Exploring the Structure and Biochemistry of Oxidation-Mediated Inhibitation of the Peptidyl-Prolyl Isomerase PIN1

Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that has been shown to be neuroprotective in aging-related neurodegenerative diseases such as Alzheimer\u27s disease (AD). However, it is not active in AD brain, and a recent proteomic screen of Mild Cognitive Impairment (MCI) brain samples revealed that Pin1 is oxidized in the brains of these pre-AD patients. This suggests that this oxidation may be the cause of the loss of the neuroprotective Pin1 function in AD. The Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pKa, making it highly susceptible to oxidation. We hypothesize that Pin1 is inhibited by oxidation of this cysteine. This hypothesis was tested using enzyme activity assays, mutational analysis, and structural studies. We conclude that the loss of Pin1 activity due to oxidation can be isolated to the oxidative modification of Cys113, and reveals a potential mechanism for countering this loss of function

scClustViz – Single-cell RNAseq cluster assessment and visualization [version 2; peer review: 2 approved]

Single-cell RNA sequencing (scRNAseq) represents a new kind of microscope that can measure the transcriptome profiles of thousands of individual cells from complex cellular mixtures, such as in a tissue, in a single experiment. This technology is particularly valuable for characterization of tissue heterogeneity because it can be used to identify and classify all cell types in a tissue. This is generally done by clustering the data, based on the assumption that cells of a particular type share similar transcriptomes, distinct from other cell types in the tissue. However, nearly all clustering algorithms have tunable parameters which affect the number of clusters they will identify in data. The R Shiny software tool described here, scClustViz, provides a simple interactive graphical user interface for exploring scRNAseq data and assessing the biological relevance of clustering results. Given that cell types are expected to have distinct gene expression patterns, scClustViz uses differential gene expression between clusters as a metric for assessing the fit of a clustering result to the data at multiple cluster resolution levels. This helps select a clustering parameter for further analysis. scClustViz also provides interactive visualisation of: cluster-specific distributions of technical factors, such as predicted cell cycle stage and other metadata; cluster-wise gene expression statistics to simplify annotation of cell types and identification of cell type specific marker genes; and gene expression distributions over all cells and cell types. scClustViz provides an interactive interface for visualisation, assessment, and biological interpretation of cell-type classifications in scRNAseq experiments that can be easily added to existing analysis pipelines, enabling customization by bioinformaticians while enabling biologists to explore their results without the need for computational expertise. It is available at https://baderlab.github.io/scClustViz/

Creative destruction in science

Drawing on the concept of a gale of creative destruction in a capitalistic economy, we argue that initiatives to assess the robustness of findings in the organizational literature should aim to simultaneously test competing ideas operating in the same theoretical space. In other words, replication efforts should seek not just to support or question the original findings, but also to replace them with revised, stronger theories with greater explanatory power. Achieving this will typically require adding new measures, conditions, and subject populations to research designs, in order to carry out conceptual tests of multiple theories in addition to directly replicating the original findings. To illustrate the value of the creative destruction approach for theory pruning in organizational scholarship, we describe recent replication initiatives re-examining culture and work morality, working parents\u2019 reasoning about day care options, and gender discrimination in hiring decisions. Significance statement It is becoming increasingly clear that many, if not most, published research findings across scientific fields are not readily replicable when the same method is repeated. Although extremely valuable, failed replications risk leaving a theoretical void\u2014 reducing confidence the original theoretical prediction is true, but not replacing it with positive evidence in favor of an alternative theory. We introduce the creative destruction approach to replication, which combines theory pruning methods from the field of management with emerging best practices from the open science movement, with the aim of making replications as generative as possible. In effect, we advocate for a Replication 2.0 movement in which the goal shifts from checking on the reliability of past findings to actively engaging in competitive theory testing and theory building. Scientific transparency statement The materials, code, and data for this article are posted publicly on the Open Science Framework, with links provided in the article

A prenylated dsRNA sensor protects against severe COVID-19

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that OAS1, through RNase L, potently inhibits SARS-CoV-2. We show that a common splice-acceptor SNP (Rs10774671) governs whether people express prenylated OAS1 isoforms that are membrane-associated and sense specific regions of SARS-CoV-2 RNAs, or only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. Importantly, in hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting this antiviral defense is a major component of a protective antiviral response

Tutorial: guidelines for annotating single-cell transcriptomic maps using automated and manual methods

Single-cell transcriptomics can profile thousands of cells in a single experiment and identify novel cell types, states and dynamics in a wide variety of tissues and organisms. Standard experimental protocols and analysis workflows have been developed to create single-cell transcriptomic maps from tissues. This tutorial focuses on how to interpret these data to identify cell types, states and other biologically relevant patterns with the objective of creating an annotated map of cells. We recommend a three-step workflow including automatic cell annotation (wherever possible), manual cell annotation and verification. Frequently encountered challenges are discussed, as well as strategies to address them. Guiding principles and specific recommendations for software tools and resources that can be used for each step are covered, and an R notebook is included to help run the recommended workflow. Basic familiarity with computer software is assumed, and basic knowledge of programming (e.g., in the R language) is recommended

Noncovalent binding of a cyclic peptide inhibitor to the peptidyl-prolyl isomerase Pin1, explored by hydrogen exchange mass spectrometry

© 2015 Published by NRC Research Press. Pin1 is a peptidyl-prolyl isomerase (PPIase) that plays a central role in eukaryotic cell cycle regulation, making this protein an interesting target for cancer therapy. Pin1 exhibits high specificity for substrates where proline is preceded by phosphoserine or phosphothreonine. The protein comprises an N-terminal WW (tryptophan-tryptophan) domain and a C-terminal PPIase domain. The cyclic peptide [CRYPEVEIC] (square brackets are used to denote the cyclic structure) represents a lead compound for a new class of nonphosphorylated Pin1 inhibitors. Unfortunately, it has not been possible thus far to characterize the Pin1-[CRYPEVEIC] complex by X-ray crystallography. Thus, the exact binding mode remains unknown. The current work employs hydrogen/deuterium exchange mass spectrometry for gaining insights into the Pin1-[CRYPEVEIC] interactions. The WW domain shows extensive conformational dynamics, both in the presence and in the absence of ligand. In contrast, profound changes in deuteration kinetics are observed in the PPIase domain after the addition of [CRYPEVEIC]. The secondary structure elements 2,3, and 4 exhibit markedly reduced deuteration, consistent with their postulated involvement in ligand binding. Unexpectedly, [CRYPEVEIC] destabilizes the range of residues 61-86, a segment that comprises basic side chains that normally interact with the substrate phosphate. This destabilization is likely caused by steric clashes with Y3 or E5 of the inhibitor. Ligand-induced destabilization has previously been reported for a few other proteins, but effects of this type are not very common. Our findings suggest that future crystallization trials on Pin1 variants deleted for residues in the 61-86 range might provide a path towards high-resolution X-ray structures of Pin1 bound to cyclic peptide inhibitors

Peroxide-mediated oxidation and inhibition of the peptidyl-prolyl isomerase Pin1

© 2015. Pin1 is a phosphorylation-dependent peptidyl-prolyl isomerase that plays a critical role in mediating protein conformational changes involved in signaling processes related to cell cycle control. Pin1 has also been implicated as being neuroprotective in aging-related neurodegenerative disorders including Alzheimer\u27s disease where Pin1 activity is diminished. Notably, recent proteomic analysis of brain samples from patients with mild cognitive impairment revealed that Pin1 is oxidized and also displays reduced activity. Since the Pin1 active site contains a functionally critical cysteine residue (Cys113) with a low predicted pKa, we hypothesized that Cys113 is sensitive to oxidation. Consistent with this hypothesis, we observed that treatment of Pin1 with hydrogen peroxide results in a 32Da mass increase, likely resulting from the oxidation of Cys113 to sulfinic acid (Cys-SO2H). This modification results in loss of peptidyl-prolyl isomerase activity. Notably, Pin1 with Cys113 substituted by aspartic acid retains activity and is no longer sensitive to oxidation. Structural studies by X-ray crystallography revealed increased electron density surrounding Cys113 following hydrogen peroxide treatment. At lower concentrations of hydrogen peroxide, oxidative inhibition of Pin1 can be partially reversed by treatment with dithiothreitol, suggesting that oxidation could be a reversible modification with a regulatory role. We conclude that the loss of Pin1 activity upon oxidation results from oxidative modification of the Cys113 sulfhydryl to sulfenic (Cys-SOH) or sulfinic acid (Cys-SO2H). Given the involvement of Pin1 in pathological processes related to neurodegenerative diseases and to cancer, these findings could have implications for the prevention or treatment of disease

Developmental Emergence of Adult Neural Stem Cells as Revealed by Single-Cell Transcriptional Profiling

Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single-cell RNA sequencing at multiple developmental time points to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that, by E13.5, RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model wherein cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood and wherein the transition to a quiescent adult NSC occurs during late neurogenesis

Single-cell transcriptomic profiling of the aging mouse brain

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand–receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process