Johns Hopkins scientists have discovered internal
"shipping labels" that allow — and perhaps force
— hundreds if not thousands of proteins to get to the
surface of cells and stay there.
Two natural proteins that use one of these "tags" are
the ion channel that lets heart cells contract on cue, and
the docking point that allows HIV, the virus that causes
AIDS, into cells, the researchers discovered.
Because proteins on the cell surface are "lock-on"
sites for drugs and other molecules, as well as triggers of
immune reactions, the findings, described in the Sept. 11
advance online publication of Nature Cell Biology,
might revolutionize efforts in drug and vaccine
development, the Johns Hopkins team says.
"A typical step in drug development is to get cells in
a dish to express the protein you want to target with
drugs, and then to test thousands of molecules to see which
ones interact with the protein and have the effect you
want," said the study's senior author, Min Li, a professor
neuroscience at the High Throughput Biology Center of
the Johns Hopkins Institute for Basic Biomedical
"But if you can't get the protein to the cell surface,
you can't use this screening technique. If we can force
proteins to the cell surface, we can overcome obstacles
that have prevented laboratory study of some really
important proteins," Li said. The application of these
surface tags to force protein transportation to the cell
surface is the subject of a Patent Cooperation Treaty
patent application submitted by The Johns Hopkins
From among 25 billion randomly created,
eight-building-block-long protein bits, postdoctoral fellow
Sojin Shikano uncovered 65 that forced a normal protein to
leave the cell's protein-building factory and go to the
cell surface. By searching sequences of known human
proteins, the researchers then identified those that use
variations of the most potent tag they'd found, dubbed
SWTY, shorthand for the four building blocks at the end of
its protein sequence — serine, tryptophan, threonine
and tyrosine, in that order.
"This particular tag and its closest relatives
actually mark normal proteins for delivery to the cell
surface," Li said. "In some diseases, a protein that should
be on the cell surface isn't, and in the lab, sometimes
it's proven impossible to get a protein to the cell surface
in order to study it. The tags we've found might help us
force proteins to the surface, which offers real hope for
overcoming these hurdles."
Laboratory studies in which the tags might be used to
force a protein of interest to the cell surface are likely
to be widely used fairly quickly, but Li cautions that any
potential clinical applications will require understanding
exactly how the tag helps the protein's transportation.
Among the "problem proteins" are those that detect
odors in the nose, and the protein that's faulty in cystic
fibrosis. Being able to force these to the cell surface in
laboratory dishes might enable identification of more
potent scents or ways to help people who can't smell or
help uncover new strategies for treating CF.
Although many scientists would say that failure to get
these proteins to the cell surface means the proteins
weren't assembled properly in the cell, Li says that how
and where proteins are made have a lot to do with the
difficulties researchers have had.
For one, proteins are made deep inside the cell; the
genetic instructions for building proteins are in the cell
nucleus, and proteins are assembled in a nearby "factory"
in the cell. Also, scientists have long known that proteins
prefer to stay put in this factory, the endoplasmic
reticulum, unless they contain specific transportation
instructions, much like an internal shipping label.
To figure out what tiny sequences might label the
protein for delivery to the cell surface, Shikano added
randomly generated eight-building-block-long tags onto one
end of a particular protein. He then evaluated whether the
protein ended up on the cell surface instead of remaining
inside the cell. The researchers found three major classes
of such tags, grouped according to similarities in their
sequences of building blocks, and delved into the most
potent of them.
By using a computer program developed by graduate
student Brian Coblitz to probe proteins' sequences, the
researchers discovered that, by fairly stringent criteria,
roughly 4 percent of all human proteins contain SWTY or a
very close relative. The eight-block-long tag itself is
part of the so-called C-terminal end of these proteins, and
its existence helps explain why some engineered proteins
don't go where they're supposed to go, Li said.
"If you remove a small part of the very end of a
protein, it seems unlikely to disrupt how the rest of the
protein folds in a three-dimensional structure, but that's
what most scientists think goes wrong if a protein doesn't
go to the surface," Li said. "But now we know 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. But Li chose this size to study in
part because naturally occurring proteins were already
known to use similar-size bits for recognition and
signaling. For example, she said, "the immune system uses
ones that are seven to nine blocks long to identify viral
proteins or other immune triggers."
Also, the number of possible combinations of
eight-block-long protein segments provides a "reasonable
number" to sort through — 25 billion or so —
given today's high throughput technologies. To make it even
easier, Shikano developed a system that would separate the
wheat from the chaff before the analysis began; if the
protein wasn't taken to the cell surface by the tag, the
"If the protein went to the cell surface, the cell was
in the mix, and if the cell wasn't there to be analyzed, we
knew we didn't want it anyway," Li said.
Authors on the paper are Shikano, Coblitz, Li and
Haiyan Sun. The researchers were funded by the National
Institute of General Medical Sciences, the National
Institute of Neurological Diseases and Stroke and the
American Heart Association.