Abstract
Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.
Highlights
Parkin is a RING E3 ubiquitin ligase [1,2,3] which, in conjunction with a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), catalyses the attachment of ubiquitin to itself and to multiple putative substrates [4,5,6]
In order to determine the effects of each peptide tag on the conformation and stability of Parkin, each protein was subjected to a limited proteolysis using subtilisin. cMyc- or FLAG-tagged Parkin are proteolysed in a similar pattern to wild type Parkin, suggesting that these tags do not impact the proteolytic susceptibility of Parkin (Figure 2A)
A thermal denaturation experiment revealed the melting temperature of wild type, cMyc, FLAG- and HA-tagged Parkin to be 56.5uC, 54.5uC, 56.0uC and 54.0uC respectively, revealing that cMyc- and HA-Parkin have reduced thermal stability in comparison to wild type Parkin (Figure 2B). These results indicate that the cMyc, FLAG- and HA-tagged epitope tagged Parkin species are not in a fully native conformation as assessed by susceptibility to proteolysis and thermal denaturation
Summary
Parkin is a RING E3 ubiquitin ligase [1,2,3] which, in conjunction with a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), catalyses the attachment of ubiquitin to itself and to multiple putative substrates [4,5,6]. Fusion tags can vary greatly in size, from large tags such as Glutathione-S-Transferade (GST), Maltose Binding Protein (MBP) and Small Ubiquitin-like Modifier (SUMO), 26 kDa, 43 kDa and 13 kDa respectively, to small peptide tags including cMyc, FLAG and HA, each around 1 kDa (Figure 1). We observed that MBP-, GST-, or SUMO-tagged Parkin was constitutively active, suggesting a disruption of the autoinhibited state. This was perhaps unsurprising given the large size of the fusion tags. The majority of cell-based studies of Parkin utilise small peptide epitope tags and the effect of these tags on Parkin activity and stability is unknown. We report here that small epitope tags fused to the N-terminus of Parkin disrupt Parkin autoinhibition and Parkin stability
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