β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Recent efforts to structurally characterize β1ARs have focused on the ligand binding sites in transmembrane helices and effector docking sites in the intracellular loops and C-terminus. The notion that the β1AR extracellular N-terminus (which is a substrate for both N- and O-linked glycosylation and a target for proteolytic cleavage) functions as a structural determinant of β1AR activation has never been considered. This study takes advantage of site-directed mutagenesis and expression studies in Chinese Hamster ovary cells that harbor a reversible glycosylation defect to map β1ARs O-glycosylation sites to Ser37/Ser41 and show that O-glycosylation at these sites prevents β1AR N-terminal cleavage. We then used an adenoviral overexpression approach to show that both full-length fully-glycosylated β1ARs (β1AR-FL) and N-terminally truncated glycosylation-defective β1ARs (β1AR-Δ52) increase cAMP accumulation and activate the ERK-MAPK signaling cascade in cardiomyocytes. However, isoproterenol-dependent cAMP accumulation is markedly enhanced, and ERK activation is markedly attenuated, in cardiomyocytes that express β1AR-Δ52, compared with β1AR-FL cultures. The conclusion that β1ARs are stabilized in a conformation that is biased toward the cAMP pathway as a result of N-terminal truncation gains further support from additional studies showing that agonistic β1AR autoantibodies induce a more rapid and robust increase in membrane PKA activity in cells that express glycosylation-defective β1ARs, compared to cells that express full-length glycosylated β1ARs. These results identify a novel role for O-glycosylation as a structural determinant of β1AR responsiveness in the heart.
Diabetes mellitus is a debilitating metabolic disorder affecting millions of people worldwide. One of the hallmark symptoms of diabetes is hyperglycemia (elevated blood glucose levels; HG). At least 60% of the diabetic patient develop hypertension and become susceptible to both macro and microvascular complications, including stroke. HG is associated with excessive vascular tone and dysfunction of the blood vessels. Recently, we demonstrated that activation of protein kinase A (PKA) leads to phosphorylation and potentiation of L‐type Ca V 1.2 channels in vascular smooth muscle resulting is increased vasoconstriction during HG. A fundamental gap in knowledge that remains unexplored is the upstream mechanisms underlying PKA activation. Here, we describe for the first time a critical role for local cAMP production by adenylyl cyclase (AC) isoform 5, upstream of PKA, in regulation and potentiation of Ca V 1.2 channels leading to vasoconstriction during HG. Consistent with this, membrane‐targeted fluorescence resonance energy transfer (FRET) biosensor for cAMP revealed a small but significant increase in cAMP production by HG, in agreement with local cAMP production. Treatment with forskolin (global cAMP activator) resulted in a significantly larger cAMP signal, aligned with cell wide global cAMP increase. In the presence of the specific AC5 and AC6 inhibitor 2,5,DDA or in vascular smooth muscle from AC5−/− mice, the HG‐induced cAMP increase was abolished. Yet, the forskolin‐induced global increase in cAMP production remained intact. Electrophysiology and pressure‐myography experiments demonstrated that the 2,5 DDA compound and genetic ablation of AC5, but not AC6, abrogated the increased in L‐type Ca V 1.2 channel activity and vasoconstriction in response to HG. Super‐resolution microscopy and proximity ligation assay confirmed close association between AC5 and L‐type Ca V 1.2 channels. Taken together, these finding demonstrate a novel role of local AC5 signaling in HG‐induced vasoconstriction due to increased Ca V 1.2 activity leading to vasoconstriction during diabetic hyperglycemia. Support or Funding Information NIH R01HL098200, R01HL121059 and NIH T32‐HL086350 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the β1AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the β1AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies β1AR N-terminal O-glycosylation at Ser37/Ser41 as a mechanism that prevents β1AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated β1ARs and N-terminally truncated glycosylation-defective β1ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes β1ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of β1AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.
Previously we found that CFTR regulates the contraction rate of spontaneously beating neonatal ventricular cardiomyocytes. In this study we examined if CFTR is involved in β‐adrenergic receptor (β‐AR) stimulated increases in contraction rate. Spontaneous beating cardiomyocytes were generated by isolation and culture of mouse neonatal ventricular myocytes. Isoproterenol dose‐dependently increased contraction rates with an EC50 of 16.6 nM. Inhibition or knockout of CFTR led to a loss of submaximal β‐AR stimulation, resulting in a right‐shift of the dose response curve with an EC50 of 66.7 nM and 31.2 nM, respectively. This targeted response to submaximal β‐AR stimulated contraction rates was specific to CFTR as inhibition of volume‐ or Ca 2+ ‐activated Cl − channels affected both submaximal and maximal concentrations. Furthermore, pre‐activation of CFTR with genistein or PG‐01 potentiated submaximal isoproterenol stimulated contraction rates, resulting in a left‐shift of the dose response curve with an EC50 of 1.1 nM and 0.6 nM, respectively. Our results indicate that CFTR plays a distinct role in regulating contraction rate responses to submaximal, but not maximal, β‐AR stimulation. Future work will be necessary to determine if individuals with alterations in CFTR activity (i.e. cystic fibrosis patients) may be at increased risk for cardiac abnormalities. Funded by the AHA, NIH, University of Illinois.
Summary Synaptic vesicle and active zone proteins are required for synaptogenesis. The molecular mechanisms for coordinated synthesis of these proteins are not understood. Using forward genetic screens, we identified the conserved THO nuclear export C omplex (THOC) as master regulator of presynapse development in C.elegans dopaminergic neurons. In THOC mutants, synaptic messenger RNAs are trapped in the nucleus, resulting in dramatic decrease of synaptic protein expression, near complete loss of synapses and compromised dopamine function. cAMP-responsive element binding protein (CREB) interacts with THOC to mark activity-dependent transcripts for efficient nuclear export. Deletion of the THOC subunit Thoc5 in mouse dopaminergic neurons causes severe defects in synapse maintenance and subsequent neuronal death in the Substantia Nigra compacta (SNc). These cellular defects lead to abrogated dopamine release, ataxia and animal death. Together, our results argue that nuclear export mechanisms can select specific mRNAs and be a rate-limiting step for synapse development and neuronal survival. Highlights Dopaminergic presynapses are severely impaired in thoc mutant worms and mice THOC specifically controls the nuclear export of synaptic transcripts CREB recruits THOC onto activity-dependent synaptic transcripts for efficient export Dopamine neurons in the SNc degenerate upon conditional knock-out of thoc5
Ferroptosis is a non-apoptotic mode of programmed cell death characterized by iron dependence and lipid peroxidation. Since the ferroptosis was proposed, researchers have revealed the mechanisms of its formation and continue to explore effective inhibitors of ferroptosis in disease. Recent studies have shown a correlation between ferroptosis and the pathological mechanisms of neurodegenerative diseases, as well as diseases involving tissue or organ damage. Acting on ferroptosis-related targets may provide new strategies for the treatment of ferroptosis-mediated diseases. This article specifically describes the metabolic pathways of ferroptosis and summarizes the reported mechanisms of action of natural and synthetic small molecule inhibitors of ferroptosis and their efficacy in disease. The paper also describes ferroptosis treatments such as gene therapy, cell therapy, and nanotechnology, and summarises the challenges encountered in the clinical translation of ferroptosis inhibitors. Finally, the relationship between ferroptosis and other modes of cell death is discussed, hopefully paving the way for future drug design and discovery.
The levels of serum S100B were elevated in patients with ischemic stroke (IS), which may be a novel biomarker for diagnosing IS. The aim of this study was to investigate the association of S100B polymorphisms and serum S100B with IS risk. We genotyped the S100B polymorphisms rs9722, rs9984765, rs2839356, rs1051169 and rs2186358 in 396 IS patients and 398 controls using polymerase chain reaction-single base extension (SBE-PCR). Serum S100B levels were measured by enzyme-linked immunosorbent assay (ELISA). Rs9722 was associated with an increased risk of IS (AA vs. GG: adjusted OR = 2.172, 95% CI, 1.175-4.014, P = 0.013; dominant: adjusted OR = 1.507, 95% CI, 1.071-2.123, P = 0.019; recessive: adjusted OR = 1.846, 95% CI, 1.025-3.323, P = 0.041; additive: adjusted OR=1.371, 95% CI, 1.109-1.694, P = 0.003). The A-C-C-C-A haplotype was associated with an increased risk of IS (OR = 1.325, 95% CI, 1.035-1.696, P = 0.025). In addition, individuals carrying the rs9722 GA/AA genotypes had a higher serum S100B compared with the rs9722 GG genotype in IS patients (P = 0.018). Our results suggest that the S100B gene rs9722 polymorphism may contribute to the susceptibility of IS, probably by promoting the expression of serum S100B.
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