An important element for efficient cell surface modification: reaction pH

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Tools to study retrograde transport

The different analysis methods to study retrograde transport are listed in Table 3. The advantage and disadvantage of those methods are also shown. The currently most popular tool in cell biology is immunofuorescence to study the intracellular distribution of toxins by microscopy, followed by an analysis of their relocalization after inhibition of retrograde transport (Johannes, Tenza et al. 1997; Amessou, Popoff et al. 2006). The toxins can be labeled with fluorescent dyes, after which they can be followed without a need for immunolabeling. Furthermore, during the initial phase of their uptake into cells, the toxins are transported in a unidirectional manner. This is different from the pool of endogenous proteins that in most cases are at equilibrium between forward and backward movements. In most cases, people use only the receptor-binding parts of the toxins in cell biology studies. These usually preserve the intracellular transport characteristics of the holotoxins without being themselves toxic. The coupling between HRP and toxins allows the observation of the cytosolic surface of organelles by electron microscopy using a technique called the whole mount. The study of endosomal coats in the retrograde route is an example for this technique (Saint-Pol, Yelamos et al. 2004). A limitation of immunofluorescence is that it is not very quantitative. STxB-based conjugates have therefore been developed to quantitatively study the different transport steps that constitute the retrograde route. Retrograde transport to the Golgi apparatus can be measured by using a specific peptide sequence that is a recognition sequence for TGN-localized sulfotransferase. When a STxB molecule that carries a tandem of this peptide sequence, termed STxB-Sulf2, reaches the TGN by retrograde transport from the plasma membrane, sulfotransferase catalyzes the transfer of inorganic sulfate from the medium onto the conserved peptide sequence. This modification can be quantitatively detected with radioactive sulfate. This technique on which one of my strategies was based will be described in detail in the next chapter.

The functions and methods of proteomics

The most significant breakthrough in proteomics is the mass spectrometric identification of gel-separated proteins (Figure 9). The proteins to be analyzed are isolated from cell lysates by biochemical fractionation or affinity purification, often followed by gel electrophoresis. Usually, the proteins are then digested with a protease such as trypsin. The mass spectrometer is used to determine the masses of the digested peptides. Thereby, a mass map can be obtained. This mass map is then compared with predicted mass maps of proteins within the database to eventually identify the proteins.
The basic components of all mass spectrometers are the same. These are composed from an ionization source, a mass analyzer, and an ion detector. The ionization source is used to convert molecules into gas phase ions. For protein analysis, the two most common types of ionization sources are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI). The development of ESI for the analysis of biological macromolecules (Kebarle and Verkerk 2009) was rewarded with the attribution of the Nobel Prize in Chemistry to John Bennett Fenn in 2002.

The two-dimensional gel approach

Two-dimensional (2D) gel is widely used to separate large amount of proteins. By combining 2D gel with different biological techniques like RNAi, the 2D gel can also be utilized to screen and identify new proteins and their functions. Cells are first treated in different conditions which cause distinguished proteomes. Crude protein mixture is obtained by harvesting cells. It is then applied to a “first dimension” gel strip that separates the proteins based on their isoelectric points. After the first separation, the strip is applied to a “second dimension” SDS-PAGE where proteins are denatured and separated in function of their size. The gels are then fixed and the proteins are visualized by silver staining. Unique spots in either stained gel could be of interest and are further recorded and characterized.

Quantitative comparison by mass spectrometry

Differential-display proteomics can also be performed using mass spectrometric quantification. Because the intensity of a peptide peak in the mass spectrum cannot be predicted, quantification is achieved by labeling one of the two conditions by stable isotopes. Such methods have been used traditionally in mass spectrometry of small molecules, and now also been introduced into the world of proteomics.
The most recent technology in mass spectrometry quantitative comparison is named stable isotope labeling with amino acids in cell culture (SILAC). In principle, SILAC relies on metabolic incorporation of a “light” or “heavy” form of the aminoacid into the proteins. Therefore, in an experiment two cell populations are grown in culture media that are identical except that one of them contains a “light” and the other a “heavy” form of a particular amino acid like 12C and 13C labeled L-lysine, respectively. When the labeled amino acid is supplied to cells in culture instead of thenatural amino acid, it is integrated into all newly synthesized proteins. After a number of cell divisions, each particular amino acid will be replaced by its isotope labeled analog. Since there is almost no chemical difference between the labeled amino acid and the natural amino acid, the cells behave exactly the same as the control cell population grown in the presence of normal amino acid. By following the similar process as traditional mass spectrometry analysis, the “heavy” and “light” peptides will be investigated in the same spectrograph and be compared quantitatively by either Maldi-MS or LC-MS. This method can be utilized to study cell signaling, post translation modification as well as gene expression regulation.

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Table of contents :

1. Intracellular transport pathways
1.1 Biosynthetic/secretory pathway
1.2 Endocytic pathways
1.2.1 Late endocytic/degradation pathway
1.2.2 Recycling pathway
1.2.3 Retrograde transport
2. Retrograde transport
2.1 The functions of retrograde transport
2.2 Pathology
2.2.1 Shiga Toxin
2.2.2 Ricin
2.2.3 Cholera toxin
2.2.4 Virus
2.2.5 Other diseases
2.3 Retrograde transport machinery
2.3.1 Clathrin coats
2.3.2 Retromer
2.4 Tools to study retrograde transport
3. Proteomics
3.1 The functions and methods of proteomics
3.1.1 Identification and analysis of proteins
3.1.2 Differential-display proteomics
a). The two-dimensional gel approach
b). Quantitative comparison by mass spectrometry
c). Protein chips
3.1.3 Protein–protein interactions
a). Purification of protein complexes
b). Yeast two-hybrid screening
c). Phage display
3.2 Proteomics in cell biology
3.2.1. A mitochondrial protein complex that links apoptosis and glycolysis
3.2.2. New component in clathrin coated vesicles
3.2.3. Kinase pathways that regulate sex-specific functions in Plasmodium
4. Reaction schemes for proteomics approaches
4.1 SNAP-tag/AGT, a DNA repair enzyme
4.2 Sulfation
4.3 Rapamycin, FK506 and FKBP
4.4 Biotin and streptavidin
4.5 N-hydroxysuccinimide in protein coupling
RESULTS
1. SNAP-tag approach
1.1 Synthesis of the BG derivative
1.2 Setting-up the experimental system
1.2.1 Expressing SNAP-tag in the Golgi apparatus
a). TGN38 and SNAP-tag hybrid
b). SNAP-tag fusion protein with GalT
1.2.2 Functional validation
a). STxB-BG endocytosis assay
b). LC-MS detection
c). Antibody uptake assay
d). Cell permeability test
e). Cell surface modification
1.3 Proteomics screening
2. Sulfation approach
2.1 The synthesis of bSuPeRs
2.1.1 bSuPeR-3b synthesis
2.1.2 Click reaction
2.1.3 bSuPeR-carbamate synthesis
2.2 Experimental setup
2.2.1 Estimation of the efficiency for cell surface modification
2.2.2 bSuPeR-carbamate characterization
2.2.3 An important element for efficient cell surface modification: reaction pH
2.2.4 A key element for efficient cell surface modification: the peptide charge
3. FKBP approach
3.1 FK506-NHS synthesis
3.2 Verification strategy
3.2.1 In vitro binding
a). STxB coupling to FK506-NHS
b). Binding assay in solution
c). Binding assay on cell surface
3.2.2 Localization of FKBP12 to the Golgi apparatus
3.2.3 Functional validation
4. Streptavidin approach
MATERIALS AND METHODS
1. Chemistry part
2. Biology part.
1. Identification of cargo candidates of the retrograde route
1.1 Integrin
1.2 Transferrin receptor
1.3 Ion transporters
1.4 Critical discussion
2. STxB as a model to verify proteomics approaches
3. Innovative proteomic approaches
3.1 Proteomics strategy for retrograde transport
3.2 Comparison with traditional proteomics approaches
3.3 Comparison of the four approaches
4. SNAP-tag for protein-protein interaction
5. Chemical probe synthesis
6 Negative charge and polyarginine
PERSPECTIVE
ACKNOWLEDGEMENTS
REFERENCES

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