Amyloid β Induces Early Changes in the Ribosomal Machinery, Cytoskeletal Organization and Oxidative Phosphorylation in Retinal Photoreceptor Cells

Amyloid β (Aβ) accumulation and its aggregation is characteristic molecular feature of the development of Alzheimer’s disease (AD). More recently, Aβ has been suggested to be associated with retinal pathology associated with AD, glaucoma and drusen deposits in age related macular degeneration (AMD). In this study, we investigated the proteins and biochemical networks that are affected by Aβ in the 661 W photoreceptor cells in culture. Time and dose dependent effects of Aβ on the photoreceptor cells were determined utilizing tandem mass tag (TMT) labeling-based quantitative mass-spectrometric approach. Bioinformatic analysis of the data revealed concentration and time dependent effects of the Aβ peptide stimulation on various key biochemical pathways that might be involved in mediating the toxicity effects of the peptide. We identified increased Tau phosphorylation, GSK3β dysregulation and reduced cell viability in cells treated with Aβ in a dose and time dependent manner. This study has delineated for the first-time molecular networks in photoreceptor cells that are impacted early upon Aβ treatment and contrasted the findings with a longer-term treatment effect. Proteins associated with ribosomal machinery homeostasis, mitochondrial function and cytoskeletal organization were affected in the initial stages of Aβ exposure, which may provide key insights into AD effects on the photoreceptors and specific molecular changes induced by Aβ peptide

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei

BackgroundTrichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.ResultsTo address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.ConclusionThis study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production

Utveckling av tekniker och metoder för kvantitativ analys av endogena substanser med mikrokolonn vätskekromatografi sammankopplad med masspektrometri

Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests. The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter. The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides. The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter. The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries. Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism

Utveckling av tekniker och metoder för kvantitativ analys av endogena substanser med mikrokolonn vätskekromatografi sammankopplad med masspektrometri

Liquid chromatography-mass spectrometry (LC-MS) is a powerful technique for the analysis of endogenous compounds. The introduction of electrospray ionization (ESI) as an interface between LC and MS has contributed strongly to a trend towards miniaturization of LC, due to the possibility to perform ESI at low flow rates. In this thesis, several aspects regarding the design of miniaturized LC systems and electrospray emitters were investigated. In addition miniaturized LC-ESI-MS have been used for the qualitative and quantitative analysis of endogenous polar compounds, peptides and protein digests. The performance of miniaturized LC-MS was compared using different electrospray emitter configurations. The results indicated that the efficiency of the LC system is rather independent of the configuration of the emitter. The lifetime of gold-coated fused silica electrospray emitters based on vapor deposited adhesion layers of titanium were investigated. The long lifetime of the emitter facilitates the use in LC-MS experiments, exemplified LC-MS by analysis of neuropeptides. The ESI voltage is shown to interfere with liquid chromatographic separations performed in packed porous graphitic carbon capillary column. This interference is ascribed to the presence of an electric field over the conductive column in absence of a ground point between the column and the ESI emitter. The solid supported enhanced microdialysis for analysis of neuropeptides were compared with conventional microdialysis. The difference between the two methodologies were evaluated by LC-MS analysis of the microdialysates. The solid supported method gave in general higher relative recoveries. Finally, a method of standard addition was developed to determine total level of tryptophan and two of its metabolites in human plasma by capillary LC-ESI tandem mass spectrometry. The method was applied in a clinical study of multiple scleroses patients treated with cytokines (IFN Beta 1a, 1b). The results show that the intervention effects the tryptophan metabolism

Micro-capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry—apowerful tool for peptide and protein identification.

In order to be able to study complex biological samples, a micro-capillary liquid chromatography system was coupled to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. The setup was tested on a tryptic digest of bovine serum albumin, which resulted in high sequence coverage (> 92% the protein

Proteome of Staphylococcus aureus Biofilm Changes Significantly with Aging

Staphylococcus aureus is a notorious biofilm-producing pathogen that is frequently isolated from implantable medical device infections. As biofilm ages, it becomes more tolerant to antimicrobial treatment leading to treatment failure and necessitating the costly removal of infected devices. In this study, we performed in-solution digestion followed by TMT-based high-throughput mass spectrometry and investigated what changes occur in the proteome of S. aureus biofilm grown for 3-days and 12-days in comparison with 24 h planktonic. It showed that proteins associated with biosynthetic processes, ABC transporter pathway, virulence proteins, and shikimate kinase pathway were significantly upregulated in a 3-day biofilm, while proteins associated with sugar transporter, degradation, and stress response were downregulated. Interestingly, in a 3-day biofilm, we observed numerous proteins involved in the central metabolism pathways which could lead to biofilm growth under diverse environments by providing an alternative metabolic route to utilize energy. In 12-day biofilms, proteins associated with peptidoglycan biosynthesis, sugar transporters, and stress responses were upregulated, whereas proteins associated with ABC transporters, DNA replication, and adhesion proteins were downregulated. Gene Ontology analysis revealed that more proteins are involved in metabolic processes in 3dwb compared with 12dwb. Furthermore, we observed significant variations in the formation of biofilms resulting from changes in the level of metabolic activity in the different growth modes of biofilms that could be a significant factor in S. aureus biofilm maturation and persistence. Collectively, potential marker proteins were identified and further characterized to understand their exact role in S. aureus biofilm development, which may shed light on possible new therapeutic regimes in the treatment of biofilm-related implant-associated infections