Decoding the Linkage Specificity of Decoding the Linkage Specificity of DeubiquitinatingDeubiquitinating Enzymes with DiEnzymes with Di-- and and TetraubiquitinTetraubiquitin: Are Longer : Are Longer PolyubiquitinPolyubiquitin Chains the Key? Chains the Key?

Nate Nate RussellRussell, Steven Feudo, , Steven Feudo, ZacZac Stiffler, Jarad Yost, Greg Costakes, Carsten Schwerdtfeger, Stiffler, Jarad Yost, Greg Costakes, Carsten Schwerdtfeger, and and Bradley Brasher Bradley Brasher ++ Boston Boston Biochem Inc., Cambridge, MA Biochem Inc., Cambridge, MA 02139 02139 +corresponding author +corresponding author

IntroductionIntroduction

BackgroundBackground

Deubiquitinating enzymes (DUBs) play an essential role in many cellular processes and their

dysfunction results in a number of human diseases. Despite this important role, the substrate

specificity and kinetic parameters of many DUBs are poorly defined. Monoubiquitin based

fluorescent substrates such as Ubiquitin-AMC (Ub-AMC) have been quite useful for DUB

characterization, though not all DUBs utilize Ub-AMC well. The use of monoubiquitin substrates

yields an incomplete picture of the preferred substrates and kinetic activity of DUBs as Ub-

AMC lacks the extended structure and isopeptide bonds that polyubiquitin (polyUb) chains

possess,. Additionally, a paucity of full-length enzymes and longer (>2 Ub) polyUb chains of

most linkage types has made it challenging to determine the types of polyUb each DUB can

process.

Here we provide an analysis of 36 DUBs (mostly full-length) for linkage specificity and possible

cleavage mechanisms against a complete set of all 8 linkages of di- and tetraubiquitin (except

K27-linked tetraubiquitin). With the exception of the K27-linked chains, all polyUb chains were

synthesized enzymatically and linkage homogeneity was verified by Absolute Quantitation of

Ubiquitin (AQUA-PRM) analysis. The combination of AQUA analysis and enzymatic synthesis

ensures our polyUb chains are identical to those found in the cell. We used the panel of polyUb

chains in DUB activity assays under similar reaction conditions to determine DUB linkage

specificity. From this data set, seven major classes of DUBs were found with regards to linkage

specificity: i) those that processed all linkages (ex. USP2, USP9x), ii) those that were unable to

process polyUb (ex. UCH DUBs), iii) those that processed only a single type of polyUb (ex.

AMSH, Otubain1), iv) those that strongly preferred a single linkage (ex. Cezanne), v) those that

were linkage selective (three or fewer linkages such as Trabid), vi) those that processed most

linkages except for linear, K27, and K29 (ex. USP28 and USP7), and vii), those that process all

linkages except K27. In general, most DUBs had limited or no ability to process linear, K27, or

K29-linked polyUb. Additionally, a longer polyUb chain played an important role in some DUB’s

abilities to process certain chain linkages. These results, especially the K29 data, had not been

observed with the shorter polyUb reagents previously available to researchers.

We followed up with specific DUBs to characterize how they processed certain polyUb chains

(exo vs. endo cleavage). A number of DUBs were found (SARS-PLpro, OTUD3, VCPIP, etc.)

where tetraubiquitin with varying linkages were processed by the same enzyme in different

ways. An expanded reagent toolbox including previously unavailable DUBs and complete sets

of di- and tetraubiquitin will greatly improve the ability to characterize DUBs.

Heat Map Analysis of DUB Activity on DiHeat Map Analysis of DUB Activity on Di-- and and TetraubiquitinTetraubiquitin ChainsChains

Figure 5. Classes of DUB activity observed. Representative SDS-PAGE analysis of each class of DUB cleavage activity. All samples were prepared and subjected to SDS-PAGE analysis as described in Figure 3. A. DUBs that

only process one type of polyUb chain linkage. B. DUBs that are unable to process any type of polyUb. C. DUBs that have a strong single linkage preference, but are capable of processing multiple linkages. D. DUBs that process

most linkages except linear, K27, and K29-linked. E. DUBs that process all polyUb chain linkages. F. DUBs that process only 3 or fewer linkages. (DUBs that cleave all linkages except K27 not shown).

SummarySummary

We analyzed 36 DUBs for their polyUb chain cleavage preferences using a complete panel of diubiquitins (all 8

possible linkages) and an almost complete panel of homogeneously linked tetraubiquitins (except for K27-linked).

This was the first time such a complete analysis had been performed against tetraubiquitin substrates.

While confirming most previously reported data against diubiquitin substrates, the tetraubiquitin data provided a

much clearer picture of how DUBs processed the various linkages than had been previously reported.

Seven classes of DUB activity were observed: i) DUBs that processed all linkages, ii) DUBs unable to process

polyUb of any type, iii) DUBs that processed a single linkage only, iv) DUBs that strongly preferred a single linkage,

v) DUBs that were linkage selective for three or fewer linkages, vi) DUBs that had no or low activity against linear,

K27, and K29 polyUb chains, and vii), DUBs that processed all linkages except K27.

In a number of cases, the same DUB processed different forms of tetraubiquitin in quite different ways. (Ex. SARS-

PLpro processing K48-linked polyUb in an endo manner while processing K63 and other linkages in an exo manner.)

Varying the concentration of selected DUBs used over two orders of magnitude showed that some DUBs become

more promiscuous at high concentrations, obscuring what their preferred substrates are.

Interestingly, an overnight incubation of 10 nM DUB (compared to 45 minutes) resulted in increased polyUb chain

processing for some enzymes (AMSH-LP), while others showed no difference (Cezanne, CYLD, SARS-PLpro) while

Trabid and USP28 showed both results depending on what linkage was being digested.

Overall, tetraubiquitin is the preferred substrate for obtaining a better understanding of how DUBs process polyUb

chains.

Determining Preferred DUB Substrates Relies on Determining Preferred DUB Substrates Relies on

Using Appropriate DUB concentrationsUsing Appropriate DUB concentrations

Figure 1: Ubiquitin forms different types of polyUb chains: Ubiquitin can form polyUb

chains via isopeptide linkages using the 7 lysines in ubiquitin or be linked in a linear fashion

via the N and C-termini for a total of 8 possible linkages. All have been detected in vivo.

Structures of diubiquitin have been solved for many of these linkages. The above figure

demonstrates the many ways that ubiquitin can be conjugated to itself, highlighting the

different structures that have been observed in these chains, and that multiple linkages can

be present in a single chain.

Figure 2: DUBs disassemble the various forms of polyUb chains found in the cell:

DUBs are used to process polyUb in a variety of ways. They are used to process proubiquitin

as it is being translated, edit the length of polyUb chains attached to a ubiquitinated substrate,

completely remove a polyUb chain as a substrate is being degraded, and disassemble free

polyUb chains to recycle the chains back into the cellular monoUb pool.

Figure 4: Identified classes of DUB activity against polyUb chain substrates. Seven

classes of DUB activity were observed. The class that each of the 36 DUBs analyzed

belongs to is shown above.

Individual DUBs Process Individual DUBs Process TetraubiquitinTetraubiquitin via Different via Different

Enzymatic Mechanisms That are Linkage DependentEnzymatic Mechanisms That are Linkage Dependent

Figure 6: Time course digestion demonstrates how the same DUB can process polyUb chains with varying

linkages in different ways. Digestions were performed as described in Figure 3 with the following changes. Digestions

were 140 l in size and 20 l was removed at 5, 10, 15, 20, 30, and 45 minutes and quenched with 5 l of 5X SDS-PAGE

sample buffer. 20 l of each time point was analyzed on an 18% 26-well Criterion SDS-PAGE gel by Coomassie staining.

All digestions shown above were performed with 1 M of the appropriate DUB except for the K48-linked tetraubiquitin

digestion by SARS-PLpro (100 nM).

Figure 7: Selected DUBs show varying levels of activity on their preferred substrates which appears to be

concentration dependent. Experiments were performed similarly to those described in Figure 3 with the following

changes. For each DUB analyzed, three enzyme concentrations were used: 10 nM, 100 nM, and 1.0 M. A second 10 nM

digestion was also allowed to incubate overnight at 37°C rather than the standard 45 minutes. All digestions for an

individual DUB against a particular type of polyUb chain were loaded next to one another for easier interpretation.

A. DUB Activity on Diubiquitin

B. DUB Activity on Tetraubiquitin

Figure 3. Heat map analysis of DUB activity on Di- and Tetraubiquitin chains. All reactions were performed under similar conditions: 20 l reactions containing 2 g of the appropriate polyUb chain and 100 nM of the appropriate

DUB. For lower activity DUBs, 1 M of enzyme was used. Reactions were brought to 20l using an assay buffer consisting of 50 mM HEPES, pH 7.5, 100 mM NaCl, and 2 mM TCEP. All DUB reactions were paired with a negative control reaction that contained only 2 g of the appropriate polyUb chain and no DUB. All reactions were incubated for 45 minutes in a 37°C water bath, reactions were then quenched with 5 l of SDS-PAGE sample buffer, and 20 l

of the quenched reaction was loaded onto an 18% Criterion 26-Well SDS-PAGE gel for analysis. After Coomassie staining of the gel, images were taken and subjected to densitometry to determine the amount of DUB cleavage that

had taken place. Quantitation from the densitometry analysis was used to generate heat maps using Plotly. Any quantitation value of <10% was treated as 0% cleavage.

A: Heat map analysis of Diubiquitin processing activity. Enzymes that were used at the 1M concentration included MYSM1, Otubain2, OTUD3CD, USP13, VCIP135, and Yod1.

B: Heat map analysis of Tetraubiquitin processing activity. No data were obtained for K27 Ub4. However, the column was included as a marker to keep the heat maps aligned for easier visual interpretation.

Classes of DUB Activity Against Classes of DUB Activity Against PolyUbPolyUb Chains: Several Different Types of Linkage PreferenceChains: Several Different Types of Linkage Preference

A. B. C.

D. E. F.

Ristic et. al (Fron. Mol. Neurosci, 2014)

Wilkinson and Fushman (The Scientist, 2012)

AMSH-LP, AMSH, Otubain1, Otulin

Cezanne, SARS-PLpro, USP30

Bap1, UCHL1, UCHL3, UCHL5, USP46/UAF1,

USP12/UAF1

USP15,USP19, USP2, USP9x, USP5

USP11, USP4

MYSM1, Otubain2, USP1/UAF1, USP12

ternary, USP13, USP28, USP46

ternary, USP7, USP8, Yod1

CYLD, OTUD3, Trabid, USP10, USP25,

VCIP135