Advertisement

If you have an ACS member number, please enter it here so we can link this account to your membership. (optional)

ACS values your privacy. By submitting your information, you are gaining access to C&EN and subscribing to our weekly newsletter. We use the information you provide to make your reading experience better, and we will never sell your data to third party members.

ENJOY UNLIMITED ACCES TO C&EN

Biological Chemistry

Ubiquitins Are Finally United

New synthetic approaches put a long-sought end to ubiquitin proteins’ unchained melody

by Stu Borman
October 11, 2010 | A version of this story appeared in Volume 88, Issue 41

Protection Scheme
[+]Enlarge
Credit: Courtesy of David Komander & Jason Chin
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.
Credit: Courtesy of David Komander & Jason Chin
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.

Chains of ubiquitin proteins play 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 mixed-linkage 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.

LINK-UP
[+]Enlarge
Credit: COURTESY OF ASHRAF BRIK
The Liu and Brik groups, working independently, joined ubiquitins using native chemical ligation—thiol attack to form a new thioester (1) and amide bond formation (2)—and then desulfurizing.
Credit: COURTESY OF ASHRAF BRIK
The Liu and Brik groups, working independently, joined ubiquitins using native chemical ligation—thiol attack to form a new thioester (1) and amide bond formation (2)—and then desulfurizing.

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.

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.”

Cambridge Crew Komander
[+]Enlarge
Credit: Neil Grant/Medical Research Council, Cambridge
(from left), Chin, and postdoc Satpal Virdee, first
author of the group’s Nature Chemical Biology paper.
Credit: Neil Grant/Medical Research Council, Cambridge
(from left), Chin, and postdoc Satpal Virdee, first
author of the group’s Nature Chemical Biology paper.

With all the homogeneous diubiqui­tins now in hand, there will be lots to do. Researchers will want to synthesize longer 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 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.

Israel Group
[+]Enlarge
Credit: Sawsan Brik
Lesly A. Erlich (from left), K. S. Ajish Kumar, Ashraf Brik,and Sudhir N. Bavikar with some agave plants on the campus of Ben-GurionUniversity of the Negev.
Credit: Sawsan Brik
Lesly A. Erlich (from left), K. S. Ajish Kumar, Ashraf Brik,and Sudhir N. Bavikar with some agave plants on the campus of Ben-GurionUniversity of the Negev.

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 Fushman 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.

Singapore Team
[+]Enlarge
Credit: Chuan-Fa Liu
Xue-Wei Liu (from left), Kalyan Kumar Pasunooti, RenliangYang, Fupeng Li, and Chuan-Fa Liu in front of two bushes reminiscent oflinked ubiquitins.
Credit: Chuan-Fa Liu
Xue-Wei Liu (from left), Kalyan Kumar Pasunooti, RenliangYang, Fupeng Li, and Chuan-Fa Liu in front of two bushes reminiscent oflinked ubiquitins.

The Liu and Brik teams synthesized diubiquitins using approaches the two groups devised last year (C&EN Online, Nov. 16, 2009). They derivatized the C-terminal 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.

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 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 investigating 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.”

Article:

This article has been sent to the following recipient:

0 /1 FREE ARTICLES LEFT THIS MONTH Remaining
Chemistry matters. Join us to get the news you need.