[Show abstract][Hide abstract] ABSTRACT: The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility towards proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome.
Journal of Biological Chemistry 04/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The engineering of catalytic function in antibodies requires precise information on their structure. Here, results are presented that show how the antibody domain structure affects its functionality. The previously designed organophosphate-metabolizing reactibody A17 has been re-engineered by replacing its constant κ light chain by the λ chain (A17λ), and the X-ray structure of A17λ has been determined at 1.95 Å resolution. It was found that compared with A17κ the active centre of A17λ is displaced, stabilized and made more rigid owing to interdomain interactions involving the CDR loops from the VL and VH domains. These VL/VH domains also have lower mobility, as deduced from the atomic displacement parameters of the crystal structure. The antibody elbow angle is decreased to 126° compared with 138° in A17κ. These structural differences account for the subtle changes in catalytic efficiency and thermodynamic parameters determined with two organophosphate ligands, as well as in the affinity for peptide substrates selected from a combinatorial cyclic peptide library, between the A17κ and A17λ variants. The data presented will be of interest and relevance to researchers dealing with the design of antibodies with tailor-made functions.
[Show abstract][Hide abstract] ABSTRACT: The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein–Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.
[Show abstract][Hide abstract] ABSTRACT: We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.
BioMed Research International 01/2014; 2014:926394. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteasome is a multisubunit protein complex that exhibits proteolytic activity and is present in the nuclei and cytoplasm of cells. The 20S proteasome, which has a molecular weight of 700 kDa and a sedimentation coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome. The degradation of proteins in the cell is regulated by the ubiquitinylation system, which marks the old or defective protein molecules for their recognition by the proteasome and subsequent proteolysis. One of the key biological functions of the proteasome is the hydrolysis of intracellular proteins to the antigenic peptides, which are then presented on the cell surface on the major histocompatibility complex class I molecules. Recent studies indicate the existence of a molecular mechanism by which the peptides generated by the proteasome can also be presented on the major histocompatibility complex class II molecules. The catalytic activity of a constitutive proteasome is mediated by three subunits, β1, β2, and β5, which are constitutively expressed in cells. The proteasome, which contains corresponding immunosubunits β1i, β2i, and β5i the catalytic center, is called the immunoproteasome and is significantly different from the constitutive proteasome in its activity and substrate specificity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic proteins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies, rheumatoid arthritis, autoimmune colitis, Alzheimer’s disease, and Huntington disease). One of the most common and socially significant autoimmune diseases is multiple sclerosis (MS), which is characterized by the destruction of the myelin sheath of nerve fibers. Myelin basic protein (MBP) is a major autoantigen in multiple sclerosis. At present, the molecular mechanisms underlying the development of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immunoproteasome in the development of this disease.
Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomyelitis (EAE), an experimental model of MS. During further development of this research, we created a model to study the intracellular proteolysis of MBP in mammalian cells. Here we show that the intracellular
hydrolysis of MBP is significantly accelerated when the proteasome–immunoproteasome balance is shifted toward the latter.
Doklady Biochemistry and Biophysics 11/2013; 453(1):300-3. · 0.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
Proceedings of the National Academy of Sciences 01/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Butyrylcholinesterase (BChE) is a serine hydrolase (EC 220.127.116.11) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.
[Show abstract][Hide abstract] ABSTRACT: Among the strategies aimed at biocompatible means for organophosphorus nerve agents neutralization, immunoglobulins have attracted attention in the 1990's and 2000's both for their ability to immobilize the toxicants, but also for their ability to be turned into enzymatically active antibodies known as catalytic antibodies or abzymes (antibodies - enzymes). We will present here a critical review of the successive strategies used for the selection of these nerve agent-hydrolyzing abzymes, based on hapten design, namely antibodies raised against a wide variety of transition state analogues, and eventually the strategies based on anti-idiotypic antibodies and reactibodies.
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.-Belogurov, A. A., Jr., Stepanov, A. V., Smirnov, I. V., Melamed, D., Bacon, A., Mamedov, A. E., Boitsov, V. M., Sashchenko, L. P., Ponomarenko, N. A., Sharanova, S. N., Boyko, A. N., Dubina, M. V., Friboulet, A., Genkin, D. D., Gabibov, A. G. Liposome-encapsulated peptides protect against experimental allergic encephalitis.
[Show abstract][Hide abstract] ABSTRACT: Development of new ways of creating catalytic antibodies possessing defined substrate specificity towards artificial substrates has important fundamental and practical aspects. Low immunogenicity combined with high stability of immunoglobulins in the blood stream makes abzymes potent remedies. A good example is the cocaine-hydrolyzing antibody that has successfully passed clinical trials. Creation of an effective antidote against organophosphate compounds, which are very toxic substances, is a very realistic goal. The most promising antidotes are based on cholinesterases. These antidotes are now expensive, and their production methods are inefficient. Recombinant antibodies are widely applied in clinics and have some advantage compared to enzymatic drugs. A new potential abzyme antidote will combine effective catalysis comparable to enzymes with high stability and the ability to switch on effector mechanisms specific for antibodies. Examples of abzymes metabolizing organophosphate substrates are discussed in this review.
[Show abstract][Hide abstract] ABSTRACT: The unique hypervariability of the immunoglobulin (Ig) superfamily provides a means to create both binding and catalytic antibodies with almost any desired specificity and activity. The diversity of antigens and concept of adaptive response suggest that it is possible to find an antigen pair to any raised Ig. In the current review we discuss combinatorial approaches, which makes it possible to obtain an antibody with predefined properties, followed by 3D structure-based rational design to enhance or dramatically change its characteristics. A similar strategy, but applied to the second partner of the antibody-antigen pair, may result in selection of complementary substrates to the chosen Ig. Finally, 2D screening may be performed solving the "Chicken and Egg" problem when neither antibody nor antigen is known.
[Show abstract][Hide abstract] ABSTRACT: Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
Proceedings of the National Academy of Sciences 09/2011; 108(38):15954-9. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.
The FASEB Journal 08/2011; 25(12):4211-21. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.
PLoS ONE 01/2011; 6(6):e20991. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.
[Show abstract][Hide abstract] ABSTRACT: 105 Biocatalysis is one of the most important functions of polymers. Understanding of the mechanisms and structural-functional interactions of biopolymers that ensure their catalytic properties is a nationwide direction of modern biochemistry, molecular biology, and molecular medicine. Such knowledge can help to closely come to the creation of biocatalysts de novo with preset features, which is so essential for the fundamental aspects of the science about life and for the solution of specific problems in biotechnology. Earlier, we were the first to propose a method to create biocatalysts using the hypervariable features of immunoglobulins as antiidiotypic antibodies to the active sites of enzymes [1, 2]. Being an alternative of the known method of obtaining abzymes (catalytic antibodies) to chemical structures, analogues of transient states of enzymatic reactions , the suggested approach often ensures a higher specificity and efficiency of catalysis . Despite the acknowledged advances in obtaining specific esterase antibodies [5‐7], a real breakthrough in the application of the proposed approach would be the creation of proteases with preset features. Construction of catalytic vaccines that are based on such proteases and are capable of target disintegration of proteins responsible for development of certain pathologies could become one of the ways of obtaining drugs of the future. Recently, we have managed to obtain a proteolytic antibody using subtilisin Carlsberg as a primary antigen [8, 9]. In this work, we have described the structural and functional properties of this abzyme and the substrate specificity of the monoclonal antibody obtained by us and the single-chain recombinant antibody corresponding to it by the gene sequence, which was expressed in a heterologous system. It was concluded that any structural similarity between the antiidiotypic antibody and
Doklady Biochemistry and Biophysics 01/2008; 420:105-7. · 0.32 Impact Factor