Hallmarks of protein oxidative damage in neurodegenerative diseases:Focus on Alzheimer's disease

The pathogenesis of several neurodegenerative diseases, including Alzheimer's disease, has been linked to a condition of oxidative and nitrosative stress, arising from the imbalance between increased reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and antioxidant defences or efficiency of repair or removal systems. The effects of free radicals are expressed by the accumulation of oxidative damage to biomolecules: nucleic acids, lipids and proteins. In this review we focused our attention on the large body of evidence of oxidative damage to protein in Alzheimer's disease brain and peripheral cells as well as in their role in signalling pathways. The progress in the understanding of the molecular alterations underlying Alzheimer's disease will be useful in developing successful preventive and therapeutic strategies, since available drugs can only temporarily stabilize the disease, but are not able to block the neurodegenerative process. © 2007 Springer-Verlag.

Oxidation of protein in human low-density lipoprotein exposed to peroxyl radicals facilitates uptake by monocytes; protection by antioxidants in vitro

Generation of neoepitopes on apolipoprotein B within oxidised low-density lipoprotein (LDL) is important in the unregulated uptake of LDL by monocytic scavenger receptors (CD36, SR-AI, LOX-1). Freshly isolated LDL was oxidised by peroxyl radicals generated from the thermal decomposition of an aqueous azo-compound. We describe that formation of carbonyl groups on the protein component is early as protein oxidation was seen after 90min. This is associated with an increased propensity for LDL uptake by U937 monocytes. Three classes of antioxidants (quercetin, dehydroepiandrosterone (DHEA) and ascorbic acid) have been examined for their capacity to inhibit AAPH-induced protein oxidation, (protein carbonyls, Δ electrophoretic mobility and LDL uptake by U937 monocytes). CD36 expression was assessed by flow cytometry and was seen to be unaltered by oxidised LDL uptake. All three classes were effective antioxidants, quercetin (P<0.01), ascorbic acid (P<0.01), DHEA (P<0.05). As LDL protein is the control point for LDL metabolism, the degree of oxidation and protection by antioxidants is likely to be of great importance for (patho)-physiological uptake of LDL by monocytes. © 2003 Elsevier B.V. All rights reserved.

Modelling the growth of prion protein aggregates in the brain in variant Creutzfeldt-Jakob disease

Deposition of insoluble prion protein (PrP) in the brain in the form of protein aggregates or deposits is characteristic of the ‘transmissible spongiform encephalopathies’ (TSEs). Understanding the growth and development of these PrP aggregates is important both in attempting to the elucidate of the pathogenesis of prion disease and in the development of treatments designed to prevent or inhibit the spread of prion pathology within the brain. Aggregation and disaggregation of proteins and the diffusion of substances into the developing aggregates (surface diffusion) are important factors in the development of protein aggregates. Mathematical models suggest that if aggregation/disaggregation or surface diffusion is the predominant factor, the size frequency distribution of the resulting protein aggregates in the brain should be described by either a power-law or a log-normal model respectively. This study tested this hypothesis for two different types of PrP deposit, viz., the diffuse and florid-type PrP deposits in patients with variant Creutzfeldt-Jakob disease (vCJD). The size distributions of the florid and diffuse plaques were fitted by a power-law function in 100% and 42% of brain areas studied respectively. By contrast, the size distributions of both types of plaque deviated significantly from a log-normal model in all brain areas. Hence, protein aggregation and disaggregation may be the predominant factor in the development of the florid plaques. A more complex combination of factors appears to be involved in the pathogenesis of the diffuse plaques. These results may be useful in the design of treatments to inhibit the development of protein aggregates in vCJD.

Quantification of vacuolation ('spongiform change'), surviving neurones and prion protein deposition in eleven cases of variant Creutzfeldt-Jakob disease

Vacuolation ('spongiform change') and prion protein (PrP) deposition were quantified in the cerebral cortex, hippocampus, dentate gyrus and molecular layer of the cerebellum in 11 cases of variant Creutzfeldt-Jakob disease (vCJD). The density of vacuoles was greater in the cerebral cortex compared to the hippocampus, dentate gyrus and cerebellum. Within the cortex, vacuole density was significantly greater in the occipital compared to the temporal lobe and the density of surviving neurones was greatest in the occipital lobe. The density of the non-florid PrP plaques was greater in the cerebellum compared to the other brain areas. There were significantly more florid-type PrP plaques in the cerebral cortex compared to the hippocampus and the molecular layer of the cerebellum. No significant correlations were observed between the densities of the vacuoles and the PrP plaques. The densities of vacuoles in the parietal cortex and the non-florid plaques in the frontal cortex were positively correlated with the density of surviving neurones. The densities of the florid and the non-florid plaques were positively correlated in the parietal cortex, occipital cortex, inferior temporal gyrus and dentate gyrus. The data suggest: (i) vacuolation throughout the cerebral cortex, especially in the occipital lobe, but less evident in the hippocampus and molecular layer of the cerebellum; (ii) the non-florid plaques are more common than the florid plaques and predominate in the molecular layer of the cerebellum; and (iii) either the florid plaques develop from the non-florid plaques or both types are morphological variants resulting from the same degenerative process.

Toward the discovery of vaccine adjuvants:coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Fluorescent microplate-based analysis of protein-DNA interactions I:immobilized protein

A simple protein-DNA interaction analysis has been developed using a high-affinity/high-specificity zinc finger protein. In essence, purified protein samples are immobilized directly onto the surface of microplate wells, and fluorescently labeled DNA is added in solution. After incubation and washing, bound DNA is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.2 nM DNA. Since the detection of bound DNA is noninvasive and the protein-DNA interaction is not disrupted during detection, iterative readings may be taken from the same well, after successive alterations in interaction conditions, if required. In this respect, the assay may therefore be considered real time and permits appropriate interaction conditions to be determined quantitatively. The assay format is ideally suited to investigate the interactions of purified unlabeled DNA binding proteins in a high-throughput format.

Signaling pathways initiated by beta-hydroxy-beta-methylbutyrate to attenuate the depression of protein synthesis in skeletal muscle in response to cachectic stimuli

To investigate the mechanism by which beta-hydroxy-beta-methylbutyrate (HMB) attenuates the depression of protein synthesis in the skeletal muscle of cachectic mice, a study has been carried out in murine myotubes in the presence of proteolysis-inducing factor (PIF). PIF inhibited protein synthesis by 50% within 4 h, and this was effectively attenuated by HMB (25-50 muM). HMB (50 muM) alone stimulated protein synthesis, and this was attenuated by rapamycin (27 nM), an inhibitor of mammalian target of rapamycin (mTOR). Further evidence for an involvement of this pathway was shown by an increased phosphorylation of mTOR, the 70-kDa ribosomal S6 kinase (p70(S6k)), and initiation factor 4E-binding protein (4E-BP1) and an increased association of eukaryotic initiation factor 2 (eIF4E) with eIF4G. PIF alone induced a transient (1-2 h) stimulation of phosphorylation of mTOR and p70(S6k). However, in the presence of HMB, phosphorylation of mTOR, p70(S6k), and 4E-BP1 was increased, and inactive 4E-BP1-eIF4E complex was reduced, whereas the active eIF4G.eIF4E complex was increased, suggesting continual stimulation of protein synthesis. HMB alone reduced phosphorylation of elongation factor 2, but this effect was not seen in the presence of PIF. PIF induced autophosphorylation of the double-strand RNA-dependent protein kinase (PKR), leading to phosphorylation of eIF2 on the alpha-subunit, which would inhibit protein synthesis. However, in the presence of HMB, phosphorylation of PKR and eIF2alpha was attenuated, and this was also observed in skeletal muscle of cachectic mice administered HMB (0.25 g/kg). These results suggest that HMB attenuates the depression of protein synthesis by PIF in myotubes through multiple mechanisms.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of eicosapentaenoic acid, protein and amino acids on protein synthesis and degradation in skeletal muscle of cachectic mice

Atrophy of skeletal muscle reduces both the quality and quantity of life of patients with cancer cachexia. Loss of muscle mass is thought to arise from a reduction in protein synthesis combined with an enhanced rate of protein degradation, and few treatments are available to counteract this process. Eicosapentaenoic acid (EPA) has been shown to attenuate the enhanced protein degradation, but to have no effect on protein synthesis. This study examines the effect of EPA combined with a protein and amino-acid supplementation on protein synthesis and degradation in gastrocnemius muscle of mice bearing the cachexia-inducing MAC16 tumour. Muscles from cachectic mice showed an 80% reduction in protein synthesis and about a 50-fold increase in protein degradation compared with muscles from nontumour-bearing mice of the same age and weight. Treatment with EPA (1 g kg-1) daily reduced protein degradation by 88%, but had no effect on protein synthesis. Combination of EPA with casein (5.35 g kg-1) also had no effect on protein synthesis, but when combined with the amino acids leucine, arginine and methionine there was almost a doubling of protein synthesis. The addition of carbohydrate (10.7 g kg-1) to stimulate insulin release had no additional effect. The combination involving the amino acids produced almost a doubling of the ratio of protein synthesis to protein degradation in gastrocnemius muscle over that of EPA alone. No treatment had a significant effect on tumour growth rate, but the inclusion of amino acids had a more significant effect on weight loss induced by the MAC16 tumour than that of EPA alone. The results suggest that combination therapy of cancer cachexia involving both inhibition of the enhanced protein degradation and stimulation of the reduced protein synthesis may be more effective than either treatment alone. © 2004 Cancer Research UK.

Role of protein kinase C and NF-kappa B in proteolysis-inducing factor-induced proteasome expression in C2C12 myotubes

Proteolysis-inducing factor (PIF) is a sulphated glycoprotein produced by cachexia-inducing tumours, which initiates muscle protein degradation through an increased expression of the ubiquitin–proteasome proteolytic pathway. The role of kinase C (PKC) in PIF-induced proteasome expression has been studied in murine myotubes as a surrogate model of skeletal muscle. Proteasome expression induced by PIF was attenuated by 4alpha-phorbol 12-myristate 13-acetate (100 nM) and by the PKC inhibitors Ro31-8220 (10 muM), staurosporine (300 nM), calphostin C (300 nM) and Gö 6976 (200 muM). Proteolysis-inducing factor-induced activation of PKCalpha, with translocation from the cytosol to the membrane at the same concentration as that inducing proteasome expression, and this effect was attenuated by calphostin C. Myotubes transfected with a constitutively active PKCalpha (pCO2) showed increased expression of proteasome activity, and a longer time course, compared with their wild-type counterparts. In contrast, myotubes transfected with a dominant-negative PKCalpha (pKS1), which showed no activation of PKCalpha in response to PIF, exhibited no increase in proteasome activity at any time point. Proteolysis-inducing factor-induced proteasome expression has been suggested to involve the transcription factor nuclear factor-kappaB (NF-kappaB), which may be activated through PKC. Proteolysis-inducing factor induced a decrease in cytosolic I-kappaBalpha and an increase in nuclear binding of NF-kappaB in pCO2, but not in pKS1, and the effect in wild-type cells was attenuated by calphostin C, confirming that it was mediated through PKC. This suggests that PKC may be involved in the phosphorylation and degradation of I-kappaBalpha, induced by PIF, necessary for the release of NF-kappaB from its inactive cytosolic complex.

Induction of protein catabolism in myotubes by 15(S)-hydroxyeicosatetraenoic acid through increased expression of the ubiquitin-proteasome pathway

The potential role of 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) as an intracellular signal for increased protein catabolism and induction of the expression of key components of the ubiquitin-proteasome proteolytic pathway induced by a tumour cachectic factor, proteolysis-inducing factor has been studied in murine C2C12 myotubes. 15(S)-HETE induced protein degradation in these cells with a maximal effect at concentrations between 78 and 312 nM. The effect was attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). There was an increase in 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the proteasome, in the same concentration range as that inducing total protein degradation, and this effect was also attenuated by EPA. 15(S)-hydroxyeicosatetraenoic acid also increased maximal expression of mRNA for proteasome subunits C2 and C5, as well as the ubiquitin-conjugating enzyme, E214k, after 4 h incubation, as determined by quantitative competitive RT-PCR. The concentrations of 15-HETE affecting gene expression were the same as those inducing protein degradation. Western blotting of cellular supernatants of myotubes treated with 15(S)-HETE for 24 h showed increased expression of p42, an ATPase subunit of the regulatory complex at similar concentrations, as well as a decrease in expression of myosin in the same concentration range. 15(S)-hydroxyeicosatetraenoic acid activated binding of nuclear factor-κB (NF-κB) in the myotube nucleus and stimulated degradation of 1-κBα. The effect on the NF-κB/1-κBα system was attenuated by EPA. In addition, the NF-κB inhibitor peptide SN50 attenuated the increased chymotrypsin-like enzyme activity in the presence of 15(S)-HETE. These results suggest that 15(S)-HETE induces degradation of myofibrillar proteins in differentiated myotubes through an induction of an increased expression of the regulatory components of the ubiquitin-proteasome proteolytic pathway possibly through the intervention of the nuclear transcription factor NF-κB, and that this process is inhibited by EPA. © 2003 Cancer Research UK.

Characterisation of protein stability in rod-insert vaginal rings

A major goal in vaccine development is elimination of the 'cold chain', the transport and storage system for maintenance and distribution of the vaccine product. This is particularly pertinent to liquid formulation of vaccines. We have previously described the rod-insert vaginal ring (RiR) device, comprising an elastomeric body into which are inserted lyophilised, rod-shaped, solid drug dosage forms, and having potential for sustained mucosal delivery of biomacromolecules, such as HIV envelope protein-based vaccine candidates. Given the solid, lyophilised nature of these insert dosage forms, we hypothesised that antigen stability may be significantly increased compared with more conventional solubilised vaginal gel format. In this study, we prepared and tested vaginal ring devices fitted with lyophilised rod inserts containing the model antigen bovine serum albumin (BSA). Both the RiRs and the gels that were freeze-dried to prepare the inserts were evaluated for BSA stability using PAGE, turbidimetry, microbial load, MALDI-TOF and qualitative precipitate solubility measurements. When stored at 4°C, but not when stored at 40°C/75% RH, the RiR formulation offered protection against structural and conformational changes to BSA. The insert also retained matrix integrity and release characteristics. The results demonstrate that lypophilised gels can provide relative protection against degradation at lower temperatures compared to semi-solid gels. The major mechanism of degradation at 40°C/75% RH was shown to be protein aggregation. Finally, in a preliminary study, we found that addition of trehalose to the formulation significantly reduces the rate of BSA degradation compared to the original formulation when stored at 40°C/75% RH. Establishing the mechanism of degradation, and finding that degradation is decelerated in the presence of trehalose, will help inform further development of RiRs specifically and polymer based freeze-dried systems in general.

Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.

LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

Fluorescent microplate-based analysis of protein-DNA interactions II:immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.