Scannella, Alessandra (2010) Characterization of multifunctional proteins from Helicobacter pylori. [Tesi di dottorato] (Unpublished)

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Abstract

This thesis reports the cloning, overexpression and characterization of two interesting proteins from Helicobacter pylori: Heat Shock protein A (HspA), and CoaX (Type III Pantothenate kinase). Helicobacter pylori produces an unusual GroES homologue protein referred as to HspA (Heat-shock protein A). Besides its classical co-chaperone activity, HspA plays additional roles being involved in nickel binding. It also exhibits an extended subcellular localization, ranging from cytoplasm to bacterial cell surface. In fact, unlike its homologue proteins, HspA is highly immunogenic being also present in the extra cellular media. For this reason it is considered a target for new therapeutic strategies. HspA consists of two domains: an N-terminal domain (domain A, residues 1-90), that is homologous with other GroES bacterial proteins and a C-terminal domain (domain B, residues 91-118), which other GroES-like proteins lack. Domain B is unique to HspA and contains 8 histidines and 4 cysteines which have been suggested to be involved in nickel binding.This study points on a unique characteristic of HspA among all GroES proteins: a high content of cysteine residues. Cysteine is the less represented residue in all known GroES proteins examined. In this context we have produced and characterized a recombinant HspA and its mutants Cys94Ala and Cys94Ala/Cys111Ala and we have identified the disulphide bridge pattern of the protein. In particular the study has been addressed on the Cys oxidised/reduced state; the disulphide bridge pattern has been assigned by integrating classical biochemical methodologies with mass spectrometry. We found that the cysteines (two from domain A and four from domain B) are engaged in three disulphide bonds between residues Cys51/Cys53, Cys94/Cys111 and Cys95/Cys112. Our results suggest that two of the disulphide bridges, located in the B domain, force the C-terminal domain to adopt a unique closed loop structure that would be optimal for binding to 2 Ni ions as suggested by the different redox environments that the protein experiences inside and outside the bacterial cell.H.pylori also produces a new Pantothenate Kinase isoform (HpCoaX) encoded by the CoaX gene referred as to HP0862, involved in a critical pathway for pathogen survival: that of coenzyme A (CoA) biosynthesis. In particular, pantothenate kinase catalyzes the first step of the universal five-step CoA biosynthetic pathway. HpCoaX enzyme, belonging to the type III class, differs in sequence, structure and enzymatic properties from the previously characterized type I, found essentially in bacteria, and type II forms found in eukarya as well as in some bacteria.In this context we have cloned the HpCoaX gene in pRoEX-HTc vector and expressed the recombinant protein in E.coli BL21( DE3) cells. We have optimized the experimental conditions for purification and stability of the expressed protein. The purified HpCoax was analyzed by gel filtration chromatography and dynamic light scattering to investigate its state of oligomerization. Results show that HpCoaX exists as a homodimer, as predicted by its homology with the other PanK III bacterial proteins. H. pylori infections remain a significant global public health problem. Vaccine and antagonist compound development against this infection appears to be a preferable strategy. Actually there is a substantial requirement for new drug targets as alternative strategies for the treatment of H. pylori infections, since there is often resistance against traditional antibiotic therapy.For the all above reasons, both HpCoaX and HspA can be considered to be a good targets for new therapeutic strategies. Our study belongs to a wide research line with the aim of identifying, characterizing and clarifying the role of specific targets, in particular of multifunctional uncharacterized proteins involved in critical pathway for the pathogen survival.

Item Type: Tesi di dottorato
Uncontrolled Keywords: Multifunctional; Heat schok proteins; antibiotic resistance; Nickel metabolism in pathogens; Coenzyme A metabolism
Depositing User: Francesca Migliorini
Date Deposited: 03 Dec 2010 10:38
Last Modified: 30 Apr 2014 19:44
URI: http://www.fedoa.unina.it/id/eprint/7993

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