SCIENCE & TECHNOLOGY
UBIQUITINS ARE
FINALLY UNITED
New synthetic approaches put a long-sought end
to ubiquitin proteins’ UNCHAINED MELODY
STU BORMAN, C&EN WASHINGTON
key roles in cellular chemistry, including
directing unwanted proteins to the proteasome for disposal. That function is carried
out by a type of ubiquitin chain called K48.
Another type, K11, marks proteins for turnover in the cell cycle, and one called K63
has a variety of functions such as activating
protein kinases.
But in addition to these three types of
homogeneous ubiquitin chains—so called
because each is linked internally in only
one way—there are four others. And there
are innumerable combinations of mixedlinkage chains. Scientists would like to be
able to understand what each of these different types of chains does in cells. But it’s
been hard to do this because most of the
chains have been impossible to make by
chemical or enzymatic means.
Now, in work that could advance the
field of ubiquitin research, three groups,
working independently, have overcome
that problem.
Ubiquitin specialist David Komander,
nonnatural amino acid mutagenesis aficionado Jason W. Chin, and coworkers at
the MRC Laboratory of Molecular Biology,
in Cambridge, England, used genetic engineering and synthetic chemistry to create
two of the previously inaccessible homogeneous ubiquitin chains, K6 and K29 (Nat.
Chem. Biol., DOI: 10.1038/nchembio.426).
Biopolymer chemical biologist Chuan-Fa
Liu of Nanyang Technological University,
Singapore, and coworkers used a technique
called native chemical ligation to chemically synthesize K48 (Chem. Commun.,
DOI: 10.1039/C0CC01382J). And protein
synthesis expert Ashraf Brik of Ben-Gurion
University of the Negev, Israel, and coworkers, also using native chemical ligation, synthesized all seven homogeneous
ubiquitin chains, including the four—K6,
K27, K29, and K33—that were previously
unavailable (Angew. Chem. Int. Ed., DOI:
10.1002/anie.201003763).
The ubiquitin chains created in each
of the studies were a mere two ubiquitins
long. But the approaches can potentially
be extended to longer chains, and two-unit
chains may turn out to be all that are needed for biological functionality in any case.
PROTECTION SCHEME In the Cambridge group’s synthesis, C-terminal thioester
in amine-protected donor ubiquitin (orange) reacts with deprotected lysine in acceptor
ubiquitin (yellow) to form native isopeptide-bonded product, which is deprotected to
yield diubiquitin (orange and yellow). Protecting groups are pink. Thioester, deprotected
lysine, and the isopeptide bond are shown in ball-and-stick representations.
+
Without access to most of the chains
until now, researchers have had a great deal
of trouble answering just that kind of question—what length the chains must have to
express in vivo activity. “We know that for
K48 chains, you need to have at least four
ubiquitin units,” says synthetic protein
chemist Tom W. Muir of Rockefeller University, in New York City, “but for the other
ones, it may be that two is enough. It’s a
completely open question.”
WITH ALL THE homogeneous diubiqui-
tins now in hand, there will be lots to do.
Researchers will want to synthesize longer
location, such as K6 or K11, in the adjacent
ubiquitin. Ubiquitin-binding proteins and
deubiquitinating enzymes recognize the
different conformations of the various
chain types, a process that underlies the
chains’ varied biological functions.
The functions of K11, K48, and K63 ubiquitin chains are known because enzymes
that catalyze their biosynthesis are known
and have been used to make them available
for study. “The problem is that we don’t
know enzymes that make the other types
specifically, which has made them very difficult to obtain,” says molecular biophysicist and ubiquitin chain expert David Fush-
“This is high-impact stuff because the field just has
not had access to these reagents—period.”
WWW.CEN-ONLINE.ORG
36
OCTOBER 11, 2010
COURTESY OF DAVID KOMANDER & JASON CHIN
CHAINS OF UBIQUITIN proteins play
chains, construct mixed-type chains, structurally analyze and determine the biological roles of the different chain types, screen
deubiquitinating enzymes to determine
their selectivity of action toward specific
chains, and make antibodies that recognize
the chains.
“There will be a lot of interest in the
ubiquitin community in getting access to
these chains,” Muir says. “I suspect the
authors are going to get a barrage of phone
calls from would-be collaborators. Among
biologists, this is high-impact stuff because
the field just has not had access to these
reagents—period.”
The chains are named for their connectivity. Each ubiquitin has 76 amino acid
residues, seven of which are lysines (symbolized by “K”). In diubiquitins and higher
oligomers, the C-terminal glycine of one
ubiquitin is linked via an isopeptide bond
to a lysine side chain at a specific numbered
working independently, joined
ubiquitins using native chemical
ligation—thiol attack to form a
new thioester (1) and amide bond
formation (2)—and then desulfurizing.
O
2
SR
1
HS
NH2
SR–
O
Isopeptide
bond
NH
THE GROUPS have already studied some
of the new diubiquitins. Komander, Chin,
and coworkers determined the crystal
structure of K6 diubiquitin and found that
its conformation is completely different
from those of K48 and K63 diubiquitins,
which had been structurally analyzed
previously—suggesting that K6’s biological
functions may be distinct.
By screening 10% of human deubiquitinases against their new K6 and K29 diubiquitins, Komander, Chin, and coworkers
also found that an enzyme called TRABID
hydrolyzes K29 chains unexpectedly
well. “TRABID has been thought to be a
vestigating the selectivity with which two
deubiquitinating hydrolases disassemble
them. Such studies are “the icing on the
cake,” Fushman says, “showing that once
you have chains you can start answering
questions that people could not address
properly before.”
Muir cautions that he’s “not convinced
any of these techniques are going to be
picked up by rank-and-file biologists to
make ubiquitin chains. It’s just going to be
beyond the expertise of many people. The
techniques are like 25-step natural product
syntheses—not something everyone is going to be able to do right now.” However, he
notes, “that may not be a problem if some
vendor picks up these diubiquitins and
sells them. Then people can have access to
them.”
In any case, “I’m a fan of all the papers,”
Muir adds. “They’re absolutely timely.
They’re going to get attention from a lot of
biochemists who work in this field, and this
is a huge field. The chemistry is highly technical, but it’s clever. And you get enough
product to actually do stuff with—which is
always nice to see.” ■
LINK-UP The Liu and Brik groups,
COU RTESY OF ASHRAF BRIK
man of the University of Maryland. The
new studies “give us the ability to bypass
enzymes and just make them all chemically, and that’s very exciting.”
In their study, Komander, Chin, and
coworkers used protein expression techniques and genetic code expansion to create a “donor” ubiquitin with a C-terminal
thioester and an “acceptor” ubiquitin with
its position-6 or -29 lysine replaced with
a protecting group. They protected all the
other amines in both ubiquitins with a second protecting group and removed the first
protecting group from position 6 or 29 to
reveal a single free amino group in the acceptor. They then reacted donor and acceptor and deprotected the product, yielding
native K6 or K29 diubiquitin.
The Liu and Brik teams synthesized
diubiquitins using approaches the two
groups devised last year (C&EN Online,
Nov. 16, 2009). They derivatized the Cterminal glycine of a donor ubiquitin with a
thioester and converted a specific lysine to
thiolysine on an acceptor ubiquitin. They
used native chemical ligation to link donor
and acceptor and then desulfurized the
product, yielding native isopeptide bonds
at the connection point. Liu’s group made
one chain type (K48), and Brik’s group synthesized all seven types.
Making every possible homogeneous
diubiquitin chain “is really impressive,”
Muir says. “It’s a tour de force of peptide
and protein chemistry.”
Ubiquitin chains carry out their functional roles in cells by tagging specific
proteins, and the three groups’ techniques
may also be useful for studying that
process by making it possible to attach
ubiquitin chains to specific proteins via
isopeptide bonds. The new syntheses “are
going to push the field amazingly,” Komander says.
K63-specific deubiquitinating enzyme,”
Komander says. “It does cleave K63 chains,
but it is 40 times more active against K29
chains. By just having this chain type for
the first time, we were instantly able to
identify an enzyme with selectivity for it. It
will now make some sense to mutate this
enzyme or take it out of cells to help study
the function of K29 chains.”
Brik and coworkers also investigated
the new diubiquitins—in their case by in-
WWW.CEN-ONLINE.ORG
37
OCTOBER 11, 2010