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Gabibov Alexander mechanisms of antigen degradation

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  • 5
  • основе действия ФОТ лежит необратимое связывание токсина с ацетилхолинэстеразой, что приводит к накоплению ацетилхолина в холинергических синапсах и коллапсу периферической и центральной нервной системы. Различные данные указывают на роль бутирилхолинэстеразы, в предотвращении холинергического кризиса путем связывания и выведения ФОТ из организма. Поэтому БуХэ представляется преспективным средством андотной профилактики и лечения отравлений ФОТ. На сегодняшний день для получения одной дозы БуХЭ необходимо 400 литров человеческой крови. Существующие системы экспресии БуХэ не являются достаточно эффективными
  • MS is well-known chronic neurodegenerative disease. Long period of time it was postulated that the main aggressors in MS are autoreactive T-Cells specific to major component of myelin membrane – myelin basic protein. However nowadays there is no doubt that B-cells and autoantibodies also have their own important position in attackers team of neurodegenerative process are thought to be one of the key-players in MS.
  • При рассеянном склерозе одна из причин демиелинизации – это атака цитотоксических лимфоцитов на олигодендроциты в центральной нервной системе. Нами было показано, что подобная атака может быть связана с презентацией на поверхности олигодендроцитов фрагментов основного белка миелина, генерируемых особым энзиматическим комплексом – иммунопротеасомой. Данный переход в свою очередь связан с переходом протеасома – иммунопротеасома, индуцируемым интерфероном. Ингибирование иммунопротеасомы позволит предотвратить некорректную презентацию и тем самым защитить олигодендроциты от цитотоксических лимфоцитов.
  • Transcript

    • 1. Alexander Gabibov Mechanisms of Antigen degradation Workshop "Biomedical technologies at Skokovo. Opportunities and challenges"
    • 2. Antigen degradation machinery
    • 3. A nti B odies Catalytic Antibodies EN zymes ABzymes
    • 4. Catalytic Antibodies . Historical Background
    • 5. Five ways to obtain catalytic antibodies Immunization by transition state analog of reaction Production of antiidiotypic antibody Reactive immunization Induction of autoimmune disease Screening of phage- display libraries Belogurov et al, BioEssays 2009
    • 6. Organophosphorous poisons
      • The major target of organophosphorous toxins are cholinesterase-like enzymes ;
      • Extremely low LD 50 value
      • OPC-associated mortality is 200,000 people per year ;
      • There are real threats of acts of terrorism, for example sarin attack in Tokyo underground at 20 March of 1995
    • 7. 9A8 may covalently accept and destroy anti-acetylcholine esterase poisons from blood stream Kolesnikov et al, PNAS 2000 3D Structure of the 9A8 Antiidiotypic Antibody Active Site
      • Superposition of the active sites of esterolytic abzymes 9A8 (green) and 17E8 (blue).
      • Ser99 - His35 diades are indicated.
      • Hydrogene bonds are indicated by dashed lines.
    • 8. Soman r9A8 interact with soman-MCA RFU Time, h
    • 9. Combinatorial approach Rational design High resolution 3D Effective screening Directed evolution
    • 10. -streptavidin molecule -BSA molecule -Biotin group -Phosphonate group Control well Experimental well Phage pull after reaction with Bt-X phosphonate Nonspecific sorbsion Nonspecific and specific sorbsion Trypsin elution Phage amplification Next rounds of selection Bt-X Reactive (covalent) selection
    • 11. A.17 Catalytic Antibody Biotin-X-phosphonate Griffin.1 scFv library + Screening for biotin-X binding Conversion into the full-size human antibody crystal Reshetnyak et al, JACS 2007
    • 12. A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base
    • 13. A.17 antibody has unusual deep cavity with nucleophilic tyrosine at its base Comparison of active site cavities of natural and created de novo biocatalysts. Chemically selected reactibody A.17 possess deepest substrate binding niche. Cross-section views of the active center of esterolytic antibodies 49G7, TEPC15, aldolase antibody 33F12, choline esterases AChE and BChE and antibody A.17 complexed with their ligands.
    • 14. The pre-existing primitive active site of the A.17 antibody stereo-selectively interacts with P(R)-isomer of the phosphonate molecule Tyr59 Tyr53 Tyr33 Tyr34 Trp109 Tyr37 Trp92 Trp48 Phe100 OP compound Tyr 37 OH P(R) Tyr 37 P(S) SN2 Tyr59 Tyr53 Tyr33 Tyr34 Trp109 Asn105 Ala107 Phe100 Trp92 Trp48 Cl - 2.5 Å 3.3 Å 3.2 Å Ser51 2.8 Å Tyr37
    • 15. A.17 antibody hydrolyzes organophosphorus pesticide – paraoxon by multi-step covalent catalysis. Covalent intermediate Amount of released p-nitrophenol in case of the reaction with paraoxon is evidently higher than concentration of active sites The reaction rate increased linearly with hydroxylamine concentration. It allows to define that dephosphorylation is rate limiting step k 2 = 1.1 ± 0.1 x10 -1 min -1 k 3 = 1.6 ± 0.2 x10 -2 min -1 k 4 = 1.26 ± 0.09 x10 -3 min -1 Tyr Tyr + Tyr Tyr Tyr Tyr + +
    • 16. The reaction mechanism of paraoxon hydrolysis by A.17 antibody proceeds through the phosphotyrosine covalent intermediate. 1578.5 m/z=30 1578.5 m/z=30 1583.0 m/z=30 Δ m=135 ( ) (unmodified Fab A.17) (phosphonylated Fab A.17) (unmodified Fab A.17)
    • 17. Perspectives: generation of artificial biocatalysts in vivo
    • 18. MESSAGE
      • Basically ALL Autoantigens may serve as substrates for autoantibodies .
      • Kolesnikov et al. PNAS, 2000
      • Ponomarenko et al. Meth. Immunol., 2002
      • Kozyr et al. Imm.Lett., 2002
      • Ponomarenko et al. PNAS, 2006
      • Ponomarenko et al. Biochemistry 2006
      • Ponomarenko et al. Biochemistry, 2007
      • Belogurov et al. J.Immunology, 2008
      • Durova et al. Molecular Immunology, 2009
      • Belogurov et al. Autoimmunity, 2009
      Belogurov et al, BioEssays 2009
    • 19. CDRH3 DNA Possible catalytic residues Schuster et al, Science, 1992, Gololobov et al, PNAS, 1995; Gololobov et al. Mol Immunol. 1997. Structural Similarity Between BV04-01 and MRL-4 anti-DNA Autoantibodies: DNA-binding and DNA-cleaving Activities are Germline-Encoded
    • 20. Do Myelin-Directed Antibodies Predict Multiple Sclerosis? N EJM, 2003 The B-Cell – Old Player, New Position on the Team NEJM, 2008 Multiple sclerosis B-cells as one of the key players in the MS Environmental hypothesis of MS induction. EBV virus involvement.
    • 21. Environmental hypothesis of MS induction. EBV virus involvement. Antibodies selected from MS Phage-display library are crossreactive towards both, Myelin Basic Protein and EBV latent membrane protein 1.
    • 22. Abzymatic Site-specific MBP hydrolysis The cleavage sites are localized inside the encephalitogenic epitopes MBP Musse et al, PNAS 2006 Ponomarenko et al, PNAS 2006
    • 23. Specific B-Cells depletion in MS Toxin Autoreactive B-Cell B-Cell Receptor (BCR), fragments of myelin basic protein (MBP) BCR MBP CD 4 CD 25High TOLERANCE
    • 24. C D A B Autoantibody binding pattern
    • 25. Specific B-Cells depletion in MS Fc-based immunotoxins are shown to be the best in the presented set due to the excellent specific/unspecific cytotoxicity ratio
    • 26. MDS score versus days post disease induction (surrounding pictures). Peptides were applied at days 7-11 (120 μg/rat per day) after disease induction by nasal route. Maximum clinical score in each group of rats, median and 95% confidential interval (in the middle). NS - not significant Belogurov et. al Autoimmunity Administration of MBP peptides to DA rats with EAE
    • 27.
      • SUV liposomes 50-90 nm mimicking virus particles
      • Interaction with APC cells
      • Dose-dependent effect
      Clinical trials of MBP46-62 formulation in DA rats
    • 28. P IP
    • 29. The level of IP in the brain is dramatically elevated during EAE development in SJL/J mice.
    • 30. Immunoproteasome localization in mice brain. LMP2 and LMP7 are carried by different cells.
    • 31. MBP hydrolysis by proteasome and enzymes. LC-ESI approach.
    • 32. Enhanced release of encephalitogenic peptide by immunoproteasome. LC-ESI approach with isotope labeled peptide.
    • 33.
      • Immunoproteasome marks oligodendrocytes for CTL (immune system)
      • Some kind of target designation
      • How can we prevent this process? The answer is Inhibitors or siRNA
    • 34. Expression of LMP7 immunosubunit is significantly decreased by siRNA administration
    • 35. Low-weight proteasome inhibitors
    • 36. Low-weight inhibitors efficiently inhibit immunoproteasome in vitro and ameliorate EAE in vivo
    • 37. Immunoproteasome as a target for MS treatment
    • 38. Der Mensch als Industriepalast (Man as Industrial Palace) Stuttgart, 1926. Chromolithograph. National Library of Medicine. Fritz Kahn (1888-1968) Kahn’s modernist visualization of the digestive and respiratory system as "industrial palace," really a chemical plant
    • 39. M.M. Shemyakin & Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Authors: Alexandre G. Gabibov , Alexey A. Belogurov Jr., Ivan V. Smirnov, Inna N. Kurkova, Ekaterina Kuzina, Alexey Kononikhin, Alexey Stepanov, Natalie A. Ponomarenko, Andrey V. Reshetnyak
    • 40. COLLABORATION RUSSIA Prof. Eugene Nikolaev Institute of Biochemical Physics RAS Prof. Alexey Boyko Moscow MS Center Prof. Dmitry Knorre , Prof. Olga Fedorova, Dr. Nikita Kuznetsov Dr. Dmitry Genkin Dr. Dobroslav Melamed USA Prof. Al Tramontano , UC Davis, Medical school National Institute of Allergy and Infectious Diseases National Institutes of Health Dr. Herbert C. Morse III ; Ciphergen Biosystems, Inc FRANCE Prof. Daniel Thomas, Alain Friboulet, Drs. Dominidue Pillet, Marjorie Paon, Berangere Avalle . The University of Technology, Compiegne Prof. Patrick Masson , Drs Eugénie Carletti, Florian Nachon Département de Toxicologie – CRSSA Institut de Recherche Biomédicale des Armées