Peripheral priming induces plastic transcriptomic and proteomic responses in circulating neutrophils required for pathogen containment.

Neutrophils rapidly respond to inflammation and infection, but to which degree their functional trajectories after mobilization from the bone marrow are shaped within the circulation remains vague. Experimental limitations have so far hampered neutrophil research in human disease. Here, using innovative fixation and single-cell-based toolsets, we profile human and murine neutrophil transcriptomes and proteomes during steady state and bacterial infection. We find that peripheral priming of circulating neutrophils leads to dynamic shifts dominated by conserved up-regulation of antimicrobial genes across neutrophil substates, facilitating pathogen containment. We show the TLR4/NF-κB signaling-dependent up-regulation of canonical neutrophil activation markers like CD177/NB-1 during acute inflammation, resulting in functional shifts in vivo. Blocking de novo RNA synthesis in circulating neutrophils abrogates these plastic shifts and prevents the adaptation of antibacterial neutrophil programs by up-regulation of distinct effector molecules upon infection. These data underline transcriptional plasticity as a relevant mechanism of functional neutrophil reprogramming during acute infection to foster bacterial containment within the circulation.

Ion mobility-tandem mass spectrometry of mucin-type O-glycans.

The dense O-glycosylation of mucins plays an important role in the defensive properties of the mucus hydrogel. Aberrant glycosylation is often correlated with inflammation and pathology such as COPD, cancer, and Crohn's disease. The inherent complexity of glycans and the diversity in the O-core structure constitute fundamental challenges for the analysis of mucin-type O-glycans. Due to coexistence of multiple isomers, multidimensional workflows such as LC-MS are required. To separate the highly polar carbohydrates, porous graphitized carbon is often used as a stationary phase. However, LC-MS workflows are time-consuming and lack reproducibility. Here we present a rapid alternative for separating and identifying O-glycans released from mucins based on trapped ion mobility mass spectrometry. Compared to established LC-MS, the acquisition time is reduced from an hour to two minutes. To test the validity, the developed workflow was applied to sputum samples from cystic fibrosis patients to map O-glycosylation features associated with disease.

Mice with renal-specific alterations of stem cell-associated signaling develop symptoms of chronic kidney disease but surprisingly no tumors.

Previously, we found that Wnt and Notch signaling govern stem cells of clear cell kidney cancer (ccRCC) in patients. To mimic stem cell responses in the normal kidney in vitro in a marker-unbiased fashion, we have established tubular organoids (tubuloids) from total single adult mouse kidney epithelial cells in Matrigel and serum-free conditions. Deep proteomic and phosphoproteomic analyses revealed that tubuloids resembled renewal of adult kidney tubular epithelia, since tubuloid cells displayed activity of Wnt and Notch signaling, long-term proliferation and expression of markers of proximal and distal nephron lineages. In our wish to model stem cell-derived human ccRCC, we have generated two types of genetic double kidney mutants in mice: Wnt-ß-catenin-GOF together with Notch-GOF and Wnt-ß-catenin-GOF together with a most common alteration in ccRCC, Vhl-LOF. An inducible Pax8-rtTA-LC1-Cre was used to drive recombination specifically in adult kidney epithelial cells. We confirmed mutagenesis of ß-catenin, Notch and Vhl alleles on DNA, protein and mRNA target gene levels. Surprisingly, we observed symptoms of chronic kidney disease (CKD) in mutant mice, but no increased proliferation and tumorigenesis. Thus, the responses of kidney stem cells in the tubuloid and genetic systems produced different phenotypes, i.e. enhanced renewal versus CKD.

Herpes simplex virus type 1 modifies the protein composition of extracellular vesicles to promote neurite outgrowth and neuroinfection.

The highly prevalent herpes simplex virus type 1 (HSV-1) causes a range of diseases, including cold sores, blinding keratitis, and life-threatening encephalitis. HSV-1 initially replicates in epithelial cells, enters the peripheral nervous system via neurites, and establishes lifelong infection in the neuronal cell bodies. Neurites are highly dynamic structures that grow or retract in response to attractive or repulsive cues, respectively. Here, we show that infection with HSV-1, but not with a mutant virus lacking glycoprotein G (gG), reduced the repulsive effect of epithelial cells on neurite outgrowth and facilitated HSV-1 invasion of neurons. HSV-1 gG was required and sufficient to induce neurite outgrowth by modifying the protein composition of extracellular vesicles, increasing the amount of neurotrophic and neuroprotective proteins, including galectin-1. Antibodies directed against galectin-1 neutralized the capacity of extracellular vesicles released from HSV-1-infected cells to promote neurite outgrowth. Our study provides new insights into the neurotropism of HSV-1 and identifies a viral protein that modifies the protein composition of extracellular vesicles to stimulate neurite outgrowth and invasion of the nervous system.IMPORTANCEHerpes simplex virus type 1 (HSV-1) must infect neurites (or nerve endings) to establish a chronic infection in neurons. Neurites are highly dynamic structures that retract or grow in the presence of repulsive or attractive proteins. Some of these proteins are released by epithelial cells in extracellular vesicles and act upon interaction with their receptor present on neurites. We show here that HSV-1 infection of epithelial cells modulated their effect on neurites, increasing neurite growth. Mechanistically, HSV-1 glycoprotein G (gG) modifies the protein composition of extracellular vesicles released by epithelial cells, increasing the amount of attractive proteins that enhance neurite outgrowth and facilitate neuronal infection. These results could inform of therapeutic strategies to block HSV-1 induction of neurite outgrowth and, thereby, neuronal infection.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Prdm16 mutation determines sex-specific cardiac metabolism and identifies two novel cardiac metabolic regulators.

AIMS: Mutation of the PRDM16 gene causes human dilated and non-compaction cardiomyopathy. The PRDM16 protein is a transcriptional regulator that affects cardiac development via Tbx5 and Hand1, thus regulating myocardial structure. The biallelic inactivation of Prdm16 induces severe cardiac dysfunction with post-natal lethality and hypertrophy in mice. The early pathological events that occur upon Prdm16 inactivation have not been explored. METHODS AND RESULTS: This study performed in-depth pathophysiological and molecular analyses of male and female Prdm16csp1/wt mice that carry systemic, monoallelic Prdm16 gene inactivation. We systematically assessed early molecular changes through transcriptomics, proteomics, and metabolomics. Kinetic modelling of cardiac metabolism was performed in silico with CARDIOKIN. Prdm16csp1/wt mice are viable up to 8 months, develop hypoplastic hearts, and diminished systolic performance that is more pronounced in female mice. Prdm16csp1/wt cardiac tissue of both sexes showed reductions in metabolites associated with amino acid as well as glycerol metabolism, glycolysis, and the tricarboxylic acid cycle. Prdm16csp1/wt cardiac tissue revealed diminished glutathione (GSH) and increased inosine monophosphate (IMP) levels indicating oxidative stress and a dysregulated energetics, respectively. An accumulation of triacylglycerides exclusively in male Prdm16csp1/wt hearts suggests a sex-specific metabolic adaptation. Metabolic modelling using CARDIOKIN identified a reduction in fatty acid utilization in males as well as lower glucose utilization in female Prdm16csp1/wt cardiac tissue. On the level of transcripts and protein expression, Prdm16csp1/wt hearts demonstrate an up-regulation of pyridine nucleotide-disulphide oxidoreductase domain 2 (Pyroxd2) and the transcriptional regulator pre-B-cell leukaemia transcription factor interacting protein 1 (Pbxip1). The strongest concordant transcriptional up-regulation was detected for Prdm16 itself, probably through an autoregulatory mechanism. CONCLUSIONS: Monoallelic, global Prdm16 mutation diminishes cardiac performance in Prdm16csp1/wt mice. Metabolic alterations and transcriptional dysregulation in Prdm16csp1/wt affect cardiac tissue. Female Prdm16csp1/wt mice develop a more pronounced phenotype, indicating sexual dimorphism at this early pathological window. This study suggests that metabolic dysregulation is an early event in the PRDM16 associated cardiac pathology.

Direct-to-biology, automated, nano-scale synthesis, and phenotypic screening-enabled E3 ligase modulator discovery.

Thalidomide and its analogs are molecular glues (MGs) that lead to targeted ubiquitination and degradation of key cancer proteins via the cereblon (CRBN) E3 ligase. Here, we develop a direct-to-biology (D2B) approach for accelerated discovery of MGs. In this platform, automated, high throughput, and nano scale synthesis of hundreds of pomalidomide-based MGs was combined with rapid phenotypic screening, enabling an unprecedented fast identification of potent CRBN-acting MGs. The small molecules were further validated by degradation profiling and anti-cancer activity. This revealed E14 as a potent MG degrader targeting IKZF1/3, GSPT1 and 2 with profound effects on a panel of cancer cells. In a more generalized view, integration of automated, nanoscale synthesis with phenotypic assays has the potential to accelerate MGs discovery.

Leveraging Ligand Affinity and Properties: Discovery of Novel Benzamide-Type Cereblon Binders for the Design of PROTACs.

Immunomodulatory imide drugs (IMiDs) such as thalidomide, pomalidomide, and lenalidomide are the most common cereblon (CRBN) recruiters in proteolysis-targeting chimera (PROTAC) design. However, these CRBN ligands induce the degradation of IMiD neosubstrates and are inherently unstable, degrading hydrolytically under moderate conditions. In this work, we simultaneously optimized physiochemical properties, stability, on-target affinity, and off-target neosubstrate modulation features to develop novel nonphthalimide CRBN binders. These efforts led to the discovery of conformationally locked benzamide-type derivatives that replicate the interactions of the natural CRBN degron, exhibit enhanced chemical stability, and display a favorable selectivity profile in terms of neosubstrate recruitment. The utility of the most potent ligands was demonstrated by their transformation into potent degraders of BRD4 and HDAC6 that outperform previously described reference PROTACs. Together with their significantly decreased neomorphic ligase activity on IKZF1/3 and SALL4, these ligands provide opportunities for the design of highly selective and potent chemically inert proximity-inducing compounds.

Frequent ZNF217 mutations lead to transcriptional deregulation of interferon signal transduction via altered chromatin accessibility in B cell lymphoma.

Recent exome-wide studies discovered frequent somatic mutations in the epigenetic modifier ZNF217 in primary mediastinal B cell lymphoma (PMBCL) and related disorders. As functional consequences of ZNF217 alterations remain unknown, we comprehensively evaluated their impact in PMBCL. Targeted sequencing identified genetic lesions affecting ZNF217 in 33% of 157 PMBCL patients. Subsequent gene expression profiling (n = 120) revealed changes in cytokine and interferon signal transduction in ZNF217-aberrant PMBCL cases. In vitro, knockout of ZNF217 led to changes in chromatin accessibility interfering with binding motifs for crucial lymphoma-associated transcription factors. This led to disturbed expression of interferon-responsive and inflammation-associated genes, altered cell behavior, and aberrant differentiation. Mass spectrometry demonstrates that ZNF217 acts within a histone modifier complex containing LSD1, CoREST and HDAC and interferes with H3K4 methylation and H3K27 acetylation. Concluding, our data suggest non-catalytic activity of ZNF217, which directs histone modifier complex function and controls B cell differentiation-associated patterns of chromatin structure.

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on sputum viscoelastic properties, airway infection and inflammation in patients with cystic fibrosis.

BACKGROUND: Recent studies demonstrated that the triple combination cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and reduces pulmonary exacerbations in cystic fibrosis (CF) patients with at least one F508del allele. However, effects of ETI on downstream consequences of CFTR dysfunction, i.e. abnormal viscoelastic properties of airway mucus, chronic airway infection and inflammation have not been studied. The aim of this study was to determine the longitudinal effects of ETI on airway mucus rheology, microbiome and inflammation in CF patients with one or two F508del alleles aged ≥12 years throughout the first 12 months of therapy. METHODS: In this prospective observational study, we assessed sputum rheology, the microbiome, inflammation markers and proteome before and 1, 3 and 12 months after initiation of ETI. RESULTS: In total, 79 patients with CF and at least one F508del allele and 10 healthy controls were enrolled in this study. ETI improved the elastic modulus and viscous modulus of CF sputum at 3 and 12 months after initiation (all p<0.01). Furthermore, ETI decreased the relative abundance of Pseudomonas aeruginosa in CF sputum at 3 months and increased the microbiome α-diversity at all time points. In addition, ETI reduced interleukin-8 at 3 months (p<0.05) and free neutrophil elastase activity at all time points (all p<0.001), and shifted the CF sputum proteome towards healthy. CONCLUSIONS: Our data demonstrate that restoration of CFTR function by ETI improves sputum viscoelastic properties, chronic airway infection and inflammation in CF patients with at least one F508del allele over the first 12 months of therapy; however, levels close to healthy were not reached.

Defining the landscape of circular RNAs in neuroblastoma unveils a global suppressive function of MYCN.

Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.

Carm1-arginine methylation of the transcription factor C/EBPα regulates transdifferentiation velocity.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.

Neurofibromatosis type 1-dependent alterations in mouse microglia function are not cell-intrinsic.

We previously discovered a sex-by-genotype defect in microglia function using a heterozygous germline knockout mouse model of Neurofibromatosis type 1 (Nf1 ± mice), in which only microglia from male Nf1 ± mice exhibited defects in purinergic signaling. Herein, we leveraged an unbiased proteomic approach to demonstrate that male, but not female, heterozygous Nf1 ± microglia exhibit differences in protein expression, which largely reflect pathways involved in cytoskeletal organization. In keeping with these predicted defects in cytoskeletal function, only male Nf1 ± microglia had reduced process arborization and surveillance capacity. To determine whether these microglial defects were cell autonomous or reflected adaptive responses to Nf1 heterozygosity in other cells in the brain, we generated conditional microglia Nf1-mutant knockout mice by intercrossing Nf1flox/flox with Cx3cr1-CreER mice (Nf1flox/wt; Cx3cr1-CreER mice, Nf1MG ± mice). Surprisingly, neither male nor female Nf1MG ± mouse microglia had impaired process arborization or surveillance capacity. In contrast, when Nf1 heterozygosity was generated in neurons, astrocytes and oligodendrocytes by intercrossing Nf1flox/flox with hGFAP-Cre mice (Nf1flox/wt; hGFAP-Cre mice, Nf1GFAP ± mice), the microglia defects found in Nf1 ± mice were recapitulated. Collectively, these data reveal that Nf1 ± sexually dimorphic microglia abnormalities are likely not cell-intrinsic properties, but rather reflect a response to Nf1 heterozygosity in other brain cells.

Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

Evolutionary origins and interactomes of human, young microproteins and small peptides translated from short open reading frames.

All species continuously evolve short open reading frames (sORFs) that can be templated for protein synthesis and may provide raw materials for evolutionary adaptation. We analyzed the evolutionary origins of 7,264 recently cataloged human sORFs and found that most were evolutionarily young and had emerged de novo. We additionally identified 221 previously missed sORFs potentially translated into peptides of up to 15 amino acids-all of which are smaller than the smallest human microprotein annotated to date. To investigate the bioactivity of sORF-encoded small peptides and young microproteins, we subjected 266 candidates to a mass-spectrometry-based interactome screen with motif resolution. Based on these interactomes and additional cellular assays, we can associate several candidates with mRNA splicing, translational regulation, and endocytosis. Our work provides insights into the evolutionary origins and interaction potential of young and small proteins, thereby helping to elucidate this underexplored territory of the human proteome.

Massively parallel identification of mRNA localization elements in primary cortical neurons.

Cells adopt highly polarized shapes and form distinct subcellular compartments in many cases due to the localization of many mRNAs to specific areas, where they are translated into proteins with local functions. This mRNA localization is mediated by specific cis-regulatory elements in mRNAs, commonly called 'zipcodes'. Although there are hundreds of localized mRNAs, only a few zipcodes have been characterized. Here we describe a novel neuronal zipcode identification protocol (N-zip) that can identify zipcodes across hundreds of 3' untranslated regions. This approach combines a method of separating the principal subcellular compartments of neurons-cell bodies and neurites-with a massively parallel reporter assay. N-zip identifies the let-7 binding site and (AU)n motif as de novo zipcodes in mouse primary cortical neurons. Our analysis also provides, to our knowledge, the first demonstration of an miRNA affecting mRNA localization and suggests a strategy for detecting many more zipcodes.

Disease- and sex-specific differences in patients with heart valve disease: a proteome study.

Pressure overload in patients with aortic valve stenosis and volume overload in mitral valve regurgitation trigger specific forms of cardiac remodeling; however, little is known about similarities and differences in myocardial proteome regulation. We performed proteome profiling of 75 human left ventricular myocardial biopsies (aortic stenosis = 41, mitral regurgitation = 17, and controls = 17) using high-resolution tandem mass spectrometry next to clinical and hemodynamic parameter acquisition. In patients of both disease groups, proteins related to ECM and cytoskeleton were more abundant, whereas those related to energy metabolism and proteostasis were less abundant compared with controls. In addition, disease group-specific and sex-specific differences have been observed. Male patients with aortic stenosis showed more proteins related to fibrosis and less to energy metabolism, whereas female patients showed strong reduction in proteostasis-related proteins. Clinical imaging was in line with proteomic findings, showing elevation of fibrosis in both patient groups and sex differences. Disease- and sex-specific proteomic profiles provide insight into cardiac remodeling in patients with heart valve disease and might help improve the understanding of molecular mechanisms and the development of individualized treatment strategies.

SUMOylation inhibition overcomes proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM cell lines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor-resistant MM.

Pan-claudin family interactome analysis reveals shared and specific interactions.

Claudins are a family of transmembrane proteins expressed in epithelial tissues and are the major components of tight junctions (TJs), which define barrier properties in epithelia and maintain cell polarity. How claudins regulate the formation of TJs and which functions they exert outside of them is not entirely understood. Although the long and unstructured C-terminal tail is essential for regulation, it is unclear how it is involved in these functions beyond interacting with TJ-associated proteins such as TJ protein ZO-1 (TJP1). Here, we present an interactome study of the pan-claudin family in Madin-Darby canine kidney (MDCK)-C7 cells by combining two complementary mass spectrometry-based pull-down techniques creating an interaction landscape of the entire claudin family. The interaction partners of the claudins' C termini reveal their possible implications in localized biological processes in epithelial cells and their regulation by post-translational modifications (PTMs).