mechanistic basis
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2022 ◽  
Vol 119 (3) ◽  
pp. e2105898119
Yiji Liao ◽  
Chen-Hao Chen ◽  
Tengfei Xiao ◽  
Bárbara de la Peña Avalos ◽  
Eloise V. Dray ◽  

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9–mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

2022 ◽  
Vol 5 (4) ◽  
pp. e202101315
Stefanie Dichtl ◽  
David E Sanin ◽  
Carolin K Koss ◽  
Sebastian Willenborg ◽  
Andreas Petzold ◽  

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn’s Disease. Previously, we and others found that TNF blocks the emergence and function of alternative-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balance of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages, we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis, and signaling pathway deconvolution. We found that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way, dependent on JNK signaling via the type 1 TNF receptor on specific populations of alternative-activated macrophages. We further determined that JNK signaling has a profound and broad effect on activated macrophage gene expression. Our findings suggest that TNF’s anti-M2 effects evolved to specifically modulate components of tissue and reparative M2 macrophages and TNF is therefore a context-specific modulator of M2 macrophages rather than a pan-M2 inhibitor.

2022 ◽  
Placheril J. John ◽  
Navneet Kumar

Abstract Arsenic, a toxic metalloid, provokes many detrimental consequences to human health. It is prevalent in earth's crust and poses a major threat to humans globally. Inorganic arsenic exposure occurs mainly via drinking water or food and is metabolized in mammals to form organic metabolites/ end products. Chronic exposure to arsenic causes lung, skin and urinary bladder cancers and increases the risks of liver, kidney and prostate cancers. Arsenic-induced ROS generation, disturbances in several signaling pathways, DNA repair inhibition, chromosomal aberrations, and epigenetic changes including alterations in DNA methylation, histone modifications and differential miRNA expression profiles are involved in cancer progression, and malignant transformation. However, details of arsenic-induced carcinogenesis and molecular mechanisms involved are still remaining obscure. MicroRNAs are post-transcriptional gene expression regulators and themselves may act as oncogenes and tumor suppressor genes. Differential miRNA expression is implicated in several human cancers. This review covers general mechanistic basis of arsenic-induced carcinogenesis, explores recent in-vitro, in-vivo and cohort studies on differential miRNA expression profiles and shares associated molecular mechanistic data on miRNA dysregulation and their functional consequences leading to arsenic induced tumorigenesis, metastasis and cancer, also discusses the future directions.

2022 ◽  
Mithu Baidya ◽  
Madhu Chaturvedi ◽  
Hemlata Dwivedi-Agnihotri ◽  
Ashutosh Ranjan ◽  
Dominic Devost ◽  

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.

2022 ◽  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml

The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.

2022 ◽  
Willow Coyote-Maestas ◽  
David Nedrud ◽  
Yungui He ◽  
Daniel Schmidt

A longstanding goal in protein science and clinical genetics is to develop quantitative models of sequence, structure, and function relationships and delineate the mechanisms by which mutations cause disease. Deep Mutational Scanning (DMS) is a promising strategy to map how amino acids contribute to protein structure and function and to advance clinical variant interpretation. Here, we introduce 7,429 single residue missense mutation into the Inward Rectifier K+ channel Kir2.1 and determine how this affects folding, assembly, and trafficking, as well as regulation by allosteric ligands and ion conduction. Our data provide high-resolution information on a cotranslationally-folded biogenic unit, trafficking and quality control signals, and segregated roles of different structural elements in fold-stability and function. We show that Kir2.1 trafficking mutants are underrepresented in variant effect databases, which has implications for clinical practice. By comparing fitness scores with expert-reviewed variant effects, we can predict the pathogenicity of variants of unknown significance and disease mechanisms of know pathogenic mutations. Our study in Kir2.1 provides a blueprint for how multiparametric DMS can help us understand the mechanistic basis of genetic disorders and the structure-function relationships of proteins.

2022 ◽  
Maria Elena De Obaldia ◽  
Takeshi Morita ◽  
Laura C Dedmon ◽  
Daniel J Boehmler ◽  
Caroline S Jiang ◽  

Female Aedes aegypti mosquitoes feed on human blood, which they use to develop their eggs. It has been widely noted that some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. Here we tested mosquito attraction to skin odor collected from human subjects and identified people who are exceptionally attractive or unattractive to mosquitoes. Notably, these preferences were stable over several years, indicating consistent longitudinal differences in skin odor between subjects. We carried out gas chromatography/quadrupole time of flight-mass spectrometry to analyze the chemical composition of human skin odor in these subjects and discovered that highly attractive people produce significantly increased levels of carboxylic acids. Consistent with the hypothesis that odor concentration is a major driver of differential attraction, mosquitoes could reliably distinguish a highly attractive human from their weakly attractive counterparts unless we substantially diluted the odor of the most attractive subject. Our work suggests that an increased abundance of mosquito attractants on the preferred subject explains differential attraction, rather than the non-preferred skin odor blend being repellent. Mosquitoes detect carboxylic acids with a large family of odor-gated ion channels encoded by the Ionotropic Receptor gene superfamily. Mutant mosquitoes lacking any of the Ionotropic Receptor (IR) co-receptors Ir8a, Ir25a, and Ir76b, were severely impaired in attraction to human scent but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in mosquito-magnet human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others will provide insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.

2022 ◽  
Smita Yadav ◽  
Sujin Byeon ◽  
Bailey Werner ◽  
Reilly Falter ◽  
Kristian Davidsen ◽  

Septins are a family of cytoskeletal proteins that regulate several important aspects of neuronal development. Septin 7 (Sept7) is enriched at the base of dendritic spines in excitatory neurons and mediates both spine formation and spine-synapse maturation. Phosphorylation at a conserved C-terminal tail residue of Sept7 mediates its translocation into the dendritic spine head to allow spine-synapse maturation. The mechanistic basis for postsynaptic stability and compartmentalization conferred by phosphorylated Sept7, however, is not known. We report herein the proteomic identification of Sept7 phosphorylation dependent neuronal interactors. Using Sept7 C-terminal phosphopeptide pulldown and biochemical assays, we show that the 14-3-3 family of proteins specifically interact with Sept7 when phosphorylated at the T426 residue. Biochemically, we validate the interaction between Sept7 and 14-3-3 isoform gamma, and show that 14-3-3 gamma is also enriched in mature dendritic spine head. Further, we demonstrate that interaction of phosphorylated Sept7 with 14-3-3 protects it from dephosphorylation, as expression of a 14-3-3 antagonist significantly decreases phosphorylated Sept7 in neurons. This study identifies 14-3-3 proteins as an important physiological regulator of Sept7 function in neuronal development.

2022 ◽  
Tinna Reynisdottir ◽  
Kimberley Anderson ◽  
Leandros Boukas ◽  
Hans Bjornsson

Wiedemann-Steiner syndrome (WSS) is a neurodevelopmental disorder caused by de novo variants in KMT2A, which encodes a multi–domain histone methyltransferase. To gain insight into the currently unknown pathogenesis of WSS, we examined the spatial distribution of likely WSS–causing variants across the 15 different domains of KMT2A. Compared to variants in healthy controls, WSS variants exhibit a 64.1–fold overrepresentation within the CXXC domain – which mediates binding to unmethylated CpGs – suggesting a major role for this domain in mediating the phenotype. In contrast, we find no significant overrepresentation within the catalytic SET domain. Corroborating these results, we find that hippocampal neurons from Kmt2a–deficient mice demonstrate disrupted H3K4me1 preferentially at CpG-rich regions, but this has no systematic impact on gene expression. Motivated by these results, we combine accurate prediction of the CXXC domain structure by AlphaFold2 with prior biological knowledge to develop a classification scheme for missense variants in the CXXC domain. Our classifier achieved 96.0% positive and 92.3% negative predictive value on a hold–out test set. This classification performance enabled us to subsequently perform an in silico saturation mutagenesis and classify a total of 445 variants according to their functional effects. Our results yield a novel insight into the mechanistic basis of WSS and provide an example of how AlphaFold2 can contribute to the in silico characterization of variant effects with very high accuracy, establishing a paradigm potentially applicable to many other Mendelian disorders.

eLife ◽  
2021 ◽  
Vol 10 ◽  
Marcia A Munoz ◽  
Emma K Fletcher ◽  
Oliver P Skinner ◽  
Julie Jurczyluk ◽  
Esther Kristianto ◽  

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.

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