CRISPR Mediated Target Validation of ERCC3 as the Target of Triptolide

Exome sequencing of Triptolide-resistant haploid cells identified mutations in TFIIH complex, with a majority of them in the ERCC3 subunit. These mutations turned out to be recessive, therefore difficult to validate using conventional cDNA overexpression approach. Here we adopted a novel CRISPR-based knock-in strategy which makes it very straightforward to re-introduce the recessive point mutations that confer drug resistance phenotype in cells. Coupled with mutagenesis and next generation sequencing, this novel gene-editing strategy can expedite small molecule target identification and validation in mammalian cells

Deubiquitinase FAM/USP9X interacts with the E3 ubiquitin ligase SMURF1 and protects it from ligase activity-dependent self-degradation.

Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ligase activity. This modular architecture allows for interactions with other proteins which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is auto-ubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taking together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability

A novel family of highly conserved antigens that induce protective immunity against Staphylococcus aureus

ABSTRACT In the human pathogen Staphylococcus aureus there exists an enormous diversity of proteins containing domains of unknown function (DUF). Here, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to S. aureus. The 18 Csa paralogs in S. aureus Newman are highly similar at the sequence level yet were found to be expressed in multiple cellular localizations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed a dynamic complex formation of Csa1A with several Csa paralogs regulated by metal ions. Interestingly, the paralogs presented various modes of interaction with Csa1A, suggesting that the proteins are involved in the same cellular process in which each paralog might contribute with a particular role. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins, confirming the uniqueness of this family. Since immunization with Csa proteins protected mice from lethal challenge with S. aureus, we propose these antigens as potential vaccine candidates