scholarly journals Is aldehyde dehydrogenase inhibited by sulfur compounds? In vitro and in vivo studies

2018 ◽  
Vol 65 (1) ◽  
pp. 125-132 ◽  
Author(s):  
Malgorzata Iciek ◽  
Magdalena Górny ◽  
Anna Bilska-Wilkosz ◽  
Danuta Kowalczyk-Pachel
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Ethanol Metabolism ◽  
Aldh Activity ◽  
Sulfur Species ◽  
Reactive Sulfur Species ◽  
Allyl Sulfides ◽  
Sulfur Containing

Aldehyde dehydrogenase (ALDH) catalyzes the critical step of ethanol metabolism, i.e.  transformation of toxic acetaldehyde to acetic acid. It is a redox sensitive protein with the key Cys in its active site. Recently, it has been documented that activity of some proteins can be modified by sulfur-containing molecules called reactive sulfur species leading to the formation of hydropersulfides. The aim of the present study was to examine whether ALDH activity can be modified through this way.Studies were performed in vitro using yeast ALDH and various reactive sulfur species, including Na2S, GSSH, K2Sx, Na2S2O3, and garlic-derived allyl sulfides. The effect of garlic-derived trisulfide on ALDH activity was  also studied  in vivo in the rat liver.The obtained results clearly demonstrated that ALDH could be regulated by sulfur species which inhibited its enzymatic activity. The results also suggested that not H2S but polysulfides or hydropersulfides were the oxidizing species responsible for this modification. This process was easily reversible by reducing agents. After the treatment with polysulfides or hydropersulfides the level of protein-bound sulfur increased, while the activity of the enzyme dramatically decreased. Moreover, the study demonstrated that ALDH activity was inhibited in vivo in the rat liver after garlic-derived trisulfide administration. This is the first study reporting the regulation of  ALDH activity by sulfane sulfur species and the results suggest that it led to the inhibition of the enzyme.

Reactive sulfur species inactivate Ca2+/calmodulin-dependent protein kinase IV via S-polysulfidation of its active-site cysteine residue

2017 ◽  
Vol 474 (15) ◽  
pp. 2547-2562 ◽  
Author(s):  
Tsuyoshi Takata ◽  
Hideshi Ihara ◽  
Naoya Hatano ◽  
Yukihiro Tsuchiya ◽  
Takaaki Akaike ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Er Stress ◽  
Active Site ◽  
Cysteine Residue ◽  
Dependent Protein Kinase ◽  
Sulfur Species ◽  
Active Site Cysteine ◽  
Dependent Protein ◽  
Reactive Sulfur Species

Reactive sulfur species (RSS) modulate protein functions via S-polysulfidation of reactive Cys residues. Here, we report that Ca2+/calmodulin (CaM)-dependent protein kinase IV (CaMKIV) was reversibly inactivated by RSS via polysulfidation of the active-site Cys residue. CaMKIV is phosphorylated at Thr196 by its upstream CaMK kinase (CaMKK), resulting in the induction of its full activity. In vitro incubation of CaMKIV with the exogenous RSS donors Na2Sn (n = 2–4) resulted in dose-dependent inhibition of the CaMKK-induced phospho-Thr196 and consequent inactivation of the enzyme activity. Conversely, mutated CaMKIV (C198V) was refractory to the Na2Sn-induced enzyme inhibition. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that Cys198 in CaMKIV represents a target for S-polysulfidation. Furthermore, phosho-Thr196 and CaMKIV activity were inhibited by incubation with cysteine hydropersulfide, a newly identified RSS that is generated from cystine by cystathionine-γ-lyase. In transfected cells expressing CaMKIV, ionomycin-induced CaMKIV phosphorylation at Thr196 was decreased upon treatment with either Na2S4 or the endoplasmic reticulum (ER) stress inducer thapsigargin, whereas cells expressing mutant CaMKIV (C198V) were resistant to this treatment. In addition, the ionomycin-induced phospho-Thr196 of endogenous CaMKIV was also inhibited by treatment either with Na2S4 or thapsigargin in Jurkat T lymphocytes. Taken together, these data define a novel signaling function for intracellular RSS in inhibiting CaMKIV activity via S-polysulfidation of its Cys198 during the response to ER stress.

scholarly journals Effect of Static Compressive Force on Aldehyde Dehydrogenase Activity in Periodontal Ligament Fibroblasts

2021 ◽  
Vol 15 (1) ◽  
pp. 417-423
Author(s):  
Fabio Schemann-Miguel ◽  
Antonio Carlos Aloise ◽  
Silvana Gaiba ◽  
Lydia Masako Ferreira
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Periodontal Ligament ◽  
Tooth Movement ◽  
Compressive Force ◽  
Control Group ◽  
Aldh Activity ◽  
Periodontal Ligament Fibroblasts ◽  
Normal Tissues

Background: The application of static compressive forces to periodontal ligament fibroblasts (PDLFs) in vivo or in vitro has been linked to the expression of biochemical agents and local tissue modifications that could be involved in maintaining homeostasis during orthodontic movement. An approach used for identifying mesenchymal cells, or a subpopulation of progenitor cells in both tumoral and normal tissues, involves determining the activity of aldehyde dehydrogenase (ALDH). However, the role of subpopulations of PDLF-derived undifferentiated cells in maintaining homeostasis during tooth movement remains unclear. Objective: This study aimed at analyzing the effect of applying a static compressive force to PDLFs on the activity of ALDH in these cells. Methods: PDLFs were distributed into two groups: control group (CG), where fibroblasts were not submitted to compression, and experimental group (EG), where fibroblasts were submitted to a static compressive force of 4 g/mm2 for 6 hours. The compressive force was applied directly to the cells using a custom-built device. ALDH activity in the PDLFs was evaluated by a flow cytometry assay. Results: ALDH activity was observed in both groups, but was significantly lower in EG than in CG after the application of a static compressive force in the former. Conclusion: Application of a static compressive force to PDLFs decreased ALDH activity.

scholarly journals ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ

2020 ◽  
Vol 144 (2) ◽  
pp. 98-104
Author(s):  
О. В. Кислова
Keyword(s):  
Alcohol Dehydrogenase ◽  
Aldehyde Dehydrogenase ◽  
Ethanol Oxidation ◽  
The Body ◽  
Inhibition Constant ◽  
Ethanol Metabolism ◽  
Dehydrogenase Reaction ◽  
Metabolism Enzymes

To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of  main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase  interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.

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.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

scholarly journals Bioinspired organometallic chemistry

2002 ◽  
Vol 74 (1) ◽  
pp. 115-122 ◽  
Author(s):  
Lanny S. Liebeskind ◽  
Jiri Srogl ◽  
Cecile Savarin ◽  
Concepcion Polanco
Keyword(s):  
Organometallic Chemistry ◽  
Refractory Metal ◽  
Redox Active ◽  
The Stability ◽  
New Procedures ◽  
Sulfur Containing ◽  
Insight Into

Given the stability of the bond between a mercaptide ligand and various redox-active metals, it is of interest that Nature has evolved significant metalloenzymatic processes that involve key interactions of sulfur-containing functionalities with metals such as Ni, Co, Cu, and Fe. From a chemical perspective, it is striking that these metals can function as robust biocatalysts in vivo, even though they are often "poisoned" as catalysts in vitro through formation of refractory metal thiolates. Insight into the nature of this chemical discrepancy is under study in order to open new procedures in synthetic organic and organometallic chemistry.

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

Sirtuin-dependent reversible lysine acetylation controls the activity of acetyl-Coenzyme A synthetase in Campylobacter jejuni

2021 ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Campylobacter Jejuni ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Protein Function ◽  
Metabolic Enzyme ◽  
Lysine Acetylation ◽  
Class Iii ◽  
Acetyl Coa

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group ( N ε ) of an active-site lysine of the AMP-forming acetyl-CoA synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, the alluded active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N -acetyltransferases (GNATs). Acs activity is lost as a result of acetylation, and restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c , cj1715, and cj1050c loci, namely the AMP-forming acetate:CoA ligase ( Cj Acs), a type IV GCN5-type lysine acetyltransferase (GNAT, hereafter Cj LatA), and a NAD + -dependent (class III) sirtuin deacylase ( Cj CobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of Cj Acs. IMPORTANCE This work is important because it provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase ( Cj Acs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of Cj Acs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

scholarly journals An active site mutant allele of human Pol ε is a mutator in vitro and in vivo

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Anderson Ayuk Agbor ◽  
Zachary F Pursell
Keyword(s):  
Active Site ◽  
Mutant Allele
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