells, a human promyelocytic leukemia cell line. HL-60 cells treated with 1uM ATRA for 0,24 and 48 hours.
• GST- E6AP Protein expression and purification:
• GST-E6AP Pull down:

Objectives:

1) Expression of GST and GST-E6AP plasmids in BL21 strain of E.Coli

2) Purification of GST and GST-E6AP proteins from BL21 strain of E.Coli

3) Validation of expression through western blotting

4) To detect GST-E6AP protein interaction with whole cell lysates of HL-60 cells treated with 1μM ATRA for 0, 24 and 48 hrs

GOMTI NAGAR EXTENSION, LUCKNOW