scholarly journals Investigation of Antidepressant Properties of Yohimbine by Employing Structure-Based Computational Assessments

2021 ◽  
Vol 43 (3) ◽  
pp. 1805-1827
Author(s):  
Munazzah Tasleem ◽  
Abdulwahed Alrehaily ◽  
Tahani M. Almeleebia ◽  
Mohammad Y. Alshahrani ◽  
Irfan Ahmad ◽  
...  
Keyword(s):  
Side Effects ◽  
Conformational Changes ◽  
Antidepressant Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Hydroxytryptamine Receptors ◽  
Hydrogen Bond Interactions ◽  
Ancient Times ◽  
Serotonergic Receptor ◽  
Neurological Conditions

The use of pharmaceuticals to treat Major Depressive Disorder (MDD) has several drawbacks, including severe side effects. Natural compounds with great efficacy and few side effects are in high demand due to the global rise in MDD and ineffective treatment. Yohimbine, a natural compound, has been used to treat various ailments, including neurological conditions, since ancient times. Serotonergic neurotransmission plays a crucial role in the pathogenesis of depression; thus, serotonergic receptor agonist/antagonistic drugs are promising anti-depressants. Yohimbine was investigated in this study to determine its antidepressant activity using molecular docking and pharmacokinetic analyses. Additionally, the in silico mutational study was carried out to understand the increase in therapeutic efficiency using site-directed mutagenesis. Conformational changes and fluctuations occurring during wild type and mutant serotonergic receptor, 5-hydroxytryptamine receptors 1A (5HT1A) and yohimbine were assessed by molecular dynamics MD simulation studies. Yohimbine was found to satisfy all the parameters for drug-likeness and pharmacokinetics analysis. It was found to possess a good dock score and hydrogen-bond interactions with wild type 5HT1A structure. Our findings elaborate the substantial efficacy of yohimbine against MDD; however, further bench work studies may be carried out to prove the same.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

scholarly journals Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

2016 ◽  
Vol 113 (37) ◽  
pp. 10358-10363 ◽  
Author(s):  
Hüseyin Ilgü ◽  
Jean-Marc Jeckelmann ◽  
Vytautas Gapsys ◽  
Zöhre Ucurum ◽  
Bert L. de Groot ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Radioligand Binding ◽  
Substrate Binding ◽  
Acid Resistance ◽  
Enteric Bacteria ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Roles ◽  
Dynamics Simulations ◽  
Substrate Binding Sites

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acid-resistance responses, such as the arginine-dependent acid resistance system. In this response, l-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the l-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of l-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type l-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli.

scholarly journals Critical role of Arg433 in rat transketolase activity as probed by site-directed mutagenesis

1998 ◽  
Vol 333 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Yunjo SOH ◽  
Byoung J. SONG ◽  
Jiingjau JENG ◽  
Abraham T. KALLARAKAL
Keyword(s):  
Critical Role ◽  
Immunoblot Analysis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Hydrogen Bond Interactions ◽  
Apparent Homogeneity ◽  
Arginine Residues ◽  
Biochemical Analyses

It has been shown that one arginine per monomer at an unknown position is essential for enzyme activity of the homodimeric transketolase (TK) [Kremer, Egan and Sable (1980) J. Biol. Chem. 255, 2405–2410]. To identify the critical arginine, four highly conserved arginine residues of rat TK (Arg102, Arg350, Arg433 and Arg506) were replaced with alanine by site-directed mutagenesis. Wild-type and mutant TK proteins were produced in Escherichia coli and characterized. The Arg102 → Ala mutant exhibited similar catalytic activity to the wild-type enzyme, whereas Arg350 → Ala, Arg506 → Ala and Arg433 → Ala mutants exhibited 36.7, 37.0 and 6.1% of the wild-type activity respectively. Three recombinant proteins (wild-type, Arg350 → Ala and Arg433 → Ala) were purified to apparent homogeneity using Ni2+-affinity chromatography and further characterized. All these proteins were able to form homodimers (148 kDa), as shown by immunoblot analysis subsequent to non-denaturing gel electrophoresis. The Arg433 → Ala mutant protein was less stable than the wild-type and Arg350 → Ala proteins at 55 °C. Kinetic analyses revealed that both Vmax and Km values were markedly affected in the Arg433 → Ala mutant. The Km values for two substrates xylulose 5-phosphate and ribose 5-phosphate were 11.5- and 24.3-fold higher respectively. The kcat/Km values of the Arg433 → Ala mutant for the two substrates were less than 1% of those of the wild-type protein. Molecular modelling of the rat TK revealed that Arg433 of one monomer has three potential hydrogen-bond interactions with the catalytically important highly conserved loop of the other monomer. Thus, our biochemical analyses and modelling data suggest the critical role of the previously uncharacterized Arg433 in TK activity.

Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
Author(s):  
S Ueshima ◽  
P Holvoet ◽  
H R Lijnen ◽  
L Nelles ◽  
V Seghers ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Solvent Accessibility ◽  
Site Directed Mutagenesis ◽  
Clot Lysis ◽  
Wild Type ◽  
Single Chain ◽  
Charged Amino Acids ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

Molecular Basis of Glucose Transport Mechanism in Plants

2019 ◽  
Author(s):  
Balaji Selvam ◽  
Ya-Chi Yu ◽  
Liqing Chen ◽  
Diwakar Shukla
Keyword(s):  
Molecular Dynamics ◽  
Free Energy ◽  
Molecular Dynamics Simulations ◽  
Conformational Changes ◽  
Transport Mechanism ◽  
Conformational Dynamics ◽  
Site Directed Mutagenesis ◽  
Free Energy Barrier ◽  
Transport Cycle ◽  
Dynamics Simulations

<p>The SWEET family belongs to a class of transporters in plants that undergoes large conformational changes to facilitate transport of sugar molecules across the cell membrane. However, the structures of their functionally relevant conformational states in the transport cycle have not been reported. In this study, we have characterized the conformational dynamics and complete transport cycle of glucose in OsSWEET2b transporter using extensive molecular dynamics simulations. Using Markov state models, we estimated the free energy barrier associated with different states as well as 1 for the glucose the transport mechanism. SWEETs undergoes structural transition to outward-facing (OF), Occluded (OC) and inward-facing (IF) and strongly support alternate access transport mechanism. The glucose diffuses freely from outside to inside the cell without causing major conformational changes which means that the conformations of glucose unbound and bound snapshots are exactly same for OF, OC and IF states. We identified a network of hydrophobic core residues at the center of the transporter that restricts the glucose entry to the cytoplasmic side and act as an intracellular hydrophobic gate. The mechanistic predictions from molecular dynamics simulations are validated using site-directed mutagenesis experiments. Our simulation also revealed hourglass like intermediate states making the pore radius narrower at the center. This work provides new fundamental insights into how substrate-transporter interactions actively change the free energy landscape of the transport cycle to facilitate enhanced transport activity.</p>

Sleep and epilepsy—chicken or egg?

2018 ◽  
Author(s):  
Dora A. Lozsadi
Keyword(s):  
Side Effects ◽  
Sleep Disorders ◽  
Learning Disability ◽  
Excessive Daytime Sleepiness ◽  
Daytime Sleepiness ◽  
Seizure Control ◽  
Subjective Sleep ◽  
Neurological Conditions ◽  
The Uk ◽  
Patients With Epilepsy

Epilepsy is the commonest serious chronic neurological condition, affecting 0.5% of the population in the UK. Subjective sleep disturbance and excessive daytime sleepiness are reported to be 50% more frequent in those with epilepsy than in controls. Causes are multiple. Both poor seizure control and nocturnal attacks are known to contribute to such sleep disorders. Epilepsy also increases the risk of associated sleep disorders, and additional neurological conditions, such as dementia, learning disability, and depression. These all affect sleep hygiene. Prescribed anti-epileptic drugs will further aggravate the problem. Side-effects will include drowsiness. Sedating benzodiazepines and barbiturates are considered worst offenders. Others affect sleep architecture to varying degrees and/or cause insomnia. While hyper-somnolence in patients with epilepsy will raise the possibility of any of the above issues, sleep deprivation is one of the commonest seizure triggers. This chapter will shed more light on the intricate relationship between sleep and epilepsy.

scholarly journals Suberoylanilide hydroxamic acid enhances the radiosensitivity of lung cancer cells through acetylated wild-type and mutant p53-dependent modulation of mitochondrial apoptosis

2021 ◽  
Vol 49 (2) ◽  
pp. 030006052098154
Author(s):  
Kan Wu ◽  
Xueqin Chen ◽  
Xufeng Chen ◽  
Shirong Zhang ◽  
Yasi Xu ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cancer Cells ◽  
Hydroxamic Acid ◽  
Site Directed Mutagenesis ◽  
Lung Cancer Cells ◽  
Mutant P53 ◽  
Suberoylanilide Hydroxamic Acid ◽  
Mitochondrial Apoptosis ◽  
Wild Type ◽  
Clonogenic Cell

Objective Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has shown potential as a candidate radiosensitizer for many types of cancers. This study aimed to explore the radiosensitization mechanism of SAHA in lung cancer cells. Methods Mutations in p53 were generated by site-directed mutagenesis using polymerase chain reaction. Transfection was performed to generate H1299 cells carrying wild-type or mutant p53. The radiosensitizing enhancement ratio was determined by clonogenic assays. Mitochondrial apoptosis was detected using JC-1 staining and flow cytometry analysis. Results Our results showed that SAHA induced radiosensitization in H1299 cells expressing wild-type p53, p53R175H or p53P223L, but this enhanced clonogenic cell death was not observed in parental H1299 (p53-null) cells or H1299 cells expressing p53 with K120R, A161T and V274R mutations. In SAHA-sensitized cells, mitochondrial apoptosis was induced following exposure to irradiation. Additionally, we observed that a secondary mutation at K120 (K120R) could eliminate p53-mediated radiosensitization and mitochondrial apoptosis. Conclusions The results of this study suggest that wild-type and specific mutant forms of p53 mediate SAHA-induced radiosensitization by regulating mitochondrial apoptosis, and the stabilization of K120 acetylation by SAHA is the molecular basis contributing to radiosensitization in lung cancer cells.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals N-terminal fatty acylation of the α-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation

1994 ◽  
Vol 303 (3) ◽  
pp. 697-700 ◽  
Author(s):  
F Galbiati ◽  
F Guzzi ◽  
A I Magee ◽  
G Milligan ◽  
M Parenti
Keyword(s):  
Palmitic Acid ◽  
G Protein ◽  
Fatty Acyl ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Α Subunit ◽  
Metabolic Labelling ◽  
Fatty Acylation ◽  
Absolute Requirement ◽  
Cdna Construct

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

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