Structural and biochemical characterization of VCPIP1 and VCP complex

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Project Abstract/Summary The ubiquitin proteasome system (UPS) is an integral part of determining protein fate. Proteins are marked with ubiquitin for degradation by the proteasome through the activity of an enzymatic cascade (ubiquitination). Deubiquitinating enzymes (DUBs) remove ubiquitin from proteins rescuing them from degradation1. Together ubiquitination and deubiquitination control protein stability and homeostasis and are essential for metazoan development and aberrant regulation implicated in disease such as cancer. Inhibitors of the UPS, such as Bortezomib, are effective cancer therapeutics. With knowledge of the structure and function of DUBs, inhibitors for DUBs can be developed as precision therapies. An example of an application of DUB inhibitors in cancer biology is stabilization of oncoproteins, such as c-Myc1, that are stabilized by a DUB. Valosin containing protein p97/p47 complex interacting protein 1 (VCPIP1), a member of the OTU family of DUBs2, is a cysteine protease that, like many of the DUBs, has been identified as a therapeutic vulnerability in human cancers3. Its known close interactor, valosin containing protein (VCP)4, is a key player in the UPS system as it unfolds a variety of poly-ubiquitinated substrates prior to their degradation by the proteosome, and is itself a promising therapeutic target5. The protein unfolding by VCP is remarkable and unique as it is able to unfold ubiquitin, a notoriously stable protein that initiates the unfolding of poly-ubiquitinated substrates6 prior to DUB activity at the bottom of the channel. There is no knowledge as to how and why VCPIP1 binds to VCP. Preliminary data from a co-purification from mammalian cells and immuno-precipitation mass spectrometry (IP-MS) confirm that VCPIP1 interacts with VCP. Using cryo-electron microscopy (cryo-EM), initial data collection using a Talos Arctica and data processing with cryoSPARC7 resulted in high quality 2D classes with secondary structure features and, ultimately, a 3.3 Å 3D reconstruction. Titan Krios data collection of chemically crosslinked sample resulted in a 3.0 Å reconstruction of the complex without substrate. This provided initial insights into the interaction site of VCPIP1 and VCP and the general stoichiometry of the complex. Using structural and biochemical methods, this proposal aims to elucidate the structure of VCPIP1 and its mechanism of interaction with VCP, why VCPIP1 forms a complex with VCP and how it acts on clients. This effort together with initial hit compounds will enable the development of inhibitors for VCPIP1. Specifically, Specific Aim 1 aims to solve a high-resolution structure of VCPIP1 bound to VCP using cryo-EM and to biochemically characterize the interaction between VCPIP1 and VCP by structure-function studies. Specific Aim 2 aims to determine a high-resolution structure of the VCPIP1/VCP complex bound to a substrate and adaptors, fluorescence/enzymology assays will be used to demonstrate deubiquitylation by VCPIP1 after substrate unfolding by VCP. Specific Aim 3 aims to use structure-based drug design (SBDD), both computationally and experimentally, to discover and improve VCPIP1 inhibitors.