EDITORS: The research reported in this release will be published in two professional journals (publication dates of June 15, 1997). The four articles (two in each publication) were refereed by the editors of each publication. They can be contacted for comments on the significance and implications of Dr. Gupta's findings. The authors and titles of each paper appear at the end of this news release.
The editors are: J. Thomas August, Archives of Biochemistry and Biophysics, (410) 955-3985 (for virus papers), and J.B. Lingel, Journal of Biological Chemistry, (513) 558-5324 (p67 transcription)
Lincoln (Neb.) - June 14, 1997 - A tiny complex of proteins and carbohydrates which stands guard over the production of proteins in animal cells "raises intriguing possibilities" for its use as an antiviral agent, a University of Nebraska-Lincoln scientist has found.
Naba Gupta, W.W. Marshall Professor of Biotechnology, and his research team summarized their research and its applications as a means of overcoming viral infections in two papers to be published June 15 in Archives of Biochemistry and Biophysics. In these articles, Gupta and his associates describe in detail their research involving the glycoprotein complex, labeled p67 on the basis of its weight in kilodaltons (one dalton is approximately equal to the weight of a hydrogen atom).
First, they recap how p67 is regulated in normal animal cells and then summarize their observations of the glycoprotein's behavior in cells infected with a virus, where they found that the level of p67 correlates directly with the cells' resistance to the virus.
Gupta discovered p67 more than a decade ago while studying a peptide initiation factor known as eIF-2. In 1972, Gupta's laboratory recognized eIF-2 as the initiator of protein synthesis in animal cells. When allowed to carry out this function, the specific genes needed to produce a hormone, a certain tissue or a specific organ are assembled and synthesis is carried out.
Protein synthesis is a highly regulated process in which phosphorylation, or inactivation of eIF-2 is a key regulatory mechanism. The breakup of the peptide chain is not, however, a single, isolated operation. A number of conditions, all with their own regulatory mechanisms, must be present in order for eIF-2 to start protein synthesis and a number of things have to happen to shut the process down. Much effort by Gupta's team was aimed at determining the conditions under which eIF-2 is allowed to initiate protein synthesis and the conditions that prevail when protein synthesis is forced to a halt.
When he began his studies, Gupta already knew that there were certain enzymes in cells called kinases that had the potential to "phosphorylate" eIF-2, rendering it inactive and thereby inhibiting protein synthesis. Accepted thinking was that the eIF-2 kinases were inactive in cells until it was time for protein synthesis to stop. Then the heretofore dormant kinases were activated somehow.
Gupta found that the kinases were never inactive. Instead, it was found, another substance, the p67 discovered in Gupta's laboratory, held the kinases in check while eIF-2 played out its initiating role in the protein synthesis process.
Much of Gupta's research team's efforts over the past decade have been devoted to providing evidence that p67 binds to eIF-2 to prevent phosphorylation by the kinases, thereby promoting protein synthesis. In so doing, they also found that the converse was true - when protein synthesis slowed, the kinases gained an upper hand and began phosphorylating eIF-2.
The process is much like a battle between two armies, in which first one of the combatants, p67, has the upper hand but eventually the tide turns. Somehow, p67 loses in numerical superiority as its phosphorylating opponent gains the upper hand. Thus, p67's defensive perimeter is breached, and eIF-2 kinases fall upon the eIF-2 initiation factor, deactivating it and forcing protein synthesis to a halt..
It was this activity that led Gupta to suspect that if p67's effectiveness was due to a numerical superiority in the battle with kinases, then wouldn't p67 also serve as a biological means of combating viral infections?
In cells infected with a virus, he said, the virus tends to increase the activity of the eIF-2 inhibiting kinases. As these kinases carry out their inhibiting function, the virus is able to take over a cell's reproductive machinery and put it to work reproducing the virus itself. Therefore, Gupta speculated, wouldn't encouraging the growth of p67 to hold eIF-2 kinases in check be a useful means of preventing the virus from taking over the cell's reproductive machinery?
Establishing p67 as an anti-viral agent as been a long, arduous process carried out over the past several years in Gupta's laboratory.
First, as he described in the papers, he followed two distinct mechanisms by which p67 activity is regulated in animal cells.
One regulatory mechanism is at the gene transcription level, where, at the resting level, cells shut off transcription of p67. At this level, Gupta said it doesn't take much to alter the protein synthesis rate in a cell. To restore p67 synthesis in resting cells, he added a mitogen, a substance that stimulates p67 transcription and subsequent protein synthesis.
Gupta said this finding was of great importance, for it "illustrates one of the most unique characteristics of any reported protein." Alteration of a single protein such as p67 alters the total protein synthesis rate in cells."
The other regulatory mechanism that Gupta said animal cells "apparently use for irreversible loss of cellular protein synthesis activity" is through activation of a protein that Gupta labeled p67-DG, short for p67-deglycosylase. This protein acts on p67 and deglycosolates it. This inactivates p67, leading the way to eIF-2 phosphorylation and inhibition of proteinsynthesis.
The p67-DG is activated "under certain physiological conditions," Gupta said. "In our laboratory, we have found that this mechanism is operative during viral infection and is responsible for shut-off of host-protein synthesis."
Gupta used two different viruses in his studies. One was a vaccinia virus and the other a baculovirus. Studies elsewhere have shown that in vaccinia virus infections, these viruses increase eIF-2 phosphorylation and inhibit host protein synthesis. In Gupta's laboratory, it was observed that after either vaccinia or baculovirus infection, a p67 deglycosolase is activated, and p67 can no longer protect eIF-2 from eIF-2 kinases. Cellular protein synthesis is thereby inhibited.
"An important observation to be made," Gupta said, "is that the p67 level in cells competes with viral concentrations. One of the cell lines we studied, (KRC-7, a human hepatoma cell line), contains unusually high levels of p67 compared to several other cell lines.
"While all of the cell lines are resistant to vaccinia viral infection at low viral concentrations", Gupta said, "the p67 level can be altered by growing the cells under different physiological conditions.
"This raises the intriguing possibility that p67 can be used as an antiviral agent," Gupta said.
Gupta's research team, in addition to showing p67 to be an antiviral agent, also showed that p67 level inhibits deglycosylation of p67 by p67-DG.
The researchers found that genetic expression of the p67 gene could be "significantly enhanced" by the addition of the blood crystal hemin to virus-infected insect cells. "We suggest," the researchers said, "that hemin prevents the conversion of an active p67-DG into an active form."
And, with p67-DG remaining inactive and p67 gene expression enhanced, the level of p67 in a cell increases, thereby increasing its ability to act as an anti-viral agent.
The papers published in the Archives of Biochemistry and Biophysics are: (1) "Viral Infection: Hemin Induces Overexpression of p67 As It Partially Prevents Appearance of an Active p67 Deglycosylase in Baculovirus-Infected Insect Cells," by Dababrata Saha, Shiyong Wu, Avirup Bose, Nabendu Chatterjee, Arup Chakraborty. Madhumita Chatterjee, and N. Gupta; (2) "Viral Infection: Regulation of Protein Synthesis during Vaccinia Viral Infection of Animal Cells," by Bose, Saha, and Gupta.
Two other papers accepted for publication in the Journal of Biological Chemistry also deal with Gupta's p67 research results. One is titled "p67 Transcription Regulates Translation in Serum-starved and Mitogen-activated KRC-7 Cells," Swato Gupta, Bose, N. Chatterjee, Saba, Shiyong Wu and N. Gupta. The other article is titled "Cloning and Characterization of the Promoter Region of a Gene Encoding a 67kDa Glycoprotein," N. Chatterjee, Cheng Zout, John Osterman and N. Gupta).