| Abstract |
Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the
mitogenic, migratory and pro-inflammatory pathways responsible for the
development of neointimal hyperplasia (NIH), a key contributor to the
failure of vascular reconstructive procedures. However, the protein levels
of SOCS3, and therefore its potential to reduce NIH, is limited by its
ubiquitylation and high turnover by the proteasome. I hypothesised that
stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the
potential to limit vascular inflammation and NIH. Consequently, the aim of
this PhD was to identify the mechanisms promoting the rapid turnover of
SOCS3. Initial experiments involved the identification of residues
involved in regulating the turnover of SOCS3 at the proteasome. I assessed
the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation
mutants and identified a C-terminal 44 amino acid region required for
SOCS3 ubiquitylation. This region localised to the SOCS box which is
involved in binding Elongin B/C and the formation of a functional E3
ubiquitin ligase complex. However, the single lysine residue at position
173, located within this 44 amino acid region, was not required for
ubiquitylation. Moreover, Emetine chase assays revealed that loss of
either Lys173 or Lys6 (as documented in the literature) had no significant
effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis
studies failed to identify key sites of ubiquitylation responsible for
targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-
immunoprecipitate was employed. These data were searched for the presence
of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of
ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206)
on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys
residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in
which all 8 Lys residues were mutated to Arg, would be resistant to
ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to
be completely resistant to ubiquitylation, and significantly more stable
than WT SOCS3. These changes occurred in the absence of any detrimental
effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C
components required to generate a functional E3 ligase complex. In
addition, both WT and Lys-less SOCS3 were equally capable of inhibiting
cytokine-stimulated STAT3 phosphorylation upon co-expression with a
chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I
generated an L189A SOCS3 mutant that could no longer bind the Elongins and
therefore form the E3 ligase complex required for ubiquitylation. A
denaturing IP to assess the ubiquitylation status of this mutant was
performed and revealed that, despite an inability to bind the Elongins,
the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together
these data suggested that SOCS3 does not auto-ubiquitylate and that a
separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought
to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling
the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to
screen an E3 ligase/DUB library, using an siARRAY, to sequentially
knockdown all known E3 ligases in the presence of a SOCS3-luciferase
fusion protein or endogenous SOCS3 in a high content imaging screening
platform. However, due to a poor assay window (<2) and non-specific
immunoreactivity of SOCS3 antibodies available, these methods were deemed
unsuitable for screening purposes. In the absence of a suitable tool to
screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-
IP) was investigated. I performed a SOCS3 under conditions which preserved
protein-protein interactions, with the aim of identifying novel E3 ligase
and/or DUBs that could potentially interact with SOCS3. These data were
searched for E3 ligase or DUB enzymes that may interact with SOCS3 in
HEK293 cells and identified two promising candidates i) an E3 ligase known
as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that
in the presence of HectD1 overexpression, a slight increase in K63-linked
polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that
an N-terminal region of SOCS3 may act as a repressor of this interaction
with HectD1. Additionally, USP15 was shown to reduce SOCS3
polyubiquitylation in a HEK293 overexpression system suggesting this may
act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to
play a major role in the interaction with USP15. The original hypothesis
of this thesis was that stabilisation of endogenous SOCS3 by inhibiting
its ubiquitylation has the potential to limit vascular inflammation and
NIH. Consistent with this hypothesis, immunohistochemistry visualisation
of SOCS3, in human saphenous vein tissue derived from CABG patients,
revealed that while SOCS3 was present throughout the media of these
vessels the levels of SOCS3 within the neointima was reduced. Finally,
preliminary data supporting the hypothesis that SOCS3 overexpression may
limit the proliferation, but not migration, of human saphenous vein smooth
muscle cells (HSVSMCs) is presented. It is expected that multiple E3
ligases and DUBs will contribute to the regulation of SOCS3 turnover.
However, the identification of candidate E3 ligases or DUBs that play a
significant role in SOCS3 turnover may facilitate the development of
peptide disruptors or gene therapy targets to attenuate pathological SMC
proliferation. A targeted approach, inhibiting the interaction between
SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would
be expected to reduce the undesirable effects associated with global
inhibition of the E3 ligase involved.
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