Johns Hopkins scientists have discovered internal "shipping labels"that allow -- and perhaps force -- hundreds if not thousands ofproteins to get to the surface of cells and stay there. Two naturalproteins that use one of these "tags" are the ion channel that letsheart cells contract on cue, and the docking point that allows HIV, thevirus that causes AIDS, into cells, the researchers discovered.
Because proteins on the cell surface are "lock-on" sites for drugsand other molecules, as well as triggers of immune reactions, thefindings, described in the Sept. 11 advance online publications ofNature Cell Biology, might revolutionize efforts in drug and vaccinedevelopment, says the Hopkins team.
"A typical step in drug development is to get cells in a dishto express the protein you want to target with drugs, and then to testthousands of molecules to see which ones interact with the protein andhave the effect you want," says the study's senior author, Min Li,Ph.D., a professor of neuroscience at the High Throughput BiologyCenter of the Johns Hopkins' Institute for Basic Biomedical Sciences.
"But if you can't get the protein to the cell surface, youcan't use this screening technique. If we can force proteins to thecell surface, we can overcome obstacles that have prevented laboratorystudy of some really important proteins," says Li. The application ofthese surface tags to force protein transportation to the cell surfaceis the subject of a Patent Cooperation Treaty (PCT) patent applicationsubmitted by The Johns Hopkins University.
From among 25 billion randomly created,eight-building-block-long protein bits, postdoctoral fellow SojinShikano uncovered 65 that forced a normal protein to leave the cell'sprotein-building factory and go to the cell surface. By searchingsequences of known human proteins, the researchers then identifiedthose that use variations of the most potent tag they'd found, dubbedSWTY, shorthand for the four building blocks at the end of its proteinsequence -- serine, tryptophan, threonine and tyrosine, in that order.
"This particular tag and its closest relatives actually marknormal proteins for delivery to the cell surface," says Li. "In somediseases, a protein that should be on the cell surface isn't, and inthe lab, sometimes it's proven impossible to get a protein to the cellsurface in order to study it. The tags we've found might help us forceproteins to the surface, which offers real hope for overcoming thesehurdles."
Laboratory studies in which the tags might be used to force aprotein of interest to the cell surface are likely to be widely usedfairly quickly, but Li cautions that any potential clinicalapplications will require understanding exactly how the tag helps theprotein's transportation.
Among the "problem proteins" are those that detect odors inthe nose, and the protein that's faulty in cystic fibrosis. Being ableto force these to the cell surface in laboratory dishes might enableidentification of more potent scents or ways to help people who can'tsmell, or help uncover new strategies for treating CF.
Although many scientists would say that failure to get theseproteins to the cell surface means the proteins weren't assembledproperly in the cell, Li says that how and where proteins are made hasa lot to do with the difficulties researchers have had.
For one, proteins are made deep inside the cell; the geneticinstructions for building proteins are in the cell nucleus, andproteins are assembled in a nearby "factory" in the cell. Also,scientists have long known that proteins prefer to stay put in thisfactory, the endoplasmic reticulum, unless they contain specifictransportation instructions, much like an internal shipping label.
To figure out what tiny sequences might label the protein fordelivery to the cell surface, Shikano added randomly generatedeight-building-block long tags onto one end of a particular protein. Hethen evaluated whether the protein ended up on the cell surface insteadof remaining inside the cell. The researchers found three major classesof such tags, grouped according to similarities in their sequences ofbuilding blocks, and delved into the most potent of them.
By using a computer program developed by graduate student BrianCoblitz to probe proteins' sequences, the researchers discovered that,by fairly stringent criteria, roughly 4 percent of all human proteinscontain SWTY or a very close relative. The eight-block-long tag itselfis part of the so-called C-terminal end of these proteins, and itsexistence helps explain why some engineered proteins don't go wherethey're supposed to go, Li says.
"If you remove a small part of the very end of a protein, itseems unlikely to disrupt how the rest of the protein folds in athree-dimensional structure, but that's what most scientists think goeswrong if a protein doesn't go to the surface," says Li. "But now weknow the problem might just be a faulty transportation signal."
Given that proteins can be thousands of building blocks long,the final eight building blocks may not seem to be very important. ButLi chose this size to study in part because naturally occurringproteins were already known to use similar-size bits for recognitionand signaling.
"The immune system uses ones that are seven to nine blocks longto identify viral proteins or other immune triggers," explains Li.
Also, the number of possible combinations of eight-block-longprotein segments provides a "reasonable number" to sort through -- 25billion or so -- given today's high throughput technologies. To make iteven easier, Shikano developed a system that would separate the wheatfrom the chaff before the analysis began -- if the protein wasn't takento the cell surface by the tag, the cell died.
"If the protein went to the cell surface, the cell was in themix, and if the cell wasn't there to be analyzed, we knew we didn'twant it anyway," says Li.
Authors on the paper are Shikano, Coblitz, Sun, and Li. Theresearchers were funded by the National Institute of General MedicalSciences, the National Institute of Neurological Diseases and Strokeand the American Heart Association.
On the Web:www.nature.com/ncb/
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