Ubiquitinated PCNA Drives USP1 Synthetic Lethality in Cancer.

CRISPR Cas9-based screening is a powerful approach for identifying and characterizing novel drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacologic inhibition of USP1 leads to decreased DNA synthesis concomitant with S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identify RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition is associated with reduced PCNA protein levels. Ectopic expression of WT or ubiquitin-dead K164R PCNA reverses USP1 inhibitor sensitivity. Our results show, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to selected BRCA1/2 WT cancer cell lines enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant tumors. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to posttranslational modifications of PCNA. Taken together, USP1 inhibition may represent a novel therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

Effect of RpoN, RpoS and LuxS Pathways on the Biofilm Formation and Antibiotic Sensitivity of Borrelia Burgdorferi.

Borrelia burgdorferi, the causative agent of Lyme disease, is capable of forming biofilm in vivo and in vitro, a structure well known for its resistance to antimicrobial agents. For the formation of biofilm, signaling processes are required to communicate with the surrounding environment such as it was shown for the RpoN-RpoS alternative sigma factor and for the LuxS quorum-sensing pathways. Therefore, in this study, the wild-type B. burgdorferi and different mutant strains lacking RpoN, RpoS, and LuxS genes were studied for their growth characteristic and development of biofilm structures and markers as well as for their antibiotic sensitivity. Our results showed that all three mutants formed small, loosely formed aggregates, which expressed previously identified Borrelia biofilm markers such as alginate, extracellular DNA, and calcium. All three mutants had significantly different sensitivity to doxycyline in the early log phase spirochete cultures; however, in the biofilm rich stationary cultures, only LuxS mutant showed increased sensitivity to doxycyline compared to the wild-type strain. Our findings indicate that all three mutants have some effect on Borrelia biofilm, but the most dramatic effect was found with LuxS mutant, suggesting that the quorum-sensing pathway plays an important role of Borrelia biofilm formation and antibiotic sensitivity.

Characterization of biofilm formation by Borrelia burgdorferi in vitro.

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.