scholarly journals Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation

2017 ◽  
Vol 114 (28) ◽  
pp. E5549-E5558 ◽  
Author(s):  
Michael V. Airola ◽  
Prajna Shanbhogue ◽  
Achraf A. Shamseddine ◽  
Kip E. Guja ◽  
Can E. Senkal ◽  
...  
Keyword(s):  
Stress Responses ◽  
Catalytic Domain ◽  
Competitive Inhibitor ◽  
Activation Mechanism ◽  
Allosteric Activation ◽  
Anionic Phospholipid ◽  
Juxtamembrane Region ◽  
Key Enzyme ◽  
Mechanistic Basis ◽  
Ceramide Generation

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I–type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the “DK switch.” Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

scholarly journals Novel Substrates for Kinases Involved in the Biosynthesis of Inositol Pyrophosphates and Their Enhancement of ATPase Activity of a Kinase

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3601
Author(s):  
Raja Mohanrao ◽  
Ruth Manorama ◽  
Shubhra Ganguli ◽  
Mithun C. Madhusudhanan ◽  
Rashna Bhandari ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Catalytic Domain ◽  
Inositol Phosphate ◽  
Substrate Recognition ◽  
Competitive Inhibitor ◽  
Inositol Polyphosphates ◽  
Inositol Pyrophosphate ◽  
Phosphate Donor ◽  
Inositol Pyrophosphates

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo-inositol polyphosphates—scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5—from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

scholarly journals Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

2017 ◽  
Vol 474 (3) ◽  
pp. 385-398 ◽  
Author(s):  
Ryan P. Emptage ◽  
Mark A. Lemmon ◽  
Kathryn M. Ferguson
Keyword(s):  
Membrane Surface ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Disease States ◽  
C Terminus ◽  
Regulatory Module ◽  
Correct Location ◽  
Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

scholarly journals S-Nitrosoglutathione Reductase—The Master Regulator of Protein S-Nitrosation in Plant NO Signaling

Plants ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 48 ◽  
Author(s):  
Jana Jahnová ◽  
Lenka Luhová ◽  
Marek Petřivalský
Keyword(s):  
Stress Responses ◽  
Current Knowledge ◽  
Protein S ◽  
Defense Responses ◽  
Reactive Nitrogen ◽  
Cellular Processes ◽  
Protein Thiols ◽  
Key Enzyme ◽  
No Signaling ◽  
Cysteine Thiols

S-nitrosation has been recognized as an important mechanism of protein posttranslational regulations, based on the attachment of a nitroso group to cysteine thiols. Reversible S-nitrosation, similarly to other redox-base modifications of protein thiols, has a profound effect on protein structure and activity and is considered as a convergence of signaling pathways of reactive nitrogen and oxygen species. In plant, S-nitrosation is involved in a wide array of cellular processes during normal development and stress responses. This review summarizes current knowledge on S-nitrosoglutathione reductase (GSNOR), a key enzyme which regulates intracellular levels of S-nitrosoglutathione (GSNO) and indirectly also of protein S-nitrosothiols. GSNOR functions are mediated by its enzymatic activity, which catalyzes irreversible GSNO conversion to oxidized glutathione within the cellular catabolism of nitric oxide. GSNOR is involved in the maintenance of balanced levels of reactive nitrogen species and in the control of cellular redox state. Multiple functions of GSNOR in plant development via NO-dependent and -independent signaling mechanisms and in plant defense responses to abiotic and biotic stress conditions have been uncovered. Extensive studies of plants with down- and upregulated GSNOR, together with application of transcriptomics and proteomics approaches, seem promising for new insights into plant S-nitrosothiol metabolism and its regulation.

Computational Insight into the Allosteric Activation Mechanism of Farnesoid X Receptor

2020 ◽  
Vol 60 (3) ◽  
pp. 1540-1550
Author(s):  
Yue Chen ◽  
Junhao Li ◽  
Zengrui Wu ◽  
Guixia Liu ◽  
Honglin Li ◽  
...  
Keyword(s):  
Farnesoid X Receptor ◽  
Activation Mechanism ◽  
Allosteric Activation ◽  
Insight Into

Ocular complications of Garcinia cambogia extract diet pills: Case report

2019 ◽  
Vol 30 (6) ◽  
pp. NP21-NP26 ◽  
Author(s):  
Hyun-kyung Cho ◽  
Yong Seop Han ◽  
Jong Moon Park
Keyword(s):  
Visual Disturbance ◽  
Competitive Inhibitor ◽  
Optic Disk ◽  
Oral Steroid ◽  
Fiber Layer ◽  
Ocular Complications ◽  
Garcinia Cambogia ◽  
Hydroxycitric Acid ◽  
First Case ◽  
Key Enzyme

Background: Garcinia cambogia contains hydroxycitric acid. Hydroxycitric acid is a potent competitive inhibitor of adenosine triphosphate citrate lyase which is a key enzyme in the synthesis of fatty acids. Hydroxycitric acid also regulates the level of serotonin. In these regards, hydroxycitric acid has been reported to exhibit weight loss activity. Adverse reactions of G. cambogia from numerous clinical studies demonstrated relatively mild reactions. However, there are some complications of G. cambogia reported in the past: acute liver injury, acute hepatitis, and hepatic failure. However, ocular complications of G. cambogia have not been reported yet. Case presentation: A 35-year-old female visited our clinic with decreased vision in the left eye and ocular pain in both eyes for the last 6 days. She also complained of headache, dizziness, and nausea. She had taken G. cambogia extract more than the recommended dose. There was myopic shift with anterior chamber shallowing in both eyes, especially in the left eye. Moreover, swelling and retinal folds of peripapillary retinal nerve fiber layer and macula were observed in both eyes. These ocular complications of G. cambogia extract resolved after discontinuation of the extract and topical and oral steroid treatment. Herein, we report the first case of ocular complications of G. cambogia extract diet pill assessed with optical coherence tomography of optic disk and macula along with dual Scheimpflug analyzer. Conclusion: It is necessary that physicians dealing with obesity advice patients about possible visual disturbance of this extract when taken in overdose so that they can see an ophthalmologist immediately.

scholarly journals The allosteric activation mechanism of a phospholipase A2-like toxin from Bothrops jararacussu venom: a dynamic description

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Antoniel A. S. Gomes ◽  
Fabio F. Cardoso ◽  
Maximilia F. Souza ◽  
Cristiano L. P. Oliveira ◽  
David Perahia ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Activation Mechanism ◽  
Activation Process ◽  
Structural Determinants ◽  
Allosteric Activation ◽  
Bothrops Jararacussu ◽  
Inactive State ◽  
Dynamic Description ◽  
Comprehensive Study

Abstract The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT, which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results.

scholarly journals Mechanisms of vasopressin-induced intracellular Ca2+ oscillations in rat inner medullary collecting duct

2011 ◽  
Vol 300 (2) ◽  
pp. F540-F548 ◽  
Author(s):  
Kay-Pong Yip ◽  
James S. K. Sham
Keyword(s):  
Flash Photolysis ◽  
Collecting Duct ◽  
Ryanodine Receptors ◽  
Competitive Inhibitor ◽  
Free Medium ◽  
Inner Medullary Collecting Duct ◽  
Oscillatory Pattern ◽  
Intracellular Ca ◽  
Medullary Collecting Duct ◽  
Mechanistic Basis

Arginine vasopressin (AVP) causes increase in intracellular Ca2+ concentration with an oscillatory pattern. Ca2+ mobilization is required for AVP-stimulated apical exocytosis in inner medullary collecting duct (IMCD). The mechanistic basis of these Ca2+ oscillations was investigated by confocal fluorescence microscopy and flash photolysis of caged molecules in perfused IMCD. Photorelease of caged cAMP and direct activation of ryanodine receptors (RyRs) by photorelease of caged cyclic ADP-ribose (cADPR) both mimicked the AVP-induced Ca2+ oscillations. Preincubation of IMCD with 100 μM 8-bromo-cADPR (a competitive inhibitor of cADPR) delayed the onset and attenuated the magnitude of AVP-induced Ca2+ oscillations. These observations indicate that the cADPR/RyR pathway is capable of supporting Ca2+ oscillations and endogenous cADPR plays a major role in the AVP-induced Ca2+ oscillations in IMCD. In contrast, photorelease of caged inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release but did not maintain sustained Ca2+ oscillations. Removal of extracellular Ca2+ halted ongoing AVP-mediated Ca2+ oscillation, suggesting that it requires extracellular Ca2+ entry. AVP-induced Ca2+ oscillation was unaffected by nifedipine. Intracellular Ca2+ store depletion induced by 20 μM thapsigargin in Ca2+-free medium triggered store-operated Ca2+ entry (SOCE) in IMCD, which was attenuated by 1 μM GdCl3 and 50 μM SKF-96365. After incubation of IMCD with 1 nM AVP in Ca2+-free medium, application of extracellular Ca2+ also triggered Ca2+ influx, which was sensitive to GdCl3 and SKF-96365. In summary, our observations are consistent with the notion that AVP-induced Ca2+ oscillations in IMCD are mediated by the interplay of Ca2+ release from RyRs and a Ca2+ influx mechanism involving nonselective cation channels that resembles SOCE.

scholarly journals Catalytic Domain Plasticity of MKK7 Reveals Structural Mechanisms of Allosteric Activation and Diverse Targeting Opportunities

2020 ◽  
Vol 27 (10) ◽  
pp. 1285-1295.e4 ◽  
Author(s):  
Martin Schröder ◽  
Li Tan ◽  
Jinhua Wang ◽  
Yanke Liang ◽  
Nathanael S. Gray ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Allosteric Activation ◽  
Structural Mechanisms

scholarly journals Heme Oxygenase-1, a Key Enzyme for the Cytoprotective Actions of Halophenols by Upregulating Nrf2 Expression via Activating Erk1/2 and PI3K/Akt in EA.hy926 Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Xiu E. Feng ◽  
Tai Gang Liang ◽  
Jie Gao ◽  
De Peng Kong ◽  
Rui Ge ◽  
...  
Keyword(s):  
Heme Oxygenase ◽  
Antioxidant Activities ◽  
Heme Oxygenase 1 ◽  
Protective Effects ◽  
Key Enzyme ◽  
Ea.Hy926 Cells ◽  
Anti Inflammatory ◽  
Mechanistic Basis

Increasing evidence has demonstrated that heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress, exhibiting cytoprotective, antioxidant, and anti-inflammatory abilities. Previously, we prepared a series of novel active halophenols possessing strong antioxidant activities in vitro and in vivo. In the present study, we demonstrated that these halophenols exhibited significant protective effects against H2O2-induced injury in EA.hy926 cells by inhibition of apoptosis and ROS and TNF-αproduction, as well as induction of the upregulation of HO-1, the magnitude of which correlated with their cytoprotective actions. Further experiments which aimed to determine the mechanistic basis of these actions indicated that the halophenols induced the activation of Nrf2, Erk1/2, and PI3K/Akt without obvious effects on the phosphorylation of p38, JNK, or the expression of PKC-δ. This was validated with the use of PD98059 and Wortmannin, specific inhibitors of Erk1/2 and PI3K, respectively. Overall, our study is the first to demonstrate that the cytoprotective actions of halophenols involve their antiapoptotic, antioxidant, and anti-inflammatory abilities, which are mediated by the upregulation of Nrf2-dependent HO-1 expression and reductions in ROS and TNF-αgeneration via the activation of Erk1/2 and PI3K/Akt in EA.hy926 cells. HO-1 may thus be an important potential target for further research into the cytoprotective actions of halophenols.

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