Biomedical subjects

Alexander Kaplun

Publications and source records attributed to Alexander Kaplun.

9 recordsLinked to original sources

PubMed2006

Distinct activities of the related protein kinases Cdk1 and Ime2.

In budding yeast, commitment to DNA replication during the normal cell cycle requires degradation of the cyclin-dependent kinase (CDK) inhibitor Sic1. The G1 cyclin-CDK complexes Cln1-Cdk1 and Cln2-Cdk1 initiate the process of Sic1 removal by directly catalyzing Sic1 phosphorylation at multiple sites. Commitment to DNA replication during meiosis also appears to require Sic1 degradation, but the G1 cyclin-CDK complexes are not involved. It has been proposed that the meiosis-specific protein kinase Ime2 functionally replaces the G1 cyclin-CDK complexes to promote Sic1 destruction. To investigate this possibility, we compared Cln2-Cdk1 and Ime2 protein kinase activities in vitro. Both enzyme preparations were capable of catalyzing phosphorylation of a GST-Sic1 fusion protein, but the phosphoisomers generated by the two activities had significantly different electrophoretic mobilities. Furthermore, mutation of consensus CDK phosphorylation sites in Sic1 affected Cln2-Cdk1- but not Ime2-dependent phosphorylation. Phosphoamino acid analysis and phosphopeptide mapping provided additional evidence that Cln2-Cdk1 and Ime2 targeted different residues within Sic1. Examination of other substrates both in vitro and in vivo also revealed differing specificities. These results indicate that Ime2 does not simply replace G1 cyclin-CDK complexes in promoting Sic1 degradation during meiosis.

CDC2 Protein Kinase↗

PubMed2006

Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli.

The enzyme acetohydroxyacid synthase (AHAS) catalyses the first common step in the biosynthesis of the three branched-chain amino acids. Enzymes in the AHAS family generally consist of regulatory and catalytic subunits. Here, we describe the first crystal structure of an AHAS regulatory subunit, the ilvH polypeptide, determined at a resolution of 1.75 A. IlvH is the regulatory subunit of one of three AHAS isozymes expressed in Escherichia coli, AHAS III. The protein is a dimer, with two beta alpha beta beta alpha beta ferredoxin domains in each monomer. The two N-terminal domains assemble to form an ACT domain structure remarkably close to the one predicted by us on the basis of the regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH). The two C-terminal domains combine so that their beta-sheets are roughly positioned back-to-back and perpendicular to the extended beta-sheet of the N-terminal ACT domain. On the basis of the properties of mutants and a comparison with 3PGDH, the effector (valine) binding sites can be located tentatively in two symmetrically related positions in the interface between a pair of N-terminal domains. The properties of mutants of the ilvH polypeptide outside the putative effector-binding site provide further insight into the functioning of the holoenzyme. The results of this study open avenues for further studies aimed at understanding the mechanism of regulation of AHAS by small-molecule effectors.

Acetolactate Synthase↗

PubMed2006

Identification of novel in vivo Raf-1 phosphorylation sites mediating positive feedback Raf-1 regulation by extracellular signal-regulated kinase.

The Ras-Raf-mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.

Amino Acid Sequence↗

PubMed2005

Drug design using the example of the complement system inhibitors' development.

Undesired activation of the complement system, a part of the immune system, is a major pathogenic factor contributing to various diseases, such as ischemia-reperfusion injury, sepsis, asthma, allergic reactions, rheumatoid arthritis, Alzheimer's disease, myasthenia, multiple sclerosis and others. The history of the development of complement system inhibitors, preventing its destructive action on the body, represents the evolution of the main methods of drug design. This review illustrates the main approaches of drug design, ranging from screening and modification of natural products to structure-based ligand design, on the basis of complement inhibitors' creation. The current status of the field of complement inhibitors is also discussed.

Animals↗

PubMed2005

Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties.

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of "typical" AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.

Acetolactate Synthase↗

PubMed2005

Monitoring the acetohydroxy acid synthase reaction and related carboligations by circular dichroism spectroscopy.

Acetohydroxy acid synthase (AHAS) and related enzymes catalyze the production of chiral compounds [(S)-acetolactate, (S)-acetohydroxybutyrate, or (R)-phenylacetylcarbinol] from achiral substrates (pyruvate, 2-ketobutyrate, or benzaldehyde). The common methods for the determination of AHAS activity have shortcomings. The colorimetric method for detection of acyloins formed from the products is tedious and does not allow time-resolved measurements. The continuous assay for consumption of pyruvate based on its absorbance at 333 nm, though convenient, is limited by the extremely small extinction coefficient of pyruvate, which results in a low signal-to-noise ratio and sensitivity to interfering absorbing compounds. Here, we report the use of circular dichroism spectroscopy for monitoring AHAS activity. This method, which exploits the optical activity of reaction products, displays a high signal-to-noise ratio and is easy to perform both in time-resolved and in commercial modes. In addition to AHAS, we examined the determination of activity of glyoxylate carboligase. This enzyme catalyzes the condensation of two molecules of glyoxylate to chiral tartronic acid semialdehyde. The use of circular dichroism also identifies the product of glyoxylate carboligase as being in the (R) configuration.

Acetolactate Synthase↗

PubMed2005

Inhibition of classical pathway of complement activation with negative charged derivatives of bisphenol A and bisphenol disulphates.

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C1r /C1s components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.

Animals↗

PubMed2004

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme.

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.

Acetolactate Synthase↗

PubMed2002

Isoleucine starvation caused by sulfometuron methyl in Salmonella typhimurium measured by translational frameshifting.

The authors have developed a tool for the study of inhibitor-induced amino acid starvation in bacteria which exploits the phenomenon of translational frameshifting. The inhibition of acetohydroxyacid synthase II by the herbicide sulfometuron methyl (SMM) has complex effects on branched-chain amino acid biosynthesis. Experiments were done with Salmonella typhimurium containing a plasmid with an isoleucine codon in a 'shifty' region, prone to translational frameshifting. SMM did not cause translational frameshifting in minimal medium under conditions that inhibit growth. A 20-fold higher concentration of SMM was required to cause starvation for isoleucine, e.g. in the presence of valine. This starvation was reflected in translational frameshifting correlated with inhibition of growth. These observations support the authors' previous suggestions based on other techniques. The method used here could be generalized for the study of complex metabolic effects related to amino acids.

Culture Media↗