CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H2O2) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells. © 2019Peer reviewe

Glyphosate does not substitute for glycine in proteins of actively dividing mammalian cells

Glyphosate (N-phosphonomethyl glycine) and its commercial herbicide formulations have been shown to exert toxicity via various mechanisms. It has been asserted that glyphosate substitutes for glycine in polypeptide chains leading to protein misfolding and toxicity. However, as no direct evidence exists for glycine to glyphosate substitution in proteins, including in mammalian organisms, we tested this claim by conducting a proteomics analysis of MDA-MB-231 human breast cancer cells grown in the presence of 100 mg/L glyphosate for 6 days. Protein extracts from three treated and three untreated cell cultures were analysed as one TMT-6plex labelled sample, to highlight a specific pattern (+/+/+/−/−/−) of reporter intensities for peptides bearing true glyphosate treatment induced-post translational modifications as well as allowing an investigation of the total proteome

Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.

We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a cell-free translation system. Here this method is combined with the conformational sensitivity of 19F NMR to monitor the folded state of the expressed protein. This report describes the optimisation of 6-fluorotryptophan incorporation in a His-tagged human serum retinol-binding protein (RBP), a disulphide bonded beta-barrel protein. Appropriate reagent concentrations for producing fluorine labelled RBP in a cell-free translation system are described. It is shown that 19F NMR is a suitable method for monitoring the production of correctly folded protein from a high-throughput expression system

H Nuclear Magnetic Resonance (NMR) Metabolomic Study of Chronic Organophosphate Exposure in Rats

metabolite

Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

Bacterial inclusion bodies (IBs) have long been considered as inactive, unfolded waste material produced by heterologous overexpression of recombinant genes. In industrial applications, they are occasionally used as an alternative in cases where a protein cannot be expressed in soluble form and in high enough amounts. Then, however, refolding approaches are needed to transform inactive IBs into active soluble protein. While anecdotal reports about IBs themselves showing catalytic functionality/activity (CatIB) are found throughout literature, only recently, the use of protein engineering methods has facilitated the on-demand production of CatIBs. CatIB formation is induced usually by fusing short peptide tags or aggregation-inducing protein domains to a target protein. The resulting proteinaceous particles formed by heterologous expression of the respective genes can be regarded as a biologically produced bionanomaterial or, if enzymes are used as target protein, carrier-free enzyme immobilizates. In the present contribution, we review general concepts important for CatIB production, processing, and application