An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP

2019 ◽  
Author(s):  
Luke A. Perera ◽  
Claudia Rato ◽  
Yahui Yan ◽  
Lisa Neidhardt ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Active Site ◽  
Covalent Modification ◽  
Energy Status ◽  
Oligomeric State ◽  
Dimer Interface ◽  
Er Chaperone ◽  
State Dependent ◽  
Repressive Effect

AbstractAMPylation is an inactivating modification that matches the activity of the major endoplasmic reticulum (ER) chaperone BiP to the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD’s activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface or in residues tracing an inhibitory relay from the dimer interface to the enzyme’s active site favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically-propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Whereas, a reciprocal signal propagated from the nucleotide binding site, provides a mechanism for coupling the oligomeric-state and enzymatic activity of FICD to the energy status of the ER.Impact StatementUnique amongst known chaperones, the endoplasmic reticulum (ER)-localized Hsp70, BiP, is subject to transient inactivation under conditions of low ER stress by reversible, covalent modification – AMPylation. The enzyme responsible for this modification, FICD, is in fact a bifunctional enzyme with a single active site capable of both AMPylation and deAMPylation. Here we elucidate, by biochemical, biophysical and structural means, the mechanism by which this enzyme is able to switch enzymatic modality: by regulation of its oligomeric state. The oligomeric state-dependent reciprocal regulation of FICD activity is, in turn, sensitive to the ATP/ADP ratio. This allosteric pathway potentially facilitates the sensing of unfolded protein load in the ER and permits the transduction of this signal into a post-translational buffering of ER chaperone activity.

scholarly journals FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

2016 ◽  
Author(s):  
Steffen Preissler ◽  
Claudia Rato ◽  
Luke Perera ◽  
Vladimir Saudek ◽  
David Ron
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Covalent Modification ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
Er Chaperone

Significance statementSome 25 years ago it was discovered that the activity of the ER chaperone BiP is regulated by covalent modification, the nature of which, AMPylation (not ADPribosylation, as had long been thought) and the enzyme responsible, FICD, have only recently been identified. Genetic inactivation of FICD and in vitro studies of the purified enzyme and substrate have done much to clarify the biochemical consequences of the modification and its underlying logic: As ER stress wanes, FICD uses ATP to AMPylate Thr518 of BiP locking BiP in a relatively inactive conformation. As ER stress levels re-mount the cells draw on this pool of inactive chaperone, which is de-AMPylated and restored to its fully active state.Here we report on the identity of the de-AMPylating enzyme - and with it on the surprising finding that both AMPylation and de-AMPylation of BiP are carried out by the same polypeptide (FICD) using the same active site, both in vivo and in vitro. Analysis of the reaction products reveals that de-AMPylation does not involve trivial concentration-dependent micro-reversibility of an enzymatic reaction, but rather a switch in the active site of FICD that facilitates two antagonistic thermodynamically favored reactions.Surprisingly BiP de-AMPylation (not AMPylation) is the default activity of FICD. The side-chain of a single regulatory residue, E234, toggles the enzyme between de-AMPylation and AMPylation in vitro. Our studies thereby uncover an active mechanism that must exist in the ER for coupling waning levels of unfolded protein stress to the conversion of FICD from its default de-AMPylation mode to BiP AMPylation. Whilst the details of this active switch remain to be discovered, we are able to suggest a plausible mechanism by which it may come about.Identification of the enzyme that de-modifies BiP to reactivate it will be of interest to cell biologists, whereas the novel features of FICD as a dualfunctioning enzyme with a single bi-functional active site will be of broad interest to enzymologists and molecular biologists.AbstractProtein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoanspecific FIC-domain containing ER-localized enzyme, FICD/HYPE, rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here it is shown that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD−/− cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

Penicillins and cephalosporins are active site-directed acylating agents: evidence in support of the substrate analogue hypothesis

1980 ◽  
Vol 289 (1036) ◽  
pp. 257-271 ◽  
Keyword(s):  
Cell Wall ◽  
Active Site ◽  
Structural Similarity ◽  
Structural Features ◽  
Covalent Modification ◽  
Cross Linking ◽  
Cell Wall Biosynthesis ◽  
Substrate Analogue ◽  
Bactericidal Effects

Penicillin and related β-lactam antibiotics are known to exert their bactericidal effects by inhibiting the cross-linking step (transpeptidation) of bacterial cell wall biosynthesis. Evidence is presented in support of the hypothesis that this inhibition results from covalent modification of the active site of sensitive enzymes as a consequence of the structural similarity between penicillin and the acyl-D-alanyl-D-alanine terminus of nascent peptidoglycan strands. Several predictions of this proposal have been verified experimentally. Penicillin-sensitive enzymes are inactivated, with the formation of a covalent, stoichiometric penicilloyl-enzyme complex in vitro . Acylenzyme intermediates have been trapped with several of these enzymes by using cell wall-related substrates. Sequence analysis of the peptides derived from active site-labelled enzymes has established that both penicilloyl and an acyl moiety derived from substrate are covalently bound to the same site, as an ester of serine 36, as predicted by the substrate analogue hypothesis. Sequences near the active site serine are homologous to sequences found in four β-lactamases, supporting the proposal that penicillinsensitive D-alanine carboxypeptidases and penicillin-inactivating β-lactamases are evolutionarily related. Structural features important for the specific and potent inhibitory properties of β-lactam antibiotics are discussed in terms of the original substrate analogue hypothesis.

scholarly journals AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Steffen Preissler ◽  
Cláudia Rato ◽  
Ruming Chen ◽  
Robin Antrobus ◽  
Shujing Ding ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Unfolded Protein ◽  
Peptide Dissociation

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

scholarly journals Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases

2004 ◽  
Vol 382 (3) ◽  
pp. 905-912 ◽  
Author(s):  
Mark R. GUY ◽  
Petr A. ILLARIONOV ◽  
Sudagar S. GURCHA ◽  
Lynn G. DOVER ◽  
Kevin J. C. GIBSON ◽  
...  
Keyword(s):  
Active Site ◽  
Covalent Modification ◽  
Biosynthetic Pathways ◽  
Specific Inhibition ◽  
Enzyme Active Site ◽  
Substrate Analogues ◽  
Glycosyl Donor ◽  
Derivatives Of ◽  
Polyprenyl Phosphates

PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an α(1→6)LM intermediate through a PPM-dependent α(1→6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a ‘natural’ C75 polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial α(1→6)mannosyltransferase, to a synthetic Manp(1→6)-Manp-O-C10:1 disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis α(1→6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.

scholarly journals Lipid-derived electrophiles induce covalent modification and aggregation of Cu,Zn-superoxide dismutase in a hydrophobicity-dependent manner

2019 ◽  
Author(s):  
Lucas S. Dantas ◽  
Lucas G. Viviani ◽  
Alex Inague ◽  
Erika Piccirillo ◽  
Leandro de Rezende ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Critical Factor ◽  
Covalent Modification ◽  
Protein Sequencing ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Dimer Interface ◽  
Electrostatic Loop

ABSTRACTLipid peroxidation generates a huge number of reactive electrophilic aldehyde products. These reactive aldehydes can modify macromolecules such as proteins, resulting in loss of function and/or aggregation. The accumulation of Cu,Zn-superoxide dismutase (SOD1) aggregates is associated with familial cases of amyotrophic lateral sclerosis (ALS). Recent studies have shown that lipid and its oxidized derivatives may play a role in this process. Here we aimed to compare and characterize the ability of lipid-derived electrophiles with different hydrophobicities to induce SOD1 modification and aggregation in vitro. SOD1 was incubated with 4-hydroxy-2-hexenal (HHE), 4-hydroxy- 2-nonenal (HNE), 2-hexen-1-al (HEX), 2,4-nonadienal (NON), 2,4-decadienal (DEC) or secosterol aldehydes (Seco-A or Seco-B) at 37°C for 24 h. Size exclusion chromatography analysis showed that hydrophobic aldehydes smarkedly enhances apo- SOD1 aggregation. More importantly, aggregation level was positively correlated to calculated aldehyde hydrophobicities (LogP). Protein sequencing by LC-MS/MS showed that aldehydes covalently modifies SOD1 at aggregation prone regions. For instance, specific lysine residues located mainly nearby the dimer interface (K3, K9) and at the electrostatic loop (K122, K128, K136) were ubiquitously modified by all aldehydes. The α,β-unsaturated aldehydes also promoted modifications on histidine and cysteine residues, with H120 and C6 being the most commonly modified residues. Overall, our data suggest that electrophile’s hydrophobicity is a critical factor that strongly influences protein aggregation propensity.Graphical abstractHighlights- Aldehyde hydrophobicity is positively correlated to SOD1 aggregation;- Lys residues located nearby the SOD1 dimer interface and electrostatic loop are ubiquitously modified by all aldehydes;- Hydrophobic aldehydes increase the lipophilic potential surface of the region where they bind;

In Vitro Induction of Crystals of MT Protein by Vinblastine-Colcemid in Mouse Spermatids

1974 ◽  
Vol 32 ◽  
pp. 132-133
Author(s):  
John J. Wolosewick ◽  
John H. D. Bryan
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Nuclear Ring ◽  
Rough Er ◽  
Distal Direction ◽  
In Vitro Induction

Early in spermiogenesis the manchette is rapidly assembled in a distal direction from the nuclear-ring-densities. The association of vesicles of smooth endoplasmic reticulum (SER) and the manchette microtubules (MTS) has been reported. In the mouse, osmophilic densities at the distal ends of the manchette are the organizing centers (MTOCS), and are associated with the SER. Rapid MT assembly and the lack of rough ER suggests that there is an existing pool of MT protein. Colcemid potentiates the reaction of vinblastine with tubulin and was used in this investigation to detect this protein.

Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

1998 ◽  
Vol 79 (05) ◽  
pp. 1041-1047 ◽  
Author(s):  
Kathleen M. Donnelly ◽  
Michael E. Bromberg ◽  
Aaron Milstone ◽  
Jennifer Madison McNiff ◽  
Gordon Terwilliger ◽  
...  
Keyword(s):  
Active Site ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Melanoma Cells ◽  
Factor V ◽  
Ancylostoma Caninum ◽  
Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Structure-Based Design, Synthesis, Biological Evaluation and Molecular Docking Study of 4-Hydroxy-N'-methylenebenzohydrazide Derivatives Acting as Tyrosinase Inhibitors with Potentiate Anti-Melanogenesis Activities

2020 ◽  
Vol 16 (7) ◽  
pp. 892-902 ◽  
Author(s):  
Aida Iraji ◽  
Mahsima Khoshneviszadeh ◽  
Pegah Bakhshizadeh ◽  
Najmeh Edraki ◽  
Mehdi Khoshneviszadeh
Keyword(s):  
Molecular Docking ◽  
Active Site ◽  
Competitive Inhibition ◽  
Docking Study ◽  
Biological Evaluation ◽  
Primary Response ◽  
Ratio Method ◽  
Molecular Docking Study ◽  
Metal Chelating

Background: Melanogenesis is a process of melanin synthesis, which is a primary response for the pigmentation of human skin. Tyrosinase is a key enzyme, which catalyzes a ratelimiting step of the melanin formation. Natural products have shown potent inhibitors, but some of these possess toxicity. Numerous synthetic inhibitors have been developed in recent years may lead to the potent anti– tyrosinase agents. Objective: A number of 4-hydroxy-N'-methylenebenzohydrazide analogues with related structure to chalcone and tyrosine were constructed with various substituents at the benzyl ring of the molecule and evaluate as a tyrosinase inhibitor. In addition, computational analysis and metal chelating potential have been evaluated. Methods: Design and synthesized compounds were evaluated for activity against mushroom tyrosinase. The metal chelating capacity of the potent compound was examined using the mole ratio method. Molecular docking of the synthesized compounds was carried out into the tyrosine active site. Results: Novel 4-hydroxy-N'-methylenebenzohydrazide derivatives were synthesized. The two compounds 4c and 4g showed an IC50 near the positive control, led to a drastic inhibition of tyrosinase. Confirming in vitro results were performed via the molecular docking analysis demonstrating hydrogen bound interactions of potent compounds with histatidine-Cu+2 residues with in the active site. Kinetic study of compound 4g showed competitive inhibition towards tyrosinase. Metal chelating assay indicates the mole fraction of 1:2 stoichiometry of the 4g-Cu2+ complex. Conclusion: The findings in the present study demonstrate that 4-Hydroxy-N'- methylenebenzohydrazide scaffold could be regarded as a bioactive core inhibitor of tyrosinase and can be used as an inspiration for further studies in this area.

Novel coumarin containing dithiocarbamate derivatives as potent α-glucosidase inhibitors for management of type 2 diabetes

2020 ◽  
Vol 16 ◽  
Author(s):  
Marjan Mollazadeh ◽  
Maryam Mohammadi-Khanaposhtani ◽  
Yousef Valizadeh ◽  
Afsaneh Zonouzi ◽  
Mohammad Ali Faramarzi ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Kinetic Study ◽  
Active Site ◽  
Docking Study ◽  
Secondary Amines ◽  
Molecular Docking Study ◽  
Glucosidase Inhibitors ◽  
Dithiocarbamate Derivatives

Background: α-Glucosidase is a hydrolyze enzyme that plays a crucial role in degradation of carbohydrates and starch to glucose. Hence, α-glucosidase is an important target in the carbohydrate mediated diseases such as diabetes mellitus. Objective: In this study, novel coumarin containing dithiocarbamate derivatives 4a-n were synthesized and evaluated against α-glucosidase in vitro and in silico. Methods: These compounds were obtained of reaction between 4-(bromomethyl)-7-methoxy-2H-chromen-2-one 1, carbon disulfide 2, and primary or secondary amines 3a-n in the presence potassium hydroxide and ethanol at room temperature. In vitro α-glucosidase inhibition and kinetic study of these compounds were performed. Furthermore, docking study of the most potent compounds was also performed by Auto Dock Tools (version 1.5.6). Results: Obtained results showed that all the synthesized compounds exhibited prominent inhibitory activities (IC50 = 85.0 ± 4.0-566.6 ± 8.6 μM) in comparison to acarbose as standard inhibitor (IC50 = 750.0 ± 9.0 µM). Among them, secondary amine derivative 4d with pendant indole group was the most potent inhibitor. Enzyme kinetic study of the compound 4d revealed that this compound compete with substrate to connect to the active site of α-glucosidase and therefore is a competitive inhibitor. Also, molecular docking study predicted that this compound as well interacted with α-glucosidase active site pocket. Conclusion: Our results suggest that the coumarin-dithiocarbamate scaffold can be a promising lead structure for design potent α-glucosidase inhibitors for treatment of type 2 diabetes.

The Molecular and Enzyme Kinetic Basis for Altered Activity of Three Cytochrome P450 2C19 Variants Found in the Chinese Population

2020 ◽  
Vol 13 (3) ◽  
pp. 233-244
Author(s):  
Amelia Nathania Dong ◽  
Nafees Ahemad ◽  
Yan Pan ◽  
Uma Devi Palanisamy ◽  
Beow Chin Yiap ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Molecular Docking ◽  
Active Site ◽  
Chinese Population ◽  
Accessible Surface Area ◽  
Solvent Accessible Surface Area ◽  
In Vitro Assays ◽  
Access Channel ◽  
Cytochrome P450 2C19

Background: There is a large inter-individual variation in cytochrome P450 2C19 (CYP2C19) activity. The variability can be caused by the genetic polymorphism of CYP2C19 gene. This study aimed to investigate the molecular and kinetics basis for activity changes in three alleles including CYP2C19*23, CYP2C19*24 and CYP2C19*25found in the Chinese population. Methods: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking. Results: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays. Conclusion: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.

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