Autoinhibitory Peptide Research Articles

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

Background: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. Methods: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. Results: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm−1s−1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [−0.1; 0.03], p = 0.387). Conclusions: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

Silencing of spontaneous activity at α4β1/3δ GABAA receptors in hippocampal granule cells reveals different ligand pharmacology

The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy. Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed. Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1 -/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells. Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced.

Skd3 (human ClpB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations.

Cells have evolved specialized protein disaggregases to reverse toxic protein aggregation and restore protein functionality. In nonmetazoan eukaryotes, the AAA+ disaggregase Hsp78 resolubilizes and reactivates proteins in mitochondria. Curiously, metazoa lack Hsp78. Hence, whether metazoan mitochondria reactivate aggregated proteins is unknown. Here, we establish that a mitochondrial AAA+ protein, Skd3 (human ClpB), couples ATP hydrolysis to protein disaggregation and reactivation. The Skd3 ankyrin-repeat domain combines with conserved AAA+ elements to enable stand-alone disaggregase activity. A mitochondrial inner-membrane protease, PARL, removes an autoinhibitory peptide from Skd3 to greatly enhance disaggregase activity. Indeed, PARL-activated Skd3 solubilizes α-synuclein fibrils connected to Parkinson's disease. Human cells lacking Skd3 exhibit reduced solubility of various mitochondrial proteins, including anti-apoptotic Hax1. Importantly, Skd3 variants linked to 3-methylglutaconic aciduria, a severe mitochondrial disorder, display diminished disaggregase activity (but not always reduced ATPase activity), which predicts disease severity. Thus, Skd3 is a potent protein disaggregase critical for human health.

Monitoring GPCR Induced Atypical MAPK p38 Signaling to Identify Key Regulators of Vascular Inflammation

Activation of the mitogen activated protein kinase (MAPK) p38 is a critical regulatory step in pro‐inflammatory signaling and a key target for therapeutic intervention in vascular diseases, including chronic inflammation and lung disease. Our recent studies defined a conserved ubiquitin driven pathway that regulates atypical p38 signaling by a subset of G protein‐coupled receptors (GPCR). Atypical p38 signaling is initiated by GPCR induced c‐Src phosphorylation of an autoinhibitory peptide in the E3 ubiquitin ligase NEDD4‐2. In turn, phospho‐activation releases the autoinhibitory peptide from the catalytic site of NEDD4‐2, a step which is critical for stress induced GPCR ubiquitination and amplification of atypical p38 signaling and inflammation in the vasculature. Despite the critical role of GPCR induced p38 signaling in the onset and progression of many pathophysiological responses, atypical p38 signaling is still relatively understudied. Here we report the development of Försters Resonance Energy Transfer (FRET) biosensors to map the spatiotemporal activation of p38 signaling by multiple GPCRs in vascular endothelial cells. These FRET sensors create an ideal platform for a small‐scale screening pipeline to identify molecular modulators of the atypical p38 signaling pathway. Our studies have already identified multiple regulatory proteins that block the induction and/or magnitude of GPCR induced p38 signaling, perturbing pro‐inflammatory signaling in the vasculature.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cyclosporin A attenuating morphine tolerance through inhibiting NO/ERK signaling pathway in human glioblastoma cell line: the involvement of calcineurin.

Cyclosporin A (CsA) is known to have an immunosuppressive action. However, it is also attracting attention due to its effects on the nervous system, such as inhibiting the development and expression of morphine-induced tolerance and dependence through unknown mechanisms. It has been shown that CsA modulates the nitric oxide (NO) synthesis and extracellular signal-regulated kinases (ERK) activation, which are potentially involved in signaling pathways in morphine-induced tolerance in cellular models. Therefore, the current study was designed to evaluate the modulatory role of CsA on the MOR tolerance, by targeting the downstream signaling pathway of NO and ERK using an in vitro model. For this purpose, T98G cells were pretreated with CsA, calcineurin autoinhibitory peptide (CAIP), and NG-nitro-l-arginine methyl ester (L-NAME) 30 min before 18 h exposure to MOR. Then, we analyzed the intracellular cyclic adenosine monophosphate (cAMP) levels and also the expression of phosphorylated ERK and nitric oxide synthase (nNOS) proteins. Our results showed that CsA (1 nM, 10 nM, and 100 nM) and CAIP (50 µM) have significantly reduced cAMP and nitrite levels as compared to MOR-treated (2.5 µM) T98G cells. This clearly revealed the attenuation of MOR tolerance by CsA. The expression of nNOS and p-ERK proteins were down-regulated when the T98G cells were pretreated with CsA (1 nM, 10 nM, and 100 nM), CAIP (50 µM), and L-NAME (0.1 mM) as compared to MOR. In conclusion, the CsA pretreatment had a modulatory role in MOR-induced tolerance, which was possibly mediated through NO/ERK signaling pathway.

Charge-Triggered Membrane Insertion of Matrix Metalloproteinase-7, Supporter of Innate Immunity and Tumors

Matrix metalloproteinase-7 (MMP-7) sheds signaling proteins from cell surfaces to activate bacterial killing, wound healing, and tumorigenesis. The mechanism targeting soluble MMP-7 to membranes has been investigated. Nuclear magnetic resonance structures of the zymogen, free and bound to membrane mimics without and with anionic lipid, reveal peripheral binding to bilayers through paramagnetic relaxation enhancements. Addition of cholesterol sulfate partially embeds the protease in the bilayer, restricts its diffusion, and tips the active site away from the bilayer. Its insertion of hydrophobic residues organizes the lipids, pushing the head groups and sterol sulfate outward toward the enzyme's positive charge on the periphery of the enlarged interface. Fluorescence probing demonstrates a similar mode of binding to plasma membranes and internalized vesicles of colon cancer cells. Binding of bilayered micelles induces allosteric activation and conformational change in the auto-inhibitory peptide and the adjacent scissile site, illustrating a potential intermediate in the activation of the zymogen.

Steady-state modulation of voltage-gated K+ channels in rat arterial smooth muscle by cyclic AMP-dependent protein kinase and protein phosphatase 2B.

Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone.

Targeting rho guanine nucleotide exchange factor ARHGEF5/TIM with auto-inhibitory peptides in human breast cancer.

The oncogenic protein ARHGEF5/TIM has long been known to express specifically in human breast cancer and other tumors, which is an important member of Rho guanine nucleotide exchange factors that activate Rho-family GTPases by promoting GTP/GDP exchange. The activation capability of TIM is auto-inhibited by a putative helix N-terminal to Dbl homology (DH) domain, which is stabilized by intramolecular interaction of Src homology 3 domain with a poly-proline sequence that locates between the helix and DH domain. Here, we attempted to target TIM DH domain using the modified versions of its auto-inhibitory helix. In the procedure, bioinformatics techniques were used to investigate the intramolecular interaction of DH domain with auto-inhibitory helix and, based on obtained knowledge, to optimize physicochemical property and structural conformation for the helix. We also performed affinity assay to determine the binding strength of modified peptides to DH domain. Consequently, two modified peptides, namely, DALYEEYNLVV and EVLYEEYQLVV were found as good binders of DH domain with dissociation constants K d of 0.35 and 2 µM, respectively. Structural analysis revealed that the charge neutralization and electrostatic interaction confer additional stability for these two peptide complexes with DH domain.

CONTROL OF ATRIAL GAP JUNCTION CONDUCTANCE BY INTRACELLULAR PHOSPHATASES

Myocardial action potential conduction velocity is a function of gap junction (GJ) electrical conductance (Gj). The phosphorylation state of GJ proteins, in particular Cx43 determines Gj and the role of protein phosphatases (PPs) remains unclear. This study aims to investigate the role of serine-threonine PPs in regulating Gj, in particular the Ca2+-dependent PP, calcineurin, and the Ca2+-independent PPs, PP1 and PP2A. Left atrial strips were dissected from male, Dunkin-Hartley guinea pigs to estimate Gj by measuring the frequency-dependent intracellular conductance1 in control solution (Na=149.4 mM), or low-Na solution (Na=29.4 mM) to raise [Ca2+]i. Inhibitors to calcinuerin, (cyclosporine-A; 5 µM; calcineurin auto-inhibitory peptide; CAIP, 50 µM); PPI (tautomycin, 5 nM) or PP2A (okadaic acid, 2 nM; fostreicin 100 nM) were used. Western blots were conducted on inhibitor perfused tissue. Values are mean±SEM, normalised to control. Differences were tested by ANOVA; null hypotheses rejected at p Low-Na significantly and reversibly decreased Gj, and in control conditions individual inhibitors had no effect. Cyclosporine-A and CAIP diminished the effects of low-Na (Gj: low-Na, 55±5.8% low-Na+ cyclosporine-A (n=6): 85±9.7%; low-Na+CAIP (n=3): 104±10.2% control). Fostreicin also reduced the low-Na effect (low-Na: 54±2.5%; low-Na+fostreicin (n=6): 77±6.1% control) but not tautomycin (low-Na: 65±4.9%; low-Na+TTM (n=7): 66±3.9%). Phosphorylation of Cx43 during low-Na was diminished by CysA, CAIP and FST. The phosphatases calcineurin and PP2A mediate the decrease of Gj in the left atrium during elevated [Ca2+]i. Connexin43 is one of the targets for calcineurin and PP2A.

Calcineurin upregulates local Ca(2+) signaling through ryanodine receptor-1 in airway smooth muscle cells.

Local Ca(2+) signals (Ca(2+) sparks) play an important role in multiple cellular functions in airway smooth muscle cells (ASMCs). Protein kinase Cϵ is known to downregulate ASMC Ca(2+) sparks and contraction; however, no complementary phosphatase has been shown to produce opposite effects. Here, we for the first time report that treatment with a specific calcineurin (CaN) autoinhibitory peptide (CAIP) to block CaN activity decreases, whereas application of nickel to activate CaN increases, Ca(2+) sparks in both the presence and absence of extracellular Ca(2+). Treatment with xestospogin-C to eliminate functional inositol 1,4,5-trisphosphate receptors does not prevent CAIP from inhibiting local Ca(2+) signaling. However, high ryanodine treatment almost completely blocks spark formation and prevents the nickel-mediated increase in sparks. Unlike CAIP, the protein phosphatase 2A inhibitor endothall has no effect. Local Ca(2+) signaling is lower in CaN catalytic subunit Aα gene knockout (CaN-Aα(-/-)) mouse ASMCs. The effects of CAIP and nickel are completely lost in CaN-Aα(-/-) ASMCs. Neither CAIP nor nickel produces an effect on Ca(2+) sparks in type 1 ryanodine receptor heterozygous knockout (RyR1(-/+)) mouse ASMCs. However, their effects are not altered in RyR2(-/+) or RyR3(-/-) mouse ASMCs. CaN inhibition decreases methacholine-induced contraction in isolated RyR1(+/+) but not RyR1(-/+) mouse tracheal rings. Supportively, muscarinic contractile responses are also reduced in CaN-Aα(-/+) mouse tracheal rings. Taken together, these results provide novel evidence that CaN regulates ASMC Ca(2+) sparks specifically through RyR1, which plays an important role in the control of Ca(2+) signaling and contraction in ASMCs.

Calcineurin and CRTC2 mediate FSH and TGFβ1 upregulation of Cyp19a1 and Nr5a in ovary granulosa cells.

Estrogens are essential for female reproduction and overall well-being, and estrogens in the circulation are largely synthesized in ovarian granulosa cells. Using primary cultures of ovarian granulosa cells from gonadotropin-primed immature rats, we have recently discovered that pituitary FSH and ovarian cytokine transforming growth factor beta 1 (TGFβ1) induce calcineurin-mediated dephosphorylation-activation of cAMP-response element-binding protein (CREB)-regulated transcription coactivator (CRTC2) to modulate the expression of Star, Cyp11a1, and Hsd3b leading to increased production of progesterone. This study explored the role of calcineurin and CRTC2 in FSH and TGFβ1 regulation of Cyp19a1 expression in granulosa cells. Ovarian granulosa cells treated with FSH displayed increased aromatase protein at 24 h post-treatment, which subsided by 48 h, while TGFβ1 acting through its type 1 receptor augmented the action of FSH with a greater and longer effects. It is known that the ovary-specific Cyp19a1 PII-promoter contains crucial response elements for CREB and nuclear receptor NR5A subfamily liver receptor homolog 1 (LRH1/NR5A2) and steroidogenic factor 1 (SF1/NR5A1), and that the Nr5a2 promoter also has a potential CREB-binding site. Herein, we demonstrate that FSH+TGFβ1 increased LRH1 and SF1 protein levels, and their binding to the Cyp19a1 PII-promoter evidenced, determined by chromatin immunoprecipitation analysis. Moreover, pretreatment with calcineurin auto-inhibitory peptide (CNI) abolished the FSH+TGFβ1-upregulated but not FSH-upregulated aromatase activity at 48 h, and the corresponding mRNA changes in Cyp19a1, and Nr5a2 and Nr5a1 at 24 h. In addition, FSH and TGFβ1 increased CRTC2 binding to the Cyp19a1 PII-promoter and Nr5a2 promoter at 24 h, with CREB bound constitutively. In summary, the results of this study indicate that calcineurin and CRTC2 have important roles in mediating FSH and TGFβ1 collateral upregulation of Cyp19a1 expression together with its transcription regulators Nr5a2 and Nr5a1 in ovarian granulosa cells.

Energy Landscapes and Catalysis in Nitric-oxide Synthase

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.

Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6.

Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/β-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3. DOI: http://dx.doi.org/10.7554/eLife.01998.001.

Probing the Ca2+/CaM-induced secondary structural and conformational changes in calcineurin

Calcineurin (CN) is a Ca2+/CaM-dependent Ser/Thr protein phosphatase that plays a critical role in coupling Ca2+ signals to a cellular response. Various methods have been applied to explore CN activation. A widely accepted model involves CaM binding to the CaM-binding domain (CN 389–413), inducing displacement of the CN autoinhibitory peptide (CN 467–486) from the active site. However, almost the entire regulatory region (CN 374–521), except the autoinhibitory peptide, is not visible in the electron density map of the reported structures. In the present study, we determined the overall secondary structure of CN in the presence or absence of Ca2+/CaM using FT-IR, and the Ca2+/CaM-induced structural dynamics and conformational changes were monitored by hydrogen–deuterium exchange experiments. The results revealed that the regulatory domain possessed some intrinsic structure. The binding of Ca2+ and subsequent binding of CaM generated a sequential folding of CN, transforming it into a more constrained, less flexible conformation.

FK506-Binding Protein 12 Modulatesμ-Opioid Receptor Phosphorylation and Protein Kinase Cε–Dependent Signaling by Its Direct Interaction with the Receptor

Protein kinase C (PKC) activation plays an important role in morphine-induced μ-opioid receptor (OPRM1) desensitization and tolerance development. It was recently shown that receptor phosphorylation by G protein-coupled receptor kinase regulates agonist-dependent selective signaling and that inefficient phosphorylation of OPRM1 leads to PKCε activation and subsequent responses. Here, we demonstrate that such receptor phosphorylation and PKCε activation can be modulated by FK506-binding protein 12 (FKBP12). Using a yeast two-hybrid screen, FKBP12 was identified as specifically interacting with OPRM1 at the Pro(353) residue. In human embryonic kidney 293 cells expressing OPRM1, the association of FKBP12 with OPRM1 decreased the agonist-induced receptor phosphorylation at Ser(375). The morphine-induced PKCε activation and the recruitment of PKCε to the OPRM1 signaling complex were attenuated both by FKBP12 short interfering RNA (siRNA) treatment and in cells expressing OPRM1 with a P353A mutation (OPRM1P353A), which leads to diminished activation of PKC-dependent extracellular signal-regulated kinases. Meanwhile, the overexpression of FKBP12 enabled etorphine to activate PKCε. Further analysis of the receptor complex demonstrated that morphine treatment enhanced the association of FKBP12 and calcineurin with the receptor. The blockade of the FKBP12 association with the receptor by the siRNA-mediated knockdown of endogenous FKBP12 or the mutation of Pro(353) to Ala resulted in a reduction in PKCε and calcineurin recruitment to the receptor signaling complex. The receptor-associated calcineurin modulates OPRM1 phosphorylation, as demonstrated by the ability of the calcineurin autoinhibitory peptide to increase the receptor phosphorylation. Thus, the association of FKBP12 with OPRM1 attenuates the phosphorylation of the receptor and triggers the recruitment and activation of PKCε.

Dissect Kif5b in nuclear positioning during myogenesis: The light chain binding domain and the autoinhibitory peptide are both indispensable

The microtubule motor kinesin-1 is responsible for the nuclear positioning during myogenesis. Here we show that the coiled-coil stalk/tail domain containing the kinesin light chain (KLC) binding sites targets to the perinuclear region like endogenous Kif5b, while the globular tail domain cannot. To investigate which fragments of kinesin heavy chain (Kif5b) is responsible for the myonuclear positioning, we transfect Kif5b expression constructs into Kif5b deficient myoblasts and test their ability to rescue the myonuclear phenotype. We find that the KLC binding domain and the autoinhibitory peptide in the globular tail region are both indispensable for the nuclear membrane localization of Kif5b and the kinesin-1-mediated myonuclear positioning. These results suggest that while the KLC binding domain may directly targets Kif5b to the myonuclear membrane, the autoinhibitory peptide may play an indirect role in regulating the kinesin-1-mediated myonuclear positioning.

Crucial Involvement of CREB-Regulated Transcription Coactivator (CRTC2) and Calcineurin in FSH and TGFβ1 Upregulation of Cyp19 Expression.

The steroid hormone estrogen governs female reproductive competence and whole-body physiology. Imbalance of estrogen often exacerbates diseases including cancer and cardiovascular risks. Estrogen is produced mainly in ovarian granulosa cells, where CYP19 (aromatase) catalyzes the conversion of theca cell-derived androgen to estrogen. The pituitary follicle-stimulating hormone (FSH) acts in concert with ovarian local factor tansforming growth factor (TGFβ1) to stimulate granulosa cell steroidogenesis, and this includes upregulation of Cyp19. However, molecular mechanisms underlying such regulation awaits further exploration. The goal of this study was to investigate FSH and TGFβ1 temporal regulation of Cyp19 expression, and the associated molecular mechanism. Primary granulosa cell culture from equine chorionic gonadotropin-primed immature rats was used. We first characterized FSH and TGFβ1 regulation of Cyp19 expression and activity. Under short-term treatment (4 h and 6 h), FSH±TGFβ1 increased Cyp19 mRNA and protein levels, while TGFβ1 alone had no effect. Under long-term treatment (24 h and 48 h) however, TGFβ1 further potentiated FSH-induced Cyp19 expression and activity. CRTC2 is a Ca2+- and cAMP-regulated CREB coactivator. Ca2+ activation of calcineurin and cAMP inhibition of salt-inducible kinase (SIK) induce dephosphorylation of CRTC2, which in turn translocates from the cytoplasm to the nucleus where it coactivates CREB. Recently, we demonstrated calcineurin and CRTC2 as crucial mediators in FSH and TGFβ1 upregulation of steroidogenic proteins StAR, P450scc and 3β-HSD enzymes. Here, we further explored the potential involvement of calcineurin/CRTC2 pathway in FSH and TGFβ1 regulation of Cyp19 expression in granulosa cells. Calcineurin autoinhibitory peptide (CNI) blocked the short-term FSH and long-term FSH+TGFβ1 effect. Moreover, chromatin immunoprecipitation analysis revealed that FSH and TGFβ1 increased the binding of CRTC2 and CREB to Cyp19 promoter at relevant post-treatment times. In all, the present study demonstrates that calcineurin and CRTC2 are crucial mediators in FSH and TGFβ1 upregulation of Cyp19 expression in ovarian granulosa cells. This study was supported by National Science Council of Taiwan. WAL was supported by an award from National Health Research Institutes, Taiwan.

Extensions of PSD-95/discs large/ZO-1 (PDZ) domains influence lipid binding and membrane targeting of syntenin-1

Syntenin-1 is a PDZ protein involved in receptor recycling and clustering. Its two PDZ domains interact with various receptors and phosphoinositides, and are flanked by N- and C-terminal regions. Here, we report the identification of an autoinhibitory peptide stretch in the N-terminus that might be regulated by phosphorylation. We further establish that basic residues in the C-terminal region mediate electrostatic interactions with reconstituted liposomes and contribute to the plasma membrane targeting. Our study adds new components to the multi-dentate membrane targeting mechanism and highlights the role of N- and C-terminal PDZ extensions in the regulation of syntenin-1 plasma membrane localization. Structured summary of protein interactionsPDZ1-PDZ2 and peptide bind by fluorescence technology (View Interaction: 1, 2, 3, 4).

Cerebellar long‐term depression requires dephosphorylation of TARP in Purkinje cells

Cerebellar long-term depression (LTD) at parallel fiber (PF)-Purkinje cell synapses is thought to play an essential role in certain forms of motor learning. Like hippocampal LTD, cerebellar LTD is mediated by the endocytosis of AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptors at postsynaptic sites. However, similar sets of kinases and phosphatases have opposite regulatory effects on hippocampal and cerebellar LTD, although the mechanisms responsible for this difference remain largely unclear. Activity-dependent dephosphorylation of stargazin (an AMPA receptor auxiliary protein) by calcineurin regulates hippocampal LTD, but whether and how stargazin is involved in cerebellar LTD is unknown. In this study, we showed that stargazin is highly phosphorylated at basal states and is dephosphorylated by the application of high KCl plus glutamate (K-glu) or of a metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine (DHPG), both of which chemically induced LTD in cerebellar slices. This chemically induced dephosphorylation of stargazin was specifically blocked by a calcineurin inhibitor. Indeed, inclusion of the calcineurin auto-inhibitory peptide in the patch pipette solution completely inhibited the LTD induced by the conjunctive stimulation of PFs and Purkinje cells. Furthermore, in Purkinje cells expressing stargazin-9D, in which all nine serine residues are mutated to aspartate, neither conjunctive stimulus nor DHPG treatment induced LTD. Finally, immunohistochemical analyses revealed that neither K-glu nor DHPG induced the endocytosis of AMPA receptors in Purkinje cells expressing stargazin-9D. Together, these results indicate that hippocampal and cerebellar LTD share a common pathway, namely dephosphorylation of stargazin by calcineurin.

The N-terminal Peptide of Mammalian GTP Cyclohydrolase I Is an Autoinhibitory Control Element and Contributes to Binding the Allosteric Regulatory Protein GFRP

GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), an obligate cofactor for NO synthases and aromatic amino acid hydroxylases. BH4 can limit its own synthesis by triggering decameric GTPCH to assemble in an inhibitory complex with two GTPCH feedback regulatory protein (GFRP) pentamers. Subsequent phenylalanine binding to the GTPCH·GFRP inhibitory complex converts it to a stimulatory complex. An N-terminal inhibitory peptide in GTPCH may also contribute to autoregulation of GTPCH activity, but mechanisms are undefined. To characterize potential regulatory actions of the N-terminal peptide in rat GTPCH, we expressed, purified, and characterized a truncation mutant, devoid of 45 N-terminal amino acids (Δ45-GTPCH) and contrasted its catalytic and GFRP binding properties to wild type GTPCH (wt-GTPCH). Contrary to prior reports, we show that GFRP binds wt-GTPCH in the absence of any small molecule effector, resulting in allosteric stimulation of GTPCH activity: a 20% increase in Vmax, 50% decrease in KmGTP, and increase in Hill coefficient to 1.6, from 1.0. These features of GFRP-stimulated wt-GTPCH activity were phenocopied by Δ45-GTPCH in the absence of bound GFRP. Addition of GFRP to Δ45-GTPCH failed to elicit complex formation or a substantial further increase in GTPCH catalytic activity. Expression of Δ45-GTPCH in HEK-293 cells elicited 3-fold greater BH4 accumulation than an equivalent of wt-GTPCH. Together, results indicate that the N-terminal peptide exerts autoinhibitory control over rat GTPCH and is required for GFRP binding on its own. Displacement of the autoinhibitory peptide provides a molecular mechanism for physiological up-regulation of GTPCH activity.