Protein interactions, in vivo

Johnsson, N., and Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91, 10340-10344. 

Niranjanakumari, S., Lasda, E., Brazas, R., and Garcia-Bianco, M. A. (2002). Reversible cross-linking combined with immunoprecipitation to study RNA-protein interactions in vivo. Methods 26, 182—190. 

Recently, Vasilescu et al. demonstrated the use of formaldehyde to preserve protein interactions in vivo followed by immunoaffinity purification of a targeted complex, cross-link reversal via heating at 95°C, separation by SDS-PAGE, and identification of bands by LC-MS/MS.7 Tagwerker et al. utilized formaldehyde cross-linking in conjunction with a novel tag-based affinity purification method.36... 

The following protocol is designed for treating cells with the PIR reagent to study protein interactions in vivo. It is based on the method of Tang et al. (2005). The use of the PIR compound to treat intact cells results in the crosslinking of proteins both on the cell surface and within the cell, which indicates that the reagent is able to cross the cell membrane. 

Piehler, J. (2005) New methodologies for measuring protein interactions in vivo and in vitro. Curr. Opin. Struct. Biol. 15(1), 4-14. 

Raciborska DA, Charlton MP (1999) Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions a new hypothesis to explain persistence of botulinum A poisoning. Can J Physiol Pharmacol 77 679-88 Raciborska DA, Trimble WS, Charlton MP (1998) Presynaptic protein interactions in vivo evidence from botulinum A, C, D and E action at frog neuromuscular junction. Eur J Neurosci 10 2617-28... 

Using FRET to Explore Protein-Protein Interactions in Vivo Figure 12-8 shows the interaction between j8-arrestin and the /1-adrenergic receptor. How would you use FRET (see Box 12-3) to demonstrate this interaction in living cells Which proteins would you fuse Which wavelengths would you use to illuminate the cells, and which would you monitor What would you expect to observe if the interaction occurred If it did not occur How might you explain the failure of this approach to demonstrate this interaction ... 

The calculated BRET ratio indicates the occurrence of protein-protein interaction in vivo. In Figure 14.11, the blank corrected BRET2 ratios for both negative and positive controls are shown and were determined as... 

A/PI4Ka 1 (Figure 3) is probably the major enzyme contributing to the previously reported F-actin associated Ptdlns 4-kinase activity (Tan and Boss, 1992 Xu et al., 1992). While it was also reported that PIP5K activity was associated with the actin-enriched fraction from plants, the specific isoform of the enzyme has not been identified (Tan and Boss, 1992). As more specific antibodies and molecular probes become available, it will be important to monitor lipid protein interaction in vivo and in vitro. 

Fig. 8. Protein-protein interaction study based on split intein. In order to monitor the protein interaction in vivo, the N- and C-terminal halves of the intein (N-intein and C-intein) are fused to N- and C-terminal halves of EGFP (A), or luciferase (B). Each of these fusion proteins is linked to the protein of interest (protein A) and its target protein (protein B). Upon protein A-protein B cooperation, the closely oriented intein fragments mediate intein splicing. The measurement of fluorescence intensity originated from the reconstituted mature EGFP protein or measurement of luciferase luminescence is possible.

Ozawa, T., Nogami, S., Sato, M., Ohya, Y., and Umezawa, Y. (2000) A fluorescent indicator for detecting protein-protein interactions in vivo based on protein splicing. AnflZ. Chem. 72, 5151-5157. 

SenGupta DJ, Zhang B, Kraemer B, Pochart P, Fields S, Wickens M. (1996). A three-hybrid system to detect RNA-protein interactions in vivo. Proc. Natl. Acad. Sci. U.S.A. 1996 93 8496-8501. Jaeger S, Eriani G, Martin F. Results and prospects of the yeast three-hybrid system. FEES Lett. 2004 556 7-12. 

Since the conception of the two-hybrid assay to detect protein-protein interactions in vivo at the end of the 1980s, key modifications to this assay have expanded its scope to detect DNA-, RNA-, and small molecule-protein interactions in so-called n-hybrid assays. More recently, n-hybrid assays have also been used to detect enzyme catalysis, where enzyme activity is linked to cell survival via transcription of a reporter gene. Here we look at the initial publications that moved the two-hybrid assay into each of these new directions. 

Farnesylated peptides, N- or S-substituted peptides with the farnesyl (Fam) moiety. Based on the hydrophobicity of this Cis unit, isoprenoid farnesylated peptides are characterized by increased bioavailability. Farnesylated peptides are useftd building blocks for the synthesis of special biologi-cal/pharmaceutical model targets, since the farnesyl group in proteins may participate in specific protein-protein interactions. In vivo, farnesylation is performed by the... 

Lee JW, Lee SK (2004) Mammalian two-hybrid assay for detecting protein-protein interactions in vivo. Methods Mol Biol 261 327-336... 

The yeast two-hybrid system is a powerful genetic technique that has been used successfully to study protein-protein interactions in vivo from a wide variety of biological sources, including yeast, plant, and mammalian (Fields and Song, 1989). The two-hybrid system provides a sensitive method to detect relatively weak and transient protein-protein interactions that may not be biochemically detectable. The principle of this assay is based on the reconstitution of transcriptional factors that promotes the expression reporter genes, which then allow the proliferation of yeast under restrictive conditions. Using the two-hybrid system, we successfully isolated IPCEFl and a novel kinesin motor proteins from a rat brain cDNA library as interactors of cytohesin 2 ARF GEF and centaurin-al ARF GAP, respectively (Venkateswarlu, 2003). The procedure used to map the binding domains of cytohesin 2 and IPCEFl, and to characterize the cytohesin 2 and IPCEFl selectivity is outlined here. 

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