Cover Page
The handle http://hdl.handle.net/1887/28466 holds various files of this Leiden University
dissertation
Author: Hendriks, Ivo Alexander
Title: Global and site-specific characterization of the SUMO proteome by mass
spectrometry
Issue Date: 2014-09-03
List of Abbreviations
LIST OF ABBREVIATIONS
[VIL]KxE
[Valine, Isoleucine or Leucine] – Lysine – Any – Glutamic Acid
53BP1
Tumor suppressor p53-binding protein 1
ABC
Ammonium bicarbonate
ACTB
Actin, Cytoplasmic 1
ADP
Adenosine diphosphate
ADSM
Acetylation Dependent SUMOylation Motif
AGC
Automatic gain control
APC4
Anaphase-Promoting Complex Subunit 4
As2O3
Arsenic trioxide
ATG12
Autophagy-Related Protein 12
ATG8
Autophagy-Related Protein 8
ATP
Adenosine triphosphate
BCA
Bicinchoninic acid assay
BHC
BRAF-HDAC Complex
BHC110
Lysine-Specific Histone Demethylase 1A
BL21
Escherichia coli BL21, genotype: fhuA2 [lon] ompT gal [dcm] ΔhsdS
BLM
Bloom Syndrome Protein
BRAF35
SWI/SNF-Related Matrix-Associated Actin-Dependent Regulator of Chromatin Subfamily E Member 1-Related
BRCA1
Breast Cancer Type 1 Susceptibility Protein
BRCA2
Breast Cancer Type 2 Susceptibility Protein
BrdU
5-bromo-2'-deoxyuridine
BSA
Bovine serum albumin
BTB
Broad-Complex, Tramtrack and Bric-a-brac
C18
Octadecyl carbon chain
CAAChloroacetamide
CAPNS1
Calpain Small Subunit 1
CBP
CREB-Binding Protein
CBX3
Chromobox Protein Homolog 3
CDK1
Cyclin-Dependent Kinase 1
cDNA
Circular DNA
CFP
Cyan Fluorescent Protein
CID
Collision-induced dissociation
CMVCytomegalovirus
CORUM
Comprehensive Resource of Mammalian Protein Complexes
CPTCamptothecin
DAPI4',6-diamidino-2-phenylindole
DAXX
Death Domain-Associated Protein 6
DDR
DNA Damage Response
DIC
Differential interference contrast
DMEM
Dulbecco's Modified Eagle Medium
DMSO
Dimethyl sulfoxide
DNA
Deoxyribonucleic acid
DTTDithiothreitol
DUB
Deubiquitylating enzyme
DUSP
Domain present in Ubiquitin-Specific Proteases
DxK
Aspartic Acid – Any – Lysine
ECL
Enhanced Chemiluminescence
EDTA
Ethylene diamine tetraacetic acid
EEF1A1
Eukaryotic Translation Elongation Factor 1 alpha 1
eGFP
Enhanced Green Fluorescent Protein
EGTA
Ethylene glycol tetraacetic acid
ELISA
Enzyme-Linked Immunosorbent Assay
ETV6
ETS-Related Protein Tel1
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List of Abbreviations
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ExK
Glutamic Acid – Any – Lysine
FACS
Fluorescence-Activated Cell Sorting
FAT10
Ubiquitin D
FAU
Ubiquitin-like protein FUBI
FBN1Fibrillin-1
FBS
Fetal Bovine Serum
FDR
False discovery rate
FKH
Forkhead winged helix DNA binding domain
FOS
Proto-Oncogene c-Fos
FoxM1
Forkhead Box Protein M1
GAL4
Yeast transcription activator protein Gal4
GFP
Green Fluorescent Protein
GO
Gene Ontology
GOBP
GO Biological Processes
GOCC
GO Cellular Compartments
GOMF
GO Molecular Functions
GSEA
Gene Set Enrichment Analysis
GST
Glutathione S-Transferase
GTF2I
General Transcription Factor II-I
H3K23ac
Histone H3 acetyl Lysine-23
H3K4me2
Histone H3 dimethyl Lysine-4
H3K4me3
Histone H3 trimethyl Lysine-4
H3K9me2
Histone H3 dimethyl Lysine-9
H3K9me3
Histone H3 trimethyl Lysine-9
H4K16ac
Histone H4 acetyl Lysine-16
HCD
Higher-Collisional Dissociation
HCl
Hydrochloric acid
HCSM
Hydrophobic Cluster SUMOylation Motif
HDAC1
Histone Deacetylase 1
HDAC2
Histone Deacetylase 2
HEK293
Human Embryonic Kidney 293
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
HERC2
E3 ubiquitin-protein ligase HERC2, HECT domain and RCC1-like domain-containing protein 2
HIF1α
Hypoxia-Inducible Factor 1-alpha
HIF2α
Hypoxia-Inducible Factor 2-alpha
His-tag
Polyhistidine-tag
HMBS
Porphobilinogen Deaminase
HP1
Chromobox Protein Homolog 3
HRD1
E3 Ubiquitin-Protein Ligase Synoviolin
HRP
Horseradish peroxidase
HSF1
Heat Shock Factor Protein 1
HU
Hydroxyurea
IMAC
Immobilized metal affinity chromatography
IP
Immunoprecipitation
IPTG
Isopropyl-β-D-thiogalactopyranoside
IRES
Internal Ribosome Entry Site
ISCM
Inverted SUMO Consensus Motif
ISG15
Interferon-induced 15 kDa protein
iTRAQ
Isobaric Tag for Relative and Absolute Quantitation
JARID1B
Lysine-Specific Demethylase 5B
JARID1C
Lysine-Specific Demethylase 5C
JUN
Transcription Factor AP-1
K0
Lysine-deficient
K0/R0
[12C614N2]lysine/[12C6,14N4]arginine
K4/R6
[2H4,12C6,14N2]lysine/[13C6,14N4]arginine
K8/R10
[13C6,15N2]lysine/[13C6,15N4]arginine
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List of Abbreviations
KDM5B
Lysine-Specific Demethylase 5B
KDM5C
Lysine-Specific Demethylase 5C
KEGG
Kyoto Encyclopedia of Genes and Genomes
KRAB
Krüppel Associated Box
KxD
Lysine – Any – Aspartic Acid
KxE
Lysine – Any – Glutamic Acid
LacZ
Beta-Galactosidase LacZ
LARC
LCR-Associated Remodeling Complex
LC
Liquid Chromatography
LC-MS/MS
Liquid chromatography - tandem mass spectrometry
LDS
Lithium Dodecyl Sulphate
LFQ
Label-Free Quantification
LTQ
Linear Ion Trap Mass Spectrometer
Lys-C
Lysyl Endopeptidase
MBD1
Methyl-CpG-Binding Domain Protein 1
MBP
Maltose-Binding Protein
MCF7
Michigan Cancer Foundation-7
MCL1
Induced Myeloid Leukemia Cell Differentiation Protein Mcl-1
MCODE
Molecular Complex Detection
MDC1
Mediator of DNA damage checkpoint protein 1
MeCP1
MeCP1 Histone Deacetylase Complex
MES
2-(N-morpholino)ethanesulfonic acid
MGC
Mammalian Gene Collection
MgCl2
Magnesium chloride
MIS18BP1
Mis18-Binding Protein 1
MITF
Microphthalmia-Associated Transcription Factor
MMS
Methyl Methanesulfonate
MOPS
3-(N-morpholino)propanesulfonic acid
MRE11
Double-Strand Break Repair Protein MRE11A
mRNA
Messenger ribonucleic acid
MS
Mass spectrometry
MS/MS
Tandem mass spectrometry
Myc
Myc Proto-Oncogene Protein
Na2HPO4
Disodium phosphate
NaCl
Sodium chloride
NaH2PO4
Monosodium phosphate
NaOH
Sodium hydroxide
NDSM
Negatively charged amino acid-Dependent SUMOylation Motif
NEDD8
Neural precursor cell expressed developmentally down-regulated protein 8
Ni-NTA
Nickel - Nitrilotriacetic acid
NKTR
NK-Tumor Recognition Protein
NOP58
Nucleolar Protein 58
NP-40
Nonidet P40, octylphenoxypolyethoxyethanol
NRD
N-terminal repressor domain
P/S
Penicillin and streptomycin
p21
Cyclin-Dependent Kinase Inhibitor 1
p300
Histone Acetyltransferase p300
p53
Cellular Tumor Antigen p53
PAGE
Polyacrylamide gel electrophoresis
PARP
Poly ADP Ribose Polymerase
PBS
Phosphate buffered saline
PBST
Phosphate Buffered Saline containing Tween-20
PCNA
Proliferating cellular nuclear antigen 1
PCR
Polymerase Chain Reaction
PDPulldown
PDSM
Phosphorylation Dependent SUMOylation Motif
PEIPolyethylenimine
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List of Abbreviations
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PEP
Posterior error probability
Pfam
Protein Families
PHD
Plant Homeo Domain
PHEM
PIPES, HEPES, EGTA, and MgCl2
PHYRE2
Protein Homology/analogY Recognition Engine V2.0
PIPES
Piperazine-N,N′-bis(2-ethanesulfonic acid)
PLA
Proximity Ligation Assay
PLK1
Polo-Like Kinase 1
PML
Promyelocytic Leukemia Protein
ppm
Parts per million
PRISM
Protease-Reliant Identification of SUMO Modification
PSPPhosphoSitePlus
PTM
Post-translational modification
Pyro-QTGG
Pyroglutamate-glutamine-threonine-glycine-glycine
QQTGG
Glutamine-glutamine-threonine-glycine-glycine
RAD51
DNA Repair Protein RAD51 Homolog 1
RanBP2
E3 SUMO-Protein Ligase Ran-Binding Protein 2
RanGAP1
Ran GTPase-Activating Protein 1
RARα
Retinoic Acid Receptor alpha
RCF
Relative Centrifugal Force
RFP1
E3 ubiquitin-protein ligase complex slx8-rfp subunit, RING finger protein 1
RFP2
E3 ubiquitin-protein ligase complex slx8-rfp subunit, RING finger protein 2
RING
Really Interesting New Gene
RNF111
RING Finger protein 111
RNF168
RING Finger Protein 168
RNF4
RING Finger Protein 4
RRM1
Ribonucleotide Reductase M1
SAE1
SUMO-Activating Enzyme Subunit 1
SAE2
SUMO-Activating Enzyme Subunit 2
SAFB2
Scaffold Attachment Factor B2
SART1
U4/U6.U5 tri-snRNP-Associated Protein 1
SCE1
SUMO Conjugating Enzyme 1
SD
Standard Deviation
SDS
Sodium dodecyl sulphate
SEM
Standard error of the mean
SENP
SUMO-Specific Protease
SENP1
Sentrin-Specific Protease 1
SENP2
Sentrin-Specific Protease 2
SETDB1
Histone-Lysine N-Methyltransferase SET Domain Bifurcated 1
shRNA
Short hairpin ribonucleic acid
SILAC
Stable Isotope Labeling of Amino Acids in Culture
SIM
SUMO Interaction Motif
Slx5
E3 ubiquitin-protein ligase complex SLX5-SLX8 subunit SLX5, Synthetic lethal of unknown function protein 5
Slx8
E3 ubiquitin-protein ligase complex SLX5-SLX8 subunit SLX8, Synthetic lethal of unknown function protein 8
SMAD4
Mothers Against Decapentaplegic Homolog 4
SMRT
Nuclear Receptor Corepressor 2
Smt3
Ubiquitin-Like Protein SMT3
SNHSASulfosuccinimidyl-acetate
SNHSSSB
sulfosuccinimidyl-disulfide-biotin
SP100
Nuclear Autoantigen Speckled 100 kDa
SRPR
Signal Recognition Particle Receptor Subunit Alpha
SRS2
ATP-dependent DNA helicase SRS2
STAT1
Signal Transducer and Activator of Transcription 1-alpha/beta
STRING
Search Tool for the Retrieval of Interacting Genes/Proteins
STUbL
SUMO-targeted Ubiquitin Ligase
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List of Abbreviations
SUMO
Small Ubiquitin-Like Modifier
SUMO-1
Small Ubiquitin-Like Modifier 1
SUMO-2
Small Ubiquitin-Like Modifier 2
SUMO-3
Small Ubiquitin-Like Modifier 3
TAD
Transactivation domain
TAP
Tandem Affinity Purification
TDG
G/T mismatch-specific thymine DNA glycosylase
TiO2
Titanium dioxide
TNB
TRIS, NaCl, and blocking reagent
TOPORS
E3 Ubiquitin-Protein Ligase Topoisomerase I-binding RING Finger Protein
TR
Thyroid Hormone Receptor
TRIM24
Transcription Intermediary Factor 1-alpha
TRIM33
E3 Ubiquitin-Protein Ligase Tripartite Motif-Containing Protein 33
TRIS
Tris(hydroxymethyl)aminomethane
Triton X-100 Polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether
Tween-20
Polysorbate 20, Polyoxyethylene (20) sorbitan monolaurate
U2-OS
U-2 Osteosarcoma
UBA2
SUMO-Activating Enzyme Subunit 2
Ubc9
Ubiquitin Carrier Protein 9
UBCH5A
Ubiquitin-Conjugating Enzyme E2 D1
UBE1
Ubiquitin-Like Modifier-Activating Enzyme 1
Ubl
Ubiquitin-like
UBL5
Ubiquitin-Like Protein 5
UCH
Ubiquitin Carboxyl-terminal Hydrolase
UFM1
Ubiquitin-fold modifier 1
USP11
Ubiquitin-Specific Peptidase 11
USP25
Ubiquitin Carboxyl-Terminal Hydrolase 25
USP7
Ubiquitin Carboxyl-Terminal Hydrolase 7
USP8
Ubiquitin Carboxyl-Terminal Hydrolase 8
UVUltraviolet
WST
Water Soluble Tetrazolium
WTWild-type
XPC
Xeroderma Pigmentosum Group C-complementing Protein
YFP
Yellow Fluorescent Protein
ZBTB1
Zinc Finger and BTB Domain-Containing Protein 1
ZNF451
Zinc Finger Protein 451
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CURRICULUM VITAE
Ivo Alexander Hendriks was born in Haarlem, The Netherlands, on the 17th of
October 1983. He attended the Atheneum Hageveld in Heemstede, where he
obtained his high school diploma in 2001. Next, Ivo studied Life Science & Technology, both at the Universities of Delft and Leiden. During the Master program,
he specialized in Functional Genomics, with a focus on Science Based Business. He
performed his Bachelor internship in the lab of Dr. Gregg Siegal at the University of
Leiden, his Master internship in the lab of Dr. Jourica Brandsma at the University of
Leiden, and his Commercial Master internship at Isogen Life Science. Ivo obtained
his university Master’s degree cum laude in 2008. Ivo then spent a few months as
a practical assistant, teaching university students in gene technology at the University of Leiden. Finally, Ivo started his PhD training in January 2009, under primary
supervision of Dr. Alfred Vertegaal, at the department of Molecular Cell Biology, at
the Leiden University Medical Center in the Netherlands.
This thesis contains the efforts and results of a five year period of research.
During his training, Ivo attended a multitude of national and international research
meetings, where he presented his work predominantly through poster presentations. He also collaborated with two mass spectrometry-oriented research groups
abroad; the Max-Planck Institute for Biochemistry in Munich, Germany as well as
the Novo Nordisk Foundation Center for Protein Research in Copenhagen, Denmark.
From March 2014 on, Ivo continues his research into SUMO as a post-doctoral researcher in the same research group, and hopes to successfully publish more
SUMO-related resources and methodologies in the future.
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LIST OF PUBLICATIONS
Matic, I.*, Schimmel, J.*, Hendriks, I.A.*, van Santen, M.A.*, van de Rijke, F., van,
D.H., Gnad, F., Mann, M., and Vertegaal, A.C. (2010). Site-specific identification of
SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic
cluster SUMOylation motif. Molecular Cell 39, 641-652. (Chapter 4)
Vyas, R., Kumar, R., Clermont, F., Helfricht, A., Kalev, P., Sotiropoulou, P., Hendriks,
I.A., Radaelli, E., Hochepied, T., Blanpain, C., Sablina, A., van, A.H., Olsen, J.V.,
Jochemsen, A.G., Vertegaal, A.C., and Marine, J.C. (2013). RNF4 is required for DNA
double-strand break repair in vivo. Cell Death and Differentiation 20, 490-502.
Schimmel, J.*, Eifler, K.*, Sigurethsson, J. O.*, Cuijpers, S. A., Hendriks, I. A.,
Verlaan-de, V. M., Kelstrup, C. D., Francavilla, C., Medema, R. H., Olsen, J. V., and
Vertegaal, A. C. (2014). Uncovering SUMOylation Dynamics during Cell-Cycle
Progression Reveals FoxM1 as a Key Mitotic SUMO Target Protein. Molecular Cell
53, 1053-1066. (Chapter 5)
Hendriks, I.A., Treffers, L.W., Verlaan - de Vries, M., Olsen, J.V., and Vertegaal, A.C.O.
SUMO-2 orchestrates chromatin modifiers in response to DNA damage. Manuscript
submitted. (Chapter 2)
Hendriks, I.A.*, Schimmel, J.*, Olsen, J.V., and Vertegaal, A.C.O. USP11 counteracts
RNF4 and stabilizes PML nuclear bodies. Manuscript submitted. (Chapter 3)
Hendriks, I.A., D’Souza, R.C., Yang, B., Verlaan - de Vries, M., Mann, M., and
Vertegaal, A.C.O. Uncovering global SUMOylation signalling networks in a sitespecific manner. Manuscript submitted. (Chapter 6)
Hendriks, I.A., D’Souza, R.C., Mann, M., and Vertegaal, A.C.O. A novel method for
the identification of SUMO acceptor lysines in a site-specific manner. Manuscript
submitted. (Chapter 7)
* Equal contribution
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ACKNOWLEDGMENTS (DANKWOORD)
De laatste en belangrijkste pagina's van ieder proefschrift, en in veel gevallen de eerste (en enige)
die gelezen worden, blijven natuurlijk het dankwoord. Na vijf lange jaren zwoegen is alle informatie
gedestilleerd en samengevoegd in een klein boekje, en is er relatief gezien vrijwel geen tijd over om
diep en lang na te denken over dit cruciale einde. Dit proefschrift was nooit tot stand gekomen zonder
de fantastische hulp van iedereen om mij heen, en bij voorbaat mijn excuses voor het geval ik iemand
vergeet te bedanken!
Allereerst Alfred, mijn co-promoter. Het is door jouw enthousiasme, creatieve manier van denken, en
doeltreffende begeleiding, dat ik uiteindelijk een wetenschapper ben geworden. Aan het begin was
je mijn directe begeleider op het lab, en nu ben je een volwaardige PI die vrijwel niet meer achter zijn
bureau vandaan komt. Het is altijd jouw droom geweest om de SUMO code te kraken, en deze droom
heb je altijd zonder terughoudendheid gedeeld met mij. Bedankt!
Peter, mijn promoter. Bedankt voor de rake commentaren bij de werkbesprekingen, en voor het
spoedig doornemen van mijn werk onder grote tijdsdruk.
My more recent lab-mates and colleagues; Aleksandra, Frauke, Joel, Renier, Roman, Sabine and
Zhenyu (Minnie). Thank you for all your help, your insights, and for all the good times we had. You
all got to know me during my later and more stressful years, and as such I am thankful that you have
managed to deal with me.
My old colleagues; Bing, Christian, Guy, Sander, Ramesh, and René. Some of you I saw going through
the same process I am going through now, years ago, and perhaps I finally understand. All of you I miss
not having around the lab anymore, as you were a great source of wisdom, and moreover we had
some great times together!
De afdeling MCB. Hans T., bedankt voor de figuurlijke schop die ik nodig had om te beginnen met
schrijven. Willem en Hans V., zonder jullie was dit proefschrift met pen en papier geschreven. Joop
en Annelies, zonder jullie had ik geen idee gehad hoe een cel er nu écht uitziet. AG en David, thanks
for the very direct and honest remarks and advices during our work discussions; we all have to come
down from our cloud sometimes. En graag bedank ik ook alle andere medewerkers van MCB die ik niet
specifiek genoemd heb.
My international collaborators. Rochelle, thank you for being patient with me over the years and
teaching me a lot about mass spec. Matthias, thank you for your brilliance and for hosting my brief
stay at the MPIB. Jesper, thank you for running my mass spec samples, and active collaboration with
our group.
Mijn goede vrienden, de ‘gang’ uit Haarlem! Corné, Constantijn, Keti, Mark en Rogier. Ik ken jullie
al vele jaren, en buiten het promoveren om hebben jullie de nodige afleiding en mentale steun
geboden. De afgelopen jaren ben ik steeds meer het contact kwijtgeraakt, naarmate de stress van
het promoveren is toegenomen. Ondanks mijn afwezigheid ontvang ik nog altijd uitnodigingen voor
allerlei activiteiten, waar ik nu zeker meer gebruik van zal maken. Bedankt!
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Louise, mijn enige student tijdens mijn PhD traject. Sommige mensen begrijpen nog steeds niet hoe je
het hebt volgehouden met mij als begeleider, en zelfs al heb ik je afgeraden om als PhD te beginnen,
ben je er toch aan begonnen. Bedankt voor al je inzet, en sterkte!
Matty, in het afgelopen jaar heb jij een gigantische hoeveelheid werk voor mij verricht. Ik kan me
niet meer herinneren wanneer ik voor het laatst een blot heb gedaan. Zonder jou had ik nooit zo ver
kunnen komen als nu, en daar ben ik je eeuwig dankbaar voor!
Mijn beste collega’s, vrienden, en paranimfen; Joost en Karolin. En uiteraard Karin en Martin. Bedankt
voor de nodige afleiding en de fantastische reizen naar de Moezel.
Joost, jij was er vanaf het begin, tot aan het einde. Zo ongeveer alles wat ik heb geleerd is via jou. Je
wist (en weet) waar alles staat in het lab, hoe alles werkt, en bovendien was je altijd realistisch en
kritisch op constructieve wijze. Je bent een jaar later begonnen met promoveren dan ik, en we ronden
tegelijkertijd af. En in de tussentijd heb je buiten werk om nog veel meer dingen klaargespeeld waar ik
nog aan moet beginnen. Respect daarvoor. En bedankt voor alles!
Karo, you joined the lab when my third year just started. From that very beginning, you were the
brightest and most motivating presence I have known. You move not only me, but the whole lab is
positively affected by your selflessness, your empathy, and your natural leadership. You are always a
source of inspiration, and many of my so-called ‘Eureka!’ moments were directly or indirectly sparked
by you. More than anything, you have always been a great friend and provided a shoulder to cry on.
Danke!
Ten slotte, mijn familie. Mama, Papa, Natasja, Stefan, Mariana, Randy, Marian, Rafael en Roan. Jullie
hebben mij altijd de nodige afleiding van werk geboden, en vormden veelal mijn wereld buiten de
wetenschap. Het is bijna niet in woorden te brengen hoe belangrijk het is om af en toe een stap weg
te nemen van hetgene wat iemand dag in en dag uit bezighoudt. En in het bijzonder, mama en papa.
Jullie hebben mij altijd een veilig thuis aangeboden, een plek waar ik iedere dag maar al te graag
terugkeer. En ik heb nooit enige reden gevonden om te willen vertrekken. Bedankt voor alles!
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