USP26 regulates TGF-ß signaling by deubiquitinating and stabilizing SMAD7.

The amplitude of transforming growth factor-ß (TGF-ß) signal is tightly regulated to ensure appropriate physiological responses. As part of negative feedback loop SMAD7, a direct transcriptional target of downstream TGF-ß signaling acts as a scaffold to recruit the E3 ligase SMURF2 to target the TGF-ß receptor complex for ubiquitin-mediated degradation. Here, we identify the deubiquitinating enzyme USP26 as a novel integral component of this negative feedback loop. We demonstrate that TGF-ß rapidly enhances the expression of USP26 and reinforces SMAD7 stability by limiting the ubiquitin-mediated turnover of SMAD7. Conversely, knockdown of USP26 rapidly degrades SMAD7 resulting in TGF-ß receptor stabilization and enhanced levels of p-SMAD2. Clinically, loss of USP26 correlates with high TGF-ß activity and confers poor prognosis in glioblastoma. Our data identify USP26 as a novel negative regulator of the TGF-ß pathway and suggest that loss of USP26 expression may be an important factor in glioblastoma pathogenesis.

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15.

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 inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor ß pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation.