Stuart Gaines

Analysis of the Disulphide Interactions of AGR2 Protein IntroductionAnterior gradient-2 (AGR2) is a pro-oncogenic protein linked to metastasis, drug resistance and inhibition of the p53 checkpoint system. While it is normally expressed in secretory epithelium, elevated levels are linked to breast, oesophageal and prostate cancers. The protein has a single cysteine residue in the active site. This single cystine allows for the AGR2 monomer to form disulphide bridges with other AGR2 monomers and also other components of a cells proteome. Here we aimed to investigate the disulphide interactions of AGR2 to try and answer some questions about the function of AGR2.

Methods Examination of AGR2 Disulphide Formation when Exposed to a reducing agentOE19 and OE33 cells were exposed to NEM (a chemical to fix disulphide bonds) and lysed. A sample of lysed OE19 and OE33 cells without NEM was also produced. The NEM and non-NEM samples of each cell type were then treated with DTT (a reducing agent to prevent formation of disulphide bonds) and run on an SDS-PAGE Gel and Western Blot for AGR2 with a set of controls that were not exposed to DTT. The membrane was then re-probed for B-actin as a control.

Analysis of the Ability of AGR2 in OE19 Cells to Reform Disulphide Bonds after exposure to a reducing agent 2 sets of OE19 cells had RPMI media removed and were then treated for 5 minutes with RPMI media containing 500mM DTT to break the disulphide bonds. 1 set then was incubated in media for 10 minutes and then washed in NEM and PBS for 5 minutes and then PBS for 5 minutes, while the other was washed with NEM and PBS and then PBS straight away. A control was also set up and incubated in media without DTT for 5 minutes and then put through the same two 5 minute wash cycles. From these 3 sets of treated cells, lysates were created and a non-reducing and DTT reducing set were formed and run on an SDS-PAGE Western Blot for AGR2 and then re-probed for B-actin and PDI as a control.

Analysis of AGR2 interaction when exposed to an oxidising agent 2 sets of OE19 cells had RPMI media removed and were then treated for 5 minutes with RPMI media containing 500mM DIA to ensure formation of disulphide bonds. 1 set was incubated in media for 10 minutes and then washed in NEM and PBS for 5 minutes and then PBS for 5 minutes, while the other was washed with NEM and PBS and then PBS straight away. A control was also set up and incubated in media without DIA for 5 minutes and then put through the same two 5 minute wash cycles. From these 3 sets of treated cells, lysates were created and a non-reducing and DTT reducing set were formed and run on an SDS-PAGE Western Blot for AGR2 and then re-probed for B-actin and PDI as a control.

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Stuart Gaines

Results Examination of AGR2 Disulphide Formation when Exposed to a reducing agent

Figure 1. On the left is a Western Blot For AGR2 in OE19 and OE33 cell treated with NEM (shown by an n in the image). On the right is a B-actin re-probe of the same membrane.

The image above shows that in the presence of DTT, AGR2 in OE19s is unable to form complexes but is present in the 16KDa band at the bottom. The evidence for this is shown by the lack of bands in OE19 and OE19N lanes between 250KDa and 50KDa. However, without DTT OE19 and OE19N lanes both show the presence of complexes. OE33 cells do not express AGR2 out of the ER and because of this, they do not show the presence of AGR2 on a western blot analysis. The right hand image here is poor and suggest some experimental error while performing the re-probe. Within non-reducing conditions, B-actin is not often displayed in a western blot.

Analysis of the Ability of AGR2 in OE19 Cells to Reform Disulphide Bonds after exposure to a reducing agent

Figure 2. The left hand side shows a Western Blot of OE19 cells from 3 different treatment cycles. C is the control, T1 is treated with DTT and T2 is treated with DTT and then given 10 minutes of recovery. The righthand image shows the same set of lysates run under reducing and no reducing conditions.

The left hand image shows that when treated with DTT and given no time to recover, the AGR2 protein is able to form dimers and complexes but in very low concentrations. The T2 set though show that the recovery period in the RPMI media allows for the disulphide bonds to reform.

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Stuart Gaines The right hand image contains large amounts of ECL feedback and because of that is hard to see. However, under reducing conditions it is possible to see a lack of complex formation in the T1 and T2 lanes. Under non-reducing conditions these couples are present in higher concentrations.

Analysis of the Ability of AGR2 in OE19 Cells to Form Disulphide Bonds after exposure to an Oxidising Agent

Captures were not taken of the final western blots.

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