scholarly journals Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells

2000 ◽  
Vol 346 (2) ◽  
pp. 447-454 ◽  
Author(s):  
Robert C. GARDNER ◽  
Stephen J. ASSINDER ◽  
Gary CHRISTIE ◽  
Grant G. F. MASON ◽  
Roger MARKWELL ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Boronic Acid ◽  
Cultured Cells ◽  
Gel Filtration ◽  
Boronic Acids ◽  
Cell Extracts ◽  
Catalytic Sites ◽  
Ic50 Values ◽  
In Cells

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The Ki values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3×Ki). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFĸB activation with IC50 values comparable to their Ki values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

scholarly journals Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

1996 ◽  
Vol 7 (10) ◽  
pp. 1535-1546 ◽  
Author(s):  
J P Paccaud ◽  
W Reith ◽  
J L Carpentier ◽  
M Ravazzola ◽  
M Amherdt ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Cdna Clones ◽  
Restrictive Temperature ◽  
Rnase Protection ◽  
Cell Extracts ◽  
Exact Function ◽  
Mammalian Protein

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

scholarly journals Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells

2000 ◽  
Vol 346 (2) ◽  
pp. 447 ◽  
Author(s):  
Robert C. GARDNER ◽  
Stephen J. ASSINDER ◽  
Gary CHRISTIE ◽  
Grant G.F. MASON ◽  
Roger MARKWELL ◽  
...  
Keyword(s):  
Boronic Acid ◽  
Cultured Cells

scholarly journals Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells

1989 ◽  
Vol 258 (1) ◽  
pp. 33-40 ◽  
Author(s):  
J L Vargas ◽  
F Aniento ◽  
J Cervera ◽  
E Knecht
Keyword(s):  
Protein Degradation ◽  
Cultured Cells ◽  
Human Cells ◽  
Lysosomal Degradation ◽  
Intracellular Degradation ◽  
Cell Extracts ◽  
Nutritional Conditions ◽  
Step Down ◽  
First Time

Vanadate, at concentrations higher than 0.04 mM, inhibits the intracellular degradation of short-lived proteins in exponentially growing L-132 human cells. The inhibition is not due to a decrease in viability or in the ATP contents of the cells. Since vanadate decreases proteolysis in cell extracts, the inhibition appears to affect the proteinases which degrade these proteins. Under optimal nutritional conditions, the degradation of long-lived proteins is accelerated by vanadate, thus providing additional evidence that in exponentially growing cultured cells degradation of short- and long-lived proteins occurs by different processes. Vanadate also efficiently inhibits the lysosomal degradation of endocytosed proteins and of long-lived proteins under step-down conditions. However, this effect seems to be unrelated to the observed inhibition of degradation of short-lived proteins, because chloroquine and leupeptin, which inhibit degradation of proteins by lysosomes, do not modify the degradation of these proteins. Our results provide for the first time a probe which, owing to its opposite effects on the degradation of short- and long-lived proteins, could be useful to clarify the mechanisms involved in protein degradation in cultured cells.

scholarly journals Characterization of Hydrogenase and Reductive Dehalogenase Activities of Dehalococcoides ethenogenes Strain 195

2005 ◽  
Vol 71 (3) ◽  
pp. 1664-1667 ◽  
Author(s):  
Ivonne Nijenhuis ◽  
Stephen H. Zinder
Keyword(s):  
Reductive Dechlorination ◽  
Methyl Viologen ◽  
Hydrogenase Activity ◽  
Atpase Inhibitor ◽  
Benzyl Viologen ◽  
Reductive Dehalogenase ◽  
Cell Extracts ◽  
Dehalococcoides Ethenogenes ◽  
In Cells

ABSTRACT Dehalococcoides ethenogenes strain 195 reductively dechlorinates tetrachloroethene (PCE) and trichloroethene (TCE) to vinyl chloride and ethene using H2 as an electron donor. PCE- and TCE-reductive dehalogenase (RD) activities were mainly membrane associated, whereas only about 20% of the hydrogenase activity was membrane associated. Experiments with methyl viologen (MV) were consistent with a periplasmic location for the RDs or a component feeding electrons to them. The protonophore uncoupler tetrachlorosalicylanilide did not inhibit reductive dechlorination in cells incubated with H2 and PCE and partially restored activity in cells incubated with the ATPase inhibitor N,N′-dicyclohexylcarbodiimide. Benzyl viologen or diquat (E o′ ≈ −360 mV) supported reductive dechlorination of PCE or TCE at rates comparable to MV (−450 mV) in cell extracts.

scholarly journals Boronic Acids as Prospective Inhibitors of Metallo-β-Lactamases: Efficient Chemical Reaction in the Enzymatic Active Site Revealed by Molecular Modeling

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2026
Author(s):  
Alexandra V. Krivitskaya ◽  
Maria G. Khrenova
Keyword(s):  
Chemical Reaction ◽  
Active Site ◽  
Boronic Acid ◽  
Boronic Acids ◽  
Nucleophilic Attack ◽  
Carbonyl Carbon Atom ◽  
Antibacterial Drug ◽  
Acid Inhibitor ◽  
Fukui Indices ◽  
Ic50 Values

Boronic acids are prospective compounds in inhibition of metallo-β-lactamases as they form covalent adducts with the catalytic hydroxide anion in the enzymatic active site upon binding. We compare this chemical reaction in the active site of the New Delhi metallo-β-lactamase (NDM-1) with the hydrolysis of the antibacterial drug imipenem. The nucleophilic attack occurs with the energy barrier of 14 kcal/mol for imipenem and simultaneously upon binding a boronic acid inhibitor. A boron atom of an inhibitor exhibits stronger electrophilic properties than the carbonyl carbon atom of imipenem in a solution that is quantified by atomic Fukui indices. Upon forming the prereaction complex between NDM-1 and inhibitor, the lone electron pair of the nucleophile interacts with the vacant p-orbital of boron that facilitates the chemical reaction. We analyze a set of boronic acid compounds with the benzo[b]thiophene core complexed with the NDM-1 and propose quantitative structure-sroperty relationship (QSPR) equations that can predict IC50 values from the calculated descriptors of electron density. These relations are applied to classify other boronic acids with the same core found in the database of chemical compounds, PubChem, and proposed ourselves. We demonstrate that the IC50 values for all considered benzo[b]thiophene-containing boronic acid inhibitors are 30–70 μM.

scholarly journals Isolation and Characterization of an Aldehyde Dehydrogenase Encoded by the aldB Gene of Escherichia coli

2005 ◽  
Vol 187 (3) ◽  
pp. 1067-1073 ◽  
Author(s):  
Kwok Ki Ho ◽  
Henry Weiner
Keyword(s):  
Escherichia Coli ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Properties ◽  
Isolation And Characterization ◽  
Cell Extracts ◽  
Bacterial Enzyme ◽  
Terminal Amino ◽  
Mammalian Enzyme ◽  
In Cells

ABSTRACT An aldehyde dehydrogenase was detected in crude cell extracts of Escherichia coli DH5α. Growth studies indicated that the aldehyde dehydrogenase activity was growth phase dependent and increased in cells grown with ethanol. The N-terminal amino acid sequence of the purified enzyme identified the latter as an aldehyde dehydrogenase encoded by aldB, which was thought to play a role in the removal of aldehydes and alcohols in cells that were under stress. The purified enzyme showed an estimated molecular mass of 220 ± 8 kDa, consisting of four identical subunits, and preferred to use NADP and acetaldehyde. MgCl2 increased the activity of the NADP-dependent enzyme with various substrates. A comparison of the effect of Mg2+ ions on the bacterial enzyme with the effect of Mg2+ ions on human liver mitochondrial aldehyde dehydrogenase revealed that the bacterial enzyme shared kinetic properties with the mammalian enzyme. An R197E mutant of the bacterial enzyme appeared to retain very little NADP-dependent activity on acetaldehyde.

scholarly journals Characterization of two β-galactosidases LacZ and WspA1 from Nostoc flagelliforme with focus on the latter’s central active region

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiang Gao ◽  
Litao Liu ◽  
Lijuan Cui ◽  
Tao Zheng ◽  
Boyang Ji ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Active Form ◽  
Gel Filtration ◽  
Special Role ◽  
Nostoc Flagelliforme ◽  
Reporter Protein ◽  
Gfp Reporter ◽  
Potential Applications ◽  
Ic50 Values

AbstractThe identification and characterization of new β-galactosidases will provide diverse candidate enzymes for use in food processing industry. In this study, two β-galactosidases, Nf-LacZ and WspA1, from the terrestrial cyanobacterium Nostoc flagelliforme were heterologously expressed in Escherichia coli, followed by purification and biochemical characterization. Nf-LacZ was characterized to have an optimum activity at 40 °C and pH 6.5, different from that (45 °C and pH 8.0) of WspA1. Two enzymes had a similar Michaelis constant (Km = 0.5 mmol/liter) against the substrate o-nitrophenyl-β-D-galactopyranoside. Their activities could be inhibited by galactostatin bisulfite, with IC50 values of 0.59 µM for Nf-LacZ and 1.18 µM for WspA1, respectively. Gel filtration analysis suggested that the active form of WspA1 was a dimer, while Nf-LacZ was functional as a larger multimer. WspA1 was further characterized by the truncation test, and its minimum central region was found to be from residues 188 to 301, having both the glycosyl hydrolytic and transgalactosylation activities. Finally, transgenic analysis with the GFP reporter protein found that the N-terminus of WspA1 (35 aa) might play a special role in the export of WspA1 from cells. In summary, this study characterized two cyanobacterial β-galactosidases for potential applications in food industry.

scholarly journals Purification of β-lactamases by affinity chromatography on phenylboronic acid-agarose

1984 ◽  
Vol 221 (2) ◽  
pp. 505-512 ◽  
Author(s):  
S J Cartwright ◽  
S G Waley
Keyword(s):  
Affinity Chromatography ◽  
Boronic Acid ◽  
Phenylboronic Acid ◽  
Boronic Acids ◽  
High Yield ◽  
Beta Lactamase ◽  
Beta Lactamases ◽  
Cell Extracts ◽  
Pseudomonas Maltophilia ◽  
One Step

Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.

scholarly journals A ‘Defective’ Conjugated Porous Poly-Azo as Dual Photocatalyst

Catalysts ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 1064
Author(s):  
Ipsita Nath ◽  
Jeet Chakraborty ◽  
Sara Abednatanzi ◽  
Pascal Van Der Voort
Keyword(s):  
High Surface ◽  
High Surface Area ◽  
Catalytic Performance ◽  
Boronic Acids ◽  
Porous Polymer ◽  
Structure Property ◽  
Catalytic Sites ◽  
Oxidative Bromination ◽  
Porous Poly

A heterogeneous photocatalyst amenable to catalyze different chemical reactions is a highly enabling and sustainable material for organic synthesis. Herein we report the synthesis and characterization of an azobenzene-based organic π–conjugated porous polymer (AzoCPP) as heterogeneous dual photocatalyst manifesting net-oxidative bromination of arenes and dehydroxylation of boronic acids to corresponding phenols. Hierarchical porosity and high surface area of the nano-sized AzoCPP allowed superior catalyst-substrate contact during catalyses, whereas the inherent structural defect present in the CPP backbone resulted in low-energy sinks functioning as de facto catalytic sites. A combination of these two structure-property aspects of AzoCPP, in addition to the dielectric constant manipulation of the system, led to excellent catalytic performance. The protocols remained valid for a wide substrate scope and the catalyst was recycled multiple times without substantial loss in catalytic activity. With the aid of subsequent control experiments and analytical characterizations, mechanisms for each catalysis are proposed and duly corroborated.

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