Name: Structural and functional characterization of ubiquitin variant inhibitors of USP15
Keywords: Usp15

Dataset Information

Structural and functional characterization of ubiquitin variant inhibitors of USP15

ABSTRACT: The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. Taking advantage of the modular nature of ubiquitin, we designed a linear dimer (diUbV) consisting of a UbV that bound the DUSP domain followed by a UbV that bound the catalytic domain, which exhibited enhanced specificity and more potent inhibition of USP15 catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the TGF- pathway. Structural analyses of three distinct UbVs bound to the catalytic domain revealed that the inhibitors locked the active site in a closed, inactive conformation. The structure of a UbV-DUSP domain complex revealed that the UbV formed an unusual strand-swapped dimer that bound two DUSP domains. These inhibitors will enable the study of USP15 function in vitro and in vivo to explore the role of the enzyme in oncology, neurology, immunology and inflammation.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jonathan Krieger

LAB HEAD: Sachdev S. Sidhu

PROVIDER: PXD011840 | Pride | 2019-01-16

REPOSITORIES: Pride

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Publications

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

Teyra Joan J Singer Alex U AU Schmitges Frank W FW Jaynes Patrick P Kit Leng Lui Sarah S Polyak Maria J MJ Fodil Nassima N Krieger Jonathan R JR Tong Jiefei J Schwerdtfeger Carsten C Brasher Bradley B BB Ceccarelli Derek F J DFJ Moffat Jason J Sicheri Frank F Moran Michael F MF Gros Philippe P Eichhorn Pieter J A PJA Lenter Martin M Boehmelt Guido G Sidhu Sachdev S SS

Structure (London, England : 1993) 20190131 4

The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs i ...[more]

PMID: 30713027

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Project description:The motor neuron (MN)–hexamer complex consisting of LIM homeobox 3, Islet-1, and nuclear LIM interactor is a key determinant of motor neuron specification and differentiation. To gain insights into the transcriptional network in motor neuron development, we performed a genome-wide ChIP-sequencing analysis and found that the MN–hexamer directly regulates a wide array of motor neuron genes by binding to the HxRE (hexamer response element) shared among the target genes. Interestingly, STAT3-binding motif is highly enriched in the MN–hexamer–bound peaks in addition to the HxRE. We also found that a transcriptionally active form of STAT3 is expressed in embryonic motor neurons and that STAT3 associates with the MN–hexamer, enhancing the transcriptional activity of the MN–hexamer in an upstream signal-dependent manner. Correspondingly, STAT3 was needed for motor neuron differentiation in the developing spinal cord. Together, our studies uncover crucial gene regulatory mechanisms that couple MN–hexamer and STAT-activating extracellular signals to promote motor neuron differentiation in vertebrate spinal cord. To explain our experimental scheme briefly, we are interested in finding target sites for the dimer of transcription factors Isl1 and Lhx3. To mimic the biological activity of Isl1/Lhx3 dimer, we made Isl1-Lhx3 fusion and found that Isl1-Lhx3 has a potent biological activity in multiple systems (i.e. generation of ectopic motor neurons). Then we made ES cell line that induces Flag-tagged Isl1-Lhx3 expression upon Dox treatment. These *mouse* ES cells differentiate to motor neurons (iMN-ESCs) when treated with Dox following EB formation. To identify genomic binding sites of Isl1-Lhx3 (Flag-tagged), we performed ChIP with Flag antibody (pull down of Flag-Isl1-Lhx3) in ES cells treated with Dox. ChIP with Flag antibody in ES cells treated with vehicle (no Dox) was done as a negative control in parallel, and sequenced along with +Dox sample. We have done these experiments twice (two sets).

Dataset Information

Structural and functional characterization of ubiquitin variant inhibitors of USP15

Publications

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

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