Highlights d A new binding site for autophagy adaptors and receptors was discovered on ATG8 d This site engages UIM-like sequences rather than the canonical ATG8-interacting motif d UIM-type autophagy adaptors and receptors can be found in plants, yeast, and humans d UIM-containing UBX domain proteins mediate autophagic clearance of CDC48/p97

Dormancy is a protective state in which diverse bacteria, including Mycobacterium tuberculosis, Staphylococcus aureus, Treponema pallidum (syphilis), and Borrelia burgdorferi (Lyme disease), curtail metabolic activity to survive external stresses, including antibiotics. Evidence suggests dormancy consists of a continuum of interrelated states, including viable but nonculturable (VBNC) and persistence states. VBNC and persistence contribute to antibiotic tolerance, reemergence from latent infections, and even quorum sensing and biofilm formation. Previous studies indicate that the protein mechanisms regulating persistence and VBNC states are not well understood. We have queried the VBNC state of Micrococcus luteus NCTC 2665 (MI-2665) by quantitative proteomics combining gel electrophoresis, high-performance liquid chromatography, and tandem mass spectrometry to elucidate some of these mechanisms. MI-2665 is a nonpathogenic actinobacterium containing a small (2.5-Mb), high-GC-content genome which exhibits a well-defined VBNC state induced by nutrient deprivation. The MI-2665 VBNC state demonstrated a loss of protein diversity accompanied by increased levels of 18 proteins that are conserved across actinobacteria, 14 of which have not been previously identified in VNBC. These proteins implicate an anaplerotic strategy in the transition to VBNC, including changes in the glyoxylate shunt, redox and amino acid metabolism, and ribosomal regulatory processes. Our data suggest that MI-2665 is a viable model for dissecting the protein mechanisms underlying the VBNC stress response and provide the first protein-level signature of this state. We expect that this protein signature will enable future studies deciphering the protein mechanisms of dormancy and identify novel therapeutic strategies effective against antibiotictolerant bacterial infections.IMPORTANCE Dormancy is a protective state enabling bacteria to survive antibiotics, starvation, and the immune system. Dormancy is comprised of different states, including persistent and viable but nonculturable (VBNC) states that contribute to the spread of bacterial infections. Therefore, it is imperative to identify how bacteria utilize these different dormancy states to survive antibiotic treatment. The objective of our research is to eliminate dormancy as a route to antibiotic tolerance by understanding the proteins that control dormancy in Micrococcus luteus NCTC 2665. This bacterium has unique advantages for studying dormancy, including a small genome and a well-defined and reproducible VBNC state. Our experiments implicate four previously identified and 14 novel proteins upregulated in VBNC that may regulate this critical survival mechanism.KEYWORDS antibiotic tolerance, proteomics, stress response, systems biology D ormancy is a remarkable stress response in which bacteria curtail metabolic activity to survive potentially fatal external stresses. Dormancy has broad implications for many pathogenic bacteria, including Mycobacterium tuberculosis, Staphylococcus ...

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We have engineered the substrate specificity of chymotrypsin to cleave after Asn by high-throughput screening of large libraries created by comprehensive remodeling of the substrate binding pocket. The engineered variant (chymotrypsiN, ChyB-Asn) demonstrated an altered substrate specificity with an expanded preference for Asn-containing substrates. We confirmed that protein engineering did not compromise the stability of the enzyme by biophysical characterization. Comparison of wild-type ChyB and ChyB-Asn in profiling lysates of HEK293 cells demonstrated both qualitative and quantitative differences in the nature of the peptides and proteins identified by liquid chromatography and tandem mass spectrometry. ChyB-Asn enabled the identification of partially glycosylated Asn sites within a model glycoprotein and in the extracellular proteome of Jurkat T cells. ChymotrypsiN is a valuable addition to the toolkit of proteases to aid the mapping of N-linked glycosylation sites within proteins and proteomes.

Significance Proteins are the machinery which execute essential cellular functions. However, measuring their abundance within an organism can be difficult and resource-intensive. Cells use a variety of mechanisms to control protein synthesis from mRNA, including short open reading frames (uORFs) that lie upstream of the main coding sequence. Ribosomes can preferentially translate uORFs instead of the main coding sequence, leading to reduced translation of the main protein. In this study, we show that uORF sequence variation between individuals can lead to different rates of protein translation and thus variable protein abundances. We also demonstrate that natural variation in uORFs occurs frequently and can be linked to whole-plant phenotypes, indicating that uORF sequence variation likely contributes to plant adaptation.