An early immunoreactive folding intermediate of the tryptophan synthase β2 subunit is a ‘molten globule’

The refolding kinetics of the tryptophan synthase β2 subunit have been investigated by circular dichroism (CD) and binding of a fluorescent hydrophobic probe (ANS), using the stopped-flow technique. The kinetics of regain of the native far UV CD signal show that, upon refolding of urea denatured β2, more than half of the protein secondary structure is formed within the dead time of the CD stopped-flow apparatus (0.013 s). On the other hand, upon refolding of guanidine unfolded β2 the fluorescence of ANS passes through a maximum after about 1 s and then ‘slowly’ decreases. These results show the accumulation, in the 1–10 s time range, of an early transient folding intermediate which has a pronounced secondary structure and a high affinity for ANS. In this time range, the near UV CD remains very low. This transient intermediate thus appears to have all the characteristics of the ‘molten globule’ state [(1987) FEBS Lett. 224, 9-13]. Moreover, by comparing the intrinsic time of the disappearance of this transient intermediate (t 35 s) with the time of formation of the previously characterized [(1988) Biochemistry 27, 7633-7640] early imuno-reactive intermediate recognized by a monoclonal antibody (t 12 s), it is shown that this native-like epitope forms within the ‘molten globule’, before the tight packing of the protein side chains

Chemical analysis of Greek pollen - Antioxidant, antimicrobial and proteasome activation properties

<p>Abstract</p> <p>Background</p> <p>Pollen is a bee-product known for its medical properties from ancient times. In our days is increasingly used as health food supplement and especially as a tonic primarily with appeal to the elderly to ameliorate the effects of ageing. In order to evaluate the chemical composition and the biological activity of Greek pollen which has never been studied before, one sample with identified botanical origin from sixteen different common plant taxa of Greece has been evaluated.</p> <p>Results</p> <p>Three different extracts of the studied sample of Greek pollen, have been tested, in whether could induce proteasome activities in human fibroblasts. The water extract was found to induce a highly proteasome activity, showing interesting antioxidant properties. Due to this activity the aqueous extract was further subjected to chemical analysis and seven flavonoids have been isolated and identified by modern spectral means. From the methanolic extract, sugars, lipid acids, phenolic acids and their esters have been also identified, which mainly participate to the biosynthetic pathway of pollen phenolics. The total phenolics were estimated with the Folin-Ciocalteau reagent and the total antioxidant activity was determined by the DPPH method while the extracts and the isolated compounds were also tested for their antimicrobial activity by the dilution technique.</p> <p>Conclusions</p> <p>The Greek pollen is rich in flavonoids and phenolic acids which indicate the observed free radical scavenging activity, the effects of pollen on human fibroblasts and the interesting antimicrobial profile.</p

The antioxidant enzyme peroxiredoxin-2 is depleted in lymphocytes seven days after ultra-endurance exercise

Purpose: Peroxiredoxin-2 (PRDX-2) is an antioxidant and chaperone-like protein critical for cell function. This study examined whether the levels of lymphocyte PRDX-2 are altered over one month following ultra-endurance exercise. Methods: Nine middle-aged men undertook a single-stage, multi-day 233 km (145 mile) ultra-endurance running race. Blood was collected immediately before (PRE), upon completion/retirement (POST), and following the race at DAY 1, DAY 7 and DAY 28. Lymphocyte lysates were examined for PRDX-2 by reducing SDS-PAGE and western blotting. In a sub-group of men who completed the race (n = 4) PRDX-2 oligomeric state (indicative of redox status) was investigated. Results: Ultra-endurance exercise caused significant changes in lymphocyte PRDX-2 (F (4,32) 3.409, p=0.020, ?(2) =0.299): seven-days after the race, PRDX-2 levels in lymphocytes had fallen to 30% of pre-race values (p=0.013) and returned to near-normal levels at DAY 28. Non-reducing gels demonstrated that dimeric PRDX-2 (intracellular reduced PRDX-2 monomers) was increased in 3 of 4 race completers immediately post-race, indicative of an "antioxidant response". Moreover, monomeric PRDX-2 was also increased immediately post-race in 2 of 4 race-completing subjects, indicative of oxidative damage, which was not detectable by DAY 7. Conclusions: Lymphocyte PRDX-2 was decreased below normal levels 7 days after ultra-endurance exercise. Excessive accumulation of reactive oxygen species induced by ultra-endurance exercise may underlie depletion of lymphocyte PRDX-2 by triggering its turnover after oxidation. Low levels of lymphocyte PRDX-2 could influence cell function and might, in part, explain reports of dysregulated immunity following ultra-endurance exercise

An in silico model of the ubiquitin-proteasome system that incorporates normal homeostasis and age-related decline

BACKGROUND: The ubiquitin-proteasome system is responsible for homeostatic degradation of intact protein substrates as well as the elimination of damaged or misfolded proteins that might otherwise aggregate. During ageing there is a decline in proteasome activity and an increase in aggregated proteins. Many neurodegenerative diseases are characterised by the presence of distinctive ubiquitin-positive inclusion bodies in affected regions of the brain. These inclusions consist of insoluble, unfolded, ubiquitinated polypeptides that fail to be targeted and degraded by the proteasome. We are using a systems biology approach to try and determine the primary event in the decline in proteolytic capacity with age and whether there is in fact a vicious cycle of inhibition, with accumulating aggregates further inhibiting proteolysis, prompting accumulation of aggregates and so on. A stochastic model of the ubiquitin-proteasome system has been developed using the Systems Biology Mark-up Language (SBML). Simulations are carried out on the BASIS (Biology of Ageing e-Science Integration and Simulation) system and the model output is compared to experimental data wherein levels of ubiquitin and ubiquitinated substrates are monitored in cultured cells under various conditions. The model can be used to predict the effects of different experimental procedures such as inhibition of the proteasome or shutting down the enzyme cascade responsible for ubiquitin conjugation. RESULTS: The model output shows good agreement with experimental data under a number of different conditions. However, our model predicts that monomeric ubiquitin pools are always depleted under conditions of proteasome inhibition, whereas experimental data show that monomeric pools were depleted in IMR-90 cells but not in ts20 cells, suggesting that cell lines vary in their ability to replenish ubiquitin pools and there is the need to incorporate ubiquitin turnover into the model. Sensitivity analysis of the model revealed which parameters have an important effect on protein turnover and aggregation kinetics. CONCLUSION: We have developed a model of the ubiquitin-proteasome system using an iterative approach of model building and validation against experimental data. Using SBML to encode the model ensures that it can be easily modified and extended as more data become available. Important aspects to be included in subsequent models are details of ubiquitin turnover, models of autophagy, the inclusion of a pool of short-lived proteins and further details of the aggregation process

Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against Influenza A M2 protein

Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection

A Conformation-Sensitive Monoclonal Antibody against the A2 Domain of von Willebrand Factor Reduces Its Proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13

Essential role of proteasomes in maintaining self-renewal in neural progenitor cells

Protein turnover and homeostasis are regulated by the proteasomal system, which is critical for cell function and viability. Pluripotency of stem cells also relies on normal proteasomal activity that mitigates senescent phenotypes induced by intensive cell replications, as previously demonstrated in human bone marrow stromal cells. In this study, we investigated the role of proteasomes in self-renewal of neural progenitor cells (NPCs). Through both in vivo and in vitro analyses, we found that the expression of proteasomes was progressively decreased during aging. Likewise, proliferation and self-renewal of NPCs were also impaired in aged mice, suggesting that the down-regulation of proteasomes might be responsible for this senescent phenotype. Lowering proteasomal activity by loss-of-function manipulations mimicked the senescence of NPCs both in vitro and in vivo; conversely, enhancing proteasomal activity restored and improved self-renewal in aged NPCs. These results collectively indicate that proteasomes work as a key regulator in promoting self-renewal of NPCs. This potentially provides a promising therapeutic target for age-dependent neurodegenerative diseases