Structural and Functional Analysis of Component X from the Bovine Heart Pyruvate Dehydrogenase Complex

A comprehensive investigation of the optimal conditions for reconstitution of bovine heart pyruvate dehydrogenase complex activity (PDC) from dissociated E2/X core and E1/E3 fractions is presented, and was employed in the elucidation of the disputed involvement of the lipoyl domains of protein X in overall complex catalysis. Selective proteolysis of the lipoyl and linker regions of protein X by Arg C, in isolated E2/X core or intact PDC, resulted in a dramatic reduction in E3 binding potential and hence, complex activity (with the former, following reconstitution with dissociated components, El and E3), when compared to control complex activities. It is proposed that owing to the proximity of the cleavage site to the E3 binding domain of protein X, loss of E3 binding affinity arises from conformational changes in the aforementioned domain and/or, the specific binding site within. Partial recovery of reconstitution was achieved in the presence of excess porcine E3, and increased with increasing protein X degradation suggestive of an additional, non specific and hence, low affinity E3 binding site -- presumably the subunit binding domain of E2. Following removal of the lipoyl and linker regions of either component E2 or X, N-ethyl[2,3-14C]maleimide labelling studies confirmed previous observations of a residual (i.e., approx. 10%) complex activity, indicating the ability of either component to substitute for the lipoyl domains of the other. The physiological implications of this are discussed in relation to the unique diacetylation properties of mammalian PDC. However, as attempts to isolate and sequence the extreme C-terminal portion of protein X following the recent discovery of a dihydrolipoamide acetyltransferase active site-like motif by J.C. Neagle (University of Glasgow, unpublished observations), remained in the initial stages, it is unclear whether deacetylation of the lipoyl domains of protein X services an acetyltransferase active site on E2 or X. Precise stoichiometric determinations employing: comparisons of 14C- acetylation in E2 and X; and, densitometric scan analysis of highly purified protein X, E2 and bovine heart PDC samples resolved by SDS-PAGE and silver stained, indicated the presence of 12mol protein X/mol PDC, and of 60mol E2/mol PDC, respectively. Structural studies involving covalent N,N'-1,2-phenylene-dimaleimide (PDM), crosslinking of the lipoyl domains of protein X in E2-lipoyl domain depleted complex, revealed dimeric organisation. Presented together, this evidence is in close agreement with the 1:1 (E3:E1) binding relationship of bovine heart OGDC, and is strongly suggestive of the involvement of six protein X dimers in the binding of six dimers of E3 to the E2/X core in bovine heart PDC. Furthermore, the presence of 60 E2 subunits supports previous proposals for a non-integrated, external protein X core positioning

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8

Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity. The data analysis revealed that the presence of heparin affects all steps of the enzyme reaction: (i) it decreases 3.5-fold the k1 and 4.0-fold the k−1, (ii) it affects the acyl-enzyme accumulation with pronounced decrease in k2 (2.7-fold), and also decrease in k3 (3.5-fold). The large values of ΔG = 12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys25)-S−/(His163)-Im+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme. Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface

Peptide evidence indicating alternative exon positioning and sequence annotation

The position of ORF X-1-3917326-3920484 in the genome scaffold is indicated by a red line on the grey track at the top of the figure and this region is expanded below, the red triangle demarking the ORF length. Predicted exons are indicated as blue boxes, linked by zigzag lines to indicate the position of exon/intron boundaries. Gene is shown with two exons. ESTs are shown as dark blue or brown boxes. Peptides aligning with this region are shown in yellow. The predicted sequence for ORF X-1-3917326-3920484 is shown as an insert and sequence that matches exon 2 of gene is shown in blue. Sequence for which there is matching peptide evidence is shown in red. Purple lettering indicates the positioning of the 'intron-located' peptide, mass spectrometric evidence for which is shown in the right hand insert.<p><b>Copyright information:</b></p><p>Taken from "The proteome of : integration with the genome provides novel insights into gene expression and annotation"</p><p>http://genomebiology.com/2008/9/7/R116</p><p>Genome Biology 2008;9(7):R116-R116.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2530874.</p><p></p

The tachyzoite expressed proteome: comparison with EST and microarray expression data

A comparison of the expressed proteome of tachyzoites with EST and microarray data reveals discrepancies between protein and transcriptional data. Venn diagram comparing the correlation between the number of non-redundant release4 genes detected by EST expression from tachyzoite and bradyzoites (available from ToxoDB) and those detected by this proteome study. The number of genes unique to each intersection is indicated. Venn diagrams comparing the correlation between release4 genes obtained by this proteome study and those detected by microarray analysis of RH strain tachyzoites, including those genes with expression of ≥ 25 and ≥ 50 percentiles. Bar chart showing the number of release4 genes also detected by proteomics for each of the four percentile ranges, 0-24%, 25-49%, 50-74%, 75-100%, determined by microarray analysis.<p><b>Copyright information:</b></p><p>Taken from "The proteome of : integration with the genome provides novel insights into gene expression and annotation"</p><p>http://genomebiology.com/2008/9/7/R116</p><p>Genome Biology 2008;9(7):R116-R116.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2530874.</p><p></p

Subcellular localisation and functional categorization of the expressed tachyzoite proteome

The numbers correspond to the total number of identified proteins in each category. Protein subcellular localization information was first assigned according to gene descriptions and GO annotation provided by ToxoDB. When no information was available, protein sequences were submitted to PATS, PlasMit and WoLF PSORT. The combined results were manually assessed to obtain subcellular localization predictions. A detailed list of proteins in each subcellular localization to accompany this figure is provided in Additional data file 12. Functional categorization was constructed using the GO classifications listed on ToxoDB for each release4 gene, which were then assigned to specific MIPS categories within the FunCatDB functional catalogue. Genes without a GO classification were assigned a putative MIPS category using additional information provided by Blast, Pfam domain alignments, InterPro and from independent literature searches. Notes: protein fate includes protein folding, modification and destination. A detailed list of proteins in each functional category to accompany this figure is provided in Additional data file 13.<p><b>Copyright information:</b></p><p>Taken from "The proteome of : integration with the genome provides novel insights into gene expression and annotation"</p><p>http://genomebiology.com/2008/9/7/R116</p><p>Genome Biology 2008;9(7):R116-R116.</p><p>Published online 21 Jul 2008</p><p>PMCID:PMC2530874.</p><p></p

Kinetic rate constants of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.

<p>Kinetic rate constants of hydrolysis of Z-FR-MCA by rCPB2.8 in absence or in the presence of 40 µM heparin.</p