Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Entropy of the Monomeric Forms of Formic Acid and Acetic Acid

A recent measurement of the entropy of gaseous formic acid at its equilibrium vapor pressure is combined with vapor density data to give 60.0±0.3 as the entropy of the monomer at 25° and one atmosphere. The corresponding dimer entropy is 83.1±0.3 e.u. Pauling's residual entropy of R ln 2 per dimer weight for random orientation of hydrogen bonds in the crystal has been included in the above figures in order to allow a reasonable entropy for the torsional motion of the hydroxyl group in the monomer. The same correction has been applied to a previously published result for the acetic acid monomer to yield 70.1±1.0 as a revised value. It is highly probable that only a single significant potential minimum of undetermined breadth and depth occurs in the rotational cycle of the hydroxyl group.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/70884/2/JCPSA6-10-9-582-1.pd

Obstructive sleep apnea associated with Diabetes mellitus Type 2: a proteomic study

Background: We previously showed that Obstructive sleep apnea (OSA), a common public health concern causing deleterious cardiometabolic dysfunction, induced proteomic alterations in red blood cells (RBC) such as changes in the redox-oligomeric state of peroxiredoxin 2 (PRDX2)1-2. Herein, we aimed to investigate whether OSA patients with Type 2 Diabetes Mellitus before and after positive airway pressure (PAP) treatment present similar changes in the RBC antioxidant protein PRDX2 to better understand the molecular basic mechanisms associated with OSA and OSA outcomes. Methods: RBC samples from control snorers (n=22 being 3 diabetics) and OSA patients before and after six month of PAP-treatment (n=29 being 8 diabetics) were analysed by non-reducing western blot using antibody against PRDX2 or PRDXSO2/3 to measure the total and overoxidized levels of monomeric/dimeric/multimeric forms of PRDX2. Results: We confirmed previously data by showing that in OSA RBC the overoxidation on the monomeric forms of PRDX2 was higher compared to controls. After PAP treatment, this overoxidation decreased followed by an increase of multimeric-overoxidized forms of PRDX2 described to be associated with chaperone protective function. In contrast, the level of PRDX2 monomers in RBC diabetic OSA, although higher abundant its overoxidation level was much lower than those observed in OSA without comorbidity and did not significant change after treatment. Moreover, the level of PAP-induced PRDX2-overoxidized-multimers was also lower in these diabetic OSA patients. The level of overoxidized monomeric/dimeric forms of PRDX2 correlated negatively with levels of insulin / triglycerides and HbA1C, respectively. After PAP, the level of (overoxidized) PRDX2SO2/3 multimers correlated positively with adrenaline levels. Conclusions: The redox/oligomeric state of RBC PRDX2 that is regulated by overoxidation of the active cysteines was differentially modulated in diabetic OSA patients compared to OSA without this comorbidity. PAP-induced overoxidized oligo forms of PRDX2 that is associated with chaperone protective function showed decreased in OSA patients with diabetes. The clinical impact of these findings needs further investigation and validation.Project partially supported by Harvard Medical School-Portugal Program (HMSP-ICJ/0022/2011), ToxOmics - Centre for Toxicogenomics and Human Health (FCT-UID/BIM/00009/2013).info:eu-repo/semantics/publishedVersio

Naturally occurring autoantibodies against beta-amyloid: investigating their role in transgenic animal and in vitro models of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder primarily affecting regions of the brain responsible for higher cognitive functions. Immunization against β-amyloid (Aβ) in animal models of AD has been shown to be effective on the molecular level but also on the behavioral level. Recently, we reported naturally occurring autoantibodies against Aβ (NAbs-Aβ) being reduced in Alzheimer's disease patients. Here, we further investigated their physiological role: in epitope mapping studies, NAbs-Aβ recognized the mid-/C-terminal end of Aβ and preferentially bound to oligomers but failed to bind to monomers/fibrils. NAbs-Aβ were able to interfere with Aβ peptide toxicity, but NAbs-Aβ did not readily clear senile plaques although early fleecy-like plaques were reduced. Administration of NAbs-Aβ in transgenic mice improved the object location memory significantly, almost reaching performance levels of wild-type control mice. These findings suggest a novel physiological mechanism involving NAbs-Aβ to dispose of proteins or peptides that are prone to forming toxic aggregates

Baculovirus Per Os Infectivity Factors Form a Complex on the Surface of Occlusion-Derived Virus

Five highly conserved per os infectivity factors, PIF1, PIF2, PIF3, PIF4, and P74, have been reported to be essential for oral infectivity of baculovirus occlusion-derived virus (ODV) in insect larvae. Three of these proteins, P74, PIF1, and PIF2, were thought to function in virus binding to insect midgut cells. In this paper evidence is provided that PIF1, PIF2, and PIF3 form a stable complex on the surface of ODV particles of the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). The complex could withstand 2% SDS-5% ß-mercaptoethanol with heating at 50°C for 5 min. The complex was not formed when any of the genes for PIF1, PIF2, or PIF3 was deleted, while reinsertion of these genes into AcMNPV restored the complex. Coimmunoprecipitation analysis independently confirmed the interactions of the three PIF proteins and revealed in addition that P74 is also associated with this complex. However, deletion of the p74 gene did not affect formation of the PIF1-PIF2-PIF3 complex. Electron microscopy analysis showed that PIF1 and PIF2 are localized on the surface of the ODV with a scattered distribution. This distribution did not change for PIF1 or PIF2 when the gene for PIF2 or PIF1 protein was deleted. We propose that PIF1, PIF2, PIF3, and P74 form an evolutionarily conserved complex on the ODV surface, which has an essential function in the initial stages of baculovirus oral infectio

Immunological identification of the alternative oxidase of Acanthamoeba castellanii mitochondria

AbstractMitochondria of the protozoa Acanthamoeba castellanii possess a cyanide-insensitive oxidase cross-reacting with monoclonal antibodies raised against the plant alternative oxidase. Immunoblotting revealed three monomeric forms (38, 35, and 32 kDa) and very low amounts of a single 65 kDa dimeric form. Cross-linking studies suggest that while in plants the alternative oxidase occurs as a dimer, in amoeba it functions as a monomer. Immunologically detectable protein levels change with the age of amoeba cell culture. Increased amounts of the 35 kDa protein are accompanied by an increase in the activity of cyanide-resistant respiration

RNA aptamers generated against oligomeric Abeta40 recognize common amyloid aptatopes with low specificity but high sensitivity.

Aptamers are useful molecular recognition tools in research, diagnostics, and therapy. Despite promising results in other fields, aptamer use has remained scarce in amyloid research, including Alzheimer's disease (AD). AD is a progressive neurodegenerative disease believed to be caused by neurotoxic amyloid beta-protein (Abeta) oligomers. Abeta oligomers therefore are an attractive target for development of diagnostic and therapeutic reagents. We used covalently-stabilized oligomers of the 40-residue form of Abeta (Abeta40) for aptamer selection. Despite gradually increasing the stringency of selection conditions, the selected aptamers did not recognize Abeta40 oligomers but reacted with fibrils of Abeta40, Abeta42, and several other amyloidogenic proteins. Aptamer reactivity with amyloid fibrils showed some degree of protein-sequence dependency. Significant fibril binding also was found for the naïve library and could not be eliminated by counter-selection using Abeta40 fibrils, suggesting that aptamer binding to amyloid fibrils was RNA-sequence-independent. Aptamer binding depended on fibrillogenesis and showed a lag phase. Interestingly, aptamers detected fibril formation with > or =15-fold higher sensitivity than thioflavin T (ThT), revealing substantial beta-sheet and fibril formation undetected by ThT. The data suggest that under physiologic conditions, aptamers for oligomeric forms of amyloidogenic proteins cannot be selected due to high, non-specific affinity of oligonucleotides for amyloid fibrils. Nevertheless, the high sensitivity, whereby aptamers detect beta-sheet formation, suggests that they can serve as superior amyloid recognition tools