Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of ...identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.
β-adrenergic signaling pathways mediate key aspects of cardiac function. Its dysregulation is associated with a range of cardiac diseases, including dilated cardiomyopathy (DCM). Previously, we ...established an iPSC model of familial DCM from patients with a mutation in TNNT2, a sarcomeric protein. Here, we found that the β-adrenergic agonist isoproterenol induced mature β-adrenergic signaling in iPSC-derived cardiomyocytes (iPSC-CMs) but that this pathway was blunted in DCM iPSC-CMs. Although expression levels of several β-adrenergic signaling components were unaltered between control and DCM iPSC-CMs, we found that phosphodiesterases (PDEs) 2A and PDE3A were upregulated in DCM iPSC-CMs and that PDE2A was also upregulated in DCM patient tissue. We further discovered increased nuclear localization of mutant TNNT2 and epigenetic modifications of PDE genes in both DCM iPSC-CMs and patient tissue. Notably, pharmacologic inhibition of PDE2A and PDE3A restored cAMP levels and ameliorated the impaired β-adrenergic signaling of DCM iPSC-CMs, suggesting therapeutic potential.
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•β-AR signaling switch from β-2 AR to β-1/2 AR mode during human iPSC-CM maturation•Upregulation of PDE2/3 leads to compromised β-adrenergic regulation in DCM iPSC-CMs•Epigenetic activation of PDE2/3 is a key molecular event during pathogenesis of DCM•Nuclear localization of mutated TNNT2 contributes to epigenetic modification in DCM
In this paper, Wu et al. profiled the β-adrenergic signaling properties in human iPSC-CMs and demonstrated novel epigenetic mechanisms that underlie the compromised β-adrenergic signaling in DCM, a common cause of heart failure and cardiac transplantation. These results enhance our understanding of DCM pathogenesis and may uncover new therapeutic targets.
Material transfer is an essential form of intercellular communication to exchange information and resources between cells. Material transfer between neurons and from glia to neurons has been ...demonstrated to support neuronal survival and activity. Understanding the extent of material transfer in the healthy nervous system is limited. Here we report that in the mouse central nervous system (CNS), neurons receive nuclear and ribosomal material of Sox10-lineage cell (SOL) origin. We show that transfer of SOL-derived material to neurons is region dependent, establishes during postnatal brain maturation, and dynamically responds to LPS-induced neuroinflammation in the adult mouse brain. We identified satellite oligodendrocyte-neuron pairs with loss of plasma membrane integrity between nuclei, suggesting direct material transfer. Together, our findings provide evidence of regionally coordinated transfer of SOL-derived nuclear and ribosomal material to neurons in the mouse CNS, with potential implications for the understanding and modulation of neuronal function and treatment of neurological disorders.
Ligand receptor interactions instruct axon guidance during development. How dendrites are guided to specific targets is less understood. The
PVD sensory neuron innervates muscle-skin interface with ...its elaborate dendritic branches. Here, we found that LECT-2, the ortholog of leukocyte cell-derived chemotaxin-2 (LECT2), is secreted from the muscles and required for muscle innervation by PVD. Mosaic analyses showed that LECT-2 acted locally to guide the growth of terminal branches. Ectopic expression of LECT-2 from seam cells is sufficient to redirect the PVD dendrites onto seam cells. LECT-2 functions in a multi-protein receptor-ligand complex that also contains two transmembrane ligands on the skin, SAX-7/L1CAM and MNR-1, and the neuronal transmembrane receptor DMA-1. LECT-2 greatly enhances the binding between SAX-7, MNR-1 and DMA-1. The activation of DMA-1 strictly requires all three ligands, which establishes a combinatorial code to precisely target and pattern dendritic arbors.
Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of ...β1AR signaling in the heart.BACKGROUNDChronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β1AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β1AR signaling in the heart.We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the β1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening.METHODS AND RESULTSWe applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β1AR association with L-type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer-based biosensors to probe subcellular β1AR-PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β1AR protein levels, receptor association with L-type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P<0.05), and receptor-induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P<0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P<0.05). However, the β1AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P<0.05), and the receptor signal was minimally affected. The isoproterenol-infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β1AR-PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β1AR association with SERCA2a, the endogenous norepinephrine-induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine-induced PKA signaling at the SERCA2a and myocyte shortening.This study reveals distinct mechanisms for the downregulation of subcellular β1AR signaling in the heart under chronic adrenergic stimulation.CONCLUSIONSThis study reveals distinct mechanisms for the downregulation of subcellular β1AR signaling in the heart under chronic adrenergic stimulation.
Abstract
β
1
-adrenergic receptors (β
1
ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of ...the β
1
AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the β
1
AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies β
1
AR N-terminal O-glycosylation at Ser
37
/Ser
41
as a mechanism that prevents β
1
AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated β
1
ARs and N-terminally truncated glycosylation-defective β
1
ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes β
1
ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of β
1
AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.
Alzheimer disease (AD) is characterized by neurodegeneration marked by loss of synapses and spines associated with hyperphosphorylation of tau protein. Accumulating amyloid β peptide (Aβ) in brain is ...linked to neurofibrillary tangles composed of hyperphosphorylated tau in AD. Here, we identify β2-adrenergic receptor (β2AR) that mediates Aβ-induced tau pathology. In the prefrontal cortex (PFC) of 1-year-old transgenic mice with human familial mutant genes of presenilin 1 and amyloid precursor protein (PS1/APP), the phosphorylation of tau at Ser-214 Ser-262 and Thr-181, and the protein kinases including JNK, GSK3α/β, and Ca2+/calmodulin-dependent protein kinase II is increased significantly. Deletion of the β2AR gene in PS1/APP mice greatly decreases the phosphorylation of these proteins. Further analysis reveals that in primary PFC neurons, Aβ signals through a β2AR-PKA-JNK pathway, which is responsible for most of the phosphorylation of tau at Ser-214 and Ser-262 and a significant portion of phosphorylation at Thr-181. Aβ also induces a β2AR-dependent arrestin-ERK1/2 activity that does not participate in phosphorylation of tau. However, inhibition of the activity of MEK, an upstream enzyme of ERK1/2, partially blocks Aβ-induced tau phosphorylation at Thr-181. The density of dendritic spines and synapses is decreased in the deep layer of the PFC of 1-year-old PS1/APP mice, and the mice exhibit impairment of learning and memory in a novel object recognition paradigm. Deletion of the β2AR gene ameliorates pathological effects in these senile PS1/APP mice. The study indicates that β2AR may represent a potential therapeutic target for preventing the development of AD.
Background: Accumulating evidence indicates that β receptors (βAR) may be involved in Alzheimer disease (AD) pathology and that amyloid β peptide (Aβ) may interact with β2AR independently of presynaptic activities.
Results: β2AR, PKA, and JNK mediate Aβ-induced phosphorylation of tau in vivo and in vitro.
Conclusion: An Aβ-β2AR signaling is involved in tau pathology in AD.
Significance: This work indicates a potential mechanism for altering AD pathology by blocking β2ARs.
G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in a broad range of physiological responses and disease states. Activated GPCRs can undergo agonist-induced ...phosphorylation by G protein receptor kinases (GRKs) and second messenger-dependent protein kinases such as protein kinase A (PKA). Here, we characterize spatially segregated subpopulations of β
-adrenergic receptor (β
AR) undergoing selective phosphorylation by GRKs or PKA in a single cell. GRKs primarily label monomeric β
ARs that undergo endocytosis, whereas PKA modifies dimeric β
ARs that remain at the cell surface. In hippocampal neurons, PKA-phosphorylated β
ARs are enriched in dendrites, whereas GRK-phosphorylated β
ARs accumulate in soma, being excluded from dendrites in a neuron maturation-dependent manner. Moreover, we show that PKA-phosphorylated β
ARs are necessary to augment the activity of L-type calcium channel. Collectively, these findings provide evidence that functionally distinct subpopulations of this prototypical GPCR exist in a single cell.
Insulin and adrenergic stimulation are two divergent regulatory systems that may interact under certain pathophysiological circumstances. Here, we characterized a complex consisting of insulin ...receptor (IR) and β2-adrenergic receptor (β2AR) in the heart. The IR/β2AR complex undergoes dynamic dissociation under diverse conditions such as Langendorff perfusions of hearts with insulin or after euglycemic-hyperinsulinemic clamps in vivo. Activation of IR with insulin induces protein kinase A (PKA) and G-protein receptor kinase 2 (GRK2) phosphorylation of the β2AR, which promotes β2AR coupling to the inhibitory G-protein, Gi. The insulin-induced phosphorylation of β2AR is dependent on IRS1 and IRS2. After insulin pretreatment, the activated β2AR-Gi signaling effectively attenuates cAMP/PKA activity after β-adrenergic stimulation in cardiomyocytes and consequently inhibits PKA phosphorylation of phospholamban and contractile responses in myocytes in vitro and in Langendorff perfused hearts. These data indicate that increased IR signaling, as occurs in hyperinsulinemic states, may directly impair βAR-regulated cardiac contractility. This β2AR-dependent IR and βAR signaling cross-talk offers a molecular basis for the broad interaction between these signaling cascades in the heart and other tissues or organs that may contribute to the pathophysiology of metabolic and cardiovascular dysfunction in insulin-resistant states.
Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have favorable cardiovascular outcomes in patients with diabetes. However, whether SGLT2i can improve obesity-related cardiac dysfunction is ...unknown. Sestrin2 is a novel stress-inducible protein that regulates AMPK-mammalian target of rapamycin (mTOR) and suppresses oxidative damage. The aim of this study was to determine whether empagliflozin (EMPA) improves obesity-related cardiac dysfunction via regulating Sestrin2-mediated pathways in diet-induced obesity. C57BL/6J mice and
knockout mice were fed a high-fat diet (HFD) for 12 weeks and then treated with or without EMPA (10 mg/kg) for 8 weeks. Treating HFD-fed C57BL/6J mice with EMPA reduced body weight and whole-body fat and improved metabolic disorders. Furthermore, EMPA improved myocardial hypertrophy/fibrosis and cardiac function and reduced cardiac fat accumulation and mitochondrial injury. Additionally, EMPA significantly augmented Sestrin2 levels and increased AMPK and endothelial nitric oxide synthase phosphorylation, but inhibited Akt and mTOR phosphorylation. These beneficial effects were partially attenuated in HFD-fed
knockout mice. Intriguingly, EMPA treatment enhanced the Nrf2/HO-1-mediated oxidative stress response, suggesting antioxidant and anti-inflammatory activity. Thus, EMPA improved obesity-related cardiac dysfunction via regulating Sestrin2-mediated AMPK-mTOR signaling and maintaining redox homeostasis. These findings provide a novel mechanism for the cardiovascular protection of SGLT2i in obesity.
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