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

• Published in: Blood 2013-11, Vol.122 (21), p.4224-4224
• Main Authors: Russell, Nate, Taylor, Trevor, Parmar, Kalindi, D'Andrea, Alan D., Brasher, Bradley B, Melandri, Francesco D
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.4224.4224

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: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.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,