Glutamate dehydrogenase in brain mitochondria: do lipid modifications and transient metabolon formation influence enzyme activity?

Metabolism of glutamate, the primary excitatory neurotransmitter in brain, is complex and of paramount importance to overall brain function. Thus, understanding the regulation of enzymes involved in formation and disposal of glutamate and related metabolites is crucial to understanding glutamate metabolism. Glutamate dehydrogenase (GDH) is a pivotal enzyme that links amino acid metabolism and TCA cycle activity in brain and other tissues. The allosteric regulation of GDH has been extensively studied and characterized. Less is known about the influence of lipid modifications on GDH activity, and the participation of GDH in transient heteroenzyme complexes (metabolons) that can greatly influence metabolism by altering kinetic parameters and lead to channeling of metabolites. This review summarizes evidence for palmitoylation and acylation of GDH, information on protein binding, and information regarding the participation of GDH in transient heteroenzyme complexes. Recent studies suggest that a number of other proteins can bind to GDH altering activity and overall metabolism. It is likely that these modifications and interactions contribute additional levels of regulation of GDH activity and glutamate metabolism.     

Combinatorial anticancer effects of curcumin and 5-fluorouracil loaded thiolated chitosan nanoparticles towards colon cancer treatment

Background

Evaluation of the combinatorial anticancer effects of curcumin/5-fluorouracil loaded thiolated chitosan nanoparticles (CRC-TCS-NPs/5-FU-TCS-NPs) on colon cancer cells and the analysis of pharmacokinetics and biodistribution of CRC-TCS-NPs/5-FU-TCS-NPs in a mouse model.

Methods

CRC-TCS-NPs/5-FU-TCS-NPs were developed by ionic cross-linking. The in vitro combinatorial anticancer effect of the nanomedicine was proven by different assays. Further the pharmacokinetics and biodistribution analyses were performed in Swiss Albino mouse using HPLC.

Results

The 5-FU-TCS-NPs (size: 150 ± 40 nm, zeta potential: + 48.2 ± 5 mV) and CRC-TCS-NPs (size: 150 ± 20 nm, zeta potential: + 35.7 ± 3 mV) were proven to be compatible with blood. The in vitro drug release studies at pH 4.5 and 7.4 showed a sustained release profile over a period of 4 days, where both the systems exhibited a higher release in acidic pH. The in vitro combinatorial anticancer effects in colon cancer (HT29) cells using MTT, live/dead, mitochondrial membrane potential and cell cycle analysis measurements confirmed the enhanced anticancer effects (2.5 to 3 fold). The pharmacokinetic studies confirmed the improved plasma concentrations of 5-FU and CRC up to 72 h, unlike bare CRC and 5-FU.

Conclusions

To conclude, the combination of 5-FU-TCS-NPs and CRC-TCS-NPs showed enhanced anticancer effects on colon cancer cells in vitro and improved the bioavailability of the drugs in vivo.

General significance

The enhanced anticancer effects of combinatorial nanomedicine are advantageous in terms of reduction in the dosage of 5-FU, thereby improving the chemotherapeutic efficacy and patient compliance of colorectal cancer cases.     

Methyl or ethyl makes the difference: Synthesis and solid-state structure of 9,10-dialkyl-9,10-dihydro-9,10-digallaanthracenes

The donor-free 9,10-dialkyl-9,10-dihydro-9,10-digallaanthracenes 1 (alkyl: methyl) and 2 (alkyl: ethyl) were prepared by reaction of 1,2-di(chloromercurio)benzene with the corresponding trialkylgallium and isolated as colourless air- and moisture sensitive crystalline compounds. In the solid-state structure of 1, two slightly different monomers 1A and 1B are found, which form a dimer 1A?1B held together by “medium” strong gallium arene π-interactions. Further weak π-interactions between 1A and 1A and 1B and 1B constitute a one-dimensional coordination polymer containing strands of the composition [?(1A?1B)?(1B?1A)?]_n. In contrast, compound 2 crystallizes in the form of distinct molecular units without any further intermolecular π-interactions. The molecular units possess D_2d symmetry and are built by strong π-interactions between two digallaanthracene monomers. Two symmetrical aryl group bridges between two gallium atoms are observed for the first time in the subunits of 2. By addition of a Lewis-base (THF, Pyridine) to 2, a monomeric planar digallaanthracene framework is restored, as proven by an X-ray crystal structure analysis of 2 · 2Py. The different structures of 1 and 2 are explained on the basis of steric effects.     

Human mitochondrial function during cardiac growth and development

Little information is presently available concerning mitochondrial respiratory and oxidative phosphorylation function in the normal human heart during growth and development. We investigated the levels of specific mitochondrial enzyme activities and content during cardiac growth and development from the early neonatal period (10-20 days) to adulthood (67 years). Biochemical analysis of enzyme specific activities and content and mitochondrial DNA (mtDNA) copy number was performed with left ventricular tissues derived from 30 control individuals. The levels of cytochrome c oxidase (COX) and complex V specific activity, mtDNA copy number and COX subunit II content remained unchanged in contrast to increased citrate synthase (CS) activity and content. The developmental increase in CS activity paralleled increasing CS polypeptide content, but was neither related to overall increases in mitochondrial number nor coordinately regulated with mitochondrial respiratory enzyme activities. Our findings of unchanged levels of cardiac mitochondrial respiratory enzyme activity during the progression from early childhood to older adult contrasts with the age-specific regulation found with CS, a Krebs cycle mitochondrial enzyme.     

Improvement in the Detection of Low Concentration Protein Digests on a MALDI TOF/TOF Workstation by Reducing α-Cyano-4-hydroxycinnamic Acid Adduct Ions

Alpha-cyano-4-hydroxycinnamic acid (α-CHCA) as a matrix facilitates the ionization of proteins and peptides in a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer. The matrix itself also ionizes and so do its sodium and potassium adducts. Matrix clusters and metal ion adducts interfere with peptide ionization and peptide mass spectrum interpretation. These matrix adducts are significantly reduced with addition of ammonium monobasic phosphate or ammonium dibasic citrate to the matrix and sample deposited onto the MALDI target. The reduction of matrix adducts results in the increase of peptide intensity and signal-to-noise ratio as well as in improvement of peptide ionization for samples deposited onto the target at levels of 10 fmol or below. These improvements were particularly significant in the detection of peptides at amol levels when reduced amounts of matrix were also used.     

Zinc-mediated Reversible Multimerization of Hsp31 Enhances the Activity of Holding Chaperone

Hsp31 protein, belonging to the DJ-1/ThiJ/PfpI superfamily, increases the survival of Escherichia coli under various stresses. While it was reported as a holding chaperone, Hsp31 was also shown to exhibit the glyoxalase III activity in subsequent study. Here, we describe our finding that Hsp31 undergoes a Zn^+ 2-mediated multimerization (HMW_Zinc), resulting in an enhanced chaperone activity. Furthermore, it was shown that the formation of HMW_Zinc is reversible such that the oligomer dissociates into the native dimer by EDTA incubation. We attempted to determine the structural change involving the transition between the native dimer and HMW_Zinc by adding Ni^+ 2, which is Zn^+ 2-mimetic, producing a potential intermediate structure. An analysis of this intermediate revealed a structure with hydrophobic interior exposed, due to an unfolding of the N-terminal loop and the C-terminal β-to-α region. A treatment with hydrogen peroxide accelerated HMW_Zinc formation, so that the Hsp31^C185E mutant rendered the formation of HMW_Zinc even at 45 °C. However, the presence of Zn^+ 2 in the catalytic site antagonizes the oxidation of C185, implying a negative role. Our results suggest an unprecedented mechanism of the enhancing chaperone activity by Hsp31, in which the reversible formation of HMW_Zinc occurs in the presence of heat and Zn^+ 2 ion.     

Comparison of proteomes in various human plasma preparations by two-dimensional gel electrophoresis

Serum or plasma can be utilized in a variety of studies targeted toward the discovery of disease biomarkers. In this study, the proteome profiles of plasma samples prepared using various anticoagulants (EDTA, heparin or citrate), were compared with those of serum using two-dimensional electrophoresis (2-DE). Proteins which evidenced different levels in the plasma and serum were screened and identified using ESI-Q-TOF MS/MS. The proteins which became detectable after the removal of fibrinogen from serum were identified as pigment epithelial differentiating factor (four spots), fetuin-like protein, and the hemopexin precursor. In particular, three proteins, pre-serum amyloid P component, plasma glutathione peroxidase precursor, and tetranectin, evidenced increased volume intensity only in the plasma samples prepared with EDTA.     

Design of disulfide-linked thioredoxin dimers and multimers through analysis of crystal contacts

Disulfide bonds play an important role in protein stability and function. Here, we describe a general procedure for generating disulfide-linked dimers and multimers of proteins of known crystal structures. An algorithm was developed to predict sites in a protein compatible with intermolecular disulfide formation with neighboring molecules in the crystal lattice. A database analysis was carried out on 46 PDB coordinates to verify the general applicability of this algorithm to predict intermolecular disulfide linkages. On the basis of the predictions from this algorithm, mutants were constructed and characterized for a model protein, thioredoxin. Of the five mutants, as predicted, in solution four formed disulfide-linked dimers while one formed polymers. Thermal and chemical denaturation studies on these mutant thioredoxins showed that three of the four dimeric mutants had similar stability to wild-type thioredoxin while one had lower stability. Three of the mutant dimers crystallized readily (in four to seven days) in contrast to the wild-type protein, which is particularly difficult to crystallize and takes more than a month to form diffraction-quality crystals. In two of the three cases, the structure of the dimer was exactly as predicted by the algorithm, while in the third case the relative orientation of the monomers in the dimer was different from the predicted one. This methodology can be used to enhance protein crystallizability, modulate the oligomerization state and to produce linear chains or ordered three-dimensional protein arrays.     

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.