Elsevier

Biochimie

Volume 82, Issue 2, February 2000, Pages 95-107
Biochimie

Review
MAPs and POEP of the roads from prokaryotic to eukaryotic kingdoms

https://doi.org/10.1016/S0300-9084(00)00383-7Get rights and content

Abstract

Methionine aminopeptidases (MAPs) play important roles in protein processing. MAPs from various organisms, for example E. coli, S. typhimurium, P. furiosus, Saccharomyces cerevisiae, and porcine have been purified to homogeneity and their MAP activities have been tested in vitro and in vivo. The DNA sequence analyses of MAP genes from the above organisms reveal sequence homologies with other prokaryotic MAPs as well as with various eukaryotic homologues of rat p67. The cellular glycoprotein, p67 protects the α-subunit of eukaryotic initiation factor 2 (eIF2) from phosphorylation by its kinases. We call this POEP (protection of eIF2α phosphorylation) activity of p67. The POEP activity of p67 is observed in different stress-related situations such as during heme-deficiency of reticulocytes, serum starvation and heat-shock of mammalian cells, vaccinia virus infection of mammalian cells, baculovirus infection of insect cells, mitosis, apoptosis, and possibly during normal cell growth. The POEP activity of p67 is regulated by an enzyme, called p67-deglycosylase (p67-DG). When active, p67-DG inactivates p67 by removing its carbohydrate moieties. Remarkable amino acid sequence similarities at the C-terminus of rat p67 with its eukaryotic and prokaryotic homologues which have MAP activities, raise several important questions: i) does rat p67 have MAP activity?; and ii) if it does have MAP activity, how the two activities (POEP and MAP) of p67 are used by mammalian cells during their growth and differentiation. In this review, discussions have been made to evaluate both POEP and MAP activities of p67 and their possible involvement during normal growth and cancerous growth of mammalian cells.

Introduction

Aminopeptidases are widely distributed in living organisms. These peptidases are classified according to their substrate specificities. One such class is the methionine aminopeptidases (MAPs) which remove N-terminal leading methionine from nascent peptides during the early stages of protein synthesis. The rate of protein synthesis in mammals is largely regulated by the phosphorylation of the α-subunit of eIF2. When phosphorylated at the α-subunit, eIF2 is inactive in the initiation of protein synthesis. A cellular glycoprotein, p67, keeps eIF2 in an active form by protecting its α-subunit from phosphorylation by eIF2-kinases (POEP activity). The eukaryotic homologues of rat p67 that include yeast, mouse, and human p67, have been cloned. The cDNA sequence analyses of p67 from the above species indicate that it may also have MAP activity. It is very important to understand the physiological significance of the two activities (POEP and MAP) of p67 during mammalian cell growth.

Section snippets

Mammals

The components (protein factors, ribosomes, and tRNAs) required for protein synthesis and its overall mechanistic steps (initiation, elongation, and termination) both in bacteria and mammals are remarkably conserved. In addition to several cofactors, the presence of other enzymes, for example aminopeptidases, is detected almost in all organisms including bacteria to human. Among several aminopeptidases, MAP drew more attention because of the unique role of methionine in the initiation of

Approaches towards purifying recombinant human MAP and testing its activity

The protein encoding open reading frame from human MAP cDNA was fused with gene encoding glutathionine-S-transferase. The GST-fusion protein was purified from E. coli, and later used to test for MAP activity. In a typical MAP assay, the substrate Met-Gly-Met-Met was first labeled with a fluorescent derivatizing reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, later incubated with the enzyme (GST-MAP), and the products (Met and Gly-Met-Met) were separated by high-performance liquid

Future questions regarding biochemical characteristics of MAPs from various organisms

Significant progress has been made in identifying, purifying, characterizing, and cloning MAP genes from various organisms. Crystal structures of prokaryotic and eukaryotic MAPs are also determined. Still, satisfactory answers to several questions have not been obtained. The issue of ribosome binding of MAPs from various organisms remains unsolved, although the observation that the initiator methionine is removed from the nascent peptide of ≈ 20 amino acids long, makes it likely that MAPs

Regulation of protein synthesis initiation in mammals

Initiation of protein synthesis plays a central role in gene expression. A precise balance between proliferation and apoptosis of mammalian cells is regulated at the level of translation of the mature messages. Translation of the messages is largely regulated at the level of the formation of the initiation complex with eukaryotic initiation factor 2 (eIF2), Met-tRNAi, and 40S ribosome. This step plays a significant role in the initiation of peptide chain synthesis [63], [64]. However, this

Positive and negative regulations of p67 activity to protect the α-subunit of eIF2

When mammalian cells in culture were serum starved, the expression of p67 was inhibited significantly. Expression and POEP activity of p67 are however induced during mitogenic stimulation [79], [80], [81]. Similarly, the expression and POEP activity of p67 are upregulated at the later stages of heat shock of mammalian cells [76]. These inductions of the levels of the message and p67 protein are very reminiscent of the expression of the immediate early genes, for example c-fos and c-jun, during

Cloning of a cDNA corresponding to rat p67

In 1993 Wu et al., reported the cloning of a cDNA for rat p67 [84]. Based on the amino acid sequences corresponding to peptides that were obtained by digesting purified rabbit p67 with either trypsin or cyanogen bromide, degenerate oligonucleotides were synthesized and used as primers to perform a RT-PCR reaction in total cytoplasmic RNA isolated from rat hepatoma tumor cells (KRC-7). A ≈ 329 bp DNA fragment was obtained and this DNA fragment was later used as a probe to screen a cDNA library

Does rat p67 have MAP activity?

Our first attempt to test for the MAP activity of the cloned rat cDNA for p67 was unsuccessful. In this attempt we made a GST-fusion chimeria with rat p67 that was purified from E. coli cells. Although most of the GST-fusion protein was degraded, whatever was left in the sample did not show any detectable MAP activity (unpublished). The degradation of GST-p67 (rat) could not be prevented by using excess protease inhibitors during the purification steps. To overcome this problem of degradation

Does rat p67 bind to ribosome?

To test for ribosome binding of rat p67, an indirect approach was taken. The entire coding region of rat p67 was fused to the gene encoding green fluorescence protein (GFP). The fusion chimera was constitutively expressed in rat KRC-7 cells. Examination of the transfected cells under the microscope did not show any green signal from the nucleolus - the site for ribosome biogenesis. However, pronounced green signals were detected mostly in the cytoplasm including the Golgi apparatus - the site

1Role(s) of p67-DG in the regulation of p67 activity

The unusual post-translational modification, such as addition of O-linked GlcNAc moieties on p67, may be a major determinant in regulating its activity. The presence of an enzyme, p67-DG, in rabbit reticulocytes has been detected and reported as early as 1993 [71]. This enzyme when active, removes the glycosyl residues from p67, making it inactive in POEP activity. During stress-related situations such as virus infections, especially vaccinia virus infection of mammalian cells and baculovirus

Acknowledgements

In this review I have tried hard to bring all the wonderful work done in this field by excellent laboratories. I would also like to apologize to other investigators whose important contributions I have missed unintentionally and were not included here. I would like to thank Dr. J. Wang from Abbott Laboratories for testing his-tagged rat p67 for its MAP activity, and letting me cite his unpublished observation. I am very grateful to Professor John W.B. Hershey (University of California, Davis,

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