DOCSLIB.ORG

Wo 2009/023270 A2

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Wo 2009/023270 A2 (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date PCT 19 February 2009 (19.02.2009) WO 2009/023270 A2 (51) International Patent Classification: 94083 (US). WANG, Chia-wei [-/US]; 3444 El Camino C12P 21/00 (2006.01) A61K 38/28 (2006.01) Real #307, Santa Clara, CA 95051 (US). GEETHING, C12N 15/09 (2006.01) A61K 38/21 (2006.01) Nathan, C. [US/US]; 2009 Garzoni Place, Santa Clara, C12N 1/00 (2006.01) A61P 43/00 (2006.01) CA 95054 (US). C07H 21/00 (2006.01) C07K 14/00 (2006.01) A61K 38/16 (2006.01) C 0 40/70 (2006.01) (74) Agents: WRONG, Karen, K. et al.; Wilson Sonsini Goodrich & Rosati, 650 Page Mill Road, Palo Alto, CA (21) International Application Number: 94304-1050 (US). PCT/US2008/009787 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) International Filing Date: 15 August 2008 (15.08.2008) AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, (25) Filing Language: English EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, (26) Publication Language: English LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH, PL, PT, (30) Priority Data: RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY,TJ, 15 August 2007 (15.08.2007) US 60/956,109 TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, 60/981,073 18 October 2007 (18.10.2007) US ZW 60/986,569 8 November 2007 (08. 11.2007) US (84) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): AMU- kind of regional protection available): ARIPO (BW, GH, NIX, INC. [US/US]; 1455 Adams Drive, Suite 1120, GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, Menlo Park, CA 94025 (US). ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, (72) Inventors; and FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL, (75) Inventors/Applicants (for US only): BOGIN, Oren NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, [US/US]; 1231 Manet Dr., Sunnyvale, CA 94087 (US). CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). STEMMER, Willem, P. [NL/US]; 108 Kathy Court, Los Gatos, CA 95030 (US). SCHELLENBERGER, Volker Published: [DE/US]; 914 Moreno Avenue, Palo Alto, CA 94303 (US). — without international search report and to be republished YIN, Yong [US/US]; 107 Sunset Dr., Sunnyvale, CA upon receipt of that report (54) Title: COMPOSITIONS AND METHODS FOR MODIFYING PROPERTIES OF BIOLOGICALLY ACTIVE POLYPEP TIDES (57) Abstract: The present invention relates to biologically active polypeptides linked to one or more accessory polypeptides. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applica- tions. POSITIONS AND METHODS FOR MODIFYING PROPERTIES O F BIOLOGICALLY ACTIVE POLYPEPTIDES CROSS-REFERENCE TO RELATED APPLICATIONS [0001 ] This application claims the priority benefit of U S Provisional Application Serial Nos 60/956, 109 filed on August 15, 2007, 60/98 1,073, filed October 18, 2007 and 60/986,569, filed November 8, 2007, pending, which are hereby incorporated herein by reference in their entirety BACKGROUND O F THE INVENTION [0002] Recombinant proteins have become very attractive candidates for the development of novel therapeutics However, production of protein pharmaceuticals requires significant optimization of processes to obtain sufficient yields of specific biologically active polypeptides It is well established that the expression of recombinant proteins in the cytoplasm of Escherichia coli, in particular mammalian recombinant proteins, frequently results in the formation of insoluble aggregates known as inclusion bodies High cell density fermentation and purification of the recombinant protein from inclusion bodies of E coli are two major bottlenecks for the cost effective production of therapeutic proteins (Panda, A K, 2003, Adv Biochem Eng Biotechnol , 85, 43) Similarly, for research purposes, where hundreds of proteins may need to be screened for various activities, the expression of soluble, active protein is desirable, thereby avoiding the step of first purifying inclusion bodies and then having to denature and refold protein each separately [0003] Examples of the many pharmaceutically important proteins that form insoluble inclusion bodies when expressed in the cytoplasmic space of E coli include human Growth Hormone (hGH) (Patra, A K et al , 2000, Protein Expr Puπf, 18, 182, Khan, R H, et al , 1998, Biotechnol Prog , 14, 722), human Granulocyte-Colony Stimulating Factor (G-CSF)( Zaveckas, M et al 2007, J Chromatogr B Analyt Technol Biomed Life Sci 852, 409, Lee, A Y et al , 2003, Biotechnol Lett , 25, 205,) and Interferon alpha (IFN-alpha, Valente, C A et al , 2006, Protein Expr Puπf 45, 226) Furthermore, the immunoglobulin domains of antibodies and their fragments, including domain antibody fragments (dAb), Fv fragments, single-chain Fv fragments (scFv), Fab fragments, Fab'2 fragments, and many non-antibody proteins (such as FnIII domains) generally form inclusion bodies upon expression in the cytoplasm of bacterial hosts (Kou, G , et al , 2007, Protein Expr Purif 52, 131, Cao, P , et al 2006, Appl Microbiol Biotechnol , 73, 15 1, Chen, L H et al , 2006, Protein Expr Pun f , 46, 495 ) [0004] Human proteins typically fold using a hydrophobic core comprising a large number of hydrophobic amino acids Research has shown that proteins can aggregate and form inclusion bodies, especially when genes from one organism are expressed in another expression host, such that the protein's native binding partners are absent, so that folding help is unavailable and hydrophobic patches remain exposed This is especially true when large evolutionary distances are crossed a cDNA isolated from a eukaryote for example, when expressed as a recombinant gene in a prokaryote, has a high risk of aggregating and forming an inclusion body While the cDNA may properly code for a translatable mRNA, the protein that results will emerge in a foreign microenvironment This often results in misfolded, inactive protein that generally accumulates as aggregates if the concentration is high enough Other effectors, such as the internal microenvironment of a prokaryotic cell (pH, osmolaπty) may differ from that of the original source of the gene and affect protein folding Mechanisms for folding a protein may also be host-dependent and thus be absent in a heterologous host, and hydrophobic residues that normally would remain buried as part of the hydrophobic core instead remain exposed and available for interaction with hydrophobic sites on other proteins. Processing systems for the cleavage and removal of internal peptides of the expressed protein may also be absent in bacteria. In addition, the fine controls that may keep the concentration of a protein low will also be missing in a prokaryotic cell, and over-expression can result in filling a cell with protein that, even if it were properly folded, would precipitate by saturating its environment. [0005] The recovery of biologically active products from the aggregated state found in inclusion bodies is typically accomplished by unfolding with chaotropic agents or acids, followed by dilution or dialysis into optimized refolding buffers. However, many polypeptides (especially structurally complex oligomeric proteins and those containing multiple disulfide bonds) do not easily adopt an active conformation following chemical denaturation. [0006] Small changes in primary structure can affect solubility, presumably by altering folding pathways (Mitraki, A. et al. (1989) Bio/Technology 7, 690; Baneyx, F, et. al. 2004 Nat Biotechnol, 22, 1399; Ventura, S. 2005 Microb Cell Fact, 4, 11). In order to reduce the formation of insoluble aggregates during high-density fermentation, some groups have linked heterologous fusion proteins to the protein of interest. Examples of such fusion sequences are Glutathione-S-Transferase (GST), Protein Disulfide Isomerase (PDI), Thioredoxin (TRX), Maltose Binding Protein (MBP), His6 tag, Chitin Binding Domain (CBD) and Cellulose Binding Domain (CBD) (Sahadev, S. et al. 2007, MoI. Cell. Biochem.; Dysom, M.R. et al. 2004, BMC Biotechnol, 14, 32). In summary, these approaches were found to be protein-specific, as they do not work for all proteins. [0007] While various fusion proteins have been designed to improve folding, chemical PEGylation of proteins has also been reported to enhance protein solubility, reduce aggregation, reduce immunogenicity, and reduce proteolysis. Nonetheless, the proper folding of overproduced polypeptides remains problematic within the highly concentrated and viscous environment of the cell cytoplasm, where aggregation occurs in a concentration-dependent manner. Another approach for the expression of mammalian proteins in bacterial hosts avoids leader peptides and expresses the active protein directly in the cytoplasm of the host. However, this process tends to result in aggregation and inclusion body formation. [0008] One widely used approach for the expression of mammalian proteins in active form in bacteria is to direct the protein into the non-reducing environment of the periplasmic space of bacterial hosts such as E.coli, typically using signal- or leader-peptides to direct secretion. Secretion into the periplasm (and rarely into the media) appears to mimic the native eukaryotic process of protein secretion, folding and disulfide formation and often results in active protein.
Load more

Recommended publications
  • FABP2 Ala54thr Polymorphism and Post-Training Changes of Body Composition and Biochemical Parameters in Caucasian Women
    G C A T T A C G G C A T genes Article FABP2 Ala54Thr Polymorphism and Post-Training Changes of Body Composition and Biochemical Parameters in Caucasian Women Agata Leo ´nska-Duniec 1,*, Katarzyna Switała´ 1, Ildus I. Ahmetov 2,3 , Craig Pickering 4 , Myosotis Massidda 5 , Maciej Buryta 6, Andrzej Mastalerz 7 and Ewelina Maculewicz 7 1 Faculty of Physical Education, Gdansk University of Physical Education and Sport, 80-336 Gdansk, Poland; katarzyna.switala@awf.gda.pl 2 Laboratory of Molecular Genetics, Kazan State Medical University, 420012 Kazan, Russia; genoterra@mail.ru 3 Department of Physical Education, Plekhanov Russian University of Economics, 117997 Moscow, Russia 4 Institute of Coaching and Performance, School of Sport and Wellbeing, University of Central Lancashire, Preston PR1 2HE, UK; craigpickering1014@hotmail.com 5 Department of Life and Environmental Sciences, University of Cagliari, 09124 Cagliari, Italy; myosotis.massidda@gmail.com 6 Institute of Physical Culture Sciences, University of Szczecin, 70-453 Szczecin, Poland; maciej.buryta@usz.edu.pl 7 Faculty of Physical Education, Jozef Pilsudski University of Physical Education in Warsaw, 00-968 Warsaw, Poland; andrzej.mastalerz@awf.edu.pl (A.M.); ewelina.jask@gmail.com (E.M.) * Correspondence: agata.leonska-duniec@awf.gda.pl Abstract: The functional FABP2 Ala54Thr polymorphism (rs1799883) is strongly associated with lipid Citation: Leo´nska-Duniec,A.; Switała,´ K.; Ahmetov, I.I.; Pickering, and carbohydrate metabolism, although the function of its potential modifying effect on training- C.; Massidda, M.; Buryta, M.; induced changes in obesity-related parameters is still unknown. The aim of the present study was Mastalerz, A.; Maculewicz, E.
    [Show full text]
  • The Role of Streptococcal and Staphylococcal Exotoxins and Proteases in Human Necrotizing Soft Tissue Infections
    toxins Review The Role of Streptococcal and Staphylococcal Exotoxins and Proteases in Human Necrotizing Soft Tissue Infections Patience Shumba 1, Srikanth Mairpady Shambat 2 and Nikolai Siemens 1,* 1 Center for Functional Genomics of Microbes, Department of Molecular Genetics and Infection Biology, University of Greifswald, D-17489 Greifswald, Germany; patience.shumba@uni-greifswald.de 2 Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, CH-8091 Zurich, Switzerland; Srikanth.MairpadyShambat@usz.ch * Correspondence: nikolai.siemens@uni-greifswald.de; Tel.: +49-3834-420-5711 Received: 20 May 2019; Accepted: 10 June 2019; Published: 11 June 2019 Abstract: Necrotizing soft tissue infections (NSTIs) are critical clinical conditions characterized by extensive necrosis of any layer of the soft tissue and systemic toxicity. Group A streptococci (GAS) and Staphylococcus aureus are two major pathogens associated with monomicrobial NSTIs. In the tissue environment, both Gram-positive bacteria secrete a variety of molecules, including pore-forming exotoxins, superantigens, and proteases with cytolytic and immunomodulatory functions. The present review summarizes the current knowledge about streptococcal and staphylococcal toxins in NSTIs with a special focus on their contribution to disease progression, tissue pathology, and immune evasion strategies. Keywords: Streptococcus pyogenes; group A streptococcus; Staphylococcus aureus; skin infections; necrotizing soft tissue infections; pore-forming toxins; superantigens; immunomodulatory proteases; immune responses Key Contribution: Group A streptococcal and Staphylococcus aureus toxins manipulate host physiological and immunological responses to promote disease severity and progression. 1. Introduction Necrotizing soft tissue infections (NSTIs) are rare and represent a more severe rapidly progressing form of soft tissue infections that account for significant morbidity and mortality [1].
    [Show full text]
  • Transferrin Variants and Conjugates
    (19) TZZ Z ¥ T (11) EP 2 604 623 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 19.06.2013 Bulletin 2013/25 C07K 14/79 (2006.01) A61K 47/48 (2006.01) A61K 38/40 (2006.01) C12N 5/10 (2006.01) (21) Application number: 12189421.6 (22) Date of filing: 08.08.2008 (84) Designated Contracting States: • Friis, Esben Peter AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 2730 Herlev (DK) HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT • Hay, Joanna RO SE SI SK TR Nottingham, Nottinghamshire NG2 6QW (GB) • Finnis, Christopher John Arthur (30) Priority: 08.08.2007 EP 07114012 Nottingham, Nottinghamshire NG7 1JB (GB) 02.04.2008 EP 08153938 (74) Representative: Williams, Rachel Clare (62) Document number(s) of the earlier application(s) in Novozymes Biopharma UK Limited accordance with Art. 76 EPC: Castle Court 08787067.1 / 2 201 036 59 Castle Boulevard Nottingham (71) Applicant: Novozymes Biopharma DK A/S Nottinghamshire NG7 1FD (GB) 2880 Bagsvaerd (DK) Remarks: (72) Inventors: This application was filed on 22-10-2012 as a • Sleep, Darrell divisional application to the application mentioned Nottingham, Nottinghamshire NG2 5DS (GB) under INID code 62. (54) Transferrin variants and conjugates (57) Based on the three-dimensional structure of "thiotransferrin"). The variant polypeptide may be conju- transferrin, the inventors have designed variant polypep- gated through the sulphur atom of the Cysteine residue tides (muteins) which have one or more Cysteine resi- to a bioactive compound. dues with a free thiol group (hereinafter referred to as EP 2 604 623 A2 Printed by Jouve, 75001 PARIS (FR) EP 2 604 623 A2 Description Reference to sequence listing 5 [0001] This application contains a Sequence Listing in computer readable form.
    [Show full text]
  • ACE2 Interaction Networks in COVID-19: a Physiological Framework for Prediction of Outcome in Patients with Cardiovascular Risk Factors
    Journal of Clinical Medicine Article ACE2 Interaction Networks in COVID-19: A Physiological Framework for Prediction of Outcome in Patients with Cardiovascular Risk Factors Zofia Wicik 1,2 , Ceren Eyileten 2, Daniel Jakubik 2,Sérgio N. Simões 3, David C. Martins Jr. 1, Rodrigo Pavão 1, Jolanta M. Siller-Matula 2,4,* and Marek Postula 2 1 Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Santo Andre 09606-045, Brazil; zofiawicik@gmail.com (Z.W.); david.martins@ufabc.edu.br (D.C.M.J.); rpavao@gmail.com (R.P.) 2 Department of Experimental and Clinical Pharmacology, Medical University of Warsaw, Center for Preclinical Research and Technology CEPT, 02-091 Warsaw, Poland; ceyileten@wum.edu.pl (C.E.); djakubik@wum.edu.pl (D.J.); mpostula@wum.edu.pl (M.P.) 3 Federal Institute of Education, Science and Technology of Espírito Santo, Serra, Espírito Santo 29056-264, Brazil; sergio@ifes.edu.br 4 Department of Internal Medicine II, Division of Cardiology, Medical University of Vienna, 1090 Vienna, Austria * Correspondence: jolanta.siller-matula@meduniwien.ac.at; Tel.: +43-1-40400-46140; Fax: +43-1-40400-42160 Received: 9 October 2020; Accepted: 17 November 2020; Published: 21 November 2020 Abstract: Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (coronavirus disease 2019; COVID-19) is associated with adverse outcomes in patients with cardiovascular disease (CVD). The aim of the study was to characterize the interaction between SARS-CoV-2 and Angiotensin-Converting Enzyme 2 (ACE2) functional networks with a focus on CVD. Methods: Using the network medicine approach and publicly available datasets, we investigated ACE2 tissue expression and described ACE2 interaction networks that could be affected by SARS-CoV-2 infection in the heart, lungs and nervous system.
    [Show full text]
  • The Role of the Mtor Pathway in Developmental Reprogramming Of
    THE ROLE OF THE MTOR PATHWAY IN DEVELOPMENTAL REPROGRAMMING OF HEPATIC LIPID METABOLISM AND THE HEPATIC TRANSCRIPTOME AFTER EXPOSURE TO 2,2',4,4'- TETRABROMODIPHENYL ETHER (BDE-47) An Honors Thesis Presented By JOSEPH PAUL MCGAUNN Approved as to style and content by: ________________________________________________________** Alexander Suvorov 05/18/20 10:40 ** Chair ________________________________________________________** Laura V Danai 05/18/20 10:51 ** Committee Member ________________________________________________________** Scott C Garman 05/18/20 10:57 ** Honors Program Director ABSTRACT An emerging hypothesis links the epidemic of metabolic diseases, such as non-alcoholic fatty liver disease (NAFLD) and diabetes with chemical exposures during development. Evidence from our lab and others suggests that developmental exposure to environmentally prevalent flame-retardant BDE47 may permanently reprogram hepatic lipid metabolism, resulting in an NAFLD-like phenotype. Additionally, we have demonstrated that BDE-47 alters the activity of both mTOR complexes (mTORC1 and 2) in hepatocytes. The mTOR pathway integrates environmental information from different signaling pathways, and regulates key cellular functions such as lipid metabolism, innate immunity, and ribosome biogenesis. Thus, we hypothesized that the developmental effects of BDE-47 on liver lipid metabolism are mTOR-dependent. To assess this, we generated mice with liver-specific deletions of mTORC1 or mTORC2 and exposed these mice and their respective controls perinatally to
    [Show full text]
  • Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis
    Role of Vegetation-Associated Protease Activity in Valve Destruction in Human Infective Endocarditis Ghada Al-Salih1, Nawwar Al-Attar2,3, Sandrine Delbosc1, Liliane Louedec1, Elisabeth Corvazier1, Ste´phane Loyau1, Jean-Baptiste Michel1,3, Dominique Pidard1,4, Xavier Duval5, Olivier Meilhac1,3,6* 1 INSERM U698, Paris, France, 2 Cardiovascular Surgery Department, Bichat Hospital, AP-HP, Paris, France, 3 University Paris Diderot, Sorbonne Paris Cite´, Paris, France, 4 Institut National des Sciences du Vivant, Centre National de la Recherche Scientifique, Paris, France, 5 INSERM CIC 007, Paris, France, 6 Bichat Stroke Centre, AP-HP, Paris, France Abstract Aims: Infective endocarditis (IE) is characterized by septic thrombi (vegetations) attached on heart valves, consisting of microbial colonization of the valvular endocardium, that may eventually lead to congestive heart failure or stroke subsequent to systemic embolism. We hypothesized that host defense activation may be directly involved in tissue proteolytic aggression, in addition to pathogenic effects of bacterial colonization. Methods and Results: IE valve samples collected during surgery (n = 39) were dissected macroscopically by separating vegetations (VG) and the surrounding damaged part of the valve from the adjacent, apparently normal (N) valvular tissue. Corresponding conditioned media were prepared separately by incubation in culture medium. Histological analysis showed an accumulation of platelets and polymorphonuclear neutrophils (PMNs) at the interface between the VG and the underlying tissue. Apoptotic cells (PMNs and valvular cells) were abundantly detected in this area. Plasminogen activators (PA), including urokinase (uPA) and tissue (tPA) types were also associated with the VG. Secreted matrix metalloproteinase (MMP) 9 was also increased in VG, as was leukocyte elastase and myeloperoxidase (MPO).
    [Show full text]
  • Subtiligase-Catalyzed Peptide Ligation Amy M
    Review Cite This: Chem. Rev. 2020, 120, 3127−3160 pubs.acs.org/CR Subtiligase-Catalyzed Peptide Ligation Amy M. Weeks*,† and James A. Wells*,†,‡ † Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94143, United States ‡ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143, United States ABSTRACT: Subtiligase-catalyzed peptide ligation is a powerful approach for site- specific protein bioconjugation, synthesis and semisynthesis of proteins and peptides, and chemoproteomic analysis of cellular N termini. Here, we provide a comprehensive review of the subtiligase technology, including its development, applications, and impacts on protein science. We highlight key advantages and limitations of the tool and compare it to other peptide ligase enzymes. Finally, we provide a perspective on future applications and challenges and how they may be addressed. CONTENTS 6.1. Subtiligase-Catalyzed Thioester and Thioa- cid Synthesis for Peptide and Protein 1. Introduction 3128 Bioconjugation 3138 2. Using Proteases in Reverse for Peptide Bond 6.2. Peptide Segment Condensation 3139 Formation 3129 6.3. Peptide Cyclization 3140 2.1. Protease-Catalyzed Peptide Bond Synthesis 6.4. Total Protein Synthesis 3140 under Thermodynamic Control 3129 7. Application of Subtiligase for Site-Specific 2.2. Protease-Catalyzed Peptide Bond Synthesis Protein Bioconjugation 3141 under Kinetic Control 3129 7.1. Sequence and Structural Requirements for 3. Protein Engineering of Subtilisin for Improved N-Terminal Modification by Subtiligase 3142 Peptide Bond Synthesis 3129 7.1.1. Characterization of Sequence and 3.1. Mutation of the Catalytic Serine to Cysteine 3129 Structural Requirements 3142 3.2.
    [Show full text]
  • Differential Proteomic Analysis of the Pancreas of Diabetic Db/Db Mice Reveals the Proteins Involved in the Development of Complications of Diabetes Mellitus
    Int. J. Mol. Sci. 2014, 15, 9579-9593; doi:10.3390/ijms15069579 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article Differential Proteomic Analysis of the Pancreas of Diabetic db/db Mice Reveals the Proteins Involved in the Development of Complications of Diabetes Mellitus Victoriano Pérez-Vázquez 1,*, Juan M. Guzmán-Flores 1, Daniela Mares-Álvarez 1, Magdalena Hernández-Ortiz 2, Maciste H. Macías-Cervantes 1, Joel Ramírez-Emiliano 1 and Sergio Encarnación-Guevara 2 1 Depto. de Ciencias Médicas, División de Ciencias de la Salud, Campus León, Universidad de Guanajuato, León, Guanajuato 37320, Mexico; E-Mails: manuguz@yahoo.com.mx (J.M.G.-F.); danymares7@hotmail.com (D.M.-A.); macistehabacuc@yahoo.com.mx (M.H.M.-C.); joelre@ugto.mx (J.R.-E.) 2 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, Mexico; E-Mails: magdini@gmail.com (M.H.-O.); encarnac@ccg.unam.mx (S.E.-G.) * Author to whom correspondence should be addressed; E-Mail: vpvazquez@ugto.mx; Tel.: +52-477-7143-812; Fax: +52-477-7167-623. Received: 4 April 2014; in revised form: 14 May 2014 / Accepted: 19 May 2014 / Published: 30 May 2014 Abstract: Type 2 diabetes mellitus is characterized by hyperglycemia and insulin-resistance. Diabetes results from pancreatic inability to secrete the insulin needed to overcome this resistance. We analyzed the protein profile from the pancreas of ten-week old diabetic db/db and wild type mice through proteomics. Pancreatic proteins were separated in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and significant changes in db/db mice respect to wild type mice were observed in 27 proteins.
    [Show full text]
  • Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases
    International Journal of Molecular Sciences Review Effects of Glycosylation on the Enzymatic Activity and Mechanisms of Proteases Peter Goettig Structural Biology Group, Faculty of Molecular Biology, University of Salzburg, Billrothstrasse 11, 5020 Salzburg, Austria; peter.goettig@sbg.ac.at; Tel.: +43-662-8044-7283; Fax: +43-662-8044-7209 Academic Editor: Cheorl-Ho Kim Received: 30 July 2016; Accepted: 10 November 2016; Published: 25 November 2016 Abstract: Posttranslational modifications are an important feature of most proteases in higher organisms, such as the conversion of inactive zymogens into active proteases. To date, little information is available on the role of glycosylation and functional implications for secreted proteases. Besides a stabilizing effect and protection against proteolysis, several proteases show a significant influence of glycosylation on the catalytic activity. Glycans can alter the substrate recognition, the specificity and binding affinity, as well as the turnover rates. However, there is currently no known general pattern, since glycosylation can have both stimulating and inhibiting effects on activity. Thus, a comparative analysis of individual cases with sufficient enzyme kinetic and structural data is a first approach to describe mechanistic principles that govern the effects of glycosylation on the function of proteases. The understanding of glycan functions becomes highly significant in proteomic and glycomic studies, which demonstrated that cancer-associated proteases, such as kallikrein-related peptidase 3, exhibit strongly altered glycosylation patterns in pathological cases. Such findings can contribute to a variety of future biomedical applications. Keywords: secreted protease; sequon; N-glycosylation; O-glycosylation; core glycan; enzyme kinetics; substrate recognition; flexible loops; Michaelis constant; turnover number 1.
    [Show full text]
  • Human Neurophysin II Antibody
    Human Neurophysin II Antibody Monoclonal Mouse IgG2B Clone # 62865 Catalog Number: MAB009 DESCRIPTION Species Reactivity Human Specificity Detects human Neurophysin II in direct ELISAs. Source Monoclonal Mouse IgG2B Clone # 62865 Purification Protein A or G purified from ascites Immunogen Synthetic peptide containing human Neurophysin II Accession # P01186 Formulation Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. See Certificate of Analysis for details. *Small pack size (-SP) is supplied either lyophilized or as a 0.2 μm filtered solution in PBS. APPLICATIONS Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website. Recommended Sample Concentration Immunohistochemistry 5-25 µg/mL See Below DATA Immunohistochemistry Neurophysin II in Human Brain Hippocampus Tissue. Neurophysin II was detected in immersion fixed paraffin- embedded sections of human brain hippocampus tissue using Mouse Anti- Human Neurophysin II Monoclonal Antibody (Catalog # MAB009) at 5 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Mouse IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC001). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent- Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to neurons. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents. PREPARATION AND STORAGE Reconstitution Reconstitute at 0.5 mg/mL in sterile PBS. Shipping The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (-SP) is shipped with polar packs.
    [Show full text]
  • ONLINE SUPPLEMENTARY TABLE Table 2. Differentially Expressed
    ONLINE SUPPLEMENTARY TABLE Table 2. Differentially Expressed Probe Sets in Livers of GK Rats. A. Immune/Inflammatory (67 probe sets, 63 genes) Age Strain Probe ID Gene Name Symbol Accession Gene Function 5 WKY 1398390_at small inducible cytokine B13 precursor Cxcl13 AA892854 chemokine activity; lymph node development 5 WKY 1389581_at interleukin 33 Il33 BF390510 cytokine activity 5 WKY *1373970_at interleukin 33 Il33 AI716248 cytokine activity 5 WKY 1369171_at macrophage stimulating 1 (hepatocyte growth factor-like) Mst1; E2F2 NM_024352 serine-throenine kinase; tumor suppression 5 WKY 1388071_x_at major histocompatability antigen Mhc M24024 antigen processing and presentation 5 WKY 1385465_at sialic acid binding Ig-like lectin 5 Siglec5 BG379188 sialic acid-recognizing receptor 5 WKY 1393108_at major histocompatability antigen Mhc BM387813 antigen processing and presentation 5 WKY 1388202_at major histocompatability antigen Mhc BI395698 antigen processing and presentation 5 WKY 1371171_at major histocompatability antigen Mhc M10094 antigen processing and presentation 5 WKY 1370382_at major histocompatability antigen Mhc BI279526 antigen processing and presentation 5 WKY 1371033_at major histocompatability antigen Mhc AI715202 antigen processing and presentation 5 WKY 1383991_at leucine rich repeat containing 8 family, member E Lrrc8e BE096426 proliferation and activation of lymphocytes and monocytes. 5 WKY 1383046_at complement component factor H Cfh; Fh AA957258 regulation of complement cascade 4 WKY 1369522_a_at CD244 natural killer
    [Show full text]
  • Cysteine Proteinases of Microorganisms and Viruses
    ISSN 00062979, Biochemistry (Moscow), 2008, Vol. 73, No. 1, pp. 113. © Pleiades Publishing, Ltd., 2008. Original Russian Text © G. N. Rudenskaya, D. V. Pupov, 2008, published in Biokhimiya, 2008, Vol. 73, No. 1, pp. 317. REVIEW Cysteine Proteinases of Microorganisms and Viruses G. N. Rudenskaya1* and D. V. Pupov2 1Faculty of Chemistry and 2Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 9393181; Email: laboratoriahps@hotmail.com Received May 7, 2007 Revision received July 18, 2007 Abstract—This review considers properties of secreted cysteine proteinases of protozoa, bacteria, and viruses and presents information on the contemporary taxonomy of cysteine proteinases. Literature data on the structure and physicochemical and enzymatic properties of these enzymes are reviewed. High interest in cysteine proteinases is explained by the discovery of these enzymes mostly in pathogenic organisms. The role of the proteinases in pathogenesis of several severe diseases of human and animals is discussed. DOI: 10.1134/S000629790801001X Key words: cysteine proteinases, properties, protozoa, bacteria, viruses Classification and Catalytic Mechanism papain and related peptidases showed that the catalytic of Cysteine Proteinases residues are arranged in the following order in the polypeptide chain: Cys, His, and Asn. Also, a glutamine Cysteine proteinases are peptidyl hydrolases in residue preceding the catalytic cysteine is also important which the role of the nucleophilic group of the active site for catalysis. This residue is probably involved in the for is performed by the sulfhydryl group of a cysteine residue. mation of the oxyanion cavity of the enzyme. The cat Cysteine proteinases were first discovered and investigat alytic cysteine residue is usually followed by a residue of ed in tropic plants.
    [Show full text]