Anti-USP5 monoclonal antibody, clone FQS21565

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub(4) resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.

T-type calciumchannels are essential contributors to the transmission of nociceptive signals in the primary afferent pain pathway. Here, we show that T-type calcium channels are ubiquitinated by WWP1, a plasma-membrane- associated ubiquitin ligase that binds to the intracellular domain III-IV linker region of the Ca(v)3.2 T-type channel and modifies specific lysine residues in this region. A proteomic screen identified the deubiquitinating enzyme USP5 as a Ca(v)3.2 III-IV linker interacting partner. Knockdown of USP5 via shRNA increases Ca(v)3.2 ubiquitination, decreases Ca(v)3.2 protein levels, and reduces Ca(v)3.2 whole-cell currents. In vivo knockdown of USP5 or uncoupling USP5 from native Cav3.2 channels via intrathecal delivery of Tat peptides mediates analgesia in both inflammatory and neuropathic mouse models of mechanical hypersensitivity. Altogether, our experiments reveal a cell signaling pathway that regulates T- type channel activity and their role in nociceptive signaling.