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Affinity maturation

Oliver Bannard, Jason G Cyster
The seminal discovery by Eisen that antibodies undergo improvements in antigen-binding affinity over the course of an immune response led to a long running search for the underlying mechanism. Germinal centers in lymphoid organs are now recognized to be critically involved in this phenomenon, known as antibody affinity maturation. As well as improving in affinity for specific epitopes, some antibody responses maintain or even increase their breadth of antigen-recognition over time. This has led to another intense line of research aimed at understanding how broadly neutralizing anti-pathogen responses are generated...
January 12, 2017: Current Opinion in Immunology
Arnone Nithichanon, Louise J Gourlay, Gregory J Bancroft, Manabu Ato, Yoshimasa Takahashi, Ganjana Lertmemongkolchai
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and Northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped prevent disease. Here, we used a humanized mouse model (hu-PBL-SCID), reconstituted with human peripheral blood mononuclear cells (PBMCs) from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N-PilO2) for boosting both T- and B-cell immune responses...
January 9, 2017: Immunology
Susha S Kutteyil, Bhakti R Pathak, Smita D Mahale
Follicle-Stimulating Hormone (FSH) is essential for mammalian folliculogenesis and spermatogenesis. Common marmoset (Callithrix jacchus) is a New World primate which exhibits an unusual FSH profile across the ovarian cycle with a mid-follicular FSH peak that is not observed in Catarrhini primates like humans. Since transcription of FSH β-subunit gene (FSHβ) is a rate-limiting step in the production of mature FSH, this study aimed to investigate the regulation of marmoset FSHβ gene expression in comparison to human...
January 3, 2017: General and Comparative Endocrinology
Anirban Ghosh, Nibedita Pradhan, Swapna Bera, Aritreyee Datta, Janarthanan Krishnamoorthy, Nikhil R Jana, Anirban Bhunia
A designed non-toxic, non-hemolytic 11-residue peptide, NF11 (NAVRWSLMRPF) not only inhibits the aggregation of Amyloid beta (Aβ40) protein but also disaggregates the pre-formed oligomers and mature Aβ fibrils thereby reducing associated-toxicity. NMR experiments provide evidence of NF11's ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aβ40. NF11 has micro molar binding affinity towards both monomeric and aggregated species for efficient clearance of toxic aggregates...
January 6, 2017: ACS Chemical Neuroscience
Miguel A Soler, Alex Rodriguez, Anna Russo, Abimbola Feyisara Adedeji, Cedrix J Dongmo Foumthuim, Cristina Cantarutti, Elena Ambrosetti, Loredana Casalis, Alessandra Corazza, Giacinto Scoles, Daniela Marasco, Alessandro Laio, Sara Fortuna
The oriented immobilization of proteins, key for the development of novel responsive biomaterials, relies on the availability of effective probes. These are generally provided by standard approaches based on in vivo maturation and in vitro selection of antibodies and/or aptamers. These techniques can suffer technical problems when a non-immunogenic epitope needs to be targeted. Here we propose a strategy to circumvent this issue by in silico design. In our method molecular binders, in the form of cyclic peptides, are computationally evolved by stochastically exploring their sequence and structure space to identify high-affinity peptides for a chosen epitope of a target globular protein: here a solvent-exposed site of β2-microglobulin (β2m)...
January 6, 2017: Physical Chemistry Chemical Physics: PCCP
Patrick Koenig, Chingwei V Lee, Benjamin T Walters, Vasantharajan Janakiraman, Jeremy Stinson, Thomas W Patapoff, Germaine Fuh
Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment...
January 5, 2017: Proceedings of the National Academy of Sciences of the United States of America
Oscar D Villarreal, Lili Yu, Roberto A Rodriguez, Liao Y Chen
Computing the ligand-protein binding affinity (or the Gibbs free energy) with chemical accuracy has long been a challenge for which many methods/approaches have been developed and refined with various successful applications. False positives and, even more harmful, false negatives have been and still are a common occurrence in practical applications. Inevitable in all approaches are the errors in the force field parameters we obtain from quantum mechanical computation and/or empirical fittings for the intra- and inter-molecular interactions...
December 26, 2016: Biochemical and Biophysical Research Communications
Forough Torabi, Adel Binduraihem, David Miller
Mature spermatozoa bind hyaluronic acid in the extracellular matrix via hyaladherins. Immature spermatozoa may be unable to interact because they do not express the appropriate hyaladherins on their surface. Fresh human semen samples were fractionated using differential density gradient centrifugation (DDGC) and the ability of these fractions to bind hyaluronic acid was evaluated. The presence of sperm hyaladherins was also assessed. CD44 was located mainly on the hyaluronic and equatorial segment and became more restricted to the equatorial segment in capacitated spermatozoa...
December 15, 2016: Reproductive Biomedicine Online
Iuri G Baseia, Bianca D B Silva, Noemia K Ishikawa, João V C Soares, Isadora F França, Shuji Ushijima, Nitaro Maekawa, María P Martín
The Amazon Forest is a hotspot of biodiversity harboring an unknown number of undescribed taxa. Inventory studies are urgent, mainly in the areas most endangered by human activities such as extensive dam construction, where species could be in risk of extinction before being described and named. In 2015, intensive studies performed in a few locations in the Brazilian Amazon rainforest revealed three new species of the genus Scleroderma: S. anomalosporum, S. camassuense and S. duckei. The two first species were located in one of the many areas flooded by construction of hydroelectric dams throughout the Amazon; and the third in the Reserva Florestal Adolpho Ducke, a protected reverse by the INPA...
2016: PloS One
Timo Heinrich, Hans-Peter Buchstaller, Bertram Cezanne, Felix Rohdich, Jörg Bomke, Manja Friese-Hamim, Mireille Krier, Thorsten Knöchel, Djordje Musil, Birgitta Leuthner, Frank Zenke
The natural product fumagillin 1 and derivatives like TNP-470 2 or beloranib 3 bind to methionine aminopeptidase 2 (MetAP-2) irreversibly. This enzyme is critical for protein maturation and plays a key role in angiogenesis. In this paper we describe the synthesis, MetAP-2 binding affinity and structural analysis of reversible MetAP-2 inhibitors. Optimization of enzymatic activity of screening hit 10 (IC50: 1μM) led to the most potent compound 27 (IC50: 0.038μM), with a concomitant improvement in LLE from 2...
December 8, 2016: Bioorganic & Medicinal Chemistry Letters
Sungwon Lim, Bob Chen, Mihalis S Kariolis, Ivan K Dimov, Thomas M Baer, Jennifer R Cochran
Affinity maturation of protein-protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity...
December 20, 2016: ACS Chemical Biology
Behnaz Heydarchi, Rob J Center, Jonathan Bebbington, Jack Cuthbertson, Christopher Gonelli, Georges Khoury, Charlene Mackenzie, Marit Lichtfuss, Grant Rawlin, Brian Muller, Damian Purcell
We isolated HIV-1 Envelope (Env)-specific memory B cells from a cow that had developed high titre polyclonal immunoglobulin G (IgG) with broad neutralizing activity after a long duration vaccination with HIV-1AD8 Env gp140 trimers. We cloned the bovine IgG matched heavy (H) and light (L) chain variable (V) genes from these memory B cells and constructed IgG monoclonal antibodies (mAbs) with either a human constant (C)-region/bovine V-region chimeric or fully bovine C and V regions. Among 42 selected Ig+ memory B cells, two mAbs (6A and 8C) showed high affinity binding to gp140 Env...
December 20, 2016: MAbs
Ricky J Milne, Jai M Perroux, Anne L Rae, Anke Reinders, John M Ward, Christina E Offler, John William Patrick, Christopher P L Grof
How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high sucrose concentrations. To this end Sorghum bicolor SUTs, SbSUT1 and SbSUT5 were characterized, by determining their transport properties heterologously expressed in yeast or Xenopus oocytes, and their in planta cellular- and sub-cellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for sucrose and high sucrose affinities in Xenopus oocytes at pH 5...
December 16, 2016: Plant Physiology
Gunderao Hanumantrao Kathwate, S Mohan Karuppayil
Tramadol is a synthetic, centrally acting low-affinity agonist of μ-opioid receptors in humans. It is used as an analgesic and is shown to have local anesthetic action. In this study, we have tried to explore its anti-Candida potential. Minimum inhibitory concentration (MIC50) and minimum fungicidal concentration (MFC) values were established. MIC50 ranged from 2 to 4 mg/mL, whereas MFC was recorded at 8 mg/mL. Also, the effect of tramadol on germ tube formation, adhesion, and biofilms in Candida albicans was studied...
December 2016: Assay and Drug Development Technologies
Cátia A Bonito, Joana Nunes, João Leandro, Filipa Louro, Paula Leandro, Fátima V Ventura, Rita C Guedes
Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid β-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme's activity. The most frequent mutation in MCADD results from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein)...
December 27, 2016: Biochemistry
Martijn van Rosmalen, Brian M G Janssen, Nathalie M Hendrikse, Ardjan J van der Linden, Pascal A Pieters, Dave Wanders, Tom F A de Greef, Maarten Merkx
Meditopes are cyclic peptides that bind in a specific pocket in the antigen-binding fragment (Fab) of a therapeutic antibody such as cetuximab. Provided their moderate affinity can be enhanced, meditope peptides could be used as a specific non-covalent and paratope-independent handles in targeted drug delivery, molecular imaging and therapeutic drug monitoring. Here we show that the affinity of a recently reported meditope for cetuximab can be substantially enhanced using a combination of yeast display and deep mutational scanning...
December 14, 2016: Journal of Biological Chemistry
Sama Adnan, R Keith Reeves, Jacqueline Gillis, Fay E Wong, Yi Yu, Jeremy V Camp, Qingsheng Li, Michelle Connole, Yuan Li, Michael Piatak, Jeffrey D Lifson, Wenjun Li, Brandon F Keele, Pamela A Kozlowski, Ronald C Desrosiers, Ashley T Haase, R Paul Johnson
Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals...
December 2016: PLoS Pathogens
Sanket J Mishra, Suman Ghosh, Andrew R Stothert, Chad A Dickey, Brian S J Blagg
Glucose regulated protein 94 kDa, Grp94, is the endoplasmic reticulum (ER) localized isoform of heat shock protein 90 (Hsp90) that is responsible for the trafficking and maturation of toll-like receptors, immunoglobulins, and integrins. As a result, Grp94 has emerged as a therapeutic target to disrupt cellular communication, adhesion, and tumor proliferation, potentially with fewer side effects compared to pan-inhibitors of all Hsp90 isoforms. Although, the N-terminal ATP binding site is highly conserved among all four Hsp90 isoforms, recent cocrystal structures of Grp94 have revealed subtle differences between Grp94 and other Hsp90 isoforms that has been exploited for the development of Grp94-selective inhibitors...
December 13, 2016: ACS Chemical Biology
Klaus Lehmann-Horn, Sheng-Zhi Wang, Sharon A Sagan, Scott S Zamvil, H-Christian von Büdingen
Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing...
December 8, 2016: JCI Insight
Irina Kareva, Abdo Abou-Slaybi, Oliver Dodd, Olga Dashevsky, Giannoula Lakka Klement
Both normal wound healing and tumor angiogenesis are mitigated by the sequential, carefully orchestrated release of growth stimulators and inhibitors. These regulators are released from platelet clots formed at the sites of activated endothelium in a temporally and spatially controlled manner, and the order of their release depends on their affinity to glycosaminoglycans (GAG) such as heparan sulfate (HS) within the extracellular matrix, and platelet open canallicular system. The formation of vessel sprouts, triggered by angiogenesis regulating factors with lowest affinities for heparan sulfate (e...
2016: PloS One
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