Nonlinear dynamics of eucaryotic pyruvate dehydrogenase multienzyme complex: Decarboxylation rate, oscillations, and multiplicity

Pyruvate conversion to acetyl-CoA by the pyruvate dehydrogenase (PDH) multienzyme complex is known as a key node in affecting the metabolic fluxes of animal cell culture. However, its possible role in causing possible nonlinear dynamic behavior such as oscillations and multiplicity of animal cells has received little attention. In this work, the kinetic and dynamic behavior of PDH of eucaryotic cells has been analyzed by using both in vitro and simplified in vivo models. With the in vitro model the overall reaction rate (v(1)) of PDH is shown to be a nonlinear function of pyruvate concentration, leading to oscillations under certain conditions. All enzyme components affect v, and the nonlinearity of PDH significantly, the protein X and the core enzyme dihydrolipoamide acyltransferase (E2) being mostly predominant. By considering the synthesis rates of pyruvate and PDH components the in vitro model is expanded to emulate in vivo conditions. Analysis using the in vivo model reveals another interesting kinetic feature of the PDH system, namely, multiple steady states. Depending on the pyruvate and enzyme levels or the operation mode, either a steady state with high pyruvate decarboxylation rate or a steady state with significantly lower decarboxylation rate can be achieved under otherwise identical conditions. In general, the more efficient steady state is associated with a lower pyruvate concentration. A possible time delay in the substrate supply and enzyme synthesis can also affect the steady state to be achieved and lead's to oscillations under certain conditions. Overall, the predictions of multiplicity for the PDH system agree qualitatively well with recent experimental observations in animal cell cultures. The model analysis gives some hints for improving pyruavte metabolism in animal cell culture.

Impact of beta-galactosidase mutations on the expression of the canine lysosomal multienzyme complex

beta-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1(+/+), GLB1(+/-) and GLB1(-/-) fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total beta-galactosidase, beta-galactosidase variants and protective protein for beta-galactosidase gene (PPGB) in GLB1(+/-) and GLB1(-/-) compared to GLB1(+/+) fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant beta-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1(+/+), GLB1(+/-) and GLB1(-/-) genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.

Pten and Ndufb8 aberrations in cervical cancer tissue

Cervical cancer is one of the world's major health issues. Despite many studies in this field, the carcinogenetic events of malignant conversion in cervical tumours have not been significantly characterised. The first aim of this project was to investigate the mutation status of the tumour suppressor gene- Phosphatase and Tension Homolog (PTEN)- in cervical cancer tissue. The second aim of this study was the analysis in the same cervical cancer tissue for aberrations in the mitochondrial electron transport chain subunit gene NDUFB8, which is localised to the same chromosomal contig as PTEN. The third aim was the evaluation of the potential therapeutic anti-cancer drug 2,4-Thiazolidinediones (TZDs) and its affect in regulating the PTEN protein in a cervical cancer cell line (HeLa). To approach the aims, paraffin-embedded cancerous cervical tissue and non-cancerous cervical tissue were obtained. DNA recovered from those tissues was then used to investigate the putative genomic changes regarding the NDUFB8 gene utilising SYBR Green I Real-Time PCR. The PTEN gene was studied via Dual-Labelled probe Real-Time PCR. To investigate the protein expression change of the PTEN protein, HeLa cells were firstly treated with different concentrations of 2,4-Thiazolidinediones and the level of PTEN protein expression was then observed utilising standard protein assays. Results indicated that there were putative copy-number changes between the cancerous cervical tissue and non-cancerous cervical tissue, with regard to the PTEN locus. This implies a potential gain of the PTEN gene in cancerous cervical tissue. With regards to normal cervical tissue versus cancerous cervical tissue no significant melting temperature differences were observed with the SYBR Green I Real-Time PCR in respect to the NDUFB8 gene. A putative up-regulation of PTEN protein was observed in TZD treated HeLa cells. © 2008 Springer Science+Business Media, LLC.

Complexo multienzimático e fontes lipídicas em rações para frangos de corte

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds

Giardia lamblia is a protozoan parasite that causes giardiasis, a diarrhoeal disease affecting humans and various animal species. Nitro drugs such as the nitroimidazole metronidazole and the nitrothiazolide nitazoxanide are used for treatment of giardiasis. Nitroreductases such as GlNR1 and GlNR2 may play a role in activation or inactivation of these drugs. The aim of this work is to characterise these two enzymes using functional assays. For respective analyses recombinant analogues from GlNR1 and GlNR2 were produced in Escherichia coli. E. coli expressing GlNR1 and GlNR2 alone or together were grown in the presence of nitro compounds. Furthermore, pull-down assays were performed using HA-tagged GlNR1 and GlNR2 as baits. As expected, E. coli expressing GlNR1 were more susceptible to metronidazole under aerobic and semi-aerobic and to nitazoxanide under semi-aerobic growth conditions whereas E. coli expressing GlNR2 were susceptible to neither drug. Interestingly, expression of both nitroreductases gave the same results as expression of GlNR2 alone. In functional assays, both nitroreductases had their strongest activities on the quinone menadione (vitamin K3) and FAD, but reduction of nitro compounds including the nitro drugs metronidazole and nitazoxanidewas clearly detected. Full reduction of 7-nitrocoumarin to 7-aminocoumarin was preferentially achieved with GlNR2. Pull-down assays revealed that GlNR1 and GlNR2 interacted in vivo forming a multienzyme complex. These findings suggest that both nitroreductases are multifunctional. Their main biological role may reside in the reduction of vitamin K analogues and FAD. Activation by GlNR1 or inactivation by GlNR2 of nitro drugs may be the consequence of a secondary enzymatic activity either yielding (GlNR1) or eliminating (GlNR2) toxic intermediates after reduction of these compounds. © 2015 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license

Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus

The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-l-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.

d-β-Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

The heroin analogue 1-methyl-4-phenylpyridinium, MPP+, both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Aβ1–42, is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-β-hydroxybutyrate protected cultured mesencephalic neurons from MPP+ toxicity and hippocampal neurons from Aβ1–42 toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome

Clostridium cellulovorans uses not only cellulose but also xylan, mannan, pectin, and several other carbon sources for its growth and produces an extracellular multienzyme complex called the cellulosome, which is involved in plant cell wall degradation. Here we report a gene for a cellulosomal subunit, pectate lyase A (PelA), lying downstream of the engY gene, which codes for cellulosomal enzyme EngY. pelA is composed of an ORF of 2,742 bp and encodes a protein of 914 aa with a molecular weight of 94,458. The amino acid sequence derived from pelA revealed a multidomain structure, i.e., an N-terminal domain partially homologous to the C terminus of PelB of Erwinia chrysanthemi belonging to family 1 of pectate lyases, a putative cellulose-binding domain, a catalytic domain homologous to PelL and PelX of E. chrysanthemi that belongs to family 4 of pectate lyases, and a duplicated sequence (or dockerin) at the C terminus that is highly conserved in enzymatic subunits of the C. cellulovorans cellulosome. The recombinant truncated enzyme cleaved polygalacturonic acid to digalacturonic acid (G2) and trigalacturonic acid (G3) but did not act on G2 and G3. There have been no reports available to date on pectate lyase genes from Clostridia.

Localization of GAR transformylase in Escherichia coli and mammalian cells

Enzymes of the de novo purine biosynthetic pathway may form a multienzyme complex to facilitate substrate flux through the ten serial steps constituting the pathway. One likely strategy for complex formation is the use of a structural scaffold such as the cytoskeletal network or subcellular membrane of the cell to mediate protein–protein interactions. To ascertain whether this strategy pertains to the de novo purine enzymes, the localization pattern of the third purine enzyme, glycinamide ribonucleotide transformylase (GAR Tfase) was monitored in live Escherichia coli and mammalian cells. Genes encoding human as well as E. coli GAR Tfase fused with green fluorescent protein (GFP) were introduced into their respective cells with regulated expression of proteins and localization patterns monitored by using confocal fluorescence microscopy. In both instances images showed proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is not localized to an existing cellular architecture, so this device is probably not used to concentrate the members of the pathway. However, discrete clusters of the pathway may still exist throughout the cytoplasm.

Domain relationships in thiamine diphosphate-dependent enzymes

Three-dimensional structures have been determined for 13 different enzymes that use thiamine diphosphate (ThDP) as a cofactor. These enzymes fall into five families, where members within a family have similar structures. In different families, there are similarities between some domains that clearly point to a common ancestor for all of these enzymes. Where the enzyme structures differ, evolutionary relationships between families can be discerned. Here, I present an analysis of these families and propose an evolutionary pathway to explain the diversity of structures that are now known.

Caracterização genotípica de doentes portugueses com patologias associadas ao complexo multienzimático lisossomal: sialidose e galactosialidose

Dissertação de mestrado em Biologi apresentada à Faculdade de Ciências da Universidade do Porto, 2008

Raman spectroscopy of some complex arsenate minerals—implications for soil remediation

The application of spectroscopy to the study of contaminants in soils is important. Among the many contaminants is arsenic, which is highly labile and may leach to non-contaminated areas. Minerals of arsenate may form depending upon the availability of specific cations for example calcium and iron. Such minerals include carminite, pharmacosiderite and talmessite. Each of these arsenate minerals can be identified by its characteristic Raman spectrum enabling identification.