Detection Of Ras Raf Protein Interactions Biology Essay

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In the recent years several methods have been developed to explore the protein modifications and interactions as they play an intrinsic role in any cellular and sub-cellular processes. Several immunohistochemistry techniques were developed for proteome studies. Proximity ligation assay is an affinity based technique used to measure protein interaction and modification at single molecular level. Fredriksson et al in 2002 developed the proximity dependent DNA ligation assay for the in situ analysis of proteins. PLA technique combines the proximity ligation with rolling circle amplification that involves the binding of the target proteins by specific antibodies linked to the oligonucleotides called as PLA probes. The proximity probes serve as the template for the added connector oligos to form a complete circularized oligo by enzymatic ligation reaction. The circularized DNA strand gets amplified using one of the antibody bound oligonucleotides as the primer to form RCP (fig.1). Fluorescence labeled oligonucleotides are hybridized to the RCP, thus allowing to analyse the RCP under fluorescence microscope [3, 6].

Antibody

Fig.1: in situ PLA: Interacting proteins are bound by the antibodies with PLA probes which then serve as template for connector oligonucleotides. Ligation followed by RCA occurs to give RCP. The amplified product is further analysed under fluorescence microscope after hybridizing with fluorescence probes.

http://www.annualreviews.org/doi/pdf/10.1146/annurev-anchem-060908-155239

Proto-oncogene serine/threonine protein kinase Raf1 functions downstream of Ras protein activation in the Mitogen-activated protein kinase (MAPK) signaling pathway regulating the cell-growth, differentiation, survival and apoptosis (Fig.2). Ras family includes the proto-oncogenes HRAS, KRAS, NRAS, RRAS. Raf family includes A-Raf, B-Raf and Raf-1(Raf-C) [7]. Mutations in any of these genes leads to over-expression of growth factors ultimately leading to various cancers.

Fig.2: Raf activated by Ras protein activates MEK/ERK pathway. (Hilger et al., 2002)

In our experiment we used in situ PLA to examine the Ras-Raf interactions in HCT116 cell line CCL-247 from American Type Culture Collection (ATCC).

MATERIALS AND METHODS:

The cell slide containing the HCT116 cell line CCL-247 (from ATCC) was regained from -20°C and was placed in 1x tris-base saline (TBS). Immediately the blue areas were dried off with the help of napkin without disturbing the cell area. Within 15sec, a hydrophobic perimeter was drawn around the cell areas on the blue line and the border was dried for 3-4 seconds before placing the slide back into TBS. Then the slide was washed twice with Tween-20 supplemented TBS (TBST- 0.05%) for 2 minutes. Before the addition of any diluent to the walls in the slide, excess buffer was removed from the slide and it was ensured that hydrophobic perimeter was intact. Duolink blocking agent (2-3 drops) was added to each well and the slide was incubated for 30 minutes at 37°C in a humidity chamber followed by a 2x2 min wash in TBST. Each slide has two wells in which one will be used for negative control and the other one for live experiment. Both primary antibodies {Ras (H-,K- and N-Ras) from US biological and Raf1 (C) from RnD} was diluted in Duolink antibody diluent to form a total volume of 40µl for the live experiment whereas for negative control, one of the antibodies (anti-Raf) was omitted. Each antibody dilution (40µl) was added to the respective wells and the slide underwent 1hour incubation at 37°C followed by a 2x2 min wash in TBST. Duolink II secondary antibodies (rabbit- and mouse+) 5x was diluted in Duolink antibody diluent to form a total volume of 80µl and 40µl of this dilution was added to each well. Then the slide was incubated at 37°C for 60min followed by a 2x2 min wash in TBST.

Ligation buffer and ligase was diluted in the ratio of 1:5 and 1:40 respectively in water to make a total volume of 80µl and 40µl of this ligation mix was added to each well followed by a 30min incubation at 37°C and a 2x2 min wash in TBST. Amplification buffer and polymerase was diluted in the ratio of 1:5 and 1:80 respectively in water to make a total volume of 300µl and 150µl of this amplification mix was added to each well. The slide was then incubated at 37°C for 60min followed by a 2x2 min wash in TBST and 2x2 min wash in TBS. Since, the reagents in this step are light sensitive, the succeeding steps were shielded from light as much as possible. The slide was centrifuged for 10sec after the removal of excess buffer from it followed by an addition of 25µl Duolink II mounting medium. Immediately, the slide was covered using a cover slip and was turn upside- down on a napkin so that excess mounting media was removed. Finally, the coverslip was glued onto the slide by applying nail polish around the edges and was stored from light in -20°C. (Duolink kit from OLINK BIOSCIENCE, Uppsala).

RESULTS AND DISCUSSION:

We performed the in situ proximity ligation assay with the cell slide containing HCT116 cell line CCL-247 from American Type Culture Collection (ATCC) to determine the ras/raf interactions. The targeted proteins were bounded using two primary antibodies raised in different species followed by binding of specific secondary antibodies with oligonucleotides called PLA probes. Upon proximity the connector oligos gets hybridized to the PLA probes proceeded by enzymatic ligation and gets amplified by RCA to yield RCP. Finally the RCP is analysed by hybridizing with fluorescence labeled oligos each spot representing individual RCP.

From the figure 3, we can observe the Ras-Raf protein interactions (highlighted in green colour) in the live experiment when compared with the negative control. Few green dots were also spotted in the negative control and this may be due to handling error. This error would have happened during the removal of excess buffer from the slide before addition of any diluent.

Ras-Raf interactions play a wide role in cell cycle. In the MAPK pathway also known as Ras/Raf/MEK/ERK, the activated G-protein Ras interacts with Raf1 which in turn phosphorylates the MEK. MEK again activates ERK by phosphorylating it, important to control cell growth, differentiation and apoptosis. Mutation of any one of the above proteins leads to over expression and activation of growth factors and finally cancer. Inhibiting the above proteins has been proved to treat cancer. In Colorectal cancers which causes majority of the cancer deaths, dysregulation of MAPK pathway is thought to be a major cause. Recently several research works are being carried out to design the novel inhibitors for the above potential proteins. These studies will pave the way to improve treatment to variety of cancers [1, 4, 7].

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