杏吧原创

TAKE A PEEK at the stuff pushed towards the back of your refrigerator
shelves. Go ahead, look. Not a pretty sight, is it? Green fur on the lemon
yogurt. Slightly slimy salad. A certain, ahem, 鈥渂ouquet鈥� about the fish left
from last Monday鈥檚 dinner. Time to clean up before things get out of hand.

Your body鈥檚 cells face a similar chore. Just as you need to toss out
leftovers when they turn bad, your cells have to get rid of proteins that have
begun to do more harm than good. When cells need to stop dividing, for example,
they must destroy the proteins that trigger growth. If they fail to do this, the
unchecked cell division becomes cancer.

And that鈥檚 not the only time your fate depends on cells purging the right
proteins at the right time. The process is also important for everything from
embryonic development to the immune system鈥檚 defence against germs. And almost
everywhere protein destruction occurs, biologists find that a protein with a
certain distinctively shaped pocket is part of the wrecking crew. The
fascinating thing is that this unusual pocket occurs in many other proteins,
too, which suggests that protein destruction may be much more widespread than
anyone thought. 鈥淭his is going to lead to a whole explosion of research,鈥� says
Kim Nasmyth, a molecular biologist at the Research Institute of Molecular
Pathology in Vienna.

The pocket in question first came to researchers鈥� notice in the early 1990s,
when Paul Freemont, a biochemist at the Imperial Cancer Research Fund in London,
found a protein with a pocket that binds zinc ions. Soon he noticed an almost
identical pocket鈥攐r 鈥渇inger鈥� in biochemists鈥� parlance鈥攊n more than
two dozen other proteins. More distantly related versions of the finger started
to come to light in proteins from many different species, ranging from plants to
people. Grasping the opportunity for a good pun, Freemont called the gene for
his protein the Really Interesting New Gene, so the pocket itself
became the 鈥淩ING finger鈥�.

杏吧原创s were soon reporting more and more examples of proteins that
included RING fingers. 鈥淲hat I really find most remarkable is that during the
last ten years RING finger motifs were discovered in zillions and zillions of
proteins of all kinds, from plants to animals and God knows what,鈥� says
Alexander Varshavsky, a molecular biologist at Caltech in Pasadena. Yet no one
had any idea what they did鈥攖ill now. 鈥淪uddenly, in the last year, it
became apparent that whenever you see a RING motif, you see a component of the
protein destruction pathway. This is the grand unification,鈥� says Varshavsky.
Not everybody is quite that sweeping, but if all or even most RING fingers are
involved in clearing away unwanted protein molecules, then protein destruction
must be even more important than cell biologists thought.

Biologist have known for a decade or more that the destruction of proteins
plays a crucial part in a cell鈥檚 life cycle
(New 杏吧原创 supplement, 15 April, p 7).
When a cell decides to destroy a protein, it tags it with a
small molecule called ubiquitin. The cell鈥檚 machinery then tows the tagged
protein away to be chopped to pieces (see Diagram).
To avoid labelling the wrong proteins, a trio of enzymes called E1, E2 and E3 guide ubiquitin to its proper
target. Once E3 has attached the tag, the target is doomed: there鈥檚 no turning
back. 鈥淓3s are the key part of the pathway,鈥� says Raymond Deshaies, a molecular
biologist at Caltech.

To ensure that the right proteins get destroyed at the right time, cells
harbour many different E3 enzymes. Researchers originally thought the various
E3s would turn out to be a motley collection of widely differing molecules.
Nobody expected that all these clean-up crews would be led by members of the
same family, all acting according to a common plan. But that seems to be the
case. Over and over again, when researchers have looked at an E3 enzyme they
find it contains a protein with a pocket that is clearly a member of the RING
finger family.

Lord of the rings

As these examples flowed in, people began to wonder whether finding a RING
finger means you鈥檝e found a new case of protein destruction, and vice versa. The
first hint that this might be so came in 1998 from Nasmyth鈥檚 lab. When he and
his colleagues dissected a huge E3 enzyme called APC (for anaphase-promoting
complex) which triggers cell division in yeast by destroying a crucial group of
proteins, they found that it contains a RING finger subunit. What鈥檚 more, the
RING finger turned out to be crucial for APC to do its job. When the researchers
deleted the gene for the RING finger protein in APC, the cells quickly died.

At the time, Nasmyth and his colleagues didn鈥檛 know what to make of this
discovery. They got their answer early in 1999 when a second group of
researchers turned up a similar result. Deshaies and his team at Caltech were
studying another large protein-killing aggregate of enzymes called Skp1,
Cdc53/Cullin, F-box receptor, blessedly abbreviated to SCF. Using the same
methods as Nasmyth, Deshaies found that one of the SCF proteins includes a RING
finger very similar to the one in APC. When the RING in SCF also turned out to
be crucial for cell survival, the researchers put two and two together: they
realised that the RING must be essential for both APC and SCF to trash unwanted
proteins. 鈥淭his is a case where the second person to discover something has the
advantage. For the first person it鈥檚 too new in a way, but for the second person
it鈥檚 like `Aha!鈥� 鈥� says Deshaies.

At about the same time, Joan and Ronald Conaway, a wife and husband team at
Oklahoma Medical Research Foundation, found another example. The Conaways were
interested in the von Hippel-Lindau gene. Mutant versions of
VHL cause a variety of cancers in people, including the majority of kidney
cancers. In normal cells, the VHL protein clusters with other proteins to form a
large enzyme. Again, a small RING finger is at the centre of the action.

Cancers depend on a rich supply of blood vessels to provide them with the
food and oxygen they need to flourish. In healthy cells, a protein called HIF-1
triggers the sprouting of blood vessels when tissues do not receive enough
oxygen. But HIF-1 is usually very short-lived, because as soon as oxygen from
the blood floods the tissues another enzyme destroys it. Joan Conaway and others
now suspect that the RING finger in VHL helps to regulate the growth of blood
vessels by throwing out HIF-1 at the proper time. When VHL is mutated,
the RING finger stops working, so HIF-1 is not destroyed and keeps on signalling
to blood vessels to grow, nursing small cancers into life-threatening ones. She
is now testing this hypothesis.

Then, last autumn, Allan Weissman, a molecular biologist at the National
Cancer Institute near Washington DC, provided the most crucial piece of evidence
to link RING fingers to protein destruction. He, too, had been continually
bumping into the characteristic RING-finger sequence whenever he studied a new
aspect of protein destruction, and he wondered if this was more than just
coincidence. To find out, he turned to a genome database and pulled out a random
selection of genes of unknown function that included the RING-finger sequence.
He tested them to see whether the proteins they made could string ubiquitin
molecules together just as the E3s do when they tag proteins for
destruction.

Weissman鈥檚 leap of faith astonished his colleagues. 鈥淚n a way, the
experiments he did were almost insane,鈥� says Deshaies. But the 鈥渋nsane鈥�
experiments worked. The proteins containing the RING finger sequence all churned
out nice strings of ubiquitin, indicating that they worked like E3s. 鈥淓very
single RING we tested showed this potential,鈥� he says. 鈥淚t was amazing. We were
really shocked when it worked. It was one of these `Wow, is this really right?鈥�
moments.鈥� Up till then, no one suspected that the genes he had chosen had
anything to do with protein destruction. Other researchers agree that Weissman鈥檚
results were the clincher, and many now think the RING-finger structure is
synonymous with E3 activity. 鈥淭he fact that every single one he tried has this
activity is pretty damned suggestive that the RING might be a very general
feature in protein destruction,鈥� says Deshaies.

Some sceptics don鈥檛 accept this, however. 鈥淥ne thing that I find worrying is
that people are saying if they have a RING, it must be an E3,鈥� says Katherine
Borden, a biochemist at Mount Sinai School of Medicine in New York. 鈥淩INGs
clearly have other functions too.鈥� If scientists get too excited about RINGs鈥�
role in protein destruction, they might neglect these other important roles, she
says.

Borden works on a RING-finger protein called PML, which is disrupted in
certain forms of leukaemia. She knows that, unlike other RINGs, PML does not
participate in protein destruction. Instead, it clusters with other proteins in
the cell鈥檚 nucleus. It鈥檚 not clear what these clumps do to keep cells healthy,
but they are broken up in leukaemia and some viral infections. And Borden found
that mutations in the RING finger of PML lead to fewer, but extremely large,
clumps. She thinks the RING might therefore play a role in helping cells
assemble structures correctly so that they protect against leukaemia. In
Borden鈥檚 view, at least some RINGs help bind different kinds of proteins
together to form the cell鈥檚 huge internal protein scaffolds.

Starving tumours

Even if it turns out that not all RING fingers are involved in protein
destruction, the discovery of so many that are could have huge practical
pay-offs. If VHL is indeed responsible for destroying the blood-vessel booster
HIF-1, for example, scientists might be able to develop drugs that mimic VHL and
so cut off the blood supply that helps a tumour flourish.

Earlier this year, Weissman鈥檚 team found that a protein that includes a RING
finger regulates the destruction of p53, one of the key players in the
prevention of cancer. In effect, p53 is a guard protein鈥攊t helps to kill
cells in which the brakes that should keep cell division in check aren鈥檛
working. When p53 is absent, such cells can divide unhindered, developing into
cancer. Normally, a protein called mdm-2 makes sure that all cells get the right
amount of p53, but until now no one knew exactly how mdm-2 does this.

Now Weissman has found that the RING finger in mdm-2 helps to destroy p53. He
and his colleagues simply mixed mdm-2 and p53 in a test tube along with some
radioactive ubiquitin. Over time, the p53 molecules got larger and
radioactive鈥攃learly the result of mdm-2 tagging them with ubiquitin. In a
cell, this would mark out the p53 to be hauled off for trashing.

Some tumours have too much mdm-2, and Weissman believes this excess causes
p53 to be destroyed too quickly, allowing out-of-control cells to continue
dividing unhindered. A drug that inhibits the activity of mdm-2 or its RING
finger might be able to bring p53 levels back to normal, halting the
uncontrolled cell divisions.

Perhaps the most famous of the genes that contain a RING-finger sequence is
BRCA1. Mutations in BRCA1, especially ones that destroy the
RING finger itself, can cause breast cancer. The obvious question is: does this
cancer gene work by disrupting protein destruction? Heinz Ruffner, a molecular
biologist at the Salk Institute in San Diego, has set out to find the answer. In
as-yet unpublished test-tube experiments, he has found that normal BRCA1 protein
does indeed tag proteins for destruction. And, sure enough, BRCA1 with
a mutated RING-finger sequence yields proteins that fail to do the job. From
this, Ruffner surmises that women with the gene mutation develop breast cancer
because having defective BRCA1 means dangerous proteins aren鈥檛 destroyed.
Ruffner has not yet completed his experiments and isn鈥檛 drawing any final
conclusions. 鈥淚n living cells the situation might be much more complex,鈥� he
cautions. It may take much more work before we understand the role that BRCA1鈥檚
RING finger plays in breast cancer.

Cancer may not be the only area in which RING fingers affect human health.
鈥淭he ubiquitin system is certainly involved in regulation of a lot of
disease-related events,鈥� says Joan Conaway. Herpes simplex virus, for example,
which causes cold sores in response to summer sunshine or cold winter winds,
uses a protein that contains a RING finger to destroy the proteins that our
cells usually use to fight viral invaders. Interestingly, one of the virus鈥檚
target proteins is PML, which suggests that this RING-finger protein helps
protect not just against cancer but also against viral infection. Here, RING
fingers seem to be arming both sides in the battle鈥攖he virus that鈥檚
attacking us, and the defenders that we deploy to protect us from the
assault.

Reports of new examples of important RINGs are coming in thick and fast. 鈥淭he
story is only just beginning,鈥� says Nasmyth. Not surprisingly, many
uncertainties remain, including the key question of whether most RINGs play a
part in the ubiquitin system. 鈥淭he next step is really to figure out what all of
these RING fingers are doing鈥攚hether all of them have this function or
whether some of them do and some of them don鈥檛,鈥� says Weissman.

Whatever the answer, the new results should help us identify the clean-up
crews that destroy and clear away the proteins our cells no longer need. They
should also lead to a way to find those proteins that have passed their use-by
date and make our cells go bad. 鈥淵ou can expect the next few years to be very,
very exciting,鈥� says Weissman.