SARKAR, ANGSHUMAN

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AbstractSummary The role of the heme regulated eIF-2a kinase (also known as the heme regulated inhibitor, HRI) in regulating initiation of protein synthesis in reticulocytes and a few other mammalian cells is well established. HRI is a cyclic AMP-independent protein kinase. Upon activation it undergoes autophosphorylation and subsequently phosphorylates the 38 kDa a subunit of eIF-2 at Ser -51 residue (Levin et al., 1976, Ranu and London., 1976). Phosphorylated eIF-2 (eIF-2a P) binds to guanine nucleotide exchange factor (GEF) also known as eIF-2B or reversing factor (RF), and forms a stable complex, which sequesters RF. As a consequence, due to unavailability of RF, which is required for the exchange of GTP for GDP for recycling of eIF-2 initiation of protein synthesis ceases (Matts et al., 1983, 1986, Ochoa 1983 and Matts and London 1984). The activation of this kinase caused due to heme deficiency or a variety of other conditions, such as heat shock, heavy metal toxicity, treatment with N-ethylmalemine (NEM) and oxidized glutathione (GSSG), leads to phosphorylation of a subunit of eIF-2 which results in inhibition of protein synthesis (reviewed in London et al., 1987; Chen and London, 1995; Pal et al., 1996). HRI has been purified and characterized extensively from rabbit reticulocyte lysate. HRI is a dimer of 92 kDa polypeptide and it has a sedimentation co-efficient of 6.6S (Trachsel et al., 1978; Levin et al., 1980). Chen et al., carried out the first cloning of HRI cDNA, in 1991. The clone containing full length HRI cDNA has been reported to be 2700 bp long. In vitro translation of mRNA transcribed from HRI cDNA yields a 90 kDa polypeptide with eIF-2a kinase activity and is recognized by a monoclonal antibody raised against purified rabbit HRI (Pal et al., 1991). Subsequently using rat brain HRI cDNA Mellor et al., (1994) demonstrated the presence of HRI transcript in a number of other non-erythroid tissues. HRI cDNA has also been cloned from a number of species, namely, rabbit, rat, mouse, yeast, Plasmodium falciparum and Drosophila (Anand, 1999). Recently, the cDNA encoding human heme-regulated eukaryotic initiation factor -2? (eIF-2?) kinase was cloned from human bone marrow culture (Omasa et al., 2002). A novel eIF-2? kinase has also been cloned and characterized most recently from silkworm Bombyx mori (Dharmaprasad et al., 2003). Heavy metal ions such as, zinc, copper, lead are minor yet ubiquitous component in the biosphere. The heavy metal ions, AsO2-, Cd2+, Cu2+, Hg2+, Pb2+ and Zn2+ in particular, have been shown to inhibit protein synthesis and disaggregate polyribosomes, while these elicit stress response in a variety of eukaryotic cell types and tissues. Although there are some data available from in vitro studies that heavy metals induce HRI activity and thus inhibit protein synthesis (Hurst et al., 1987), not much is known on the regulation of HRI expression and its relevance in the regulation of protein synthesis in cells cultured in vitro or in organisms. Recently, the mouse HRI gene promoter has been cloned, sequenced and analyzed (Lu and Chen, 2002). The sequence details revealed that mouse HRI promoter does not contain a TATA box, rather it has a TATA less promoter with an initiator (Inr) site and three SP1 sites. Their data also suggested that the first 159 nucleotides (-159) were sufficient and necessary for the basal promoter activity. However, no data is available on the expression and regulation of HRI promoter activity particularly that of human HRI under stress conditions, namely, heat-shock and heavy metal toxicity. Investigations from our laboratory by Anand and Pal (1997, 2002) have revealed that HRI was almost undetectable in blood sample of normal rabbits and it increases almost 24-fold in the reticulocytes of rabbits during drug-as well as lead-induced anemia. Furthermore, when rabbits recovered from anemia, the quantity of HRI decreased significantly. These studies therefore indicated that a human HRI antibody-based detection kit might be useful for diagnosing human anemia and other hematological disorders. Therefore, there was a need to purify HRI from HeLa/K562 cells and prepare human HRI- specific monoclonal antibodies which may subsequently be used for preparation of a detection kit for anemia of various kind including lead poisoning in human patients. Further, these antibody probes may be useful for studying the regulation of protein synthesis in human, particularly during various hematological abnormalities. In the light of the above mentioned reports and observations attempts have therefore been made to achieve the following objectives as reported in this thesis: 1. Determine the effect of heat-shock and heavy metal toxicity (specifically lead) on the expression of HRI in human cells cultured in vitro and to study the regulation of HRI mediated protein synthesis. 2. Cloning of human HRI promoter and determine its regulation during heavy metal toxicity as well as heat-shock and also during hemin supplement. Investigation was also carried out on the role of heme in influencing the HRI promoter activity. 3. Purification of HRI from human cells and preparation of mouse monoclonal antibodies to human HRI. The thesis is divided into five chapters. The first two chapters deal with Introduction and Materials & Methodology. Chapters 3-5 deal with Results and Discussion. Chapter 3: This chapter deals with the objective no.1, i.e. to understand the effect of heat-shock and heavy metal toxicity (specifically lead) on the expression of HRI in human cells cultured in vitro and to study the regulation of HRI mediated protein synthesis. Although heavy metals, namely, cadmium, mercury and lead have been shown to inhibit protein synthesis by stimulating eIF-2a phosphorylation through activation of HRI in a cell-free reticulocyte lysate, nothing was known on the effect of heavy metals on HRI expression and protein synthesis in vivo. Our results indicated that in in vitro cultured human K562 cells, lead exposure caused not only activation of HRI but its overexpression as well. Further, the level of HRI expression and its activity during lead exposure were concentration-dependent. Interestingly, results of the time-course experiment of lead exposure indicated that a period of 8 h exposure induced the highest level of expression of HRI. On the other hand, during heat shock, an increased expression of HRI took place within an hour. These results thus suggest that lead exposure, although a type of stress is different from other cytoplasmic stresses, such as heat shock. The expression of HRI was determined after one hour of heat shock at 42.5oC in both HeLa and K562 cells. Although the level of HRI expression increased in HeLa cells to 3-fold, much less increase was observed in K562 cells. In vivo, under such stress conditions where HRI is known to interact with Hsps, Hsp90 and Hsp70 in particular (Pal, 1998, Anand and Pal, 2002), no significant change in their quantity was observed by Western blot analysis in such transient exposures. The level of p34cdc2 a protein kinase of cdk family, the master regulator of cell cycle, was also determined during lead exposure. At 500 mg/ml of lead concentration, this protein was synthesized more and showed a lower mobility than those in control (untreated) cells. This might be indicative of a change in the phosphorylation status of this protein, which is known to have a bearing on the regulation of cell division. The effect of lead acetate and heat-shock on newly synthesized proteins of K562 and HeLa cells were monitored by labeling the cells with both radioactive amino acid (35S-methionine)-labeling and non-radioactive (biotin-7-NHS-leucine)-labeling procedure. By either method, no distinguishable changes in the protein profile (newly synthesized) could be observed due to lead exposure as well as heat-shock. An attempt was made to determine if lead-induced decline in cell proliferation was caused due to apoptosis. A rather low level of apoptosis (9%) was detected and that might be contributing to the depletion of cell population at 500 mg/ml of lead concentration. However, at an organism level, it appears that apoptosis takes place in a significant way as an effect of lead exposure, particularly in the germ cells in rat testis (Adhikari et al., 2001). Chapter 4: This chapter deals with the second objective, i.e. cloning of human HRI promoter and understanding its regulation under various stress conditions. Results on the role of heme in the regulation of HRI promoter activity are also reported in this chapter. PCR primers for cloning the human HRI promoter was designed from the mouse HRI promoter sequence available in the literature (Lu and Chen, 2002). The human HRI promoter was cloned using genomic DNA from 293 cells (Human embryonic kidney cells). When the human promoter was sequenced, it revealed extensive homology with that of the mouse suggesting that HRI promoter is highly conserved between human and mouse. Human promoter was cloned in pGL-3Enhancer vector, in which the SV40 promoter and Enhancer drove the firefly luciferase gene expression. The positive clones were characterized by restriction digestion analysis. K562 cells were transfected with 10 ?g of plasmids using Lipofectamine 2000? for each experiment. The basal activity of HRI promoter was almost 20-fold higher in K562 (erythroid) cells than that in 293 (non-erythroid) cells. When the cells were subjected to heat shock (42.5?C for 1 h) and lead toxicity (100 ?g/ml for 8 h) almost 2-2.5 times increased promoter activity was observed. In order to determine the role of heme in modulating HRI promoter activity, transient transfection with HRI promoter construct followed by luciferase assay was carried out for 48 h in the presence of 10, 25 and 50 ?M of hemin. It was observed that HRI promoter activity decreased in a dose-dependent manner in hemin-treated cells as compared to that in control (untreated) cells. Our studies also revealed that hemin reduces lead acetate induced up regulation of HRI promoter activity in a significant manner. It was also very interesting that total quantity of iron concentration (when hemin was provided in culture medium) in K562 cells increased up to 25% after treatment with lead acetate. This indicated that lead toxicity induce an increased hemin uptake perhaps to overcome the stress effects and thereby maintain the global protein synthesis. Thus, hemin may be used as a stress protective chemotheraputic reagent to relieve the overall stress effects. Therefore, a direct relationship between the HRI promoter activity and hemin is established, in this investigation for the first time. Identification of transcription factors involved in these mechanisms are currently in progress. Chapter 5: This chater deals with the third objective of this thesis. Where an attempt was made to purify HRI from human cells and to raise anti-human HRI mAb. Human K562 cells grown in batches were pooled and from a total number of 1.2 x 109 cells, proteins were extracted. A total volume of 12-ml supernatant containing 270 mg of protein was used for purification of HRI. HRI was purified by the multistep procedures as described for purification of rabbit reticulocyte HRI (Trachsel et al., 1978; Chen et al., 1991). Monitoring of HRI after each step of purification was carried out by SDS-PAGE analysis as well as by determining heme-dependent eIF-2a kinase activity of the preparations as described in (Pal et al, 1991). Partially purified HRI preparation from human K562 cells was used as antigens for immunization of Balb/C mice. The sera of two immunized mice recognized one major protein band (by Western blot analysis) around 90 kDa region and also a few other protein bands of lower molecular weight. The splenocytes from these mice were used for cell fusion. Hybridoma cells secreting antibodies as detected by western blot were cloned two times by limiting dilution plating in 96 well plates in DMEM medium. Culture supernatant was tested for the presence of antibodies by immunoblot analysis. Supernatant containing mAb when used for the immunoblot, it reacted against a protein close to 66 kDa. Therefore, production of monoclonal antibody against human HRI was not successful.Thus, it appears that a preparation containing purified HRI is required since it is a very low abundant protein. The antibody, which reacted against a protein (close to 66 kD) in a variety of different cell extracts, is currently being characterized. In conclusions, results presented in the thesis describe the following unique findings. In brief, 1) For the first time it has been shown that in in vitro cultured human K562 cells, lead exposure causes not only activation of HRI but its overexpression as well. Results also indicate that the level of HRI expression and its activity during lead exposure was concentration-dependent. 2) These cell lines can be very usefully utilized as models to determine the molecular mechanism of lead toxicity, despite the differences in the response between cells and the organisms. 3) Expression of HRI may be used as an indicator of lead toxicity or similar stress. 4) HRI promoter sequences between mouse and human is highly conserved. 5) Increase in the HRI promoter activity during various cytoplasmic stresses (heat shock and lead toxicity) suggests the involvement of specific transcription factors that may be activated under these conditions, and therefore, characterization of such factors would help in understanding the regulation of HRI expression. 6) It has been shown that hemin works as a negative regulator of HRI promoter activity.

The Heme-Regulated Eukaryotic Initiation Factor 2a (eIF-2a) Kinase: Expression and Regulation of Protein Synthesis in Human Cells in vitro During Heat-Shock and Lead Toxicity

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The Heme-Regulated Eukaryotic Initiation Factor 2a (eIF-2a) Kinase: Expression and Regulation of Protein Synthesis in Human Cells in vitro During Heat-Shock and Lead Toxicity

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