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The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence–function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other’s activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions.

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.

Purpose. It is controversial whether donor-recipient sex mismatch is a risk factor associated with corneal graft failure. The purpose of this study was to investigate the effect of sex mismatch on corneal graft failure in high-risk and non-high-risk patients. Design. A retrospective study. Methods. The medical charts of patients who underwent corneal transplantations by one surgeon between 2012 and 2017 were reviewed. Patients were defined as high-risk for failure if they had glaucoma, ocular surface disease, or corneal vascularization. Graft failure rates were compared using the Kaplan–Meier survival curves between sex matched and mismatched subjects and between male-to-female grafting and other patients. Results. One hundred and thirteen patients with a minimum follow-up of 18 months were included. In 62 non-high-risk patients, graft failure rates were similar between the sex mismatched and the sex matched recipients (p=0.645, log-rank) and in male donor to female recipient transplantations and in the other transplantations (p=0.496, log-rank). Analysis of fifty-one eyes of 51 high-risk graft recipients (mean age of 73.4 ± 12.7 years, N = 26 females) showed that graft failure rates were significantly higher in the sex mismatched than sex matched recipients (p=0.022, log-rank) and in male donor to female recipient transplantations than in the other transplantations (p=0.002, log-rank). Conclusions. Sex matching for every patient bares logistic difficulties; however, in patients who are at high-risk for graft failure, it may be a simple way to improve outcomes and better utilize corneal grafts.

The ability of cells to coordinately migrate in groups is crucial to enable them to travel long distances during embryonic development, wound healing and tumorigenesis, but the fundamental mechanisms underlying intercellular coordination during collective cell migration remain elusive despite considerable research efforts. A novel analytical framework is introduced here to explicitly detect and quantify cell clusters that move coordinately in a monolayer. The analysis combines and associates vast amount of spatiotemporal data across multiple experiments into transparent quantitative measures to report the emergence of new modes of organized behavior during collective migration of tumor and epithelial cells in wound healing assays. First, we discovered the emergence of a wave of coordinated migration propagating backward from the wound front, which reflects formation of clusters of coordinately migrating cells that are generated further away from the wound edge and disintegrate close to the advancing front. This wave emerges in both normal and tumor cells, and is amplified by Met activation with hepatocyte growth factor/scatter factor. Second, Met activation was found to induce coinciding waves of cellular acceleration and stretching, which in turn trigger the emergence of a backward propagating wave of directional migration with about an hour phase lag. Assessments of the relations between the waves revealed that amplified coordinated migration is associated with the emergence of directional migration. Taken together, our data and simplified modeling-based assessments suggest that increased velocity leads to enhanced coordination: higher motility arises due to acceleration and stretching that seems to increase directionality by temporarily diminishing the velocity components orthogonal to the direction defined by the monolayer geometry. Spatial and temporal accumulation of directionality thus defines coordination. The findings offer new insight and suggest a basic cellular mechanism for long-term cell guidance and intercellular communication during collective cell migration.

DNA methylation is a fundamental epigenetic mark that governs chromatin organization, cell identity, and gene expression. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 207 healthy tissue samples. Replicates of the same cell-type are >99.5% identical, demonstrating robustness of cell identity programs to genetic variation and environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny, and identifies methylation patterns retained since gastrulation. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hyper-methylated loci are rare and are enriched for CpG islands, polycomb targets, and CTCF binding sites, suggesting a novel role in shaping cell type-specific chromatin looping. The atlas provides an essential resource for interpretation of disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies. Competing Interest Statement This work was supported by GRAIL, Inc. (Menlo Park, CA). GC, JB, HA, PM, PN, AA, OV, and AJ are employees, shareholders and/or founders at GRAIL, Inc. The remaining authors have declared no conflict of interest.