V. D. Knorre

Chemical polysialylation: Design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid-oxyntomodulin (PSA-OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA-OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA-OXM at 15 μmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.

Immunoproteasome enhances intracellular proteolysis of myelin basic protein

300 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 sedimenta tion coefficient of 20S, is present as a proteolytic core in a more complex particle, the 26S proteasome [1]. 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 [2]. 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 mole cules [3]. Recent studies indicate the existence of a molecular mechanism by which the peptides gener ated by the proteasome can also be presented on the major histocompatibility complex class II molecules [4]. 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 immuno proteasome and is significantly different from the con stitutive proteasome in its activity and substrate speci ficity. The set of antigenic peptides produced by the immunoproteasome differs from the set of peptides produced by the constitutive proteasome [5, 6]. It was recently shown that immunoproteasome not only changes the degradation spectrum of antigenic pro teins but also ensures the maintenance of protein homeostasis under conditions of oxidative stress caused by the action of interferons on the cell [7]. The amount of immunoproteasome in cells increases in various diseases (hematologic malignancies [8], rheu matoid arthritis [9], autoimmune colitis [10], Alzhe imer’s disease [11], and Huntington disease [12]). 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 develop ment of multiple sclerosis are being actively studied. Recent studies have demonstrated an important role of both the constitutive proteasome and the immuno proteasome in the development of this disease [13]. Earlier, we studied in vitro the proteolysis of MBP by the proteasome isolated from normal mice and mice with experimental autoimmune encephalomy elitis (EAE), an experimental model of MS [14]. Dur ing 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. It is known that the expression of the myelin basic protein in mammals is detected solely in the central and peripheral nervous systems. This protein is local ized in the membrane of specialized cells, oligoden drocytes and Schwann cells, forming the myelin sheath of axons. Unfortunately, work with primary human neuronal cultures is associated with numerous experimental difficulties (first of all, the lack of mate rial and ethical concerns). In view of this, at the first step of this work, to study the proteolysis of MBP ex vivo we created a genetic construct that made it possible to express a recombinant human MBP in mammalian cells. For this purpose, a DNA fragment 546 bp long, encoding the full length MBP, was amplificated by PCR using specific overlapping oligo nucleotides and then cloned into the pBudCE4.1/EF Immunoproteasome Enhances Intracellular Proteolysis of Myelin Basic Protein

Substrate specificity of catalytic autoantibodies in neurodegenerative processes.

61 Autoantibodies are acknowledged to play a pathological role in development of autoimmune processes [1]. The ability of autoantibodies to penetrate across the blood–brain barrier and to colocalize with neuronal antigens suggests that they may be involved in neurodegenerative processes. Recently, we have discovered the phenomenon of catalytic degradation of myelin basic protein (MBP) by antigen-specific autoantibodies isolated from blood serum of patients with multiple sclerosis (MS) and SJL mice developing experimental autoimmune encephalomyelitis (EAE) and assumed that autoantibodies may be involved in pathological demyelination [2, 3]. It was demonstrated that the catalytic activity of autoantibodies strongly correlates with the stage of progression of pathological process in MS, assessed according to the commonly used EDSS scale [4]. This consistent pattern was confirmed by other authors [5]; however, a number of principal questions regarding the discovered function of anti-MBP autoantibodies remained unanswered. In particular, it remains unclear (1) whether the catalytic autoantibodies to MBP are generated only in MS or in other neuronal pathologies as well and (2) what the specificity of MBP cleavage by autoantibodies is and whether the discovered reaction can be used as a diagnostic marker.

Functional degradation of myelin basic protein. The proteomic approach

Proteolytic degradation of autoantigens is of prime importance in current biochemistry and immunology. The fundamental issue is the functional role of peptides produced in the process of change of the hydrolysis specificity during the transition from the normal to a pathologic state. In some cases identification of specific peptide fragments can be a diagnostic and prognostic criterion of the pathology progress. The subject of this work is the comparative study of degradation peculiarities of one of the major neuroantigens, myelin basic protein, by proteases activated upon the development of a pathological demyelinating process, and by proteasomes of different origin. Comparison of the specificity of the tested biocatalysts in some cases demonstrated critical changes in the set of myelin basic protein fragments capable of being presented on the major histocompatibility complex class I upon neurodegeneration, which may promote the development of autoimmune pathological processes.

Mediators and biomarkers of inflammation in meningitis: Cytokine and peptidome profiling of cerebrospinal fluid

Differential diagnosis of bacterial and viral meningitis is an urgent problem of the modern clinical medicine. Early and accurate detection of meningitis etiology largely determines the strategy of its treatment and significantly increases the likelihood of a favorable outcome for the patient. In the present work, we analyzed the peptidome and cytokine profiles of cerebrospinal fluid (CSF) of 17 patients with meningitis of bacterial and viral etiology and of 20 neurologically healthy controls. In addition to the identified peptides (potential biomarkers), we found significant differences in the cytokine status of the CSF of the patients. We found that cut-off of 100 pg/ml of IL-1β, TNF, and GM-CSF levels discriminates bacterial and viral meningitis with 100% specificity and selectivity. We demonstrated for the first time the reduction in the level of two cytokines, IL-13 and GM-CSF, in the CSF of patients with viral meningitis in comparison with the controls. The decrease in GM-CSF level in the CSF of patients with viral meningitis can be explained by a disproportionate increase in the levels of cytokines IL-10, IFN-γ, and IL-4, which inhibit the GM-CSF expression, whereas IL-1, IL-6, and TNF activate it. These observations suggest an additional approach for differential diagnosis of bacterial and viral meningitis based on the normalized ratio IL-10/IL-1β and IL-10/TNF > 1, as well as on the ratio IFN-γ/IL-1β and IFN-γ/ TNF < 0.1. Our findings extend the panel of promising clinical and diagnostic biomarkers of viral and bacterial meningitis and reveal opposite changes in the cytokine expression in meningitis due to compensatory action of proand antiinflammatory factors.

Antibodies-antidotes against organophosphorus compounds

94 Organophosphorus toxins (OPTs) are the most toxic compounds for humans and animals known to date. This group includes some pesticides, zarin, zoman, and VX gas. The effect of OPTs is based on irreversible binding of toxin with acetylcholinesterase of higher organisms. Modern protectors and therapeutic agents used in poisoning with OPTs are imperfect. Lowmolecular-weight dots synthesized on the basis of oximes (dipriroxime, obidoxine, and pralidoxime) are toxic and interact with OPTs at high concentrations (to 50 mg/kg) [1]. Among the most promising new antidotes that can neutralize OPTs, one should distinguish butyrylcholinesterase [2]. Today, human butyrylcholinesterase is obtained by purification of blood plasma. This is an expensive procedure that must meet stringent requirements for testing the initial material owing to contamination of donor plasma with viruses. A project on obtaining a recombinant enzyme in sufficient amounts has not been realized in any leading laboratory of the world. Creating an antibody that could be able to neutralize OPTs is an attractive purpose. Currently, recombinant and humanized antibodies are widely used as vaccines and therapeutic agents. Immunoglobulin molecules have a number of advantages over enzymes as drugs, because they have a long half-life time in the blood flow. In addition, they can be efficiently eliminated from the body in the form of complexes with the antigen. In this work, we for the first time proposed to use a recombinant antibody selected from a half-synthetic human immunoglobulin library and expressed in an eukaryotic system as an antidote against OPTs. Data on specific binding of OPT analogues, obtained in this study, provide reasons to consider recombinant antibodies as potential effective OPT-neutralizing agents.

mRNA expression profile of mouse oligodendrocytes in inflammatory conditions

In this study, we performed transcriptome profiling of oligodendrocyte culture of mice treated with the remyelinating therapeutic agent benztropine in the presence and absence of interferon gamma (IFNγ). The results of this work are important for understanding the expression profile of oligodendrocytes under conditions of systemic inflammation in the central nervous system in multiple sclerosis as well as the mechanisms of cellular response to benztropine in light of its possible use for the treatment of multiple sclerosis.

Deimination of the myelin basic protein decelerates its proteasome-mediated metabolism

Deimination of myelin basic protein (MBP) by peptidylarginine deiminase (PAD) prevents its binding to the proteasome and decelerates its degradation by the proteasome in mammalian cells. Potential anticancer drug tetrazole analogue of chloramidine 2, at concentrations greater than 1 µM inhibits the enzymatic activity of PAD in vitro. The observed acceleration of proteasome hydrolysis of MBP to antigenic peptides in the presence of PAD inhibitor may increase the efficiency of lesion of the central nervous system by cytotoxic lymphocytes in multiple sclerosis. We therefore suggest that clinical trials and the introduction of PAD inhibitors in clinical practice for the treatment of malignant neoplasms should be performed only after a careful analysis of their potential effect on the induction of autoimmune neurodegeneration processes.

Mechanism-dependent selection of immunoglobulin gene library for obtaining covalent biocatalysts.

179 Covalent catalysis is a fundamental mechanism ensuring the unique properties of enzymes as the most efficient biocatalysts. Designing artificial enzymes that are capable of covalent catalysis may provide a basis for studying the evolution of biocatalytic functions and ensure their employment in pharmaceutics and biotechnology. This was the first study to use the strategy of chemical selection of a semisynthetic library of variable fragments of human immunoglobulin genes to obtain artificial biocatalysts capable of covalent catalysis. As a result, a family of artificial enzymes was obtained, which formed an active site that can covalently interact with aryl phosphonate 1, an irreversible inhibitor of serine proteases. The most active clone A.17 exhibited enzymatic properties and catalyzed the hydrolysis of the peptide bond in the low-molecularweight substrate Phe–MCA.

Cloning and characterization of serpin from red king crab Paralithodes camtschaticus

&NA; Serpins are a family of serine protease inhibitors that are involved in numerous physiological processes and are known to regulate innate immunity pathways. To advance our understanding of their role in P. camtschaticus, a commercially significant species, we cloned and characterized a serpin from this species, designated serpin PC, that has anticoagulant and anticomplement effects on human blood. We found that serpin PC is a secreted protein with a typical serpin‐like primary structure that is similar to other known crustacean serpins. Recombinant serpin PC was found to have inhibitory activity against R/K‐specific bovine cationic trypsin. The reaction proceeds through the formation of a stable covalent complex of peptidase with P1 residue R383 of serpin PC. This interaction is characterized by a relatively high overall inhibition constant kass=(2.3 ± 0.7) × 106 M−1s−1 and an SI of 4.7 ± 0.8. Protein localization by western blotting showed that serpin PC is present in the muscles and, to a lesser extent, the heart, whereas it is transcribed predominantly in hemocytes and the heart. Through peptidase activity profiling of hemocytes and plasma, we found that serpin PC inhibits at least two R/K‐specific activities and showed that it inhibits phenoloxidase (PO) activity induction in hemocytes. HighlightsA serpin PC from red king crab, P. camtschaticus was cloned and characterized.Though transcribed in hemocytes and the heart, the protein was found in the muscles and heart.Serpin PC inhibits at least two R/K‐specific peptidases in hemocytes and plasma.In hemocytes, serpin PC suppresses PO activity induction.