Chemical shift assignments of Leishmania mexicana ICP, a novel cysteine peptidase inhibitor.
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ABSTRACT:
SUBMITTER: Smith BO
PROVIDER: S-EPMC2592598 | biostudies-literature | 2006
REPOSITORIES: biostudies-literature
Publications
Chemical shift assignments of Leishmania mexicana ICP, a novel cysteine peptidase inhibitor.
Smith Brian O BO Westrop Gareth D GD Mottram Jeremy C JC Coombs Graham H GH
Journal of biomolecular NMR 20060101
Similar Datasets
Project description:Clan CA, family C1 cysteine peptidases (CPs) are important virulence factors and drug targets in parasites that cause neglected diseases. Natural CP inhibitors of the I42 family, known as ICP, occur in some protozoa and bacterial pathogens but are absent from metazoa. They are active against both parasite and mammalian CPs, despite having no sequence similarity with other classes of CP inhibitor. Recent data suggest that Leishmania mexicana ICP plays an important role in host-parasite interactions. We have now solved the structure of ICP from L. mexicana by NMR and shown that it adopts a type of immunoglobulin-like fold not previously reported in lower eukaryotes or bacteria. The structure places three loops containing highly conserved residues at one end of the molecule, one loop being highly mobile. Interaction studies with CPs confirm the importance of these loops for the interaction between ICP and CPs and suggest the mechanism of inhibition. Structure-guided mutagenesis of ICP has revealed that residues in the mobile loop are critical for CP inhibition. Data-driven docking models support the importance of the loops in the ICP-CP interaction. This study provides structural evidence for the convergent evolution from an immunoglobulin fold of CP inhibitors with a cystatin-like mechanism.
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Project description:Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana.
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Project description:Data validation plays an important role in ensuring the reliability and reproducibility of studies. NMR investigations of the functional properties, dynamics, chemical kinetics, and structures of proteins depend critically on the correctness of chemical shift assignments. We present a novel probabilistic method named ARECA for validating chemical shift assignments that relies on the nuclear Overhauser effect data . ARECA has been evaluated through its application to 26 case studies and has been shown to be complementary to, and usually more reliable than, approaches based on chemical shift databases. ARECA is available online at http://areca.nmrfam.wisc.edu/.
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Project description:ZCCHC9 is a human nuclear protein with sequence homology to yeast Air1p/Air2p proteins which are RNA-binding subunits of the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex involved in nuclear RNA quality control and degradation in yeast. The ZCCHC9 protein contains four retroviral-type zinc knuckle motifs. Here, we report the NMR spectral assignment of the zinc knuckle region of ZCCHC9. These data will allow performing NMR structural and RNA-binding studies of ZCCHC9 with the aim to investigate its role in the RNA quality control in human.
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Project description:Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5' to an abasic site, giving a 3'-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and (13)C(beta)) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein.
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Project description:NMR chemical shifts provide important local structural information for proteins. Consistent structure generation from NMR chemical shift data has recently become feasible for proteins with sizes of up to 130 residues, and such structures are of a quality comparable to those obtained with the standard NMR protocol. This study investigates the influence of the completeness of chemical shift assignments on structures generated from chemical shifts. The Chemical-Shift-Rosetta (CS-Rosetta) protocol was used for de novo protein structure generation with various degrees of completeness of the chemical shift assignment, simulated by omission of entries in the experimental chemical shift data previously used for the initial demonstration of the CS-Rosetta approach. In addition, a new CS-Rosetta protocol is described that improves robustness of the method for proteins with missing or erroneous NMR chemical shift input data. This strategy, which uses traditional Rosetta for pre-filtering of the fragment selection process, is demonstrated for two paramagnetic proteins and also for two proteins with solid-state NMR chemical shift assignments.
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Project description:The protozoan Trypanosoma brucei rhodesiense causes Human African Trypanosomiasis, also known as sleeping sickness, and penetrates the central nervous system, leading to meningoencephalitis. The Cathepsin L-like cysteine peptidase of T. b. rhodesiense has been implicated in parasite penetration of the blood-brain barrier and its activity is modulated by the chagasin-family endogenous inhibitor of cysteine peptidases (ICP). To investigate the role of ICP in T. b. rhodesiense bloodstream form, ICP-null (Δicp) mutants were generated, and lines re-expressing ICP (Δicp:ICP). Lysates of Δicp displayed increased E-64-sensitive cysteine peptidase activity and the mutant parasites traversed human brain microvascular endothelial cell (HBMEC) monolayers in vitro more efficiently. Δicp induced E-selectin in HBMECs, leading to the adherence of higher numbers of human neutrophils. In C57BL/6 mice, no Δicp parasites could be detected in the blood after 6 days, while mice infected with wild-type (WT) or Δicp:ICP displayed high parasitemia, peaking at day 12. In mice infected with Δicp, there was increased recruitment of monocytes to the site of inoculation and higher levels of IFN-γ in the spleen. At day 14, mice infected with Δicp exhibited higher preservation of the CD4+, CD8+, and CD19+ populations in the spleen, accompanied by sustained high IFN-γ, while NK1.1+ populations receded nearly to the levels of uninfected controls. We propose that ICP helps to downregulate inflammatory responses that contribute to the control of infection.
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Project description:The Ca2+ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca2+ transporters. The only published NMR chemical shift assignments for Ca2+-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca2+-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).
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Project description:A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8DeltaCTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded >3.5 mg of active enzyme per litre of E. coli culture.
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Project description:Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.
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