Qiu Zhang

Companion of Cellulose Synthase (CC), has previously been shown to sustain cellulose synthesis during salt stress. However, the role of CCs in this process is not well-understood. In this study, we show that the intrinsically disordered N-terminal domain of Arabidopsis CC1 (CC1NTD) can undergo liquid-liquid phase separation (LLPS) under defined reducing conditions in the presence of trimethylamine N-oxide (TMAO), a naturally occurring plant stress osmolyte. The observed biomolecular condensates are enriched in CC1NTD and display liquid-like behavior, including spherical morphology, fusion, and dynamic exchange with the surrounding solution. We further show that TMAO compacts CC1NTD without inducing folding, that CC1NTD condensates recruit tubulin and accelerate microtubule polymerization while retaining dynamic properties. Additionally, we also studied the intrinsically disordered N-terminal domain of CC2, a closely related paralog to CC1. We found that CC2NTD remains disordered but does not phase separate independently under the same experimental conditions. However, it does partition into CC1NTD condensates. Together, these results define a condition-dependent condensate state for CC1NTD and link that state to tubulin-related function. Finally, we propose a novel mechanism for the formation and maintenance of stress-associated cellulose synthase compartments driven by LLPS of the disordered regions of CSC and accessory proteins.

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we have designed a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibited PLpro with k_inact/K_I = 10,000 M^− 1 s^− 1, achieved sub-µM EC₅₀ values against three SARS-CoV-2 variants in mammalian cell lines, and did not inhibit a panel of human deubiquitinases at > 30 µM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validated our design strategy and established the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.