Characterization Of Full-Length, Recombinant Amsh, USP9x, and USP1: Clinically-Relevant Deubiquitinases Involved In Hematological Malignancies

Attachment of polyubiquitin to substrate proteins generates important biological signaling cues that are inherent to the linkage type of the polyubiquitin chain. For example, K48-linked polyubiquitin chains result in proteasome-mediated degradation of proteins to which they are attached, whereas K63-linked polyubiquitin chains play roles in various intracellular signaling cascades. An important feature of protein ubiquitination is that it is reversible. Substrate-anchored chains may be edited or removed from proteins by highly specialized proteases called deubiquitinating enzymes (DUBs). There are approximately 90 DUBs identified in humans and many have been identified as potential druggable targets because of their involvement in hematological malignancies such as Fanconi Anemia, human follicular lymphomas and diffuse large B-cell lymphomas. DUB activity is regulated by a variety of cues including specificity for a protein substrate(s) to which polyubiquitin chains are conjugated; the presence of protein cofactor(s) that activate or inhibit DUB function; or preference for a specific polyubiquitin linkage type(s). Thus, understanding the mechanisms, regulation, and substrate preferences for deubiquitinases is of great interest, from both academic and clinical viewpoints. To help address these needs, we produced highly purified recombinant deubiquitinase enzymes to facilitate in vitro biochemical studies and drug-discovery efforts. Herein we report the initial characterization of the following, clinically-relevant enzymes:

1. AMSH is a JAMM-class metalloprotease that specifically cleaves K63-linked polyubiquitin chains. This DUB is activated by its partner STAM at the endosome, where its activity opposes ubiquitin-dependent sorting of receptors to lysosomes. AMSH plays important roles in cell growth, and IL-2, GM-CSF, and BMP (bone morphogenetic protein) signaling pathways. Our results demonstrate that recombinant AMSH has no activity against commonly used DUB substrates such as ubiquitin-AMC and ubiquitin-rhodamine. AMSH hydrolyzed K63-linked diubiquitin substrates (but not diubiquitin of other linkage types), and this activity was increased by an order of magnitude in the presence of recombinant STAM protein.

2. USP9x is an essential component of TGFβ/BMP signaling cascades. USP9x biology is likely to be complex, as over-expression of the DUB correlates with increased MCL1 protein—a driving force in both follicular- and diffuse large B-cell lymphomas. Conversely, decreased expression of USP9x cooperates with K-RAS mutations to accelerate aggressive pancreatic tumors in mice. This DUB was reported to hydrolyze K29- and K33-linked polyubiquitins chains, as well as numerous K48-linked polyubiquitinated substrates. The full-length recombinant USP9x is highly active against ubiquitin-AMC (kcat/Km = 1.1x10⁶ M^-1 s^-1), and cleaves all polyubiquitin chain linkages other than linear- (“Met1-linked”) and K27-linked, demonstrating the potential for this deubiquitinase to act on multiple targets.

3. USP1 is a deubiquitinating enzyme of the C19 peptidase family and functions as a negative regulator of the Fanconi Anemia pathway. Reported substrates of USP1 include monoubiquitinated forms of FANCD2, PCNA and ID1. USP1 plays important roles in DNA damage responses and cancer-related processes, and inhibiting the function of this DUB sensitizes some cancer cells to chemotherapy. USP1 is nearly completely inactive in the absence of its activating partner, UAF1. We observed that recombinant USP1/UAF1 complex was able to efficiently cleave K6- and K63-linked diubiquitin chains, and hydrolyzed ubiquitin-AMC with great efficiency (kcat/Km = 1.3x10⁶ M^-1 s^-1). Potent, small-molecule inhibitors of USP1 have been identified using in vitro assays, and data from ongoing studies to determine the potency of these inhibitors against USP1/UAF1 complex will be presented.

In conclusion, we have investigated the in vitro substrate preferences and kinetic profiles of three deubiquitinases of clinical relevance. This data will be valuable in the design and analysis of assays used to identify small-molecule inhibitors of these highly specialized proteases.

Russell:Boston Biochem Inc: Employment. Taylor:Boston Biochem Inc: Employment. Brasher:Boston Biochem Inc: Employment. Melandri:Boston Biochem Inc: Employment.