Established and emerging roles in diseases

Report
The Small Ubiquitin-like Modifiers: Established and emerging roles in diseases
Mike Tatham
Ron Hay lab
Wellcome Trust Centre for Gene Regulation and Expression
University of Dundee
ELRIG/SLAS Drug Discovery Manchester 2012
Phylogenetic relationship in the ubiquitin like modifier superfamily
• SUMO system is only found in Eukaryotes
• Yeasts, flies and worms only express a single SUMO
• Vertebrates express three paralogues SUMO-1, SUMO2, SUMO-3
ELRIG/SLAS Drug Discovery Manchester 2012
The SUMO conjugation system
• SUMO system is only found in Eukaryotes
• Yeasts, flies and worms only express a single SUMO
• Vertebrates express three paralogues SUMO-1, SUMO2, SUMO-3
• Like ubiquitin SUMOs are conjugated to protein
substrates in a three step mechanism
• Most SUMO conjugation occurs within a consensus
motif yKXE/D
• SUMO conjugation can occur independent of E3s
• SUMO-2 and SUMO-3 contain consensus motifs and
can modify themselves to form polySUMO chains
• Deletion of yeast SUMO is lethal
• Deletion of Ubc9 in mice is lethal
Ubiquitination
Ubiquitin E1 (2)
Ubiquitin E2 (~20)
Ubiquitin E3 (hundreds)
U
U
U
U
Substrate
Substrate
Ubiquitin
Protease (~100)
SUMOylation
SUMO E1 (1 – SAE1/2)
SUMO E2 (1 – Ubc9)
[SUMO E3 (10-20?)]
S
yKXE
S
S
S
S
yKXD
Substrate
Substrate
SUMO
Protease (8)
ELRIG/SLAS Drug Discovery Manchester 2012
Structural overview
• SUMO system is only found in Eukaryotes
• Yeasts, flies and worms only express a single SUMO
• Vertebrates express three paralogues SUMO-1, SUMO2, SUMO-3
• Like ubiquitin SUMOs are conjugated to protein
substrates in a three step mechanism
• Most SUMO conjugation occurs within a consensus
motif yKXE/D
• SUMO conjugation can occur independent of E3s
• SUMO-2 and SUMO-3 contain consensus motifs and
can modify themselves to form polySUMO chains
• Deletion of yeast SUMO is lethal
• Deletion of Ubc9 in mice is lethal
• SUMOs have low sequence homology to ubiquitin but
high 3D structural similarity
Cellular characteristics
A
+MG132
-MG132
+MG132
+MG132
Time
(h)
Time (h) 0 1Time
2 3(h)
5 70 0 11 22 33 5 77 1 2 3 5 7
A -MG132
U
138-
138-
IB Ubiquitin
48-
48-
35.5251712.59-
35.5251712.59-
Unconjugated
Unconjugated
U
ubiquitin
ubiquitin
B
B
138-
138-
S
4835.5251712.59-
35.5251712.59-
S
S
SUMO-2 S SUMO-2
conjugates conjugates
Substrat
e
66-
66IB SUMO-2/3
48-
Unconjugated Unconjugated
S
SUMO-2
SUMO-2
C
C
66-
IB SUMO-1
48-
D
SUMO-1
SUMO-1
Substrat
conjugates conjugates
e
664835.5251712.5912.5Total
35.5251712.5912.566-
S
138-
138-
D
U
U
Ubiquitin
Ubiquitin
conjugates conjugates
Substrate
66-
66-
U
66-
extracts
Unconjugated Unconjugated
S
SUMO-1
SUMO-1
Unconjugated Unconjugated
SUMO-1
SUMO-1
ELRIG/SLAS
Drug Discovery
Manchester 2012
4848a-Tubulin
a-Tubulin
35.5-
35.5-
• SUMO system is only found in Eukaryotes
• Yeasts, flies and worms only express a single SUMO
• Vertebrates express three paralogues SUMO-1, SUMO2, SUMO-3
• Like ubiquitin SUMOs are conjugated to protein
substrates in a three step mechanism
• Most SUMO conjugation occurs within a consensus
motif yKXE/D
• SUMO conjugation can occur independent of E3s
• SUMO-2 and SUMO-3 contain consensus motifs and
can modify themselves to form polySUMO chains
• Deletion of yeast SUMO is lethal
• Deletion of Ubc9 in mice is lethal
• SUMOs have low sequence homology to ubiquitin but
high 3D structural similarity
• SUMOs are predominantly nuclear proteins
• SUMO-1 and SUMO-2/-3 have largely overlapping
protein targets with some distinctions
Molecular functions of SUMO
SUMOylation
SUMO E1 (1 – SAE1/2)
SUMO E2 (1 – Ubc9)
[SUMO E3 (10-20?)]
S
S
S
S
S
Substrate
Substrate
SUMO
Protease (8)
Altered function
Subcellular localisation
Enzymatic activity
Complex formation
Further modification
Block modifications
SUMO BP
S
S
Substrate
SUMO BP
S
Substrate
S
S
S
S
Substrate
ELRIG/SLAS Drug Discovery Manchester 2012
• SUMO system is only found in Eukaryotes
• Yeasts, flies and worms only express a single SUMO
• Vertebrates express three paralogues SUMO-1, SUMO2, SUMO-3
• Like ubiquitin SUMOs are conjugated to protein
substrates in a three step mechanism
• Most SUMO conjugation occurs within a consensus
motif yKXE/D
• SUMO conjugation can occur independent of E3s
• SUMO-2 and SUMO-3 contain consensus motifs and
can modify themselves to form polySUMO chains
• Deletion of yeast SUMO is lethal
• Deletion of Ubc9 in mice is lethal
• SUMOs have low sequence homology to ubiquitin but
high 3D structural similarity
• SUMOs are predominantly nuclear proteins
• SUMO-1 and SUMO-2/-3 have largely overlapping
protein targets with some distinctions
• SUMO conjugation does not have a common effect on
proteins, but has myriad of protein-specific
consequences mediated by SUMO Interaction Motifs
(SIMS)
Cellular functions of SUMO
350
TAP-SUMO-2 cells
300
TAG
250
SUMO
Substrate
200
150
100
50
0
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
Number of SUMO substrates
Golebiowski et al Sci Signal. 2009;2(72):ra24.
Tatham et al Sci. Signal. 2011;4(178):rs4
•Purify SUMO from cells
•Identify and quantify proteins by quantitative mass
spectrometry-based proteomics
•Identified a total of ~900 SUMO substrates
Year
~10% of cellular proteins are modified by SUMO
ELRIG/SLAS Drug Discovery Manchester 2012
SUMO and human diseases
Centromere instability,
and facial anomalies
syndrome
Dermatomyositis
Autoimmune
regulation
Immunological
disorders
Megakaryoblastic
Breast cancer leukemia
Atypical myeloproliferative
Prostate cancer
disease
Melanoma
Squamous cell carcinoma
Cancers
Renal cell carcinoma
Colon cancer
DNA viruses
RNA viruses
Ovarian cancer
Infectious
diseases
SUMO
Multiple System Atrophy
Extra and Intra-cellular bacteria
Alzheimer's disease
Congenital heart
disease
Obesity
Multiple myeloma
Protozoa
Circulatory
Diseases
Transient global and
focal cerebral ischemia
Acute Promyelocytic leukaemia
Frontotemporal
dementia
Familial dilated
cardiomyopathy
Spinal and bulbar
muscular atrophy
Heart failure
Liver damage
Cystic fibrosis
Others
Neurological
disorders
Amyotrophic lateral
sclerosis
Frontotemporal lobar
degeneration
Spinocerebellar
ataxia type 1
Parkinson's disease
Amyotrophic lateral sclerosis
Neuronal intranuclear
inclusion disease
Dementia with Lewy Bodies
Huntington's disease
Muscular
dystrophy
Rheumatoid arthritis
ELRIG/SLAS Drug Discovery Manchester 2012
SUMO and human diseases
Types of evidence linking SUMO with diseases
1. Disease protein x is modified by SUMO which alters its function.
2. SUMO conjugation is altered in disease cells
3. SUMO is abnormally distributed within disease cells
4. Enzymes of the SUMO modification system are abnormally expressed in disease cells
5. SUMO system modulation alters the disease phenotype in model cells
Immunolabelling of NII in the hippocampal subiculum of
patients with NIID.
(Takahashi-Fujigasaki et al. Neuropathology and Applied
Neurobiology (2006), 32 , 92–100)
Immunostaining of aggregates in glioma cell models for multiple
system atrophy (MSA).
(D.L. Pountney et al. Neuroscience Letters 381 (2005) 74–79)
Alzheimer’s disease
(Amyloid beta protein (derived from from APP)) Li et al. PNAS 100 (2003)
(tau) Dorval & Fraser. JBC 281 (2005)
Parkinson’s disease
(a-synuclein, DJ-1) Dorval & Fraser. JBC 281 (2005), Shinbo et al. Cell. Death Diff. (2006)
Prion disease
(PrP)
Juanes et al. JBC 284 (2009)
Polyglutamine diseases
Huntington’s (Huntingtin) Steffan et al. Science (2004)
Kennedy’s
(Androgen Rec) Mukherjee et al. JBC (2009)
Dentatorubro-pallidoluysian atrophy (Atrophin-1)
Terashima et al. Neuroreport. 13 (2002)
Spinocerebellar ataxia
(ATAXIN1, 7) Riley et al. JBC 280 (2005), Janer et al. Hum. Mol. Gen. 19 (2010)
Tauopathy
(tau) Dorval & Fraser. JBC 281 (2005)
Familial amyotrophic lateral sclerosis (SOD1)
Fei et al. BBRC 347 (2006)
ELRIG/SLAS Drug Discovery Manchester 2012
The SUMO-SIM interaction
Q. How do we take advantage of the SUMO system therapeutically?
A. It depends on what you want to do!
SIM peptide
SUMO
SUMO BP
SUMO E1 (1 – SAE1/2)
SUMO E2 (1 – Ubc9)
[SUMO E3 (10-20?)]
SUMO BP
S
yKXE
S
yKXD
Substrate
Substrate
SUMO
Protease (8)
ELRIG/SLAS Drug Discovery Manchester 2012
S
S
S
To be or not to be specific: What can parasites tell us?
Q. How do we take advantage of the SUMO system therapeutically?
A. It depends on how specific you want to be!
DNA viruses
RNA viruses
Protozoa
Infectious
diseases
Extra and Intra-cellular bacteria
Wimmer P, et al. J Virol. 2012 Jan;86(2):642-54.
ELRIG/SLAS Drug Discovery Manchester 2012
SUMO and human diseases
Centromere instability,
and facial anomalies
syndrome
Dermatomyositis
Autoimmune
regulation
Immunological
disorders
Megakaryoblastic
Breast cancer leukemia
Atypical myeloproliferative
Prostate cancer
disease
Melanoma
Squamous cell carcinoma
Cancers
Renal cell carcinoma
Colon cancer
DNA viruses
RNA viruses
Ovarian cancer
Infectious
diseases
SUMO
Multiple System Atrophy
Extra and Intra-cellular bacteria
Alzheimer's disease
Congenital heart
disease
Obesity
Multiple myeloma
Protozoa
Circulatory
Diseases
Transient global and
focal cerebral ischemia
Acute Promyelocytic leukaemia
Frontotemporal
dementia
Familial dilated
cardiomyopathy
Spinal and bulbar
muscular atrophy
Heart failure
Liver damage
Cystic fibrosis
Others
Neurological
disorders
Amyotrophic lateral
sclerosis
Frontotemporal lobar
degeneration
Spinocerebellar
ataxia type 1
Parkinson's disease
Amyotrophic lateral sclerosis
Neuronal intranuclear
inclusion disease
Dementia with Lewy Bodies
Huntington's disease
Muscular
dystrophy
Rheumatoid arthritis
ELRIG/SLAS Drug Discovery Manchester 2012
Example of a successful drug therapy involving SUMO - APL
Acute Promyelocytic Leukaemia (APL)
•A rare condition driven by a chromosomal translocation resulting in the
fusion of the PML and retinoic acid receptor a proteins (PML-RARa)
•Very malignant and charaterised by sudden hemorrhages and accumulation
of promyelocytes in blood
•Retinoic acid and arsenic trioxide treatment induce differentiation of
promyelocytes and clinical remission.
•PML-RARa and PML are known to be SUMOylated and degraded in response
to arsenic
De The et al J. Cell. Biol. 2012. 198 No.1 11-21
ELRIG/SLAS Drug Discovery Manchester 2012
Liu et al Curr. Op. Chem. Biol. 2012. 16 92-98
Example of a successful drug therapy involving SUMO - APL
Ring Finger protein 4 (RNF4) aka SNURF
Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546
ELRIG/SLAS Drug Discovery Manchester 2012
Example of a successful drug therapy involving SUMO - APL
Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546
ELRIG/SLAS Drug Discovery Manchester 2012
A model for SUMO-dependent disease remission
U
U
U
U
U S
S
ARSENIC
SUMO
conjugation
PML
S
S
S
S
S
Ubiquitin
conjugation
PML
SUMO
deconjugation
U
S
U
U SS
U
PML
Ubiquitin
deconjugation
Tatham et al 2008. Nat. Cell. Biol. 10. 5. 538-546
ELRIG/SLAS Drug Discovery Manchester 2012
The SUMO system as a therapeutic target - Summary
•SUMO is functionally highly pleiotropic affecting many important cellular pathways
•There is a range of evidence linking SUMO to significant human diseases.
•The precise role of SUMO in many diseases is not determined and so its potential as a therapeutic
target is largely unclear
•The best approach to modulating SUMO function for individual disease therapy is unclear
•There is an academic and clinical argument for small molecule effectors of the SUMO system to
help clarify these issues.
ELRIG/SLAS Drug Discovery Manchester 2012
Acknowledgements
Ron Hay
Filip Golebiowski (Glasgow)
Ellis Jaffray
Marie-Claude Geoffroy (Paris)
Ivan Matic
Amit Garg
ELRIG/SLAS Drug Discovery Manchester 2012
Jurgen Cox
Matthias Mann

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