A novel approach for selecting potent peptide inhibitors of the SARS-CoV2-Mpro protease

Here, we describe a new high throughput selection technology for identifying exceedingly specific and effective peptide inhibitors. This technology incorporates the co-expression of a cytotoxic protein and a library of peptide variants inserted directly into a loop of a carrier protein. Selection is based on the cytotoxicity neutralization by a member of a peptide library binding to and inhibiting the cytotoxic protein. Our technology provides the flexibility of screening both cyclic and linear peptides. Herein, we demonstrate the power of this technology by developing selective inhibitors of the main coronavirus protease (Mpro) in a matter of weeks by screening libraries of cyclic and linear peptides. This technology opens up an opportunity to develop inhibitors for a wide range of previously undruggable targets.

Identification of subfunctionalized aggregate-remodeling J-domain proteins in plants

Hsp70s and J-domain proteins (JDPs) are among the most critical components of the cellular protein quality control machinery, playing crucial roles in preventing and solubilizing cytotoxic protein aggregates. Bacteria, yeast and plants additionally have large, multimeric Hsp100-class disaggregases which, allow the resolubilization of otherwise “dead-end” aggregates, including amyloids. JDPs interact with aggregated proteins and specify the aggregate remodeling activities of Hsp70s and Hsp100s. Plants have a complex network of cytosolic Hsp70s and JDPs, however the aggregate remodeling properties of plant JDPs are not well understood. Here we identify evolutionary-conserved Class II JDPs in the model plant Arabidopsis thaliana with distinct aggregate remodeling functionalities. We identify eight plant orthologs of the yeast protein, Sis1, the principal JDP responsible for directing the yeast chaperone machinery for remodeling protein aggregates. Expression patterns vary dramatically among the eight paralogous proteins under a variety of stress conditions, indicating their subfunctionalization to address distinct stressors. Consistent with a role in solubilizing cytotoxic protein aggregates, six of these plant JDPs associate with heat-induced protein aggregates in vivo as well as colocalize with plant Hsp101 to distinct heat-induced protein aggregate centers. Finally, we show that these six JDPs can differentially remodel multiple model protein aggregates in yeast confirming their involvement in aggregate resolubilization. These results demonstrate that compared to complex metazoans, plants have a robust network of JDPs involved in aggregate remodeling activities with the capacity to process a variety of protein aggregate conformers.

AMP-activated protein kinase and adenylate kinase prevent the ATP catastrophe and cytotoxic protein aggregation

ABSTRACTATP is the main source of chemical energy in all life and is maintained at several millimolar in eukaryotic cells. However, the mechanisms responsible for and physiological relevance of this high and stable concentration of ATP remain unclear. We herein demonstrate that AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperate to maintain cellular ATP levels regardless of glucose concentrations. Single cell imaging of ATP-reduced yeast mutants revealed that ATP concentrations in these mutants underwent stochastic and transient depletion of ATP repeatedly, which induced the cytotoxic aggregation of endogenous proteins and pathogenic proteins, such as huntingtin and α-synuclein. Moreover, pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the accumulation of α-synuclein aggregates and its cytotoxicity. The removal of cytotoxic aggregates depended on proteasomes, and proteasomal activity cooperated with AMPK or ADK to resist proteotoxic stresses. The present results provide the first evidence to show that cellular ATP homeostasis ensures proteostasis and revealed that stochastic fluctuations in cellular ATP concentrations contribute to cytotoxic protein aggregation, implying that AMPK and ADK are important factors that prevent proteinopathies, such as neurodegenerative diseases.

USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome-Associated Quality Control

SUMMARYDefects within mRNAs or nascent chains that halt ribosomal progression can trigger ribosome-associated quality control (RQC) pathways that facilitate mRNA and nascent polypeptide destruction as well as ribosome recycling. Failure to remove defective mRNAs or nascent chains can lead to the accumulation of cytotoxic protein aggregates and proteotoxic stress. We previously established that the E3 ligase ZNF598 catalyzes regulatory ribosomal ubiquitylation of specific 40S ribosomal proteins required for downstream RQC events. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and facilitate ribosomal deubiquitylation following RQC activation. Overexpression of either USP21 or OTUD3 enhances readthrough of stall-inducing sequences as compared to knock-in cells lacking individual RRub sites suggesting that combinatorial ubiquitylation of RPS10 (eS10) and RPS20 (uS10) is required for optimal resolution of RQC events and that deubiquitylating enzymes can limit RQC activation.