Munro, Kirsten Margaret Anderson (2016)

246 Pages·2016·6.75 MB·English

Munro, Kirsten Margaret Anderson (2016) Stabilising suppressor of
cytokine signalling 3 (SOCS3) protein levels to limit neointimal
hyperplasia.PhD thesis.

http://theses.gla.ac.uk/7564/

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Enlighten:Theses
http://theses.gla.ac.uk/
[email protected]
Stabilising Suppressor of Cytokine Signalling 3 (SOCS3)
Protein Levels to Limit Neointimal Hyperplasia

Kirsten Margaret Anderson Munro
MSci, MRes

Thesis submitted in fulfilment of the requirements for the Degree of Doctor of
Philosophy

April 2016

School of Medical, Veterinary and Life Sciences
Institute of Cardiovascular and Medical Sciences

University of Glasgow

Supervisor: Professor T.M. Palmer
Co-supervisor: Professor A.H. Baker
II

Summary
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,
III

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.
IV

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.
V

Table of Contents

Summary ............................................................................................................................... II
List of Tables...................................................................................................................... XII
List of Figures ................................................................................................................... XIII
Acknowledgement............................................................................................................ XVI
Author’s Declaration ....................................................................................................... XVII
Abbreviations ................................................................................................................. XVIII
1 Introduction .................................................................................................................... 1
1.1 Cardiovascular disease ............................................................................................ 1
1.1.1 CHD-Atherosclerosis pathogenesis ...................................................................... 1
1.1.2 Revascularisation of atherosclerotic vessels ......................................................... 4
1.2 Cytokine signalling .................................................................................................. 8
1.2.1 Interleukin 6 classic vs. trans-signalling ............................................................... 8
1.3 Suppressors of cytokine signalling (SOCS) ............................................................ 9
1.3.1 SOCS3 orchestrates a negative feedback loop with the JAK-STAT pathway.... 10
1.3.2 Structural organisation of the SOCS3 protein provides an insight into its
function ........................................................................................................................ 11
1.3.3 Erythropoietin signalling ..................................................................................... 13
1.3.4 Leptin signalling ................................................................................................. 14
1.3.5 Insulin-like growth factor I signalling................................................................. 15
1.3.6 Granulocyte Colony-Stimulating Factor signalling ............................................ 15
1.3.7 The mechanism of SOCS3-mediated IL6-gp130R-JAK/STAT inhibition ......... 16
1.3.7.1 SOCS3 docks at pY757 on the intracellular domain of the gp130R ...... 16
1.3.7.2 The dual interaction of SOCS3 with the gp130R and JAK2 facilitates the
negative regulation of the JAK/STAT pathway. ....................................................... 16
1.3.7.3 JAK3 is resistant to inhibition by SOCS3 .............................................. 17
1.3.7.4 The role of the SOCS3 kinase inhibitory region (KIR) .......................... 18
1.3.7.5 SOCS3 may outcompete JAK/STAT components for phosphotyrosine
docking sites. ............................................................................................................. 19
VI

1.3.8 SOCS3 targets substrates for degradation ........................................................... 22
1.3.9 The mechanism of SOCS3 turnover ................................................................... 26
1.3.9.1 Proteasomal degradation ......................................................................... 26
1.3.9.2 Tyrosine phosphorylation regulates SOCS3 stability ............................. 27
1.3.9.3 Role of the SOCS3 PEST motif in determining stability ....................... 28
1.3.9.4 Role of calpain proteases in determining SOCS3 stability ..................... 28
1.3.9.5 SOCS2 as a regulator of SOCS3 stability .............................................. 30
1.4 Epigenetic modulation of SOCS3 expression in disease ....................................... 30
1.4.1 SOCS3 promoter hyper-methylation in cancer ................................................... 30
1.4.2 SOCS3 promoter hyper-methylation in cardiovascular disease.......................... 31
1.4.3 Histone acetylation and deacetylation regulates transcription factor access to the
SOCS3 promoter .......................................................................................................... 31
1.5 Ubiquitylation ........................................................................................................ 33
1.5.1 Ubiquitylation is an ATP dependent process involving three discrete enzymes 33
1.5.1 Deubiquitylation of substrates............................................................................. 35
1.5.2 Ubiquitin chain arrangement ............................................................................... 36
1.5.3 Lys48 polyubiquitin chains target substrates for degradation at the 26S
proteasome ................................................................................................................... 37
1.5.4 Lys63 polyubquitin chains: non-proteolytic or proteolytic ................................. 38
1.5.5 The N-terminal α-amino group of a protein may be ubiquitylated ..................... 40
1.5.6 Ubiquitin-like proteins (UBLs) ........................................................................... 40
1.5.6.1 SUMOylation .......................................................................................... 41
1.5.6.2 NEDDylation .......................................................................................... 42
1.5.7 Identifying putative sites of ubiquitylation ......................................................... 42
1.6 SOCS3 in the vasculature ...................................................................................... 43
1.6.1 JAK/STAT signalling and NIH. .......................................................................... 43
1.6.2 SOCS3 and a role for endothelial barrier function? ............................................ 43
1.6.3 SOCS expression in atherosclerosis .................................................................... 44
VII

1.6.4 Reducing inflammatory infiltrate to the intimal layer reduced neointimal lesion
growth .......................................................................................................................... 46
1.6.5 SOCS3 limits pathological angiogenesis ............................................................ 47
1.7 Migration and proliferation of VSMCs ................................................................. 48
1.7.1 SOCS3 mediated inhibition of focal adhesion kinase 1 (FAK1) prevents cellular
migration. ..................................................................................................................... 48
1.7.2 SOCS3-mediated downregulation of matrix metalloproteinases (MMPs) limits
cell migration ............................................................................................................... 49
1.7.3 IL-6 signalling and cyclin D1 expression in VSMCs ......................................... 50
1.7.4 SOCS3 induction in the vasculature and downregulation in neointimal lesions 52
1.8 Hypothesis ............................................................................................................. 54
1.9 Aims ...................................................................................................................... 55
2 Materials and Methods ................................................................................................. 56
2.1 Materials ................................................................................................................ 56
2.2 Methods ................................................................................................................. 59
2.2.1 Cell Culture ......................................................................................................... 59
2.2.1.1 Culture of HEK293 and murine embryonic fibroblasts(MEFs) ............. 59
2.2.1.2 Culture of AS-M.5.5, HSVEC and HUVECs ......................................... 59
2.2.2 Culture of HSVSMCs ......................................................................................... 59
2.2.2.1 Coating of plastic ware with poly-D-lysine for HEK293 cells ............... 60
2.2.3 Cloning of cDNA constructs in E.coli ................................................................ 60
2.2.3.1 Transformation of competent E. coli ...................................................... 60
2.2.3.2 Transformation of ultracompetent E.coli for ligations. .......................... 61
2.2.3.3 Glycerol stock preparation ...................................................................... 61
2.2.4 DNA plasmid isolation, quantification and visualisation ................................... 61
2.2.4.1 Small scale: Wizard® Plus SV Miniprep ............................................... 61
2.2.4.2 Large scale: EndoFree Plasmid Maxiprep .............................................. 62
2.2.4.3 Quantification of plasmid DNA concentration ....................................... 63
2.2.4.4 DNA gel electrophoresis ......................................................................... 63
VIII

2.2.5 SOCS3 mutagenesis ............................................................................................ 64
2.2.5.1 SOCS3 truncation mutants ..................................................................... 64
2.2.5.2 Site-directed PCR mutagenesis ............................................................... 64
2.2.5.3 Generation of a Lys-less human SOCS3 ................................................ 66
2.2.6 Transfection of cDNA ......................................................................................... 67
2.2.7 Cell protein analysis via immunoblotting ........................................................... 67
2.2.7.1 Cell harvesting ........................................................................................ 67
2.2.7.2 The bicinchoninic acid (BCA) protein assay .......................................... 68
2.2.7.3 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) and Immunoblotting ..................................................................................... 69
2.2.7.4 Blocking nitrocellulose membrane and incubation with primary antibody
69
2.2.7.5 Incubation with secondary antibodies and immunoblot visualisation .... 69
2.2.7.6 Stripping of membranes .......................................................................... 70
2.2.7.7 Gel components ...................................................................................... 71
2.2.7.8 Densitometry ........................................................................................... 74
2.2.8 Immunoprecipitation ........................................................................................... 74
2.2.8.1 Co-Immunoprecipitation (co-IP) of SOCS3-interacting proteins. .......... 75
2.2.9 Emetine chase to investigate the stability of WT vs. mutant SOCS3 ................. 76
2.2.10 Optimising the sensitivity of the Epo/Gp130 chimeric receptor (Epo/Gp130R)
assay to measure SOCS3 functionality ........................................................................ 76
2.2.11 The identification of an E3 ubiquitin ligases and DUBs controlling SOCS3
turnover: the development of a SOCS3-luciferase fusion protein as a tool to screen an
E3 ligase siRNA library ............................................................................................... 77
2.2.11.1 Investigating the proteasome as a major route of SOCS3 turnover in AS-
M human endothelial cells ........................................................................................ 77
2.2.11.2 The identification of a cell system to be used in an E3 ligase siRNA
library screen ............................................................................................................. 77
2.2.11.3 Generation of a SOCS3-Firefly Luciferase fusion protein ..................... 77
2.2.11.4 Luciferase assay ...................................................................................... 78
IX

2.2.11.5 Generation of a SOCS3-Luc lenti virus .................................................. 79
2.2.11.6 Concentration of the lentivirus particles ................................................. 79
2.2.11.7 Measuring virus particle titre .................................................................. 79
2.2.11.8 Generation of AS-M.5 clonal cell lines stably expressing SOCS3-Luc . 80
2.2.12 Immunofluorescence visualisation of SOCS3 in MEFs and HUVECs as a
method for screening an E3 ligase siRNA library. ...................................................... 81
2.2.13 Reversed-phase liquid chromatography tandem mass spectrometry (LC-MS)
screen to identify E3 ligase/DUB enzymes interacting with SOCS3. ......................... 82
2.2.13.1 IP of SOCS3 for LC-MS-MS analysis .................................................... 82
2.2.13.2 SDS-PAGE and in-gel protein visualisation. .......................................... 83
2.2.13.3 Band excision and trypsin digestion. ...................................................... 83
2.2.13.4 LC-MS-MS analysis. .............................................................................. 84
2.2.14 Investigating SOCS3 in the vasculature ............................................................ 84
2.2.14.1 Immunolocalisation of SOCS3 in HSV tissue. ....................................... 84
2.2.14.2 Optimising the infection efficiency of the Sffv-GFP LV in HSVECs. .. 86
2.2.14.3 Proliferation assay................................................................................... 86
2.2.14.4 Migration assay ....................................................................................... 86
2.2.14.5 Statistical analysis ................................................................................... 87
3 Investigating the regulation of SOCS3 stability .......................................................... 88
3.1 Introduction ........................................................................................................... 88
3.1.1 Aims .................................................................................................................... 88
3.2 Results ................................................................................................................... 89
3.2.1 Identification of key lysine residues involved in SOCS3 ubiquitylation ............ 89
3.2.2 Emetine treatment to assess the role of Lys6 on SOCS3 protein stability .......... 89
3.2.3 Mapping the site of SOCS3 ubiquitylation ......................................................... 94
3.2.4 Investigating the potential for SOCS3 to auto-ubiquitylate ................................ 96
3.2.5 Investigating whether Lys173 is a key site of SOCS3 ubiquitylation .............. 100
3.2.6 Emetine treatment to assess the role of Lys173 on SOCS3 protein stability .... 100
3.2.7 Mapping the sites of SOCS3 ubiquitylation via LC-MS-MS ........................... 105