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Abstract Whole genome and exome sequencing are reporting on hundreds of thousands of missense mutations. Taking a pan-disease approach, we explored how mutations in intrinsically disordered regions (IDRs) break or generate protein interactions mediated by short linear motifs. We created a peptide-phage display library tiling ~57,000 peptides from the IDRs of the human proteome overlapping 12,301 single nucleotide variants associated with diverse phenotypes including cancer, metabolic diseases and neurological diseases. By screening 80 human proteins, we identified 366 mutation-modulated interactions, with half of the mutations diminishing binding, and half enhancing binding or creating novel interaction interfaces. The effects of the mutations were confirmed by affinity measurements. In cellular assays, the effects of motif-disruptive mutations were validated, including loss of a nuclear localisation signal in the cell division control protein CDC45 by a mutation associated with Meier-Gorlin syndrome. The study provides insights into how disease-associated mutations may perturb and rewire the motif-based interactome.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5–52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5–52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5–52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5–52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5–52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5–52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease.

Abstract Phosphorylation is a ubiquitous post‐translation modification that regulates protein function by promoting, inhibiting or modulating protein–protein interactions. Hundreds of thousands of phosphosites have been identified but the vast majority have not been functionally characterised and it remains a challenge to decipher phosphorylation events modulating interactions. We generated a phosphomimetic proteomic peptide‐phage display library to screen for phosphosites that modulate short linear motif‐based interactions. The peptidome covers ~13,500 phospho‐serine/threonine sites found in the intrinsically disordered regions of the human proteome. Each phosphosite is represented as wild‐type and phosphomimetic variant. We screened 71 protein domains to identify 248 phosphosites that modulate motif‐mediated interactions. Affinity measurements confirmed the phospho‐modulation of 14 out of 18 tested interactions. We performed a detailed follow‐up on a phospho‐dependent interaction between clathrin and the mitotic spindle protein hepatoma‐upregulated protein (HURP), demonstrating the essentiality of the phospho‐dependency to the mitotic function of HURP. Structural characterisation of the clathrin‐HURP complex elucidated the molecular basis for the phospho‐dependency. Our work showcases the power of phosphomimetic ProP‐PD to discover novel phospho‐modulated interactions required for cellular function.

Abstract While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.

The question whether interference with the ubiquitous splicing machinery can lead to cell-type specific perturbation of cellular function is addressed here by T cell specific ablation of the general U5 snRNP assembly factor CD2BP2/U5-52K. This protein defines the family of nuclear GYF domain containing proteins that are ubiquitously expressed in eukaryotes with essential functions ascribed to early embryogenesis and organ function. Abrogating CD2BP2/U5-52K in T cells, allows us to delineate the consequences of splicing machinery interferences for T cell development and function. Increased T cell lymphopenia and T cell death are observed upon depletion of CD2BP2/U5-52K. A substantial increase in exon skipping coincides with the observed defect in the proliferation/differentiation balance in the absence of CD2BP2/U5-52K. Prominently, skipping of exon 7 in Mdm4 is observed, coinciding with upregulation of pro-apoptotic gene expression profiles upon CD2BP2/U5-52K depletion. Furthermore, we observe enhanced sensitivity of naïve T cells compared to memory T cells to changes in CD2BP2/U5-52K levels, indicating that depletion of this general splicing factor leads to modulation of T cell homeostasis. Given the recent structural characterization of the U5 snRNP and the crosslinking mass spectrometry data given here, design of inhibitors of the U5 snRNP conceivably offers new ways to manipulate T cell function in settings of disease. [ABSTRACT FROM AUTHOR]

Abstract Specific protein–protein interactions are central to all processes that underlie cell physiology. Numerous studies have together identified hundreds of thousands of human protein–protein interactions. However, many interactions remain to be discovered, and low affinity, conditional, and cell type‐specific interactions are likely to be disproportionately underrepresented. Here, we describe an optimized proteomic peptide‐phage display library that tiles all disordered regions of the human proteome and allows the screening of ~ 1,000,000 overlapping peptides in a single binding assay. We define guidelines for processing, filtering, and ranking the results and provide PepTools, a toolkit to annotate the identified hits. We uncovered >2,000 interaction pairs for 35 known short linear motif (SLiM)‐binding domains and confirmed the quality of the produced data by complementary biophysical or cell‐based assays. Finally, we show how the amino acid resolution‐binding site information can be used to pinpoint functionally important disease mutations and phosphorylation events in intrinsically disordered regions of the proteome. The optimized human disorderome library paired with PepTools represents a powerful pipeline for unbiased proteome‐wide discovery of SLiM‐based interactions.

Integrin activation is required for neutrophil functions. Impaired integrin activation on neutrophils is the hallmark of leukocyte adhesion deficiency (LAD) syndrome in humans, characterized by impaired leukocyte recruitment and recurrent infections. The Src kinase-associated phosphoprotein 2 (Skap2) is involved in integrin functions in different leukocyte subtypes. However, the role of Skap2 in β2 integrin activation and neutrophil recruitment is unknown. In this study, we demonstrate the crucial role of Skap2 in regulating actin polymerization and binding of talin-1 and kindlin-3 to the β2 integrin cytoplasmic domain, thereby being indispensable for β2 integrin activation and neutrophil recruitment. The direct interaction of Skap2 with the Wiskott-Aldrich syndrome protein via its SH3 domain is critical for integrin activation and neutrophil recruitment in vivo. Furthermore, Skap2 regulates integrin-mediated outside-in signaling events and neutrophil functions. Thus, Skap2 is essential to activate the β2 integrins, and loss of Skap2 function is sufficient to cause a LAD-like phenotype in mice.

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed “inside-out signaling”. Subsequently, ligand bound LFA-1 transmits a signal into the T cells (“outside-in signaling”) which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain-containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin-mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion-induced AKI in mice. By using genetically engineered mice and transduced Slp76(-/-) primary leukocytes, we demonstrate that ADAP as well as two N-terminal-located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase-γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin-mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion-induced AKI in humans.

In this work the potential of comparative transcriptomics was explored of Saccharomyces (S.) cerevisiae and S. pastorianus for their discrimination. This way an alternative should be demonstrated to comparative genomics, which can be difficult as a result of their aneuoploid genomes composed of mosaics of the parental genomes. Strains were selected according to their application in beer brewing, i.e. top and bottom fermenting yeasts. Comparative transcriptomics was performed for four strains each of commercially available S. cerevisiae (top fermenting) and Saccharomyces pastorianus (bottom fermenting) brewing yeasts grown at two different temperatures to mid-exponential growth phase. A non-reference based approach was chosen in the form of alignment against a de novo assembled brewery-associated pan transcriptome to exclude bias introduced by manual selection of reference genomes. The result is an analysis workflow for self-contained comparative transcriptomics of Saccharomyces yeasts including, but not limited to, the analysis of core and accessory gene expression, functional analysis and metabolic classification. The functionality of this workflow is demonstrated along the principal differentiation of accessory transcriptomes of S. cerevisiae versus S. pastorianus strains. Hence, this work provides a concept enabling studies under different brewing conditions.

JAK2-V617F-positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, [[beta].sub.1] and [[beta].sub.2], may contribute to CMN pathophysiology remained unclear. [[beta].sub.1] ([[alpha].sub.4][[beta].sub.1]; VLA-4) and [[beta].sub.2] ([[alpha].sub.L][[beta].sub.2]; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin ([JAK2.sup.+/VF] mice) to vascular cell adhesion molecule 1- (VCAM1-) and intercellular adhesion molecule 1-coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of [[beta].sub.1] and [[beta].sub.2] integrins for their respective ligands. For [[beta].sub.1] integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2- V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti-VLA-4 and anti- [[beta].sub.2] integrin antibodies suppress pathologic thrombosis as observed in [JAK2.sup.+/VF] mice. In addition, aberrant homing of [JAK2.sup.+/VF] leukocytes to the spleen was inhibited by neutralizing anti-[[beta].sub.2] antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen., Introduction Chronic myeloproliferative neoplasia (CMN) is a malignant hematopoietic disease characterized by excessive proliferation of one or more myeloid cell lineages as erythrocytes, platelets, or leukocytes. CMN comprises several sub-entities, [...]

Being able to characterize the size of raindrops is useful in a number of fields including meteorology, hydrology, agriculture and telecommunications. Associated with this article are data sets containing surface (i.e. ground-level) measurements of raindrop size from two different instruments and two different geographical locations. Students may begin to develop some sense of the character of raindrop size distributions through some basic exploratory data analysis of these data sets. Teachers of mathematical statistics students will find an example useful for discussing the beta, gamma, lognormal and Weibull probability density models, as well as fitting these by maximum likelihood and assessing the quality of fit. R software is provided by the authors to assist students in these investigations.