MUNICIPIO ORO (GR) PLATA (GR) TOTAL (GR)

To determine the subcellular distribution of the enzymes, co-localisation experiments were performed by co-expressing the OTUB1 and the E2 conjugases in mammalian cells. For this study, OTUB1 and the selected E2 proteins were transferred from pDONR223 (or pDONR207) entry vectors into fluorescent-tagged mammalian expression constructs. Although in terms of validation, it has already been shown that OTUB1 interacts with these E2s, the location in which they interact has not yet been established. As individual enzyme, OTUB1 possesses a putative nuclear localisation signal and resides in the nucleus of porcine kidney cells (Balakirev et al., 2003; Shan et al., 2009). However, recent work revealed that OTUB1 regulates p53 in the cytoplasm (Sun et al., 2012). On the other hand, analysis of the localisation of UBE2A/B, UBE2D1, UBE2D2, UBE2D3, UBE2E1, UBE2E2, UBE2N and UBE2R1 shows that the Ubc4/5 family proteins (UBE2D1, UBE2D2, and UBE2D3) specifically co-localised with c-Cbl in endosomes (Umebayashi et al., 2008). UBE2D2 was also found to co-localised with Nedd4 in the cytoplasmic periphery (Anan et al., 1998) and UBE2N has been shown to regulate p53 also in the cytoplasmic compartment (Laine et al., 2006). While UBE2D2 and UBE2N seem to be cytoplasmic proteins, UBE2E1 co- immunoprecipitates and co-localises with Ataxin-1 protein in the nucleus (Hong et al.,

2008).

In this experiment, the main objective was to evaluate whether co-expression causes a change in the distribution that could suggest a particular localisation for interaction. OTUB1 and ΔNOTUB1 were transferred into pEGFP-N2 vector that fuses an N-terminal green fluorescent-tagged to the protein. It should be noted that OTUB1 in pDONR223 does not have any stop codon (Rual et al., 2004), therefore only N-terminal tags will be suitable to OTUB1. Meanwhile, all the E2 enzymes were transferred into pG-cherry-mGR (kindly contributed by Sheila Ryan, University of Liverpool). ORF cloned in this vector will be expressed in mammalian cells as a tagged protein with C-terminal red fluorescent-tag. All clones were sequence-verified before transfecting into HeLa cells for immunofluorescence

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studies. Vectors containing gene of interests were then singly transfected or co-transfected into HeLa cells to observe any overlap in their distribution, or changes when co-expressed to suggest the potential for interaction.

5.5.1 Localisation of OTUB1, ΔNOTUB1, UBE2D2, UBE2E1 and UBE2N

Immunofluorescence studies showed that OTUB1 localised to the same cellular compartments as the E2 enzymes, which shows that they have a potential for interaction in vivo. This experiment demonstrates that OTUB1, UBE2D2 and UBE2N are localised throughout the cell while UBE2E1 is predominantly nuclear.

In Figure 5.5, OTUB1 and ΔNOTUB1 prove to have a diffuse localisation throughout the cell. Meanwhile in Figure 5.6, UBE2E1 appears to have a predominantly nuclear localisation, consistent with literature reports. Also, it can be confirmed that similar to OTUB1 and ΔN

OTUB1, UBE2D2 demonstrates a diffuse localisation throughout the cell, as does UBE2N, although it does appear to have a stronger localisation in the nucleus which would be consistent with its reported role in DNA repair (Nakada et al., 2010).

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Figure 5.5 Localisation of (A) OTUB1 and (B) ΔNOTUB1 singly transfected into HeLa cells. Both transfections show a wide distribution throughout the cell. The DAPI blue stain shows cell nuclei while OTUB1 and ΔNOTUB1 are observed in green.

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Figure 5.6 Localisation of (A) UBE2N, (B) UBE2E1 and (C) UBE2D2 in HeLa cells: UBE2N and UBE2D2 show a diffuse distribution throughout the cell while UBE2E1 shows a nuclear distribution. The DAPI blue stain shows cell nuclei while the E2s are observed in red.

OTUB1-GFP

DAPI Merge

OTUB1-GFP

ΔN

OTUB1-GFP DAPI Merge

DAPI Merge

UBE2N-mCherry

DAPI Merge

UBE2E1-mCherry

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5.5.2 Co-localisation study of OTUB1 and ΔNOTUB1, with E2 proteins

Co-localisation studies were performed to determine whether OTUB1 co-expressed with the E2s could cause any recruitment of either protein to a particular subcellular localisation, thus implying a physical interaction. As seen in Figure 5.7, when OTUB1 is co-expressed with UBE2D2 and UBE2N, there does not appear to be any change in the subcellular distribution of any of the proteins. However, when OTUB1 is co-expressed with UBE2E1, there does appear to be some recruitment of OTUB1 to the nucleus compared to when OTUB1 is singly transfected; however it is still localised throughout the cell. There is no change in the subcellular distribution of UBE2E1 compared to its single transfections where it still shows localisation predominantly to the nucleus and small punctuate structures in the cytoplasm.

In Figure 5.8, there do not appear to be any changes in the subcellular distribution of any of the proteins when co-expressed in which, ΔNOTUB1 and UBE2D2 still show a wide distribution as when singly transfected and UBE2N still appears to have a stronger expression in the nucleus. In conclusion, co-expression of the E2 enzymes UBE2D2, UBE2E1 and UB2N with both OTUB1 and ΔNOTUB1 did not alter the distribution of any of the proteins; however they still have the potential to co-localise as they are expressed within the same cellular compartments.

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Figure 5.7 Co-localisation of (A) UBE2D2, (B) UBE2E1 and (C) UBE2N with OTUB1 in HeLa cells: When compared to single transfections the subcellular distribution of OTUB1 does not change when co-expressed with UBE2D2 and UBE2N, suggesting that one does not recruit the other to certain organelles when co-expressed. However OTUB1 seems to be slightly more nuclear when co-expressed with UBE2E1. The DAPI stain shows cell nuclei.

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Figure 5.8 Co-localisation of (A) UBE2D2 and (B) UBE2N with ΔNOTUB1 in HeLa cells: There appears to be no change in subcellular distribution even though OTUB1 is missing its N-terminus. The DAPI stain shows cell nuclei.

DAPI Merge

UBE2D2-mCherry

DAPI Merge

UBE2E1-mCherry

UBE2N-mCherry DAPI Merge