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RNF169 is a DYRK1A substrate. (A) FLAG-tagged RNF169 expressed in HEK-293T cells depleted of DYRK1A by siRNA transfection was purified using an anti-FLAG Ab and subsequently used in an IVK assay in the presence of radioactive labeled ATP. Proteins were analyzed by autoradiography and in WBs probed with an anti-FLAG Ab to check for equal amounts of RNF169 protein. DYRK1A depletion was assessed in WBs of total lysates from parallel samples. Quantification is shown in Fig. S5E. (B) A radioactive IVK assay was performed using bacterially produced MBP-RNF169 in the absence or presence of GST-DYRK1A. The background DYRK1A autophosphorylation was determined by incubation of the protein alone. The Coomassie blue staining demonstrates equal loading and the arrow points to MBP-RNF169. (C) MBP-fused RNF169 was used as a substrate in IVK assays with GST-DYRK1A and the phosphorylated peptides were identified by MS analysis. The position of the phosphorylated aa is in violet and the validated residues are in blue. The peptide coverage of RNF169 is also shown (identified peptides in green), rendering a coverage of 87%. See Fig. S6E,F for validation experiments. (D) Evolutionary conservation of the DYRK1A-dependent phosphosites in RNF169. Alignment of the RNF169 proteins from different the species and human RFN168: Dr, Danio rerio; Hs, Homo sapiens; Md, Monodelphis domestica; Mm, Mus musculus; Xl, Xenopus laevis. The phosphosites validated in the IVK assays are shown in blue, with residues at P − 3 and P + 1 in the same color if matching the DYRK1A consensus phosphorylation site. The putative phosphosites assayed in IVK assays but not validated are shown in violet. The arrows indicate residues important for the interaction with the ubiquitinated nucleosome⁴¹.
Source publication
Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. More...
Citations
... As both TRAF3 and RAD54L2 are required for the expression of ACE2, we next searched for the connection between TRAF3 and RAD54L2. Review of a protein-protein interaction database suggests that DYRK1A is a candidate binding partner for both TRAF3 and RAD54L2 (28). Of note, DYRK1A has been identified as an essential gene for SARS-CoV-2 infection in other CRISPR screens (12,14,15,18) and has recently been shown to be required for ACE2 expression (22). ...
Angiotensin converting enzyme 2 (ACE2), the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is differentially expressed in a wide variety of tissues and cell types. The expression of ACE2 is under tight regulation, but the mechanisms regulating ACE2 expression have not yet been well defined. Through a genome-wide CRISPR knockout screen, we discovered that host factors TRAF3, DYRK1A, and RAD54L2 (TDR) form a complex to regulate the expression of ACE2. Knockout of TRAF3, DYRK1A, or RAD54L2 reduces the mRNA levels of ACE2 and inhibits the cellular entry of SARS-CoV-2. On the other hand, SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We have identified mutations in spike (S) (P1079T) and nucleocapsid (N) (S194L) that enhance the replication of SARS-CoV-2 in cells that express ACE2 at a low level. Our results have revealed the mechanisms for the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2.
IMPORTANCE
The expression of ACE2 is essential for the entry of SARS-CoV-2 into host cells. We identify a new complex—the TDR complex—that acts to maintain the abundance of ACE2 in host cells. The identification and characterization of the TDR complex provide new targets for the development of therapeutics against SARS-CoV-2 infection. By analysis of SARS-CoV-2 virus replicating in cells expressing low levels of ACE2, we identified mutations in spike (P1079T) and nucleocapsid (S194L) that overcome the restriction of limited ACE2. Functional analysis of these key amino acids in S and N extends our knowledge of the impact of SARS-CoV-2 variants on virus infection and transmission.
... Dyrk1a could also influence cell survival indirectly by its role in DNA repair. For example, DYRK1A associates with a DNA repair protein, RNF169, and thereby promotes homologous recombination repair (Menon et al., 2019;Roewenstrunk et al., 2019). In summary, Dyrk1a function could be essential to maintain cell survival by several mechanisms. ...
Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.
... DYRK1A phosphorylates various substrates both in the nucleus and cytoplasm and consequently acts as a regulator of the cell cycle, cell quiescence, and cell differentiation (Aranda et al, 2011;Becker & Sippl, 2011). DYRK1A is also involved in many other cellular processes such as cytoskeletal organization (Ryoo et al, 2007;Ori-McKenney et al, 2016) and DNA damage response (Guard et al, 2019;Menon et al, 2019;Roewenstrunk et al, 2019). Human DYRK1A is encoded in the Down Syndrome Critical Region in chromosome 21 Hämmerle et al, 2003), and higher expression of DYRK1A is responsible for most of the phenotypes including intellectual disability of Down syndrome patients (Altafaj et al, 2001). ...
... Our phosphoproteomic analysis identified FAM53C as a protein that makes a complex with DCAF7/WDR68 (Miyata & Nishida, 2021). Several other proteomic approaches have identified FAM53C as a DYRK1A interactor (Varjosalo et al, 2013;Guard et al, 2019;Menon et al, 2019;Roewenstrunk et al, 2019;Viard et al, 2022). These results indicate that FAM53C associates in cells with DYRK1A and/or DCAF7/WDR68. ...
The protein kinase DYRK1A encoded in human chromosome 21 is the major contributor to the multiple symptoms observed in Down syndrome patients. In addition, DYRK1A malfunction is associated with various other neurodevelopmental disorders such as autism spectrum disorder. Here, we identified FAM53C with no hitherto known biological function as a novel suppressive binding partner of DYRK1A. FAM53C is bound to the catalytic protein kinase domain of DYRK1A, whereas DCAF7/WDR68, the major DYRK1A-binding protein, binds to the N-terminal domain of DYRK1A. The binding of FAM53C inhibited autophosphorylation activity of DYRK1A and its kinase activity to an exogenous substrate, MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them, forming a tri-protein complex. DYRK1A possesses an NLS and accumulates in the nucleus when overexpressed in cells. Co-expression of FAM53C induced cytoplasmic re-localization of DYRK1A, revealing the cytoplasmic anchoring function of FAM53C to DYRK1A. Moreover, the binding of FAM53C to DYRK1A suppressed the DYRK1A-dependent nuclear localization of DCAF7/WDR68. All the results show that FAM53C binds to DYRK1A, suppresses its kinase activity, and anchors it in the cytoplasm. In addition, FAM53C is bound to the DYRK1A-related kinase DYRK1B with an Hsp90/Cdc37-independent manner. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in normal brain tissue. FAM53C-dependent regulation of the kinase activity and intracellular localization of DYRK1A may play a significant role in gene expression regulation caused by normal and aberrant levels of DYRK1A.
... From within the CRO1 duplicated region, DYRK1A was selected as a candidate gene based on its multiple reported dose-sensitive effects on brain abnormalities in DS, including neurodevelopment, retina development and Alzheimer's type neurodegeneration, 51-54 as well as its interaction with proteins involved in DNA-repair [55][56][57] and cellular quiescence. 58 We aimed to correct the copy number of DYRK1A to disomy, using CRISPR-Cas9 gene targeting, in the same way as we recently accomplished in T21 iPSCs, for the chr21 gene BACE2. ...
... As DYRK1A was recently found interacting with proteins involved in DNA repair, [55][56][57] we repeated once more the γH2AX staining on the same iPSC panel, this time co-staining with 53BP1 (NHEJ-DDR mediator) (Supplementary Figure S7). The relative profile of γH2AX levels seen in Fig. 4f was fully reproduced again in the cell lines and upon DYRK1A inhibitors treatment (Supplementary Figure S7a). ...
... This isogenic system proved that trisomic overdose of this chromosomal segment caused an increase in nuclear γH2AX foci, likely reflecting an increase in unrepaired DSBs in iPSC nuclei (Fig. 4, Supplementary Figure S7). As DYRK1A was previously seen interacting with and phosphorylating DNA repair proteins, [55][56][57] we inhibited this phosphorylation activity using two selective chemical inhibitors. Each of these inhibitors corrected the number of γH2AX foci nearly to the same level as the CRISPR/Cas9 editing (Fig. 4), showing that DYRK1A overdose is the likely main driver of this phenotype, but not completely excluding the potential additional, weaker contribution of other genes in the CRO1 duplicated segment. ...
Background:
People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down.
Methods:
Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids.
Findings:
Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage.
Interpretation:
Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies.
Funding:
Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".
... DYRK1A phosphorylates pleiotropic substrates both in the nucleus and cytoplasm, and consequently acts as a regulator of the cell cycle, cell quiescence, and cell differentiation (Aranda et al, 2011;Becker & Sippl, 2011;Galceran et al, 2003;Park et al, 2009). DYRK1A is also involved in many other cellular processes such as cytoskeletal organization (Dowjat et al, 2012;Liu et al, 2008;Ori-McKenney et al, 2016;Ryoo et al, 2007;Scales et al, 2009) and DNA damage response (Guard et al, 2019;Menon et al, 2019;Roewenstrunk et al, 2019). Human DYRK1A is encoded in the Down Syndrome Critical Region (DSCR) in chromosome 21 (Galceran et al, 2003;Hämmerle et al, 2003) and higher expression of DYRK1A is responsible for most of the phenotypes including intellectual disability of Down syndrome patients. ...
... Our phospho-proteomic analysis identified FAM53C as a protein that makes a complex with DCAF7/WDR68(Miyata & Nishida, 2021). Two other proteomic approaches have suggested FAM53C as a DYRK1A-interactor(Roewenstrunk et al, 2019) (Guard et al, 2019. ...
A protein kinase DYRK1A encoded in human chromosome-21 is the major contributor for multiple symptoms observed in Down syndrome patients. In addition, DYRK1A dysfunction has been associated with various neuronal disorders, including autism spectrum disorder and Alzheimer disease. Here we identified FAM53C as a novel suppressive binding partner of DYRK1A. FAM53C bound to the catalytic kinase domain of DYRK1A, whereas DCAF7/WDR68, the major known DYRK1A-binding protein, binds to its N-terminal domain. The binding of FAM53C inhibited the protein kinase activity of DYRK1A to itself and an exogenous substrate MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them. DYRK1A possesses a nuclear localization signal and accumulates in the nucleus when overexpressed in cells. FAM53C induced cytoplasmic re-localization of DYRK1A and DCAF7/WDR68. FAM53C is thus a binding suppressor of DYRK1A, anchoring it in an inactive state in the cytoplasm. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in the normal brain tissues. FAM53C-dependent regulation of DYRK1A may play a significant role in gene expression modification caused by DYRK1A.
... For example, the lncRNA PRLH1 was identified as an RNF169 interactor that promotes homologous recombination (HR) repair (Deng et al., 2019); RNF169 is a new interacting partner for dual-specificity tyrosine-phosphorylation-regulated kinase 1A Frontiers in Genetics frontiersin.org (DYRK1A) (Roewenstrunk et al., 2019). RNF169 was also found to bind to ubiquitylated H2A-Lys13/Lys15 in a manner that involves its canonical ubiquitin-binding helix and a pair of arginine-rich motifs (Kitevski-LeBlanc et al., 2017). ...
Background: Pancreatic adenocarcinoma (PAAD) is a highly deadly and aggressive tumour with a poor prognosis. However, the prognostic value of RNF169 and its related mechanisms in PAAD have not been elucidated. In this study, we aimed to explore prognosis-related genes, especially RNF169 in PAAD and to identify novel potential prognostic predictors of PAAD.
Methods: The GEPIA and UALCAN databases were used to investigate the expression and prognostic value of RNF169 in PAAD. The correlation between RNF169 expression and immune infiltration was determined by using TIMER and TISIDB. Correlation analysis with starBase was performed to identify a potential regulatory axis of lncRNA-miRNA-RNF169.
Results: The data showed that the level of RNF169 mRNA expression in PAAD tissues was higher than that in normal tissues. High RNF169 expression was correlated with poor prognosis in PAAD. In addition, analysis with the TISIDB and TIMER databases revealed that RNF169 expression was positively correlated with tumour immune infiltration in PAAD. Correlation analysis suggested that the long non-coding RNA (lncRNA) AL049555.1 and the microRNA (miRNA) hsa-miR-324-5p were involved in the expression of RNF169, composing a potential regulatory axis to control the progression of PAAD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that RNF169 plays a role in PAAD through pathways such as TNF, Hippo, JAK-STAT and Toll-like receptor signaling.
Conclusion: In summary, the upregulation of RNF169 expression mediated by ncRNAs might influence immune cell infiltration in the microenvironment; thus, it can be used as a prognostic biomarker and a potential therapeutic target in PAAD.
... These 226 proteins were enriched in the mitochondrial respirasome but also in "extracellular space" and in "serine-type endopeptidase inhibitor activity" (Figure 3E). Taking advantage of our previously published proteomic dataset of the hippocampus of the same type of mice, we could also compare the proteomic changes due to 2019), versus 273/3608 in the cerebellum). Indeed, we detected significant overlaps of 14%-18% corresponding to 14 proteins differentially abundant both in the cerebellum and the hippocampus when looking at the genotype contrast and 15 in the treatment contrast in TG ( Figure 4). ...
Introduction
DYRK1A is a dual-specificity kinase that is overexpressed in Down syndrome (DS) and plays a key role in neurogenesis, neuronal differentiation and function, cognitive phenotypes, and aging. Dyrk1A has also been implicated in cerebellar abnormalities observed in association with DS, and normalization of Dyrk1A dosage rescues granular and Purkinje cell densities in a trisomic DS mouse model. However, the underlying molecular mechanisms governing these processes are unknown.
Methods
To shed light on the effects of Dyrk1A overexpression in the cerebellum, here we investigated the cerebellar proteome in transgenic Dyrk1A overexpressing mice in basal conditions and after treatment with green tea extract containing epigallocatechin-3-gallate (EGCG), a DYRK1A inhibitor.
Results and Discussion
Our results showed that Dyrk1A overexpression alters oxidative phosphorylation and mitochondrial function in the cerebellum of transgenic mice. These alterations are significantly rescued upon EGCG-containing green tea extract treatment, suggesting that its effects in DS could depend in part on targeting mitochondria, as shown by the partially restoration by the treatment of the increased mtDNA copy number in TG non-treated mice.
... DYRK1A acts as a kinase for SIRT1, activates its deacetylation activity, and further promotes the inhibition of P53, thereby maintaining cancer cell survival under stressful conditions [39]. A comprehensive interaction screening based on proteomics linked DYRK1A to RNF169 and DNA damage response [40]. The results demonstrated that RNF169 is a DYRK1A substrate, and that DYRK1A binding to RNF169 is necessary for the recruitment of DYRK1A to DSB-induced foci [40]. ...
... A comprehensive interaction screening based on proteomics linked DYRK1A to RNF169 and DNA damage response [40]. The results demonstrated that RNF169 is a DYRK1A substrate, and that DYRK1A binding to RNF169 is necessary for the recruitment of DYRK1A to DSB-induced foci [40]. Our results demonstrated that knockout DYRK1A in PC cell lines might result in increased DNA damage and impaired HRR after DSBs, hence decreasing radioresistance in pancreatic cancer. ...
Simple Summary
Pancreatic cancer is the fourth leading cause of cancer-related death in Western countries. Although several therapeutic strategies have been developed for pancreatic cancer, radiation therapy has not yet yielded satisfactory results. Unraveling the mechanism of radioresistance in pancreatic cancer and developing new therapeutic targets has become a major challenge. Therefore, we applied kinome-wide CRISPR-Cas9 loss-of-function screening combined with the 3D cell culture method and identified DYRK1A as a sensitive target for radiotherapy. Additionally, we confirmed that DYRK1A-targeted inhibitors could enhance the efficacy of radiotherapy. Our results further support the use of CRISPR-Cas9 screening to identify novel therapeutic targets and develop new strategies to enhance radiotherapy efficacy in pancreatic cancer.
Abstract
Although radiation therapy has recently made great advances in cancer treatment, the majority of patients diagnosed with pancreatic cancer (PC) cannot achieve satisfactory outcomes due to intrinsic and acquired radioresistance. Identifying the molecular mechanisms that impair the efficacy of radiotherapy and targeting these pathways are essential to improve the radiation response of PC patients. Our goal is to identify sensitive targets for pancreatic cancer radiotherapy (RT) using the kinome-wide CRISPR-Cas9 loss-of-function screen and enhance the therapeutic effect through the development and application of targeted inhibitors combined with radiotherapy. We transduced pancreatic cancer cells with a protein kinase library; 2D and 3D library cells were irradiated daily with a single dose of up to 2 Gy for 4 weeks for a total of 40 Gy using an X-ray generator. Sufficient DNA was collected for next-generation deep sequencing to identify candidate genes. In this study, we identified several cell cycle checkpoint kinases and DNA damage related kinases in 2D- and 3D-cultivated cells, including DYRK1A, whose loss of function sensitizes cells to radiotherapy. Additionally, we demonstrated that the harmine-targeted suppression of DYRK1A used in conjunction with radiotherapy increases DNA double-strand breaks (DSBs) and impairs homologous repair (HR), resulting in more cancer cell death. Our results support the use of CRISPR-Cas9 screening to identify new therapeutic targets, develop radiosensitizers, and provide novel strategies for overcoming the tolerance of pancreatic cancer to radiotherapy.
... Neither APP, nor SOD1, nor USP16 were found in this segmental duplication, and none of the genes in this region have so far been shown to increase DNA damage by their over-expression alone. Among the genes in this segment, the kinase encoded by DYRK1A was recently implicated in regulating the repair of DNA breaks caused by ionizing radiation [57][58][59] , and this region contains several transcription factors and chromatin modi ers whose individual and interactive roles remain to be studied in more detail, opening up possibilities of yet undiscovered mechanisms contributing in a major way to accelerated aging in DS. ...
Cells from people with Down syndrome (DS) show faster accumulation of DNA damage and epigenetic aging marks. Causative mechanisms remain un-proven and hypotheses range from amplified chromosomal instability to actions of several supernumerary chromosome 21 genes. Plasma immunoglobulin G (IgG) glycosylation profiles are established as a reliable predictor of biological and chronological aging. We performed IgG glycan profiling of n=246 individuals with DS (208 adults and 38 children) from three European populations and compared these to age-, sex- and demography-matched general populations. We uncovered very significantly increased IgG glycosylation aging marks associated with DS. Average levels of IgG glycans without galactose (G0) and those with two galactoses (G2) as a function of age in persons with DS corresponded to levels detected in 19 years older euploid individuals. Some aging marks were significant already in children with DS. Remarkably, the IgG glycan profiles of a child with segmental duplication of only 31 genes on chromosome 21 had values similar to those of age-matched DS children, outside the normal children’s range. This is the first non-epigenetic evidence of accelerated systemic biological aging in DS, suggesting it begins very early in childhood. It points to a causative contribution of the overdose of genes in a short segment of chromosome 21, not previously linked to accelerated aging, opening a route to discovery of hitherto unrecognised mechanisms.
... Recently, several studies have revealed an additional role of DYRK1A in DNA damage repair via its interaction with the ring finger protein RNF169, a negative regulator of double-strand breaks (DBS) repair, that leads to the recruitment of the 53BP1 scaffolding protein associated with non-homologous end joining (NHEJ)-promoting factor, at the DBS sites [125][126][127]. ...
Down syndrome is the main cause of intellectual disabilities with a large set of comorbidities from developmental origins but also that appeared across life span. Investigation of the genetic overdosage found in Down syndrome, due to the trisomy of human chromosome 21, has pointed to one main driver gene, the Dual-specificity tyrosine-regulated kinase 1A (Dyrk1a). Dyrk1a is a murine homolog of the drosophila minibrain gene. It has been found to be involved in many biological processes during development and in adulthood. Further analysis showed its haploinsufficiency in mental retardation disease 7 and its involvement in Alzheimer’s disease. DYRK1A plays a role in major developmental steps of brain development, controlling the proliferation of neural progenitors, the migration of neurons, their dendritogenesis and the function of the synapse. Several strategies targeting the overdosage of DYRK1A in DS with specific kinase inhibitors have showed promising evidence that DS cognitive conditions can be alleviated. Nevertheless, providing conditions for proper temporal treatment and to tackle the neurodevelopmental and the neurodegenerative aspects of DS across life span is still an open question.
