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CELL BIOLOGY Accomplice Fingered In Cholera ToxicityThe MO—Disulfide Exchanges—Also May Be Used for Other Jobs Inside the Cell The effects of cholera, one of the most rapidly fatal diseases known, are largely due to a toxin secreted by the bacterium Vibrio cholerae that acts on intestinal epithelial cells. The cholera toxin is a deft invader, making its way backwards through the cell's own pathways for secreting and degrading proteins—endocytosing into the cell, moving through the Golgi complex to the endoplasmic reticulum, and finally into the cytosol. Once there, the toxin initiates a signaling cascade that results in chloride channels opening at the cellular membrane, causing the massive loss of water and the diarrhea associated with cholera.
The proposed chaperone mechanism of protein disulfide isomerase (PDI) is remarkably similar to known chaperones powered by ATP. In the figure at top, the chaperone Hsp 70 (crescent) binds to and releases a peptide as its binding affinity is changed by ATP hydrolysis. Below, the same action in PDI is governed by the formation and reduction of disulfide bonds. Illustration courtesy of Billy Tsai Stealth OperationA study published in the March 23 Cell has revealed one of the ways that cholera toxin hijacks some of the cell's own machinery to get into the cytosol. In uncovering part of the toxin's trail, a team led by Tom Rapoport, Howard Hughes investigator and HMS professor of cell biology, has also identified a novel mechanism for chaperoning the unfolding of proteins, one that may have broader implications for protein transport within cells.Much of how cholera toxin carries out its cellular coup has been worked out, but how it is able to get from the lumen of the ER to the cytosol remains a mystery. Like a prisoner escaping from a locked cell, the toxin slips through the ER membrane even though a protein generally cannot cross a membrane in its folded form. The team reasoned the toxin must have an accomplice, a protein within the ER that functions as an accessory. "We assumed that there has to be some enzyme in the ER lumen that can unfold the cholera toxin," said Billy Tsai, HMS research fellow in cell biology and the paper's first author. "Once it unfolds, it can travel across the membrane and get refolded."
Tom Rapoport (left) and Billy Tsai have helped uncover how cholera toxin's path to the cytosol is aided by one of the cell's own enzymes. Photo by Steve Gilbert The team, which included Wayne Lencer, HMS associate professor of pediatrics, and research fellow Chiara Rodighiero, both at Children's Hospital, used a biochemical fractionation of proteins of the lumen to weed out the one able to unfold the toxin. The culprit they found is a protein known for its ability to form and shuffle disulfide bonds, protein disulfide isomerase, or PDI. This molecule has two cysteines that can either exist as two reduced sulfhydryl groups or oxidized and paired in a disulfide bond. PDI can cycle back and forth between these two conformations by interacting with the cysteines of other proteins, and in doing so reduces or oxidizes them. Second Power SourceThe team found that PDI's ability to bind to the toxin depends on its redox state—binding to and unfolding the toxin when reduced, releasing it when oxidized. They believe that PDI acts as a new kind of protein chaperone that helps to unfold the toxin and allows it to pass through the channels of the ER membrane and into the cytosol. Chaperones that regulate the folding and unfolding of proteins function like clamps, binding to a protein at an open pocket that then clamps down before releasing it. The opening and closing of the binding site is generally driven by ATP hydrolysis. The team believes that PDI performs a remarkably similar activity, powered by disulfide exchanges rather than ATP.In identifying PDI's role in smuggling the toxin to the cytosol, the team also offers a solution to a puzzling aspect of the toxin's path. The periplasm of bacteria and the lumen of the ER are generally thought to be analogous, with similar oxidative environments that promote disulfide bond formation in proteins. But cholera toxin presents a paradox. "The question is, if two compartments are analogous," said Tsai, "why is it that in one compartment you have complete assembly, folding, and oxidation of the toxin, and in the other you have the complete opposite? It gets unfolded, it gets disassembled, and it gets reduced." The difference seems to lie in the toxin itself. After the bacterium secretes the toxin, it makes one final adjustment: it nicks a peptide bond between two of the toxin's subunits, leaving only a disulfide bond connecting the two pieces. This nicking, which is probably carried out by secreted proteases outside the bacterium, may render the toxin susceptible to being reduced and unfolded by PDI. The team engineered a mutated, "non-nickable" version of the toxin and found that it was not able to inflict its characteristic damage to epithelial cells. Day JobCholera toxin has been known to hijack the cell's own protein shuttling systems, so it makes sense that PDI may have normal chaperone functions in the ER. Rapoport's lab, which studies how proteins get transported across the ER membrane, is interested in the possibility that PDI is involved in recognizing and ejecting proteins that could not be folded properly in the ER to be degraded in the cytosol. Proteins that are taken into the ER are accompanied by signal sequences, but it is unknown how transport in the reverse direction is triggered. "The question is, how is this unfolded state recognized and how are those proteins selected and distinguished from those proteins that are not unfolded?" Rapoport asks.PDI's method of disulfide bond swapping offers a system that can carry out several functions by moving electrons and changing binding properties, similar to ATP-driven mechanisms. Jonathan Beckwith, the American Cancer Society professor of microbiology and molecular genetics at HMS, who studies the proteins in E. coli analogous to PDI, said, "This paper shows, in a dramatic way, just how flexible these proteins are," with their ability to create, cleave, and shuffle disulfide bonds in different environments. "In this case, PDI clearly does all three at different times." Disulfide bonds previously had been thought to form or be reduced spontaneously in response to their environment. The idea that proteins actively use these bonds to perform tasks within the cell is just catching on. —Courtney Humphries |
