Imetajate pseudokinaasi TRIB3 funktsioonid ja regulatsioon

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Tribbles homoloog 3 (TRIB3) on imetajate geen, mille avaldumistase suureneb mitmesuguste rakustresside, näiteks glükoosi- või aminohappepuuduse, endoplasmaatilise retiikulumi stressi, hüpoksia või oksüdatiivse stressi korral. Selle geeni poolt kodeeritav valk TRIB3 on pseudokinaas – valk, mis primaarjärjestuselt sarnaneb proteiinikinaasile, kuid sisaldab asendusi katalüütiliselt kriitilistes aminohappejääkides. TRIB3 reguleerib rakus toimuvad protsesse valk-valk interaktsioonide kaudu. Kirjeldatud on tema seondumist mitmete transkriptsioonifaktoritega, kinaasidega, ubikvitiini ligaasidega ja muud tüüpi valkudega ning sel moel mõjutab TRIB3 raku stressivastust ja rakusurma, arengulisi protsesse, põletikku ja ainevahetust. Käesolevas doktoritöös uuriti mitmeid TRIB3 geeni toimimisega seotud küsimusi nii raku kui ka organismi tasemel, rakendades muuhulgas meie töörühma poolt loodud hiireliini, mille genoomist on eemaldatud Trib3 geen (Trib3−/− hiired). Töös saadud tulemused näitavad, et inimese maksakasvaja rakuliinis muutub rakustressi korral TRIB3 mRNA isovormide hulgas domineerivaks variant, millel on lühenenud 5′-liiderjärjestus, mis võimaldab efektiivsemat valgutootmist. Nuumrakud on immuunrakud, mis viivad ellu allergilisi reaktsioone. Uurides Trib3 rolli hiire nuumrakkude kultuurides, selgus, et Trib3 avaldumist nendes rakkudes suurendab kasvufaktor interleukiin-3 ning Trib3 puudumine vähendab nuumrakkude võimet teostada immunoloogilisi reaktsioone in vitro. Analüüsides Trib3 avaldumist hiire peaajus, tuvastati, et mRNA arvukus kasvab aju lootelise arengu käigus ja sööda tarbimisel, milles puudub asendamatu aminohape. Trib3−/− hiirte aju uurimisel ilmnes, et neil on suurenenud külgmised ajuvatsakesed, kuid muid olulisi erinevusi aju ehituses ei täheldatud ning käitumiskatsete tulemused näitasid, et Trib3−/− hiirtel on tüüpiline pikaajaline ruumimälu, hirmumälu ja võimekus tunnetada sööda aminohappelist koostist. Selgitamaks TRIB3 tähtsust glükoosipuuduse korral teostati ülegenoomne geeniekspressiooni uuring ning leiti, et TRIB3 pidurdab oluliselt IGFBP2 geeni avaldumise mahasurumist glükoosipuuduse käes kannatavates rakkudes, mis on varasemalt kirjeldamata mehhanism toitainepuudusest tingitud rakusurma takistamiseks.Tribbles homolog 3 (TRIB3) is a mammalian gene that is upregulated in response to several types of cellular stress, including glucose or amino acid deprivation, endoplasmic reticulum stress, hypoxia and oxidative stress. The TRIB3 protein is a pseudokinase, i.e., a protein that displays sequence similarity to protein kinases but contains substitutions at positions that are critical for catalytic activity in canonical protein kinases. TRIB3 is known to form protein–protein interactions with several transcription factors, protein kinases, ubiquitin ligases and other proteins, and, through these interactions, TRIB3 is implicated in the regulation of the cellular stress response, cell death, developmental processes, inflammation and metabolism. In this dissertation, several aspects of TRIB3 gene regulation and function were studied at the cell and organism levels, facilitated by the generation of a Trib3 knockout mouse line by our group. To obtain a better understanding of TRIB3 induction mechanisms, comparative quantification of different TRIB3 mRNA isoforms was performed in human hepatoma cells, revealing that mRNA isoforms containing a truncated 5′-UTR become predominant in stressful conditions, enhancing the translational potential of the TRIB3 mRNA pool. Studying the role of mouse Trib3 in mast cells, tissue-resident immune cells that mediate allergic responses, it is shown that the growth factor interleukin-3 positively regulates Trib3 expression in these cells, and a lack of Trib3 impairs the immunological functions of mast cells, implicating Trib3 in the modulation of the immune response. Analysis of Trib3 expression in the mouse brain uncovered upregulation during embryonic brain development and after the consumption of an amino acid-deficient diet. Trib3 knockout mice exhibited enlarged lateral ventricles in the brain; nevertheless, long-term spatial memory, fear memory and aversion to amino acid-imbalanced diet appear unaltered by a lack of Trib3. Finally, the role of TRIB3 in the cellular stress response to glucose deficiency was investigated using genome-wide gene expression profiling. Crucially, TRIB3 substantially alleviated the repression of IGFBP2 in glucose-deprived cells, which represents a novel mechanism of deferring cell death caused by nutrient deficiency

Transcriptional Profiling of Hypoxia-Regulated Non-coding RNAs in Human Primary Endothelial Cells

Hypoxia occurs in human atherosclerotic lesions and has multiple adverse effects on endothelial cell metabolism. Recently, key roles of long non-coding RNAs (lncRNAs) in the development of atherosclerosis have begun to emerge. In this study, we investigate the lncRNA profiles of human umbilical vein endothelial cells subjected to hypoxia using global run-on sequencing (GRO-Seq). We demonstrate that hypoxia regulates the nascent transcription of ~1800 lncRNAs. Interestingly, we uncover evidence that promoter-associated lncRNAs are more likely to be induced by hypoxia compared to enhancer-associated lncRNAs, which exhibit an equal distribution of up- and downregulated transcripts. We also demonstrate that hypoxia leads to a significant induction in the activity of super-enhancers next to transcription factors and other genes implicated in angiogenesis, cell survival and adhesion, whereas super-enhancers near several negative regulators of angiogenesis were repressed. Despite the majority of lncRNAs exhibiting low detection in RNA-Seq, a subset of lncRNAs were expressed at comparable levels to mRNAs. Among these, MALAT1, HYMAI, LOC730101, KIAA1656, and LOC339803 were found differentially expressed in human atherosclerotic lesions, compared to normal vascular tissue, and may thus serve as potential biomarkers for lesion hypoxia

Exploiting Glutamine Consumption in Atherosclerotic Lesions by Positron Emission Tomography Tracer (2S,4R)-4-F-18-Fluoroglutamine

Increased glutamine metabolism by macrophages is associated with development of atherosclerotic lesions. Positron emission tomography/computed tomography (PET/CT) with a glutamine analog (2S,4R)-4-F-18-fluoroglutamine (F-18-FGln) allows quantification of glutamine consumption in vivo. Here, we investigated uptake of F-18-FGln by atherosclerotic lesions in mice and compared the results with those obtained using the glucose analog 2-deoxy-2-F-18-fluoro-D-glucose (F-18-FDG). Uptake of F-18-FGln and F-18-FDG by healthy control mice (C57BL/6JRj) and atherosclerotic low-density lipoprotein receptor-deficient mice expressing only apolipoprotein B100 (LDLR(-/-)ApoB(100/100)) was investigated. The mice were injected intravenously with F-18-FGln or F-18-FDG for in vivo PET/CT imaging. After sacrifice at 70 minutes post-injection, tracer uptake was analyzed by gamma counting of excised tissues and by autoradiography of aorta cryosections, together with histological and immunohistochemical analyses. We found that myocardial uptake of F-18-FGln was low. PET/CT detected lesions in the aortic arch, with a target-to-background ratio (SUVmax, aortic arch/SUVmean, blood) of 1.95 +/- 0.42 (mean +/- standard deviation). Gamma counting revealed that aortic uptake of F-18-FGln by LDLR(-/-)ApoB(100/100) mice (standardized uptake value [SUV], 0.35 +/- 0.06) was significantly higher than that by healthy controls (0.20 +/- 0.08, P = 0.03). More detailed analysis by autoradiography revealed that the plaque-to-healthy vessel wall ratio of F-18-FGln (2.90 +/- 0.42) was significantly higher than that of F-18-FDG (1.93 +/- 0.22, P = 0.004). Immunohistochemical staining confirmed that F-18-FGln uptake in plaques co-localized with glutamine transporter SLC7A7-positive macrophages. Collectively these data show that the F-18-FGln PET tracer detects inflamed atherosclerotic lesions. Thus, exploiting glutamine consumption using F-18-FGln PET may have translational relevance for studying atherosclerotic inflammation

Dissecting the polygenic basis of atherosclerosis via disease-associated cell state signatures

Coronary artery disease (CAD) is a pandemic disease where up to half of the risk is explained by genetic factors. Advanced insights into the genetic basis of CAD require deeper understanding of the contributions of different cell types, molecular pathways, and genes to disease heritability. Here, we investigate the biological diversity of atherosclerosis-associated cell states and interrogate their contribution to the genetic risk of CAD by using single-cell and bulk RNA sequencing (RNA-seq) of mouse and human lesions. We identified 12 disease-associated cell states that we characterized further by gene set functional profiling, ligand-receptor prediction, and transcription factor inference. Importantly, Vcam1+ smooth muscle cell state genes contributed most to SNP-based heritability of CAD. In line with this, genetic variants near smooth muscle cell state genes and regulatory elements explained the largest fraction of CAD-risk variance between individuals. Using this information for variant prioritization, we derived a hybrid polygenic risk score (PRS) that demonstrated improved performance over a classical PRS. Our results provide insights into the biological mechanisms associated with CAD risk, which could make a promising contribution to precision medicine and tailored therapeutic interventions in the future.publishedVersionPeer reviewe

Microanatomy of the Human Atherosclerotic Plaque by Single-Cell Transcriptomics

Rationale:Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed.Objective:Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis.Methods and Results:We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4(+) and CD8(+) T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells.Conclusions:This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease

Single-Cell Gene-Regulatory Networks of Advanced Symptomatic Atherosclerosis

BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis

Pharmacological or TRIB3-Mediated Suppression of ATF4 Transcriptional Activity Promotes Hepatoma Cell Resistance to Proteasome Inhibitor Bortezomib

The proteasome is an appealing target for anticancer therapy and the proteasome inhibitor bortezomib has been approved for the treatment of several types of malignancies. However, the molecular mechanisms underlying cancer cell resistance to bortezomib remain poorly understood. In the current article, we investigate how modulation of the eIF2α–ATF4 stress pathway affects hepatoma cell response to bortezomib. Transcriptome profiling revealed that many ATF4 transcriptional target genes are among the most upregulated genes in bortezomib-treated HepG2 human hepatoma cells. While pharmacological enhancement of the eIF2α–ATF4 pathway activity results in the elevation of the activities of all branches of the unfolded protein response (UPR) and sensitizes cells to bortezomib toxicity, the suppression of ATF4 induction delays bortezomib-induced cell death. The pseudokinase TRIB3, an inhibitor of ATF4, is expressed at a high basal level in hepatoma cells and is strongly upregulated in response to bortezomib. To map genome-wide chromatin binding loci of TRIB3 protein, we fused a Flag tag to endogenous TRIB3 in HepG2 cells and performed ChIP-Seq. The results demonstrate that TRIB3 predominantly colocalizes with ATF4 on chromatin and binds to genomic regions containing the C/EBP–ATF motif. Bortezomib treatment leads to a robust enrichment of TRIB3 binding near genes induced by bortezomib and involved in the ER stress response and cell death. Disruption of TRIB3 increases C/EBP–ATF-driven transcription, augments ER stress and cell death upon exposure to bortezomib, while TRIB3 overexpression enhances cell survival. Thus, TRIB3, colocalizing with ATF4 and limiting its transcriptional activity, functions as a factor increasing resistance to bortezomib, while pharmacological over-activation of eIF2α–ATF4 can overcome the endogenous restraint mechanisms and sensitize cells to bortezomib

The Power of Single-Cell RNA Sequencing in eQTL Discovery

Genome-wide association studies have successfully mapped thousands of loci associated with complex traits. During the last decade, functional genomics approaches combining genotype information with bulk RNA-sequencing data have identified genes regulated by GWAS loci through expression quantitative trait locus (eQTL) analysis. Single-cell RNA-Sequencing (scRNA-Seq) technologies have created new exciting opportunities for spatiotemporal assessment of changes in gene expression at the single-cell level in complex and inherited conditions. A growing number of studies have demonstrated the power of scRNA-Seq in eQTL mapping across different cell types, developmental stages and stimuli that could be obscured when using bulk RNA-Seq methods. In this review, we outline the methodological principles, advantages, limitations and the future experimental and analytical considerations of single-cell eQTL studies. We look forward to the explosion of single-cell eQTL studies applied to large-scale population genetics to take us one step closer to understanding the molecular mechanisms of disease