Mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in the presence of methylglyoxal.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be one of the targets of methylglyoxal (MGO), a metabolite of glycolysis that increased in diabetes. However, the mechanism of GAPDH inactivation in the presence of MGO is unclear. The purpose of the work was to study the reaction of GAPDH with MGO and to identify the products of the reaction. It was shown that incubation of recombinant human GAPDH with MGO leads to irreversible inactivation of the enzyme, which is accompanied by a decrease in SH-group content by approximately 3.3 per tetramer GAPDH. MALDI-TOF MS analysis showed that the modification of GAPDH with MGO results in the oxidation of the catalytic cysteine residues (Cys152) to form cysteine-sulfinic acid. In addition, 2 arginine residues (R80 and R234) were identified that react with MGO to form hydroimidazolones. Incubation of SH-SY5Y neuroblastoma cells with MGO resulted in the inactivation of GAPDH and inhibition of glycolysis. The mechanism of GAPDH oxidation in the presence of MGO suggests the participation of superoxide anion, which is formed during the reaction of amino groups with methylglyoxal. The role of GAPDH in protection against the damaging effect of ROS in cells in the case of inefficiency of MGO removal by the GSH-dependent glyoxalase system is discussed.

Testing a Hypothesis of 12S rRNA Methylation by Putative METTL17 Methyltransferase.

Mitochondrial ribosome assembly is a complex multi-step process involving many additional factors. Ribosome formation differs in various groups of organisms. However, there are universal steps of assembly and conservative factors that have been retained in evolutionarily distant taxa. METTL17, the object of the current study, is one of these conservative factors involved in mitochondrial ribosome assembly. It is present in both bacteria and the mitochondria of eukaryotes, in particular mice and humans. In this study, we tested a hypothesis of putative METTL17 methyltransferase activity. MALDI-TOF mass spectrometry was used to evaluate the methylation of a putative METTL17 target - a 12S rRNA region interacting with METTL17 during mitochondrial ribosome assembly. The investigation of METTL17 and other mitochondrial ribosome assembly factors is of both fundamental and practical significance, because defects in mitochondrial ribosome assembly are often associated with human mitochondrial diseases.

Products of S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase: Relation between S-nitrosylation and oxidation.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the major targets of NO in cells, especially in neurodegenerative diseases. S-Nitrosylation of GAPDH is accompanied by its translocation into the nucleus with subsequent apoptosis. The product of GAPDH modification by NO is considered to be S-nitrosylated GAPDH (GAPDH-SNO). However, this has not been confirmed by direct methods. METHODS: Products of GAPDH modification in the presence of the NO donor diethylamine NONOate were analyzed by MALDI- and ESI- mass spectrometry methods. RESULTS: The adduct between GAPDH and dimedone was detected by MALDI-MS analysis after incubation of S-nitrosylated GAPDH with dimedone, which points to the formation of cysteine-sulfenic acid (GAPDH-SOH) in the protein. Analysis of the protein hydrolysate revealed the incorporation of dimedone into the catalytic residue Cys150. An additional peak that corresponded to GAPDH-SNO was detected by ESI-MS analysis in GAPDH after the incubation with the NO donor. The content of GAPDH-SNO and GAPDH-SOH in the modified GAPDH was evaluated by different approaches and constituted 2.3 and 0.7 mol per mol GAPDH, respectively. A small fraction of GAPDH was irreversibly inactivated after NO treatment, suggesting that a minor part of the products includes cysteine-sulfinic or cysteine-sulfonic acids. CONCLUSIONS: The main products of GAPDH modification by NO are GAPDH-SNO and GAPDH-SOH that is presumably formed due to the hydrolysis of GAPDH-SNO. GENERAL SIGNIFICANCE: The obtained results are important for understanding the molecular mechanism of redox regulation of cell functions and the role of GAPDH in the development of neurodegenerative disorders.

S-glutathionylation of human glyceraldehyde-3-phosphate dehydrogenase and possible role of Cys152-Cys156 disulfide bridge in the active site of the protein.

BACKGROUND: We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of H2O2 and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated. METHODS: Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH. RESULTS: S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S. CONCLUSIONS: S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1. GENERAL SIGNIFICANCE: The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.

Aim23p Interacts with the Yeast Mitochondrial Ribosomal Small Subunit.

Protein synthesis in mitochondria is generally organized in a bacterial-like manner but, at the same time, possesses several unique traits. Translation initiation in mitochondria is regulated by two protein factors, mtIF2 and mtIF3. Previously we demonstrated that Saccharomyces cerevisiae Aim23 protein is an ortholog of IF3 in budding yeast. However, the data on the interactions between Aim23p and other proteins are limited. Here, we demonstrated that Aim23p interacts with the yeast mitochondrial ribosomal small subunit both in vivo and in vitro using co-immunoprecipitation and density gradient sedimentation.

Possible Role of Escherichia coli Protein YbgI.

Proteins containing the NIF3 domain are highly conserved and are found in bacteria, eukaryotes, and archaea. YbgI is an Escherichia coli protein whose gene is conserved among bacteria. The structure of YbgI is known; however, the function of this protein in cells remains obscure. Our studies of E. coli cells with deleted ybgI gene suggest that YbgI is involved in formation of the bacterial cell wall.

Amyloid-ß with isomerized Asp7 cytotoxicity is coupled to protein phosphorylation.

Neuronal dysfunction and loss associated with the accumulation of amyloid-ß (Aß) in the form of extracellular amyloid plaques and hyperphosphorylated tau in the form of intraneuronal neurofibrillary tangles represent key features of Alzheimer's disease (AD). Amyloid plaques found in the brains of AD patients are predominantly composed of Aß42 and its multiple chemically or structurally modified isoforms. Recently, we demonstrated that Aß42 with isomerised Asp7 (isoAß42) which is one of the most abundant Aß isoform in plaques, exhibited high neurotoxicity in human neuronal cells. Here, we show that, in SH-SY5Y neuroblastoma cells, the administration of synthetic isoAß42 rather than intact Aß42 resulted in a significantly higher level of protein phosphorylation, especially the phosphorylation of tau, tubulins, and matrin 3. IsoAß42 induced a drastic reduction of tau protein levels. Our data demonstrate, for the first time, that isoAß42, being to date the only known synthetic Aß species to cause AD-like amyloidogenesis in an animal AD model, induced cell death by disabling structural proteins in a manner characteristic of that observed in the neurons of AD patients. The data emphasize an important role of isoAß42 in AD progression and provide possible neurotoxicity paths for this particular isoform.

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme.

BACKGROUND: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. METHODS: Rabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. RESULTS: Incubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD+ and thermal stability. CONCLUSIONS: The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. GENERAL SIGNIFICANCE: The manuscript considers one of the possible mechanisms of redox regulation of cell functions.

Trastuzumab and Pertuzumab Plant Biosimilars: Modification of Asn297-linked Glycan of the mAbs Produced in a Plant with Fucosyltransferase and Xylosyltransferase Gene Knockouts.

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPB-WT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPB-ΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297-linked GnGn-type glycan contains α1,3-fucose and ß1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.

Dimerization of Tyr136Cys alpha-synuclein prevents amyloid transformation of wild type alpha-synuclein.

Expression of human alpha-synuclein in E. coli cells is known to result in a mixture of the wild type alpha-synuclein and the protein containing Tyr136Cys substitution due to the translational error. The amount of Cys136 alpha-synuclein (Cys136-AS) may reach approximately 50% of the recombinant protein. The wild-type and Cys136-containing fractions of alpha-synuclein were separated using thiol-Sepharose, and their properties were investigated. In the absence of reducing agents, Cys136-AS forms dimers due to the disulfide bonding. Both wild-type and Cys136 alpha-synuclein preparations are prone to aggregate during prolonged incubation under shaking at pH 4 and 37°C, but only the wild-type alpha-synuclein produces amyloid aggregates. The aggregates produced by either monomeric or dimeric Cys136-AS do not exhibit amyloid properties according to the test with Thioflavin T. Moreover, an admixture of dimeric Cys136-AS prevents the amyloid transformation of the wild-type alpha-synuclein. CD spectroscopy analysis revealed an enhanced content of alpha-helical structures in the aggregates produced by dimeric Cys136-AS. The admixture of Cys136-AS in preparations of human recombinant alpha-synuclein can be a source of erroneous interpretation of experiments on amyloid transformation of this protein.

[Mechanisms of perioperative corneal abrasions: alterations in tear film proteome]. / Mekhanizmy razvitiia perioperatsionnykh érozii rogovitsy: izmeneniia proteomnogo sostava sleznoi plenki.

Perioperative corneal abrasion is an ophthalmic complication commonly found in patients underwent general anesthesia. In this study, correlations between development of corneal injury and proteomic changes in tear film during general anesthesia were examined using an animal (rabbit) model. Being started after 1-h anesthesia, the process of accumulation of pathological changes in the cornea unequivocally led clinically significant abrasions following 3-6 h of the narcosis. The corneal damage was associated with alterations in profiles of major proteins of the tear film. Analysis of the tear proteome pointed to depression of lachrymal glands function, and suggested serotransferrin, serum albumin and annexin A1 as potential tear markers of the complication. The tear film alterations included fast drop of total antioxidant activity and activity of superoxide dismutase, and decrease in interleukin-4 and increase in interleukin-6 content indicating development of oxidative and pro-inflammatory responses. These findings suggest antioxidant and anti-inflammatory therapy as prospective approach for prevention/treatment of perioperative corneal abrasions. The observed anesthesia-induced effects should be considered in any study of ocular surface diseases employing anesthetized animals.

Reorganization of low-molecular-weight fraction of plasma proteins in the annual cycle of cyprinidae.

Reorganization of the low-molecular-weight fraction of cyprinid plasma was analyzed using various electrophoretic techniques (disc electrophoresis, electrophoresis in polyacrylamide concentration gradient, in polyacrylamide with urea, and in SDS-polyacrylamide). The study revealed coordinated changes in the low-molecular-weight protein fractions with seasonal dynamics and related reproductive rhythms of fishes. We used cultured species of the Cyprinidae family with sequenced genomes for the detection of these interrelations in fresh-water and anadromous cyprinid species. The common features of organization of fish low-molecular-weight plasma protein fractions made it possible to make reliable identification of their proteins. MALDI mass-spectrometry analysis revealed the presence of the same proteins (hemopexin, apolipoproteins, and serpins) in the low-molecular-weight plasma fraction in wild species and cultured species with sequenced genomes (carp, zebrafish). It is found that the proteins of the first two classes are organized as complexes made of protein oligomers. Stoichiometry of these complexes changes in concordance with the seasonal and reproductive rhythms.

Mass spectrometric approaches to study enveloped viruses: new possibilities for structural biology and prophylactic medicine.

This review considers principles of the use of mass spectrometry for the study of biological macromolecules. Some examples of protein identification, virion proteomics, testing vaccine preparations, and strain surveillance are represented. Possibilities of structural characterization of viral proteins and their posttranslational modifications are shown. The authors' studies by MALDI-MS on S-acylation of glycoproteins from various families of enveloped viruses and on oligomerization of the influenza virus hemagglutinin transmembrane domains are summarized.

Complete genome and proteome of Acholeplasma laidlawii.

We present the complete genome sequence and proteogenomic map for Acholeplasma laidlawii PG-8A (class Mollicutes, order Acholeplasmatales, family Acholeplasmataceae). The genome of A. laidlawii is represented by a single 1,496,992-bp circular chromosome with an average G+C content of 31 mol%. This is the longest genome among the Mollicutes with a known nucleotide sequence. It contains genes of polymerase type I, SOS response, and signal transduction systems, as well as RNA regulatory elements, riboswitches, and T boxes. This demonstrates a significant capability for the regulation of gene expression and mutagenic response to stress. Acholeplasma laidlawii and phytoplasmas are the only Mollicutes known to use the universal genetic code, in which UGA is a stop codon. Within the Mollicutes group, only the sterol-nonrequiring Acholeplasma has the capacity to synthesize saturated fatty acids de novo. Proteomic data were used in the primary annotation of the genome, validating expression of many predicted proteins. We also detected posttranslational modifications of A. laidlawii proteins: phosphorylation and acylation. Seventy-four candidate phosphorylated proteins were found: 16 candidates are proteins unique to A. laidlawii, and 11 of them are surface-anchored or integral membrane proteins, which implies the presence of active signaling pathways. Among 20 acylated proteins, 14 contained palmitic chains, and six contained stearic chains. No residue of linoleic or oleic acid was observed. Acylated proteins were components of mainly sugar and inorganic ion transport systems and were surface-anchored proteins with unknown functions.

Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein.

HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.

Proteomic characterization of Mycoplasma gallisepticum nanoforming.

The goal of this work was to create a model for the long persistence of Mycoplasma gallisepticum in depleted medium and under low growth temperature followed by proteomic study of the model. Nanoforms and revertants for M. gallisepticum were obtained. Proteomic maps were produced for different stages of the formation of nanoforms and revertants. It is shown that proteins responsible for essential cellular processes of glycolysis, translation elongation, and DnaK chaperone involved in the stabilization of newly synthesized proteins are crucial for the reversion of M. gallisepticum to a vegetative form. Based on the current data, it is assumed that changes in the metabolism of M. gallisepticum during nanoforming are not post-mortal, thus M. gallisepticum does not transform to uncultivable form, but remains in a reversible dormant state during prolonged unfavorable conditions.

Byproduct with altered fluorescent properties is formed during standard deprotection step of hexachlorofluorescein labeled oligonucleotides.

HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material with altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore, for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential, the careful RP-HPLC purification step is required.

Proteome of the bacterium Mycoplasma gallisepticum.

Using modern proteomic assays, we have identified the products of gene expression and posttranslational modifications of proteins of the bacterium Mycoplasma gallisepticum S6. Combinations of different technologies of protein separation by electrophoresis and mass-spectrometric analysis gave us a total of 446 proteins, i.e. 61% of the annotated proteins of this microorganism. The Pro-Q Diamond and Pro-Q Emerald dye technology was used for fluorescent detection of ten phosphoproteins and two glycoproteins. The acylation of proteins was studied by electrophoresis after in vivo labeling with different 14C-labeled fatty acids, followed by autoradiography. Sixteen acylated proteins were identified, with a quarter of them involved in plasma membrane construction and another quarter involved in cell energy metabolism.

Analysis of myelin basic protein fragmentation by proteasome.

The proteasome is a high molecular protein complex whose purpose is specific protein degradation in eukaryotic cells. One of the proteasome functions is to produce peptides, which will then be presented on the outer cell membrane using main histocompatibility complex (MHC) molecules of the first or second class. There are definite reasons to believe that proteasome directly takes part in the specific degradation of myelin basic protein (MBP), which make up to 30% of all proteins in the myelin sheath of neuronal axons. The details of the proteasomal degradation of MBP are still unclear. In this work, the features of specific MBP degradation by proteasome were studied.It was demonstrated that MBP (non-ubiquitinated) is a good substrate for 20S and for the 26S proteasome. This is the first work on detecting the sites of MBP proteolysis by proteasome from brains of SJL/J/J and Balb/C mice's lines. Substantial differences in the degradation pattern of this neuroantigen were found, which could indicate the better presentation MBP parts on MHC molecules in the case of mice predisposed to the development of experimental autoimmune encephalomyelitis.