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Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
DNA fragments incorporating the coding sequences of the genes of interest can be cloned in frame with N- or C-terminally tagged GFP and fRFP using established (e.g. pEGFP-N1, Clontech; pmKate2-N, Evrogen) or bespoke vectors. It is recommended that for each target protein, a GFP and an fRFP tagged construct be created because, in our experience, the nature of the protein-tag can have an effect on protein expression, even if the reasons for this are not necessarily clear. It is further important to keep in mind that GFP has severely diminished fluorescence intensity when in acidic compartments (Haupts et al., 1998), such as the lysosome, and that FRET will work poorly or not at all when fluorescent proteins are on opposite sides of the membrane. Therefore, it is recommended to use known protein structures or topologies to place the fluorescent tag on the protein terminus that is cytosolic for each target protein. In cases where protein topology is unknown, both the N- and C-termini should be separately tagged for each fluorescent protein. fRFP is recommended over shorter wavelength RFPs (e.g. DsRed or mCherry, Clontech) because the longer wavelength excitation spectrum of fRFP has essentially no overlap with the 488-nm laser, meaning very little background signal will arise from direct laser stimulation.
Modifications of the Cell Surface Leading to Cell Elimination
Published in Alvaro Macieira-Coelho, Molecular Basis of Aging, 2017
Oxidized proteins and oxidized lipids are preferentially degraded105 as part of a repair mechanism or cellular homeostasis. Hydroperoxylinoleoyl-containing phosphatidylcholine in lipid monolayers extends their surface area and is preferentially hydrolyzed by phospholipase A2.106 The occurrence of dityrosine in hemoglobin seems to trigger a selective proteolysis of modified hemoglobin by an endogenous protease called macroxyproteinase.104 Others immunoprecipitated the macroxyproteinase and claimed that this had no effect on a selective proteolysis of oxidized hemoglobin which yet was degraded by a metalloinsulinase.107 As the degrading enzymes also lose activity as the cell ages, modified forms of proteins tend to accumulate late in the life span. Selective degradation of modified proteins serves the purpose of repair by “de novo synthesis” in cells having an ongoing protein synthesis. Erythrocytes no longer synthesize proteins and therefore reveal oxidative damage in lipids, hemoglobin, and membrane proteins. Some of this accumulating damage induces changes in the surface protein topology and results in opsonization.
Probable role for major facilitator superfamily domain containing 6 (MFSD6) in the brain during variable energy consumption
Published in International Journal of Neuroscience, 2020
Sonchita Bagchi, Emelie Perland, Kimia Hosseini, Johanna Lundgren, Noura Al-Walai, Sania Kheder, Robert Fredriksson
The transmembrane (TM) prediction software ‘Protter’ [24] was used for interactive protein feature visualisation and integration with experimental proteomic data to obtain the protein topology of human MFSD6, MFSD8 and MFSD10. The fully automated homology Swiss-Model program [25] was used to build 3 D models, where MFS transporters were used as templates. A structurally known MFS transporter protein found in several gram-negative bacteria, YajR, [26] was used as template for the MFSD6, MFSD8 and MFSD10 models, providing GMQE scores at 27, 44 and 50 respectively. Manual inspections of the alignments were performed to verify that conserved MFS motifs, such as the MFS fold [39] were aligned to enhance credibility of the models. Tertiary structures were finalised using Swiss-Pdb Viewer [40] and Adobe Photoshop CS6.
Biological membranes in EV biogenesis, stability, uptake, and cargo transfer: an ISEV position paper arising from the ISEV membranes and EVs workshop
Published in Journal of Extracellular Vesicles, 2019
Ashley E. Russell, Alexandra Sneider, Kenneth W. Witwer, Paolo Bergese, Suvendra N. Bhattacharyya, Alexander Cocks, Emanuele Cocucci, Uta Erdbrügger, Juan M. Falcon-Perez, David W. Freeman, Thomas M. Gallagher, Shuaishuai Hu, Yiyao Huang, Steven M. Jay, Shin-ichi Kano, Gregory Lavieu, Aleksandra Leszczynska, Alicia M. Llorente, Quan Lu, Vasiliki Mahairaki, Dillon C. Muth, Nicole Noren Hooten, Matias Ostrowski, Ilaria Prada, Susmita Sahoo, Tine Hiorth Schøyen, Lifu Sheng, Deanna Tesch, Guillaume Van Niel, Roosmarijn E. Vandenbroucke, Frederik J. Verweij, Ana V. Villar, Marca Wauben, Ann M. Wehman, Hang Yin, David Raul Francisco Carter, Pieter Vader
The functional capacity of an EV is likely dependent on membrane protein topology. Overall, membrane protein composition appears to be heavily influenced by EV size, the cell type of origin and cellular activation state. One technique to study EV membrane composition is free radical incorporation or electron-dense lipid labelling. Incorporating free radicals in membrane samples can provide useful topological information on both the membrane and/or associated/integral proteins based on the preferential localization of the radical used [106,107]. On the other hand, using lipids containing radicals at different positions in the tail could help provide information about the transmembrane region itself [108]. Some new techniques to make pseudo-membranes containing differently shaped phospholipids can allow for further study of how composition can influence membrane curvature and fusion by using nuclear magnetic resonance (NMR) [109].
Covalent labeling and mass spectrometry reveal subtle higher order structural changes for antibody therapeutics
Published in mAbs, 2019
Patanachai Limpikirati, John E. Hale, Mark Hazelbaker, Yongbo Huang, Zhiguang Jia, Mahdieh Yazdani, Eric M. Graban, Robert C. Vaughan, Richard W. Vachet
More residues undergo significant changes in modification extent after heating at 55°C for 4 h, and these changes can be found in all rituximab domains, particularly in the VH and VL domains of the Fab region (Table 2). Most of the changes in each domain are increases in labeling, with an exception of the VH and CL domains where there is a similar or higher number of labeling decreases. Almost twice as many labeling changes occur at His and Lys residues upon heating to 55°C compared to heating at 45°C, although most of the changes are characterized as low or medium changes. Most of the labeling changes at Tyr, Ser, and Thr are characterized as medium or high changes. Overall, these results indicate likely changes in protein topology, although the moderate labeling changes at His and Lys residues suggest somewhat modest structural changes. In contrast to the heat stress at 45°C, after heating at 55°C, the sites whose label levels undergo significant changes are found to cluster in the Fab region of the protein, especially in VH, VL, and CL domains (Figure 3(b)). Such clustering suggests localized structural changes in the Fab region of rituximab. Similar clustering of residues undergoing labeling changes are not observed in the Fc region (Figure 3(b)).