28 results on '"Paola Lo Surdo"'
Search Results
2. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6
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Oretta Finco, Erika Bartolini, Federica Bianchi, Paola Lo Surdo, Manuele Martinelli, Francesca Buricchi, Matthew J. Bottomley, Roberta Cozzi, Daniele Veggi, Vega Masignani, Domenico Maione, and Laura Santini
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,medicine.drug_class ,Meningococcal Vaccines ,Neisseria meningitidis ,Crystallography, X-Ray ,Monoclonal antibody ,medicine.disease_cause ,Biochemistry ,Epitope ,law.invention ,Epitopes ,03 medical and health sciences ,0302 clinical medicine ,Bacterial Proteins ,Antibody Repertoire ,law ,vaccine ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Antigens, Bacterial ,biology ,Research ,Binding protein ,Antibodies, Monoclonal ,Genetic Variation ,human antibodies ,Virology ,Meningococcal Infections ,030104 developmental biology ,Immunization ,Complement Factor H ,biology.protein ,Recombinant DNA ,fHbp ,Antibody ,030217 neurology & neurosurgery ,Protein Binding ,Biotechnology - Abstract
The 4 component meningococcus B vaccine (4CMenB) vaccine is the first vaccine containing recombinant proteins licensed for the prevention of invasive meningococcal disease caused by meningococcal serogroup B strains. 4CMenB contains 3 main recombinant proteins, including the Neisseria meningitidis factor H binding protein (fHbp), a lipoprotein able to bind the human factor H. To date, over 1000 aa sequences of fHbp have been identified, and they can be divided into variant groups 1, 2, and 3, which are usually not crossprotective. Nevertheless, previous characterizations of a small set (n = 10) of mAbs generated in humans after 4CMenB immunization revealed 2 human Fabs (huFabs) (1A12, 1G3) with some crossreactivity for variants 1, 2, and 3. This unexpected result prompted us to examine a much larger set of human mAbs (n = 110), with the aim of better understanding the extent and nature of crossreactive anti-fHbp antibodies. In this study, we report an analysis of the human antibody response to fHbp, by the characterization of 110 huFabs collected from 3 adult vaccinees during a 6-mo study. Although the 4CMenB vaccine contains fHbp variant 1, 13 huFabs were also found to be crossreactive with variants 2 and 3. The crystal structure of the crossreactive huFab 1E6 in complex with fHbp variant 3 was determined, revealing a novel, highly conserved epitope distinct from the epitopes recognized by 1A12 or 1G3. Further, functional characterization shows that human mAb 1E6 is able to elicit rabbit, but not human, complement-mediated bactericidal activity against meningococci displaying fHbp from any of the 3 different variant groups. This functional and structural information about the human antibody response upon 4CMenB immunization contributes to further unraveling the immunogenic properties of fHbp. Knowledge gained about the epitope profile recognized by the human antibody repertoire could guide future vaccine design.—Bianchi, F., Veggi, D., Santini, L., Buricchi, F., Bartolini, E., Lo Surdo, P., Martinelli, M., Finco, O., Masignani, V., Bottomley, M. J., Maione, D., Cozzi, R. Cocrystal structure of meningococcal factor H binding protein variant 3 reveals a new crossprotective epitope recognized by human mAb 1E6.
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- 2019
3. Investigating the Role of Antigen Orientation on the Immune Response Elicited by Neisseria meningitidis Factor H Binding Protein on GMMA
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Renzo Alfini, Brunella Brunelli, Erika Bartolini, Martina Carducci, Enrico Luzzi, Francesca Ferlicca, Scilla Buccato, Barbara Galli, Paola Lo Surdo, Maria Scarselli, Giacomo Romagnoli, Elena Cartocci, Domenico Maione, Silvana Savino, Francesca Necchi, Isabel Delany, and Francesca Micoli
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Pharmacology ,Infectious Diseases ,Drug Discovery ,Immunology ,Pharmacology (medical) ,GMMA ,outer membrane vesicles ,Neisseria meningitidis ,conjugation - Abstract
GMMA are outer membrane vesicles (OMVs) released from Gram-negative bacteria genetically modified to enhance OMVs formation that have been shown to be optimal systems to enhance immunogenicity of protein antigens. Here, we selected Neisseria meningitidis factor H binding protein (fHbp) and used the conjugation chemistry as a tool to alter antigen orientation on GMMA. Indeed, fHbp was randomly linked to GMMA or selectively attached via the N-terminus to mimic native presentation of the protein on the bacterial surface. Interestingly, protein and peptide array analyses confirmed that antibodies induced by the selective and the random conjugates showed a pattern very similar to fHbp natively expressed on bacterial surfaces or to the recombinant protein mixed with GMMA, respectively. However, the two conjugates elicited antibodies with similar serum bactericidal activity against meningococcal strains, superior to the protein alone or physically mixed with GMMA. Presentation of fHbp on GMMA strongly enhances the functional immune response elicited by the protein but its orientation on the bacterial surface does not have an impact. This study demonstrates the flexibility of the GMMA platform as a display and delivery system for enhancing antigen immunogenicity and further supports the use of such promising technology for the development of effective vaccines.
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- 2022
4. Structural characterization of a cross-protective natural chimera of factor H binding protein from meningococcal serogroup B strain NL096
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Daniele Veggi, Enrico Malito, Paola Lo Surdo, Werner Pansegrau, Valentina Rippa, Newton Wahome, Silvana Savino, Vega Masignani, Mariagrazia Pizza, and Matthew J. Bottomley
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Structural Biology ,Genetics ,Biophysics ,Biochemistry ,Computer Science Applications ,Biotechnology - Abstract
Invasive meningococcal disease can cause fatal sepsis and meningitis and is a global health threat. Factor H binding protein (fHbp) is a protective antigen included in the two currently available vaccines against serogroup B meningococcus (MenB). FHbp is a remarkably variable surface-exposed meningococcal virulence factor with over 1300 different amino acid sequences identified so far. Based on this variability, fHbp has been classified into three variants, two subfamilies or nine modular groups, with low degrees of cross-protective activity. Here, we report the crystal structure of a natural fHbp cross-variant chimera, named variant1-2,3.x expressed by the MenB clinical isolate NL096, at 1.2 Å resolution, the highest resolution of any fHbp structure reported to date. We combined biochemical, site-directed mutagenesis and computational biophysics studies to deeply characterize this rare chimera. We determined the structure to be composed of two adjacent domains deriving from the three variants and determined the molecular basis of its stability, ability to bind Factor H and to adopt the canonical three-dimensional fHbp structure. These studies guided the design of loss-of-function mutations with potential for even greater immunogenicity. Moreover, this study represents a further step in the understanding of the fHbp biological and immunological evolution in nature. The chimeric variant1-2,3.x fHbp protein emerges as an intriguing cross-protective immunogen and suggests that identification of such naturally occurring hybrid proteins may result in stable and cross-protective immunogens when seeking to design and develop vaccines against highly variable pathogens.
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- 2021
5. Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen
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Marcin Krzysztof Bugno, Minggang Cui, Paola Lo Surdo, Yujian Zhang, Jennifer Naginskaya, Ou Ma, Avinash Chinthala, Mark R. Wilson, Zongming Fu, Nilesh Vaidya, Xiangming Li, Li Jing, Matthew Sanders, Richard Sisson, Ryan Yamagata, Jishna Ganguly, and Zihao Wang
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Cell growth ,Computer science ,Chinese hamster ovary cell ,Clone (cell biology) ,Cytomegalovirus ,Computational biology ,CHO Cells ,Quality by Design ,High-Throughput Screening Assays ,Biopharmaceutical ,Cricetulus ,Cell culture ,Batch Cell Culture Techniques ,Vaccines, Subunit ,Animals ,Critical quality attributes ,Antigens, Viral ,Selection (genetic algorithm) ,Cells, Cultured ,Biotechnology - Abstract
Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200-1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.
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- 2019
6. Crystal structure of FhuD at 1.6 Å resolution: a ferrichrome-binding protein from the animal and human pathogenStaphylococcus pseudintermedius
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Francesca Abate, Paola Lo Surdo, Domenico Maione, Matthew J. Bottomley, Roberta Cozzi, Martina Maritan, and Enrico Malito
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Models, Molecular ,0301 basic medicine ,Staphylococcus pseudintermedius ,Staphylococcus ,030106 microbiology ,Biophysics ,Siderophores ,Plasma protein binding ,Crystallography, X-Ray ,medicine.disease_cause ,Biochemistry ,Research Communications ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Structural Biology ,Genetics ,medicine ,Molecular replacement ,Amino Acid Sequence ,Peptide sequence ,Ferrichrome ,biology ,Protein Stability ,Membrane transport protein ,Binding protein ,Membrane Transport Proteins ,Condensed Matter Physics ,biology.organism_classification ,030104 developmental biology ,chemistry ,Structural Homology, Protein ,biology.protein ,Carrier Proteins ,Crystallization ,Protein Binding - Abstract
Staphylococcus pseudintermediusis a leading cause of disease in dogs, and zoonosis causes human infections. Methicillin-resistantS. pseudintermediusstrains are emerging, resembling the global health threat ofS. aureus. Therefore, it is increasingly important to characterize potential targets for intervention againstS. pseudintermedius. Here, FhuD, anS. pseudintermediussurface lipoprotein implicated in iron uptake, was characterized. It was found that FhuD bound ferrichrome in an iron-dependent manner, which increased the thermostability of FhuD by >15°C. The crystal structure of ferrichrome-free FhuD was determinedviamolecular replacement at 1.6 Å resolution. FhuD exhibits the class III solute-binding protein (SBP) fold, with a ligand-binding cavity between the N- and C-terminal lobes, which is here occupied by a PEG molecule. The two lobes of FhuD were oriented in a closed conformation. These results provide the first detailed structural characterization of FhuD, a potential therapeutic target ofS. pseudintermedius.
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- 2016
7. Non-Enzymatic and Site-Specific Glycan Shedding: A Novel Protein Degradation Pathway Observed in a Stabilized Form of RSV Prefusion F Protein
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Zihao Wang, Matthew J. Bottomley, Sai Tian, Eric Yearley, Paola Lo Surdo, Li Jing, Ying Huang, Ankita Balsaraf, Jiang Qian, Sumana Chandramouli, and Nicolas Moniotte
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0301 basic medicine ,Models, Molecular ,Glycan ,Glycosylation ,Hot Temperature ,Mutagenesis (molecular biology technique) ,Respiratory Syncytial Virus Infections ,Protein degradation ,01 natural sciences ,Epitope ,Analytical Chemistry ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,Viral Proteins ,law ,Polysaccharides ,Humans ,Neutralizing antibody ,Deamidation ,biology ,010405 organic chemistry ,Chemistry ,Protein Stability ,0104 chemical sciences ,Respiratory Syncytial Viruses ,carbohydrates (lipids) ,030104 developmental biology ,Biochemistry ,Proteolysis ,biology.protein ,Recombinant DNA - Abstract
Stability is one of the critical attributes of a protein-based therapeutic or vaccine product, which is directly linked to product quality and efficacy. Elucidating protein degradation pathways is required to obtain thorough understanding of the product and ensure degradation products are properly monitored. We observed a unique protein degradation involving nonenzyme catalyzed loss of a complete N-linked glycan under stress condition from an engineered respiratory syncytial virus (RSV) prefusion F protein (RSVPreF3). Investigations involving mass spectrometry, molecular modeling, and mutagenesis revealed that the glycan shedding was site-specific, dependent on structural elements, and required a glycine residue immediately following the site of glycosylation. The glycan loss did not negatively affect the binding between the main immunogenic epitope Site O and the neutralizing antibody D25. Further study indicated that the glycan shedding followed a similar but different mechanism than that of conventional deamidation. Since glycosylation is an important attribute for many recombinant therapeutic proteins or vaccine antigens, the finding from this study suggests the need to monitor this new type of degradation, especially when glycosylation has an impact on efficacy or safety.
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- 2018
8. Molecular Engineering of Ghfp, the Gonococcal Orthologue of Neisseria meningitidis Factor H Binding Protein
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Beatrice Aricò, Laura Santini, Brunella Brunelli, Maria Antonietta Gentile, Paola Lo Surdo, Michele Pallaoro, Francesca Cantini, Daniele Veggi, Valentina Rippa, Mariagrazia Pizza, Maria Scarselli, Giulia Iannello, Lucia Banci, Emiliano Palmieri, Eva Grassi, and Francesca Ferlicca
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Microbiology (medical) ,Blood Bactericidal Activity ,Virulence Factors ,Recombinant Fusion Proteins ,Clinical Biochemistry ,Immunology ,Heterologous ,Computational biology ,Neisseria meningitidis ,Protein Engineering ,medicine.disease_cause ,Epitope ,Immunology and Allergy ,Mice ,Bacterial Proteins ,Antigen ,medicine ,Animals ,Antigens, Bacterial ,Vaccines, Synthetic ,Vaccines ,Sequence Homology, Amino Acid ,biology ,Rational design ,Antibodies, Bacterial ,Virology ,Neisseria gonorrhoeae ,3. Good health ,Bacterial vaccine ,Epitope mapping ,Bacterial Vaccines ,biology.protein ,Antibody - Abstract
Knowledge of the sequences and structures of proteins produced by microbial pathogens is continuously increasing. Besides offering the possibility of unraveling the mechanisms of pathogenesis at the molecular level, structural information provides new tools for vaccine development, such as the opportunity to improve viral and bacterial vaccine candidates by rational design. Structure-based rational design of antigens can optimize the epitope repertoire in terms of accessibility, stability, and variability. In the present study, we used epitope mapping information on the well-characterized antigen ofNeisseria meningitidisfactor H binding protein (fHbp) to engineer its gonococcal homologue, Ghfp. Meningococcal fHbp is typically classified in three distinct antigenic variants. We introduced epitopes of fHbp variant 1 onto the surface of Ghfp, which is naturally able to protect against meningococcal strains expressing fHbp of variants 2 and 3. Heterologous epitopes were successfully transplanted, as engineered Ghfp induced functional antibodies against all three fHbp variants. These results confirm that structural vaccinology represents a successful strategy for modulating immune responses, and it is a powerful tool for investigating the extension and localization of immunodominant epitopes.
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- 2015
9. Structure of the meningococcal vaccine antigen NadA and epitope mapping of a bactericidal antibody
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Elena Cartocci, Isabella Bertoldi, Marco Biancucci, Daniele Veggi, Alessia Liguori, Giulietta Maruggi, Mariagrazia Pizza, Giacomo Romagnoli, Ilaria Ferlenghi, Irene Vercellino, Paola Lo Surdo, Vega Masignani, Maria Scarselli, Rino Rappuoli, Laura Santini, Roberto Petracca, Enrico Malito, Matthew J. Bottomley, Silvana Savino, Sara Marchi, Glen Spraggon, Nathalie Norais, and Agnese Faleri
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Models, Molecular ,Spectrometry, Mass, Electrospray Ionization ,Molecular Sequence Data ,Meningococcal Vaccines ,Meningococcal vaccine ,Neisseria meningitidis, Serogroup B ,Crystallography, X-Ray ,medicine.disease_cause ,Microbiology ,Microscopy, Electron, Transmission ,Antigen ,medicine ,Protein Isoforms ,Trimeric autotransporter adhesin ,Amino Acid Sequence ,Adhesins, Bacterial ,Antigens, Bacterial ,Multidisciplinary ,Sequence Homology, Amino Acid ,Protein Stability ,business.industry ,Neisseria meningitidis ,Temperature ,Deuterium Exchange Measurement ,Biological Sciences ,Vaccine efficacy ,Antibodies, Bacterial ,Virology ,Protein Structure, Tertiary ,Bacterial adhesin ,Epitope mapping ,Binding Sites, Antibody ,Protein Multimerization ,business ,Epitope Mapping ,Conformational epitope - Abstract
Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.
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- 2014
10. Crystal structures of human Fabs targeting the Bexsero meningococcal vaccine antigen NHBA
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Paola Lo Surdo, Roberta Cozzi, Daniele Veggi, Enrico Malito, Matthew J. Bottomley, and Martina Maritan
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0301 basic medicine ,Models, Molecular ,Protein Conformation, alpha-Helical ,Gene Expression ,mAbs ,Neisseria meningitidis ,medicine.disease_cause ,Crystallography, X-Ray ,Bexsero ,Biochemistry ,Epitope ,Fragment antigen-binding ,Research Communications ,Structural Biology ,vaccine ,Cloning, Molecular ,Immunoglobulin Fab Fragments ,Condensed Matter Physics ,Antibodies, Bacterial ,Recombinant Proteins ,Neisseria ,monoclonal antibodies ,Bacterial Outer Membrane Proteins ,Protein Binding ,medicine.drug_class ,Genetic Vectors ,Fabs ,Biophysics ,Meningococcal Vaccines ,Meningococcal vaccine ,Computational biology ,Biology ,Meningitis, Meningococcal ,Monoclonal antibody ,03 medical and health sciences ,Antigen ,Genetics ,medicine ,Escherichia coli ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Antigens, Bacterial ,Binding Sites ,biology.organism_classification ,Virology ,Intrinsically Disordered Proteins ,Kinetics ,030104 developmental biology ,HEK293 Cells ,NHBA ,Protein Conformation, beta-Strand ,fragment antigen binding ,Carrier Proteins ,Peptides - Abstract
The crystal structures and functional characterization of two anti-NHBA human Fabs provide insight into the recognition by human antibodies of one of the main components of the first vaccine against serogroup B N. meningitidis (Bexsero)., Neisserial heparin-binding antigen (NHBA) is a surface-exposed lipoprotein from Neisseria meningitidis and is a component of the meningococcus B vaccine Bexsero. As part of a study to characterize the three-dimensional structure of NHBA and the molecular basis of the human immune response to Bexsero, the crystal structures of two fragment antigen-binding domains (Fabs) isolated from human monoclonal antibodies targeting NHBA were determined. Through a high-resolution analysis of the organization and the amino-acid composition of the CDRs, these structures provide broad insights into the NHBA epitopes recognized by the human immune system. As expected, these Fabs also show remarkable structural conservation, as shown by a structural comparison of 15 structures of apo Fab 10C3 which were obtained from crystals grown in different crystallization conditions and were solved while searching for a complex with a bound NHBA fragment or epitope peptide. This study also provides indirect evidence for the intrinsically disordered nature of two N-terminal regions of NHBA.
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- 2017
11. Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice
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Elena Fattori, Claus Bendtsen, Paola Lo Surdo, Ayesha Sitlani, Andrea Carfi, Yan G. Ni, Giuseppe Mesiti, Paolo Monaci, Manuela Cappelletti, Alessandra Calzetta, and Shilpa Pandit
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QD415-436 ,Biochemistry ,Antibodies ,Mice ,Endocrinology ,In vivo ,in vivo functional validation ,Animals ,Humans ,Research Articles ,animal model systems ,Gene knockdown ,biology ,PCSK9 ,Serine Endopeptidases ,Cholesterol, LDL ,Cell Biology ,active immunization ,Proprotein convertase ,Cell biology ,HEK293 Cells ,Liver ,Receptors, LDL ,LDL receptor ,biology.protein ,Kexin ,Female ,Immunization ,Proprotein Convertases ,Target protein ,Proprotein Convertase 9 ,cholesterol lowering treatments ,Antibody - Abstract
Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.
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- 2012
12. Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH
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Yan G. Ni, Brian K. Hubbard, Paola Lo Surdo, Matthew J. Bottomley, Ethan C. Settembre, Agostino Cirillo, Shilpa Pandit, Alessandra Calzetta, Ayesha Sitlani, and Andrea Carfi
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Models, Molecular ,Down-Regulation ,Plasma protein binding ,Biochemistry ,Epidermal growth factor ,Genetics ,Humans ,cardiovascular diseases ,Molecular Biology ,Cofactor binding ,Chemistry ,PCSK9 ,Scientific Reports ,Serine Endopeptidases ,Subtilisin ,food and beverages ,nutritional and metabolic diseases ,Hydrogen-Ion Concentration ,Surface Plasmon Resonance ,Proprotein convertase ,Protein Structure, Tertiary ,Receptors, LDL ,Proteolysis ,LDL receptor ,Kexin ,lipids (amino acids, peptides, and proteins) ,Proprotein Convertases ,Proprotein Convertase 9 ,Protein Binding - Abstract
The protein PCSK9 (proprotein convertase subtilisin/kexin type 9) is a key regulator of low-density lipoprotein receptor (LDLR) levels and cardiovascular health. We have determined the crystal structure of LDLR bound to PCSK9 at neutral pH. The structure shows LDLR in a new extended conformation. The PCSK9 C-terminal domain is solvent exposed, enabling cofactor binding, whereas the catalytic domain and prodomain interact with LDLR epidermal growth factor(A) and β-propeller domains, respectively. Thus, PCSK9 seems to hold LDLR in an extended conformation and to interfere with conformational rearrangements required for LDLR recycling.
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- 2011
13. Mass spectrometry study of PRL-3 phosphatase inactivation by disulfide bond formation and cysteine into glycine conversion
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Paola Lo Surdo, Gaetano Barbato, Laura Orsatti, Federica Innocenti, and Fabio Talamo
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chemistry.chemical_classification ,Molecular Sequence Data ,Organic Chemistry ,Phosphatase ,Glycine ,Alkylation ,Mass Spectrometry ,Neoplasm Proteins ,Analytical Chemistry ,Enzyme Activation ,Enzyme ,chemistry ,Biochemistry ,Protein purification ,Humans ,Phosphorylation ,Chelation ,Amino Acid Sequence ,Cysteine ,Disulfides ,Protein Tyrosine Phosphatases ,Oxidation-Reduction ,Spectroscopy - Abstract
The Phosphatase of Regenerating Liver-3 (PRL-3) is a cysteine-based phosphatase (CBP) that is highly over-expressed in liver metastasis in colorectal cancer and suspected to be involved in the progression from tumor to metastasis. During substrate-specificity studies based on the screening of PRL-3 phosphatase activity on several phosphorylated synthetic peptides, we observed a decrease in activity depending on sample aging and storage conditions. By liquid chromatography combined with selective alkylation and mass spectrometry, we found two main PRL-3 inactivation pathways: a disulfide bond formation between the catalytic C104 and C49, blocking the enzyme in an inactive oxidized form, or the conversion of the catalytic C104 into glycine. We also found that the disulfide formation and the cysteine into glycine conversion are catalyzed by cations present in the sample after protein purification through a nickel column. By adding a cation chelator such as EDTA and de-oxygenating the sample with argon, PRL-3 phosphatase activity was preserved. These findings suggest that PRL-3, like other CBPs, is sensitive to inactivation by catalytic cysteine oxidation and this has implications for future studies of its activity and specificity.
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- 2009
14. Structural and Biochemical Characterization of the Wild Type PCSK9-EGF(AB) Complex and Natural Familial Hypercholesterolemia Mutants
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Marco Mattu, Carl P. Sparrow, Matthew J. Bottomley, Joseph C. Santoro, Lionello Ruggeri, Fabio Talamo, Paola Lo Surdo, Jennifer Baysarowich, Richard T. Cummings, Rose M. Cubbon, Raffaele De Francesco, Laura Orsatti, Ayesha Sitlani, Alessandra Calzetta, Andrea Carfi, A. Noto, Timothy S. Fisher, and Agostino Cirillo
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Models, Molecular ,EGF-like domain ,Molecular Sequence Data ,Mutant ,Familial hypercholesterolemia ,Crystallography, X-Ray ,Biochemistry ,Cell Line ,Hyperlipoproteinemia Type II ,medicine ,Humans ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,Chemistry ,Hydrogen bond ,PCSK9 ,Serine Endopeptidases ,Wild type ,Cell Biology ,medicine.disease ,Molecular biology ,Protein Structure, Tertiary ,Receptors, LDL ,Mutation ,LDL receptor ,Proprotein Convertases ,Salt bridge ,Proprotein Convertase 9 ,Protein Binding - Abstract
PCSK9 regulates low density lipoprotein receptor (LDLR) levels and consequently is a target for the prevention of atherosclerosis and coronary heart disease. Here we studied the interaction, of LDLR EGF(A/AB) repeats with PCSK9. We show that PCSK9 binds the EGF(AB) repeats in a pH-dependent manner. Although the PCSK9 C-terminal domain is not involved in LDLR binding, PCSK9 autocleavage is required. Moreover, we report the x-ray structure of the PCSK9DeltaC-EGF(AB) complex at neutral pH. Compared with the low pH PCSK9-EGF(A) structure, the new structure revealed rearrangement of the EGF(A) His-306 side chain and disruption of the salt bridge with PCSK9 Asp-374, thus suggesting the basis for enhanced interaction at low pH. In addition, the structure of PCSK9DeltaC bound to EGF(AB)(H306Y), a mutant associated with familial hypercholesterolemia (FH), reveals that the Tyr-306 side chain forms a hydrogen bond with PCSK9 Asp-374, thus mimicking His-306 in the low pH conformation. Consistently, Tyr-306 confers increased affinity for PCSK9. Importantly, we found that although the EGF(AB)(H306Y)-PCSK9 interaction is pH-independent, LDLR(H306Y) binds PCSK9 50-fold better at low pH, suggesting that factors other than His-306 contribute to the pH dependence of PCSK9-LDLR binding. Further, we determined the structures of EGF(AB) bound to PCSK9DeltaC containing the FH-associated D374Y and D374H mutations, revealing additional interactions with EGF(A) mediated by Tyr-374/His-374 and providing a rationale for their disease phenotypes. Finally, we report the inhibitory properties of EGF repeats in a cellular assay measuring LDL uptake.
- Published
- 2009
15. Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a Regulatory Structural Zinc-binding Domain
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Raffaele De Francesco, Paola Gallinari, Andrea Carfi, Paolo Di Giovine, Paola Lo Surdo, Philip Jones, Petra Neddermann, Rita Scarpelli, Matthew J. Bottomley, Christian Steinkühler, Agostino Cirillo, and Federica Ferrigno
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Repressor ,Plasma protein binding ,Crystallography, X-Ray ,Biochemistry ,Histone Deacetylases ,Cell Line ,Protein structure ,Humans ,Binding site ,Molecular Biology ,Binding Sites ,biology ,Active site ,Acetylation ,Cell Biology ,Chromatin Assembly and Disassembly ,Chromatin ,Protein Structure, Tertiary ,Repressor Proteins ,Zinc ,Histone ,Protein Structure and Folding ,biology.protein ,Protein Binding - Abstract
Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR·HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.
- Published
- 2008
16. 2-Trifluoroacetylthiophenes, a novel series of potent and selective class II histone deacetylase inhibitors
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Jesus Maria Ontoria Ontoria, Michael Rowley, Ottavia Cecchetti, Matthew J. Bottomley, Federica Ferrigno, Rita Scarpelli, Christian Steinkühler, Andrea Carfi, Paola Lo Surdo, Carsten Schultz-Fademrecht, and Philip Jones
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Stereochemistry ,medicine.drug_class ,Clinical Biochemistry ,Molecular Conformation ,Pharmaceutical Science ,Thiophenes ,Crystallography, X-Ray ,Biochemistry ,Histone Deacetylases ,Structure-Activity Relationship ,Drug Discovery ,medicine ,Structure–activity relationship ,Enzyme Inhibitors ,Binding site ,Molecular Biology ,Histidine ,chemistry.chemical_classification ,Binding Sites ,Molecular Structure ,biology ,Chemistry ,Organic Chemistry ,Histone deacetylase inhibitor ,Active site ,Histone Deacetylase Inhibitors ,Zinc ,Enzyme ,Enzyme inhibitor ,Drug Design ,biology.protein ,Molecular Medicine ,Histone deacetylase - Abstract
The identification of class II HDAC inhibitors has been hampered by lack of efficient enzyme assays, in the preceding paper two assays have been developed to improve the efficiency of these enzymes: mutating an active site histidine to tyrosine, or by the use of a trifluoroacetamide lysine substrate, allowing screening to identify class II HDAC inhibitors. Herein, 2-trifluoroacetylthiophenes have been demonstrated to inhibit class II HDACs, resulting in the development of a series of 5-(trifluoroacetyl)thiophene-2-carboxamides as novel, potent and selective class II HDAC inhibitors. X-ray crystal structures of the HDAC 4 catalytic domain with a bound inhibitor demonstrate these compounds are active site inhibitors and bind in their hydrated form.
- Published
- 2008
17. Effects of pH and Low Density Lipoprotein (LDL) on PCSK9-dependent LDL Receptor Regulation
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Joseph C. Santoro, Marco Mattu, Carl P. Sparrow, Anil Tarachandani, Raffaele De Francesco, Douglas Wisniewski, Samuel D. Wright, Paola Lo Surdo, Rose M. Cubbon, Shilpa Pandit, Timothy S. Fisher, Ayesha Sitlani, Richard T. Cummings, Paul A. Fischer, Andrea Carfi, and Alessandra Calzetta
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Low-density lipoprotein receptor gene family ,Endosome ,Hypercholesterolemia ,Mutation, Missense ,CHO Cells ,Biology ,Models, Biological ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Cricetulus ,Cricetinae ,medicine ,Animals ,Humans ,Molecular Biology ,Genes, Dominant ,PCSK9 ,Chinese hamster ovary cell ,Serine Endopeptidases ,Genetic Diseases, Inborn ,Cell Biology ,medicine.disease ,Molecular biology ,Lipoproteins, LDL ,Hypocholesterolemia ,Receptors, LDL ,chemistry ,Low-density lipoprotein ,LDL receptor ,Kexin ,lipids (amino acids, peptides, and proteins) ,Proprotein Convertases ,Proprotein Convertase 9 ,Lysosomes ,Protein Binding - Abstract
Mutations within PCSK9 (proprotein convertase subtilisin/kexin type 9) are associated with dominant forms of familial hyper- and hypocholesterolemia. Although PCSK9 controls low density lipoprotein (LDL) receptor (LDLR) levels post-transcriptionally, several questions concerning its mode of action remain unanswered. We show that purified PCSK9 protein added to the medium of human endothelial kidney 293, HepG2, and Chinese hamster ovary cell lines decreases cellular LDL uptake in a dose-dependent manner. Using this cell-based assay of PCSK9 activity, we found that the relative potencies of several PCSK9 missense mutants (S127R and D374Y, associated with hypercholesterolemia, and R46L, associated with hypocholesterolemia) correlate with LDL cholesterol levels in humans carrying such mutations. Notably, we found that in vitro wild-type PCSK9 binds LDLR with an approximately 150-fold higher affinity at an acidic endosomal pH (K(D) = 4.19 nm) compared with a neutral pH (K(D) = 628 nm). We also demonstrate that wild-type PCSK9 and mutants S127R and R46L are internalized by cells to similar levels, whereas D374Y is more efficiently internalized, consistent with their affinities for LDLR at neutral pH. Finally, we show that LDL diminishes PCSK9 binding to LDLR in vitro and partially inhibits the effects of secreted PCSK9 on LDLR degradation in cell culture. Together, the results of our biochemical and cell-based experiments suggest a model in which secreted PCSK9 binds to LDLR and directs the trafficking of LDLR to the lysosomes for degradation.
- Published
- 2007
18. Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components
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Marzia Monica Giuliani, Mariagrazia Pizza, Vega Chiron Srl Masignani, Alessia Liguori, Paola Lo Surdo, Barbara Galli, Agnese Faleri, Laura Santini, Isabella Bertoldi, and Nathalie Norais
- Subjects
0301 basic medicine ,medicine.drug_class ,Meningococcal Vaccines ,Monoclonal antibody ,medicine.disease_cause ,Biochemistry ,Epitope ,Microbiology ,03 medical and health sciences ,Epitopes ,Mice ,Antigen ,Genetics ,medicine ,Animals ,Humans ,Adhesins, Bacterial ,Molecular Biology ,biology ,Neisseria meningitidis ,Deuterium Exchange Measurement ,biology.organism_classification ,Virology ,Antibodies, Bacterial ,Bacterial adhesin ,Meningococcal Infections ,030104 developmental biology ,Epitope mapping ,biology.protein ,Neisseria ,Antibody ,Epitope Mapping ,Biotechnology - Abstract
Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation.
- Published
- 2015
19. Apo, Zn2+-bound and Mn2+-bound structures reveal ligand-binding properties of SitA from the pathogen Staphylococcus pseudintermedius
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Enrico Malito, Paola Lo Surdo, Matthew J. Bottomley, Domenico Maione, Francesca Abate, and Roberta Cozzi
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Models, Molecular ,Staphylococcus pseudintermedius ,Staphylococcus ,lcsh:Life ,lcsh:QR1-502 ,SBP, solute-binding protein ,Plasma protein binding ,Crystallography, X-Ray ,medicine.disease_cause ,Bioinformatics ,Biochemistry ,lcsh:Microbiology ,DSC, differential scanning calorimetry ,HIC, hydrophobic interaction chromatography ,Dog Diseases ,CV, column volume ,Peptide sequence ,Pathogen ,Calorimetry, Differential Scanning ,biology ,Protein Stability ,ITC, isothermal titration calorimetry ,Circular Dichroism ,ABC, ATP-binding cassette ,zinc ,class III solute binding protein ,Staphylococcal Infections ,RP-HPLC, reverse-phase HPLC ,Staphylococcus aureus ,MR, molecular replacement ,manganese ,Protein Binding ,staphylococcal disease ,SEC, size-exclusion chromatography ,rmsd, root mean square deviation ,Molecular Sequence Data ,Mutation, Missense ,Biophysics ,Staphylococcal infections ,S2 ,TEV, tobacco etch virus ,Microbiology ,Dogs ,Bacterial Proteins ,PDB, Protein Data Bank ,medicine ,Animals ,Humans ,divalent metal cation ,Amino Acid Sequence ,Protein Structure, Quaternary ,Molecular Biology ,Original Paper ,Sequence Homology, Amino Acid ,FhuD2, ferric hydroxamate uptake D2 ,Mutagenesis ,Cell Biology ,biology.organism_classification ,medicine.disease ,Protein Structure, Tertiary ,lcsh:QH501-531 ,Apolipoproteins ,transport ,Protein Multimerization - Abstract
The Gram-positive bacterium Staphylococcus pseudintermedius is a leading cause of canine bacterial pyoderma, resulting in worldwide morbidity in dogs. S. pseudintermedius also causes life-threatening human infections. Furthermore, methicillin-resistant S. pseudintermedius is emerging, resembling the human health threat of methicillin-resistant Staphylococcus aureus. Therefore it is increasingly important to characterize targets for intervention strategies to counteract S. pseudintermedius infections. Here we used biophysical methods, mutagenesis, and X-ray crystallography, to define the ligand-binding properties and structure of SitA, an S. pseudintermedius surface lipoprotein. SitA was strongly and specifically stabilized by Mn2+ and Zn2+ ions. Crystal structures of SitA complexed with Mn2+ and Zn2+ revealed a canonical class III solute-binding protein with the metal cation bound in a cavity between N- and C-terminal lobes. Unexpectedly, one crystal contained both apo- and holo-forms of SitA, revealing a large side-chain reorientation of His64, and associated structural differences accompanying ligand binding. Such conformational changes may regulate fruitful engagement of the cognate ABC (ATP-binding cassette) transporter system (SitBC) required for metal uptake. These results provide the first detailed characterization and mechanistic insights for a potential therapeutic target of the major canine pathogen S. pseudintermedius, and also shed light on homologous structures in related staphylococcal pathogens afflicting humans.
- Published
- 2014
20. Crystal Structure and Nuclear Magnetic Resonance Analyses of the SAND Domain from Glucocorticoid Modulatory Element Binding Protein-1 Reveals Deoxyribonucleic Acid and Zinc Binding Regions
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Matthew J. Bottomley, Klaus Scheffzek, Paola Lo Surdo, and Michael Sattler
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,HMG-box ,Protein Conformation ,Molecular Sequence Data ,Biology ,Crystallography, X-Ray ,Autoantigens ,DNA-binding protein ,Transactivation ,chemistry.chemical_compound ,Endocrinology ,Glucocorticoid receptor ,Nuclear magnetic resonance ,Humans ,Amino Acid Sequence ,Molecular Biology ,Binding Sites ,Sequence Homology, Amino Acid ,Nuclear Proteins ,Antigens, Nuclear ,DNA ,General Medicine ,DNA-binding domain ,Protein Structure, Tertiary ,DNA-Binding Proteins ,DNA binding site ,Zinc ,chemistry ,Structural Homology, Protein ,Mutagenesis, Site-Directed ,Transcription Factors ,Binding domain - Abstract
The glucocorticoid-modulatory element-binding proteins, GMEB1 and GMEB2, are ubiquitous, multifunctional DNA-binding proteins with important roles in the modulation of transcription upon steroid hormone activation. The GMEB proteins have intrinsic transactivation ability, but also control the glucocorticoid response via direct binding to the glucocorticoid receptor. They are also mandatory host proteins for Parvovirus replication. Here we present the 1.55 A resolution crystal structure of a central portion of GMEB1, encompassing its SAND domain, which shares 80% sequence identity with the GMEB2 SAND domain. We demonstrate that this domain, also present in numerous proteins implicated in chromatin-associated transcriptional regulation, is necessary and sufficient to bind the glucocorticoid-modulatory element (GME) DNA target. We use nuclear magnetic resonance (NMR) and binding studies to map the DNA recognition surface to an alpha-helical region exposing the conserved KDWK motif. Using site-directed mutagenesis, key residues for DNA binding are identified. In contrast to the previously determined NMR structure of the Sp100b SAND domain, we find that the GMEB1 SAND domain also comprises a zinc-binding motif. Although the zinc ion is not necessary for DNA binding, it is found to determine the C-terminal conformation of the GMEB1 SAND domain. We also show that homologous zinc-binding motifs exist in a subset of SAND domain proteins and probe the roles of this novel motif.
- Published
- 2003
21. Structure of the PH domain from Bruton's tyrosine kinase in complex with inositol 1,3,4,5-tetrakisphosphate
- Author
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Marko Hyvönen, Ronan O'Brien, Elena Baraldi, Andrew M. Riley, John E. Ladbury, Barry V. L. Potter, Paola Lo Surdo, Kristina Djinovic Carugo, and Matti Saraste
- Subjects
Models, Molecular ,crystal structure ,X Chromosome ,Inositol Phosphates ,Recombinant Fusion Proteins ,Mutant ,Molecular Sequence Data ,Calorimetry ,Crystallography, X-Ray ,Phosphatidylinositols ,Substrate Specificity ,chemistry.chemical_compound ,Membrane Lipids ,Structure-Activity Relationship ,Protein structure ,Agammaglobulinemia ,Structural Biology ,hemic and lymphatic diseases ,Agammaglobulinaemia Tyrosine Kinase ,Bruton's tyrosine kinase ,Humans ,Point Mutation ,Inositol ,human ,Phosphatidylinositol ,Amino Acid Sequence ,Binding site ,Molecular Biology ,biology ,Sequence Homology, Amino Acid ,PH domain ,Wild type ,tyrosine kinase ,Protein-Tyrosine Kinases ,Molecular biology ,Protein Structure, Tertiary ,Pleckstrin homology domain ,Biochemistry ,chemistry ,Amino Acid Substitution ,biology.protein ,immunodeficiency ,Dimerization ,Sequence Alignment - Abstract
Background: The activity of Bruton's tyrosine kinase (Btk) is important for the maturation of B cells. A variety of point mutations in this enzyme result in a severe human immunodeficiency known as X-linked agammaglobulinemia (XLA). Btk contains a pleckstrin-homology (PH) domain that specifically binds phosphatidylinositol 3,4,5-trisphosphate and, hence, responds to signalling via phosphatidylinositol 3-kinase. Point mutations in the PH domain might abolish membrane binding, preventing signalling via Btk. Results: We have determined the crystal structures of the wild-type PH domain and a gain-of-function mutant E41K in complex with D- myo -inositol 1,3,4,5-tetra-kisphosphate (Ins (1,3,4,5)P 4 ). The inositol Ins (1,3,4,5)P 4 binds to a site that is similar to the inositol 1,4,5-trisphosphate binding site in the PH domain of phospholipase C- δ . A second Ins (1,3,4,5)P 4 molecule is associated with the domain of the E41K mutant, suggesting a mechanism for its constitutive interaction with membrane. The affinities of Ins (1,3,4,5)P 4 to the wild type (K d = 40 nM), and several XLA-causing mutants have been measured using isothermal titration calorimetry. Conclusions: Our data provide an explanation for the specificity and high affinity of the interaction with phosphatidylinositol 3,4,5-trisphosphate and lead to a classification of the XLA mutations that reside in the Btk PH domain. Mis-sense mutations that do not simply destabilize the PH fold either directly affect the interaction with the phosphates of the lipid head group or change electrostatic properties of the lipid-binding site. One point mutation (Q127H) cannot be explained by these facts, suggesting that the PH domain of Btk carries an additional function such as interaction with a G α protein.
- Published
- 1999
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22. Endocytosis: How dynamin sets vesicles PHree!
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Paul C. Driscoll, Paola Lo Surdo, and Matthew J. Bottomley
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Dynamins ,endocrine system ,macromolecular substances ,GTPase ,Biology ,Cytoplasmic Granules ,Endocytosis ,General Biochemistry, Genetics and Molecular Biology ,Bulk endocytosis ,GTP Phosphohydrolases ,chemistry.chemical_compound ,Phosphatidylinositol ,Binding site ,Dynamin ,Binding Sites ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Vesicle ,Blood Proteins ,Intracellular Membranes ,Phosphoproteins ,Cell biology ,Pleckstrin homology domain ,chemistry ,biological phenomena, cell phenomena, and immunity ,General Agricultural and Biological Sciences ,Dimerization - Abstract
The dynamin GTPase is required for clathrin-dependent, receptor-mediated endocytosis. Exciting new studies have shown that dynamin's pleckstrin homology domain binds to phosphatidylinositol 4,5-bisphosphate in vivo, thus localising dynamin directly at the plasma membrane and ultimately enabling vesiculation.
- Published
- 1999
23. Mining the bacterial unknown proteome: identification and characterization of a novel family of highly conserved protective antigens in Staphylococcus aureus
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Paola Lo Surdo, Elisabeth McWhinnie, Fabio Bagnoli, Sara Marchi, Fabiana Falugi, Maria Rita Fontana, Enrico Malito, Sabrina Liberatori, Markus Schirle, Vincenzo Nardi-Dei, Mikkel Nissum, Guido Grandi, Maria Grazia De Falco, Marco Biancucci, Glen Spraggon, Ambra Marongiu, C. Daniela Rinaudo, Benedetta Lombardi, Christina Schluepen, and Matthew J. Bottomley
- Subjects
Staphylococcus aureus ,Proteome ,Structural similarity ,Human pathogen ,Mice, Inbred Strains ,Computational biology ,Biology ,medicine.disease_cause ,Staphylococcal infections ,Crystallography, X-Ray ,Biochemistry ,Mice ,Antigen ,Bacterial Proteins ,medicine ,Extracellular ,Animals ,Data Mining ,Molecular Biology ,Antigens, Bacterial ,Cell Biology ,Staphylococcal Infections ,medicine.disease ,Immunology ,Function (biology) - Abstract
In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
- Published
- 2013
24. EsiB, a novel pathogenic Escherichia coli secretory immunoglobulin A-binding protein impairing neutrophil activation
- Author
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Silvia Rossi Paccani, Xavier Daura, Mario Ferrer Navarro, Roberto Rosini, Paola Lo Surdo, Marco Soriani, Isabella Bertoldi, Ilaria Pastorello, Laura Serino, Rossella Mattera, Barbara Nesta, Valentina Rippa, Mariagrazia Pizza, Mariangela Del Vecchio, Jos A. G. van Strijp, Alexander J. Laarman, Dunja Urosev, and Danilo Gomes Moriel
- Subjects
Immunoglobulin A ,Models, Molecular ,Protein Conformation ,Virulence Factors ,Virulence ,Crystallography, X-Ray ,Microbiology ,Neutrophil Activation ,Bacterial genetics ,03 medical and health sciences ,Gene Knockout Techniques ,Mice ,Immune system ,Antigen ,Pathogenic Escherichia coli ,Virology ,Escherichia coli ,Animals ,Humans ,Pathogen ,030304 developmental biology ,Immune Evasion ,0303 health sciences ,Antigens, Bacterial ,biology ,030306 microbiology ,Escherichia coli Proteins ,biology.organism_classification ,QR1-502 ,3. Good health ,Immunology ,Immunoglobulin A, Secretory ,biology.protein ,Antibody ,Carrier Proteins ,Research Article - Abstract
In this study, we have characterized the functional properties of a novel Escherichia coli antigen named EsiB (E. coli secretory immunoglobulin A-binding protein), recently reported to protect mice from sepsis. Gene distribution analysis of a panel of 267 strains representative of different E. coli pathotypes revealed that esiB is preferentially associated with extraintestinal strains, while the gene is rarely found in either intestinal or nonpathogenic strains. These findings were supported by the presence of anti-EsiB antibodies in the sera of patients affected by urinary tract infections (UTIs). By solving its crystal structure, we observed that EsiB adopts a superhelical fold composed of Sel1-like repeats (SLRs), a feature often associated with bacterial proteins possessing immunomodulatory functions. Indeed, we found that EsiB interacts with secretory immunoglobulin A (SIgA) through a specific motif identified by an immunocapturing approach. Functional assays showed that EsiB binding to SIgA is likely to interfere with productive FcαRI signaling, by inhibiting both SIgA-induced neutrophil chemotaxis and respiratory burst. Indeed, EsiB hampers SIgA-mediated signaling events by reducing the phosphorylation status of key signal-transducer cytosolic proteins, including mitogen-activated kinases. We propose that the interference with such immune events could contribute to the capacity of the bacterium to avoid clearance by neutrophils, as well as reducing the recruitment of immune cells to the infection site., IMPORTANCE Pathogenic Escherichia coli infections have recently been exacerbated by increasing antibiotic resistance and the number of recurrent contagions. Attempts to develop preventive strategies against E. coli have not been successful, mainly due to the large antigenic and genetic variability of virulence factors, but also due to the complexity of the mechanisms used by the pathogen to evade the immune system. In this work, we elucidated the function of a recently discovered protective antigen, named EsiB, and described its capacity to interact with secretory immunoglobulin A (SIgA) and impair effector functions. This work unravels a novel strategy used by E. coli to subvert the host immune response and avoid neutrophil-dependent clearance.
- Published
- 2013
25. Structural and functional characterization of the Staphylococcus aureus virulence factor and vaccine candidate FhuD2
- Author
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Paola Lo Surdo, Marco Biancucci, Glen Spraggon, Matthew J. Bottomley, Fabio Bagnoli, Paolo Mariotti, Enrico Malito, Vincenzo Nardi-Dei, Mikkel Nissum, Silvana Savino, Guido Grandi, and Ravi Prakash Mishra
- Subjects
Models, Molecular ,Siderophore ,Staphylococcus aureus ,Virulence Factors ,Static Electricity ,Virulence ,Siderophores ,Biology ,medicine.disease_cause ,Crystallography, X-Ray ,Hydroxamic Acids ,Biochemistry ,Ferric Compounds ,Virulence factor ,Microbiology ,Bacterial genetics ,chemistry.chemical_compound ,Mice ,Antigen ,Bacterial Proteins ,medicine ,Animals ,Humans ,Molecular Biology ,Ferrichrome ,Antigens, Bacterial ,Protein Stability ,Transferrin ,Membrane Transport Proteins ,Staphylococcal Vaccines ,Cell Biology ,Ligand (biochemistry) ,chemistry ,Genes, Bacterial ,Periplasmic Binding Proteins - Abstract
Staphylococcus aureus is a human pathogen causing globally significant morbidity and mortality. The development of antibiotic resistance in S. aureus highlights the need for a preventive vaccine. In the present paper we explore the structure and function of FhuD2 (ferric-hydroxamate uptake D2), a staphylococcal surface lipoprotein mediating iron uptake during invasive infection, recently described as a promising vaccine candidate. Differential scanning fluorimetry and calorimetry studies revealed that FhuD2 is stabilized by hydroxamate siderophores. The FhuD2–ferrichrome interaction was of nanomolar affinity in surface plasmon resonance experiments and fully iron(III)-dependent. We determined the X-ray crystallographic structure of ligand-bound FhuD2 at 1.9 Å (1 Å=0.1 nm) resolution, revealing the bilobate fold of class III SBPs (solute-binding proteins). The ligand, ferrichrome, occupies a cleft between the FhuD2 N- and C-terminal lobes. Many FhuD2–siderophore interactions enable the specific recognition of ferrichrome. Biochemical data suggest that FhuD2 does not undergo significant conformational changes upon siderophore binding, supporting the hypothesis that the ligand-bound complex is essential for receptor engagement and uptake. Finally, immunizations with FhuD2 alone or FhuD2 formulated with hydroxamate siderophores were equally protective in a murine staphylococcal infection model, confirming the suitability and efficacy of apo-FhuD2 as a protective antigen, and suggesting that other class III SBPs might also be exploited as vaccine candidates.
- Published
- 2012
26. Rational design of a meningococcal antigen inducing broad protective immunity
- Author
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Barbara Brogioni, Marzia Monica Giuliani, Daniele Veggi, Maurizio Comanducci, Beatrice Aricò, Michele Pallaoro, Enrico Malito, Paola Lo Surdo, Silvana Savino, Annalisa Colaprico, Maria Scarselli, Patrizia Giannetti, Lucia Banci, Francesco Doro, Francesca Cantini, Marta Tontini, Brunella Brunelli, Mariagrazia Pizza, Elena Cartocci, Markus Hilleringmann, Rino Rappuoli, Sara Dragonetti, Matthew J. Bottomley, Federica Di Marcello, Laura Ciucchi, Vincenzo Nardi-Dei, and Emmanuelle Palumbo
- Subjects
Models, Molecular ,Magnetic Resonance Spectroscopy ,Biology ,Neisseria meningitidis ,medicine.disease_cause ,Crystallography, X-Ray ,Protein Engineering ,Protein Structure, Secondary ,Microbiology ,Mice ,Immune system ,Protein structure ,Antigen ,Bacterial Proteins ,Immunity ,medicine ,Animals ,Humans ,Peptide sequence ,Antigens, Bacterial ,General Medicine ,Protein engineering ,Virology ,Anti-Bacterial Agents ,Drug Design ,Mutation ,biology.protein ,Mutant Proteins ,Antibody - Abstract
The sequence variability of protective antigens is a major challenge to the development of vaccines. For Neisseria meningitidis, the bacterial pathogen that causes meningitis, the amino acid sequence of the protective antigen factor H binding protein (fHBP) has more than 300 variations. These sequence differences can be classified into three distinct groups of antigenic variants that do not induce cross-protective immunity. Our goal was to generate a single antigen that would induce immunity against all known sequence variants of N. meningitidis. To achieve this, we rationally designed, expressed, and purified 54 different mutants of fHBP and tested them in mice for the induction of protective immunity. We identified and determined the crystal structure of a lead chimeric antigen that was able to induce high levels of cross-protective antibodies in mice against all variant strains tested. The new fHBP antigen had a conserved backbone that carried an engineered surface containing specificities for all three variant groups. We demonstrate that the structure-based design of multiple immunodominant antigenic surfaces on a single protein scaffold is possible and represents an effective way to create broadly protective vaccines.
- Published
- 2011
27. A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake
- Author
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Joseph C. Santoro, Sookhee Ha, Paola Lo Surdo, Rose M. Cubbon, Anka G. Ehrhardt, Xun Shen, Dale Lewis, Richard T. Cummings, Douglas Wisniewski, Yan G. Ni, Brian K. Hubbard, Lionello Ruggeri, Matthew J. Bottomley, Jon H. Condra, Shilpa Pandit, Leila Njimoluh, Cinzia Volpari, Stefania Di Marco, Laura Orsatti, A. Noto, Holly A. Hammond, Timothy S. Fisher, Dana D. Wood, Ayesha Sitlani, and Andrea Carfi
- Subjects
Low-density lipoprotein receptor gene family ,media_common.quotation_subject ,Hypercholesterolemia ,Biology ,Biochemistry ,Protein Structure, Secondary ,Fragment antigen-binding ,chemistry.chemical_compound ,Immunoglobulin Fab Fragments ,Humans ,Internalization ,Molecular Biology ,media_common ,PCSK9 ,Serine Endopeptidases ,Antibodies, Monoclonal ,Hep G2 Cells ,Cell Biology ,Proprotein convertase ,Protein Structure, Tertiary ,Lipoproteins, LDL ,chemistry ,Amino Acid Substitution ,Receptors, LDL ,Low-density lipoprotein ,LDL receptor ,Mutagenesis, Site-Directed ,Kexin ,lipids (amino acids, peptides, and proteins) ,Proprotein Convertases ,Proprotein Convertase 9 - Abstract
PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.
- Published
- 2010
28. Conformation of full-length Bruton tyrosine kinase (Btk) from synchrotron X-ray solution scattering
- Author
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C. I. Edvard Smith, PT Mattsson, José A. Márquez, Matti Saraste, Marika Knekt, Paola Lo Surdo, Maxim V. Petoukhov, Anna Westlund, Klaus Scheffzek, and Dmitri I. Svergun
- Subjects
Models, Molecular ,Protein Conformation ,Sensitivity and Specificity ,General Biochemistry, Genetics and Molecular Biology ,SH3 domain ,Protein structure ,X-Ray Diffraction ,immune system diseases ,hemic and lymphatic diseases ,Agammaglobulinaemia Tyrosine Kinase ,Bruton's tyrosine kinase ,Humans ,Cloning, Molecular ,Protein kinase A ,Molecular Biology ,ABL ,General Immunology and Microbiology ,biology ,General Neuroscience ,Reproducibility of Results ,Articles ,Protein-Tyrosine Kinases ,Recombinant Proteins ,Pleckstrin homology domain ,Solutions ,Biochemistry ,Biophysics ,biology.protein ,Tyrosine kinase ,Synchrotrons ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity.
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