USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

Release of Enzymatically Active Deubiquitinating Enzymes upon Reversible Capture by Disulfide Ubiquitin Reagents.

Deubiquitinating enzymes (DUBs) catalyze the cleavage of ubiquitin from target proteins. Ubiquitin is post-translationally attached to proteins and serves as an important regulatory signal for key cellular processes. In this study, novel activity-based probes to study DUBs were synthesized that comprise a ubiquitin moiety and a novel disulfide warhead at the C-terminus. These reagents can bind DUBs covalently by forming a disulfide bridge between the active-site cysteine residue and the ubiquitin-based probe. As disulfide bridges can be broken by the addition of a reducing agent, these novel ubiquitin reagents can be used to capture and subsequently release catalytically active DUBs, whereas existing capturing agents bind irreversibly. These novel reagents allow for the study of these enzymes in their active state under various conditions.

Shear stress-dependent downregulation of the adhesion-G protein-coupled receptor CD97 on circulating leukocytes upon contact with its ligand CD55.

Adhesion G protein-coupled receptors (aGPCRs) are two-subunit molecules, consisting of an adhesive extracellular α subunit that couples noncovalently to a seven-transmembrane ß subunit. The cooperation between the two subunits and the effect of endogenous ligands on the functioning of aGPCRs is poorly understood. In this study, we investigated the interaction between the pan-leukocyte aGPCR CD97 and its ligand CD55. We found that leukocytes from CD55-deficient mice express significantly increased levels of cell surface CD97 that normalized after transfer into wild-type mice because of contact with CD55 on both leukocytes and stromal cells. Downregulation of both CD97 subunits occurred within minutes after first contact with CD55 in vivo, which correlated with an increase in plasma levels of soluble CD97. In vitro, downregulation of CD97 on CD55-deficient leukocytes cocultured with wild-type blood cells was strictly dependent on shear stress. In vivo, CD55-mediated downregulation of CD97 required an intact circulation and was not observed on cells that lack contact with the blood stream, such as microglia. Notably, de novo ligation of CD97 did not activate signaling molecules constitutively engaged by CD97 in cancer cells, such as ERK and protein kinase B/Akt. We conclude that CD55 downregulates CD97 surface expression on circulating leukocytes by a process that requires physical forces, but based on current evidence does not induce receptor signaling. This regulation can restrict CD97-CD55-mediated cell adhesion to tissue sites.

USP7: combining tools towards selectivity.

Active-site directed probes based on the ubiquitin scaffold have been successfully applied as tools to determine the levels of active deubiquitinating enzymes (DUBs). Here, we show the development of a Ub-based reagent selective for the DUB USP7. This concept can be applied for the generation of other new selective reagents.

Development of a DUB-selective fluorogenic substrate.

Ubiquitination is a post-translational modification that is involved in a plethora of cellular processes. Target proteins can be specifically modified with a single ubiquitin (Ub) molecule or with complex chains. In recent years, research has focused on deubiquitinating enzymes (DUBs) as potential therapeutic candidates in various diseases. USP16 is an emerging target due to its involvement in mitosis and stem cell self-renewal. Generally, activity-based probes (ABPs) used to study DUBs are based on the ubiquitin scaffold, thus lacking target selectivity. To overcome this issue, we designed a Ub-based activity probe bearing specific mutations to achieve selectivity for USP16, by combining structural modelling and analysis and mutational calculation predictions. We develop a fluorogenic substrate, the first of its kind, that is processed exclusively by USP16, which allows us to monitor USP16 activity in complex samples.

Diubiquitin-Based NMR Analysis: Interactions Between Lys6-Linked diUb and UBA Domain of UBXN1.

Ubiquitination is a process in which a protein is modified by the covalent attachment of the C-terminal carboxylic acid of ubiquitin (Ub) to the ε-amine of lysine or N-terminal methionine residue of a substrate protein or another Ub molecule. Each of the seven internal lysine residues and the N-terminal methionine residue of Ub can be linked to the C-terminus of another Ub moiety to form 8 distinct Ub linkages and the resulting differences in linkage types elicit different Ub signaling pathways. Cellular responses are triggered when proteins containing ubiquitin-binding domains (UBDs) recognize and bind to specific polyUb linkage types. To get more insight into the differences between polyUb chains, all of the seven lysine-linked di-ubiquitin molecules (diUbs) were prepared and used as a model to study their structural conformations in solution using NMR spectroscopy. We report the synthesis of diUb molecules, fully 15N-labeled on the distal (N-terminal) Ub moiety and revealed their structural orientation with respect to the proximal Ub. As expected, the diUb molecules exist in different conformations in solution, with multiple conformations known to exist for K6-, K48-, and K63-linked diUb molecules. These multiple conformations allow structural flexibility in binding with UBDs thereby inducing unique responses. One of the well-known but poorly understood UBD-Ub interaction is the recognition of K6 polyubiquitin by the ubiquitin-associated (UBA) domain of UBXN1 in the BRCA-mediated DNA repair pathway. Using our synthetic 15N-labeled diUbs, we establish here how a C-terminally extended UBA domain of UBXN1 confers specificity to K6 diUb while the non-extended version of the domain does not show any linkage preference. We show that the two distinct conformations of K6 diUb that exist in solution converge into a single conformation upon binding to this extended form of the UBA domain of the UBXN1 protein. It is likely that more of such extended UBA domains exist in nature and can contribute to linkage-specificity in Ub signaling. The isotopically labeled diUb compounds described here and the use of NMR to study their interactions with relevant partner molecules will help accelerate our understanding of Ub signaling pathways.

Non-hydrolyzable Diubiquitin Probes Reveal Linkage-Specific Reactivity of Deubiquitylating Enzymes Mediated by S2 Pockets.

Ubiquitin chains are important post-translational modifications that control a large number of cellular processes. Chains can be formed via different linkages, which determines the type of signal they convey. Deubiquitylating enzymes (DUBs) regulate ubiquitylation status by trimming or removing chains from attached proteins. DUBs can contain several ubiquitin-binding pockets, which confer specificity toward differently linked chains. Most tools for monitoring DUB specificity target binding pockets on opposing sides of the active site; however, some DUBs contain additional pockets. Therefore, reagents targeting additional pockets are essential to fully understand linkage specificity. We report the development of active site-directed probes and fluorogenic substrates, based on non-hydrolyzable diubiquitin, that are equipped with a C-terminal warhead or a fluorogenic activity reporter moiety. We demonstrate that various DUBs in lysates display differential reactivity toward differently linked diubiquitin probes, as exemplified by the proteasome-associated DUB USP14. In addition, OTUD2 and OTUD3 show remarkable linkage-specific reactivity with our diubiquitin-based reagents.

Catching a DUB in the act: novel ubiquitin-based active site directed probes.

Protein ubiquitylation is an important regulator of protein function, localization and half-life. It plays a key role in most cellular processes including immune signaling. Deregulation of this process is a major causative factor for many diseases. A major advancement in the identification and characterization of the enzymes that remove ubiquitin, deubiquitylases (DUBs) was made by the development of activity-based probes (ABPs). Recent advances in chemical protein synthesis and ligation methodology has yielded novel reagents for use in ubiquitylation research. We describe recent advances and discuss future directions in reagent development for studying DUBs.

E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system.

In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.

Polyubiquitin serves as a recognition signal, rather than a ratcheting molecule, during retrotranslocation of proteins across the endoplasmic reticulum membrane.

Polyubiquitination is required for retrotranslocation of proteins from the endoplasmic reticulum back into the cytosol, where they are degraded by the proteasome. We have tested whether the release of a polypeptide chain into the cytosol is caused by a ratcheting mechanism in which the attachment of polyubiquitin prevents the chain from moving back into the endoplasmic reticulum. Using a permeabilized cell system in which major histocompatibility complex class I heavy chains are retrotranslocated under the influence of the human cytomegalovirus protein US11, we demonstrate that polyubiquitination alone is insufficient to provide the driving force for retrotranslocation. Substrate release into the cytosol requires an additional ATP-dependent step. Release requires a lysine 48 linkage of ubiquitin chains. It does not occur when polyubiquitination of the substrate is carried out with glutathione S-transferase (GST)-ubiquitin, and this correlates with poly-GST-ubiquitin not being recognized by a ubiquitin-binding domain in the Ufd1-Npl4 cofactor of the ATPase p97. These data suggest that polyubiquitin does not serve as a ratcheting molecule. Rather, it may serve as a recognition signal for the p97-Ufd1-Npl4 complex, a component implicated in the movement of substrate into the cytosol.