Medzihradszky KF

References (4)

Title : The oligopeptidase B of Leishmania regulates parasite enolase and immune evasion - Swenerton_2011_J.Biol.Chem_286_429
Author(s) : Swenerton RK , Zhang S , Sajid M , Medzihradszky KF , Craik CS , Kelly BL , McKerrow JH
Ref : Journal of Biological Chemistry , 286 :429 , 2011
Abstract : Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are targets for the development of new anti-parasitic therapy. Protozoan parasites like Leishmania predominantly express Clan CA cysteine proteases for key life cycle functions. It was therefore unexpected to find a high level of serine protease activity expressed by Leishmania donovani. Purification of this activity followed by mass spectrometry identified oligopeptidase B (OPB; Clan SC, family S9A) as the responsible enzyme. This was confirmed by gene knock-out of OPB, which resulted in the disappearance of the detected serine protease activity of Leishmania extracts. To delineate the specific role of OPB in parasite physiology, proteomic analysis was carried out on OPB(-/-) versus wild type parasites. Four protein species were significantly elevated in OPB(-/-) parasites, and all four were identified by mass spectrometry as enolase. This increased enolase was enzymatically inactive and associated with the parasite membrane. Aside from its classic role in carbohydrate metabolism, enolase was recently found to localize to membranes, where it binds host plasminogen and functions as a virulence factor for several pathogens. As expected, there was a striking alteration in macrophage responses to Leishmania when OPB was deleted. Whereas wild type parasites elicited little, if any, response from infected macrophages, OPB(-/-) parasites induced a massive up-regulation in gene transcription. Additionally, these OPB(-/-) parasites displayed decreased virulence in the murine footpad infection model.
ESTHER : Swenerton_2011_J.Biol.Chem_286_429
PubMedSearch : Swenerton_2011_J.Biol.Chem_286_429
PubMedID: 20961853
Gene_locus related to this paper: leima-OPB

Title : Nicotinic agonist binding site mapped by methionine- and tyrosine-scanning coupled with azidochloropyridinyl photoaffinity labeling - Tomizawa_2009_J.Med.Chem_52_3735
Author(s) : Tomizawa M , Talley TT , Park JF , Maltby D , Medzihradszky KF , Durkin KA , Cornejo-Bravo JM , Burlingame AL , Casida JE , Taylor P
Ref : Journal of Medicinal Chemistry , 52 :3735 , 2009
Abstract : Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tyrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.
ESTHER : Tomizawa_2009_J.Med.Chem_52_3735
PubMedSearch : Tomizawa_2009_J.Med.Chem_52_3735
PubMedID: 19459645

Title : Defining nicotinic agonist binding surfaces through photoaffinity labeling - Tomizawa_2007_Biochemistry_46_8798
Author(s) : Tomizawa M , Maltby D , Medzihradszky KF , Zhang N , Durkin KA , Presley J , Talley TT , Taylor P , Burlingame AL , Casida JE
Ref : Biochemistry , 46 :8798 , 2007
Abstract : Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.
ESTHER : Tomizawa_2007_Biochemistry_46_8798
PubMedSearch : Tomizawa_2007_Biochemistry_46_8798
PubMedID: 17614369

Title : Mapping the elusive neonicotinoid binding site - Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
Author(s) : Tomizawa M , Talley TT , Maltby D , Durkin KA , Medzihradszky KF , Burlingame AL , Taylor P , Casida JE
Ref : Proc Natl Acad Sci U S A , 104 :9075 , 2007
Abstract : Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid and thiacloprid, are nonprotonated and selective for insects and their nicotinic receptors, whereas nicotinoids such as nicotine and epibatidine are cationic and selective for mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), as a structural surrogate for the extracellular ligand-binding domain of the nicotinic receptor, is similarly sensitive to neonicotinoids and nicotinoids. It therefore seemed possible that the proposed very different interactions of the neonicotinoids and nicotinoids might be examined with a single AChBP by using optimized azidochloropyridinyl photoaffinity probes. Two azidoneonicotinoids with a nitro or cyano group were compared with the corresponding desnitro or descyano azidonicotinoids. The four photoactivated nitrene probes modified AChBP with up to one agonist for each subunit based on analysis of the intact derivatized protein. Identical modification sites were observed by collision-induced dissociation analysis for the neonicotinoids and nicotinoids with similar labeling frequency of Tyr-195 of loop C and Met-116 of loop E at the subunit interface. The nitro- or cyano-substituted guanidine/amidine planes of the neonicotinoids provide a unique electronic conjugation system to interact with loop C Tyr-188. The neonicotinoid nitro oxygen and cyano nitrogen contact loop C Cys-190/Ser-189, whereas the cationic head of the corresponding nicotinoids is inverted for hydrogen-bonding and cation-pi contact with Trp-147 and Tyr-93. These structural models based on AChBP directly map the elusive neonicotinoid binding site and further describe the molecular determinants of agonists on nicotinic receptors.
ESTHER : Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
PubMedSearch : Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
PubMedID: 17485662