DYRK1A Kinase Inhibitors Promote β-Cell Survival and Insulin Homeostasis.
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ABSTRACT:
SUBMITTER: Barzowska A
PROVIDER: S-EPMC8467532 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DYRK1A (the dual specificity tyrosine phosphorylation-regulated kinase 1A) plays an important role in body growth and brain physiology. Overexpression of this kinase has been associated with the development of Down syndrome in both human and animal models, whereas single copy loss-of-function of DYRK1A leads to increased apoptosis and decreased brain size. Although more than a dozen of DYRK1A targets have been identified, the molecular basis of its involvement in neuronal development remains unclear. Here we show that DYRK1A and another pro-survival member of the DYRK family, DYRK3, promote cell survival through phosphorylation and activation of SIRT1, an NAD(+)-dependent protein deacetylase that is essential in a variety of physiological processes including stress response and energy metabolism. DYRK1A and DYRK3 directly phosphorylate SIRT1 at Thr(522), promoting deacetylation of p53. A SIRT1 phosphorylation mimetic (SIRT1 T522D) displays elevated deacetylase activity, thus inhibiting cell apoptosis. Conversely, a SIRT1 dephosphorylation mimetic (SIRT1 T522V) fails to mediate DYRK-induced deacetylation of p53 and cell survival. We show that knockdown of endogenous DYRK1A and DYRK3 leads to hypophosphorylation of SIRT1, sensitizing cells to DNA damage-induced cell death. We also provide evidence that phosphorylation of Thr(522) activates SIRT1 by promoting product release, thereby increasing its enzymatic turnover. Taken together, our findings provide a novel mechanism by which two anti-apoptotic DYRK members promote cell survival through direct modification of SIRT1. These findings may have important implications in understanding the molecular mechanisms of tumorigenesis, Down syndrome, and aging.
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Project description:Caloric restriction (CR) extends organismal life- and health-span by improving glucose homeostasis. How CR affect the structure-function of pancreatic beta cells remains unknown. We used single nucleus transcriptomics to show that CR increases the expression of genes for beta cell identity, protein processing, and organelle homeostasis. Gene regulatory network analysis reveal that CR activates transcription factors important for beta cell identity and homeostasis, while imaging metabolomics demonstrates that CR beta cells are more energetically competent. In fact, high-resolution microscopy show that CR reduces beta cell mitophagy to increase mitochondria mass and the potential for ATP generation. However, CR beta cells have impaired adaptive pro liferation in response to high fat diet feeding. Finally, we show that long-term CR delays the onset of beta cell aging and promotes cell longevity by reducing beta cell turnover. Therefore, CR could be a feasible approach to preserve compromised beta cell structure-function during aging and diabetes. 27 diabetes.
2024-09-12 | GSE276572 | GEO
Project description:Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human β cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human β cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater β cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human β cell proliferation, and we provide clarity for future efforts in structure-based drug design for human β cell regenerative drugs.
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Project description:DYRK1A has been implicated as an important drug target in various therapeutic areas, including neurological disorders and oncology. DYRK1A has more recently been shown to be involved in pathways regulating human ?-cell proliferation, thus making it a potential therapeutic target for both Type 1 and Type 2 diabetes. Our group, using a high-throughput phenotypic screen, identified harmine that is able to induce ?-cell proliferation both in vitro and in vivo. Since harmine has suboptimal kinase selectivity, we sought to expand structure-activity relationships for harmine's DYRK1A activity, to enhance selectivity, while retaining human ?-cell proliferation capability. We carried out the optimization of the 1-position of harmine and synthesized 15 harmine analogues. Six compounds showed excellent DYRK1A inhibition with IC50 in the range of 49.5-264 nM. Two compounds, 2-2 and 2-8, exhibited excellent human ?-cell proliferation at doses of 3-30 ?M, and compound 2-2 showed improved kinase selectivity as compared to harmine.
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Project description:We studied the role of protein degradation pathways in the regulation of insulin production and secretion and hypothesised that autophagy regulates proinsulin degradation, thereby modulating beta cell function.Proinsulin localisation in autophagosomes was demonstrated by confocal and electron microscopy. Autophagy was inhibited by knockdown of autophagy-related (ATG) proteins and using the H(+)-ATPase inhibitor bafilomycin-A1. Proinsulin and insulin content and secretion were assessed in static incubations by ELISA and RIA.Confocal and electron microscopy showed proinsulin localised in autophagosomes and lysosomes. Beta-Atg7 (-/-) mice had proinsulin-containing sequestosome 1 (p62 [also known as SQSTM1])(+) aggregates in beta cells, indicating proinsulin is regulated by autophagy in vivo. Short-term bafilomycin-A1 treatment and ATG5/7 knockdown increased steady-state proinsulin and hormone precursor chromogranin A content. ATG5/7 knockdown also increased glucose- and non-fuel-stimulated insulin secretion. Finally, mutated forms of proinsulin that are irreparably misfolded and trapped in the endoplasmic reticulum are more resistant to degradation by autophagy.In the beta cell, transport-competent secretory peptide precursors, including proinsulin, are regulated by autophagy, whereas efficient clearance of transport-incompetent mutated forms of proinsulin by alternative degradative pathways may be necessary to avoid beta cell proteotoxicity. Reduction of autophagic degradation of proinsulin increases its residency in the secretory pathway, followed by enhanced secretion in response to stimuli.
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Project description:Overcoming metabolic stress is a critical step for solid tumour growth. However, the underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway. Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis, possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation in vivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo.
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Project description:Type 2 Diabetes (T2DM) affects more than 300 million people worldwide. One of the hallmarks of T2DM is peripheral insulin resistance, in part due to unproductive insulin signaling through the insulin receptor. The insulin receptor (INSR) exists as two isoforms, INSR-A and INSR-B, which results from skipping or inclusion of exon 11 respectively. What determines the relative abundance of the different insulin receptor splice variants is unknown. Moreover, it is not yet clear what the physiological roles of each of the isoforms are in normal and diseased beta cells. In this study, we show that insulin induces INSR exon 11 inclusion in pancreatic beta cells in both human and mouse. This occurs through activation of the Ras-MAPK/ERK signaling pathway and up-regulation of the splicing factor SRSF1. Induction of exon 11 skipping by a splice-site competitive antisense oligonucleotide inhibited the MAPK-ERK signaling pathway downstream of the insulin receptor, sensitizing the pancreatic ?-cell line MIN6 to stress-induced apoptosis and lipotoxicity. These results assign to insulin a regulatory role in INSR alternative splicing through the Ras-MAPK/ERK signaling pathway. We suggest that in beta cells, INSR-B has a protective role, while INSR-A expression sensitizes beta cells to programmed cell death.
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Project description:The DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1A) gene encodes a proline-directed Ser/Thr kinase. Elevated expression and/or altered distribution of the kinase have been implicated in the neurological impairments associated with Down syndrome (DS) and Alzheimer's disease (AD). Consequently, DYRK1A inhibition has been of significant interest as a potential strategy for therapeutic intervention of DS and AD. Many classes of novel inhibitors have been described in the past decade. Although non-radioactive methods for analyzing DYRK1A inhibition have been developed, methods employing radioactive tracers are still commonly used for quantitative characterization of DYRK1A inhibitors. Here, we present a non-radioactive ELISA assay based on the detection of DYRK1A-phosphorylated dynamin 1a fragment using a phosphorylation site-specific antibody. The assay was verified by the use of two well-characterized DYRK1A inhibitors, epigallocatechin gallate (EGCG) and harmine. The IC 50s for EGCG and harmine determined by the ELISA method were found to be comparable to those previously measured by radioactive tracing methods. Furthermore, we determined the mode of inhibition for EGCG and harmine by a modification of the ELISA assay. This assay confirms the mode of inhibition of EGCG (non-ATP-competitive) and harmine (ATP-competitive), as previously determined. We conclude that the ELISA platform demonstrated here is a viable alternative to the traditional radioactive tracer assays for analyzing DYRK1A inhibitors.
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Project description:Rationale: Regulatory T cells (Treg cells) play an important role in maintaining peripheral tolerance by suppressing over-activation of effector T cells. The kinase PDK1 plays a pivotal role in conventional T cell development. However, whether PDK1 signaling affects the homeostasis and function of Treg cells remains elusive. Methods: In order to evaluate the role of PDK1 in Treg cells from a genetic perspective, mice carrying the floxed PDK1 allele were crossbred with Foxp3 Cre mice to efficiently deleted PDK1 in Foxp3+ Treg cells. Flow cytometry was used to detect the immune cell homeostasis of WT and PDK1fl/flFoxp3Cre mice. RNA-seq was used to assess the differences in transcriptional expression profile of WT and PDK1-deficient Treg cells. The metabolic profiles of WT and PDK1-deficient Treg cells were tested using the Glycolysis Stress Test and Mito Stress Test Kits by the Seahorse XFe96 Analyser. Results: PDK1 was essential for the establishment and maintenance of Treg cell homeostasis and function. Disruption of PDK1 in Treg cells led to a spontaneous fatal systemic autoimmune disorder and multi-tissue inflammatory damage, accompanied by a reduction in the number and function of Treg cells. The deletion of PDK1 in Treg cells destroyed the iron ion balance through regulating MEK-ERK signaling and CD71 expression, resulting in excessive production of intracellular ROS, which did not depend on the down-regulation of mTORC1 signaling. Inhibition of excessive ROS, activated MEK-Erk signaling or overload Fe2+ could partially rescue the survival of PDK1-deficient Treg cells. Conclusion: Our results defined a key finding on the mechanism by which PDK1 regulates Treg cell survival via controlling redox homeostasis.
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