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Biochemistry: Titia Sixma

TitiaSixma.jpg

Titia Sixma, Ph.D. professorHead of division, Group leader

About Titia Sixma

Research interest: Structural Biology

One of the current challenges in structural biology is to study structure and function of the large and often transient complexes that are important in cellular systems. We study structure and function of complexes in ubiquitin conjugation, DNA mismatch repair and chromatin remodeling using protein crystallography and complementary biophysical techniques. 

Ubiquitin conjugation

Ubiquitin conjugation processes are critical signaling systems for most cellular processes. They attach one or more ubiquitins to target proteins and change their cellular fate, by promoting novel interactions, controlling degradation of short-lived proteins or inducing a relocalization. Because of the importance for regulating cell cycle, chromatin regulation, apoptosis and DNA repair deregulation of ubiquitin-dependent processes often leads to cancer. The process of conjugation by ubiquitin(-like) proteins involves covalent linking of one or more 76-amino-acid ubiquitins to a target protein by an E1/E2/E3 cascade of enzymes. Correct ubiquitination requires the complex spatial arrangement of ubiquitin, E2, E3 proteins and the target simultaneously in a precise but flexible manner. As these are reversible processes the role of deubiquitinating enzymes (DUBs) in the process is equally important to maintain the balance in the system.

We are interested in the regulation of the process of ubiquitin conjugation and deconjugation. We study the specificity of E2/E3 complexes and DUBs for specific targets such as H2A and PCNA or ubiquitin itself. We are interested how these ubiquitin ligases choose their target and how their actions are regulated.

The activity of many DUBs is regulated in the cell by modulation. We study regulation of intrinsic catalytic activity of a number of different DUBs by partners, cofactors or additional domains outside the catalytic subunit. Knowledge of this type of regulation can be of interest for drug development targeting these signaling proteins. 

DNA mismatch repair

One of the most prevalent forms of human hereditary cancer, HNPCC, is caused by mutations in the genes encoding the DNA mismatch repair proteins. These proteins are the human homologs of E. coli MutS and MutL, which execute the first two steps in repair of misincorporated base pairs during DNA duplication besides playing a role in the prevention of recombination of homeologous sequences. We study the crystal structure of MutS complexed to mismatched DNA and how this asymmetric ATPase couples DNA mismatch recognition to initiation of repair. 

Nicotinic Acetylcholine Receptor homolog AChBP

In the past we have studied the molluscan Acetylcholine Binding Protein (AchBP) as a mimic for ligand binding in nicotinic acetylcholine receptors (nAChR). This glial protein has conserved all the ligand binding properties and hence has served very well as a model system for studying ion channel function for the cys-loop receptors that include the GABAa and 5HT3 serotonin receptors, particularly with respect to their ligand binding properties.

We have used AChBP as a model system to study many aspects of ligand binding in Cys-loop receptors. These studies revealed ligand binding properties for a variety of ligands, the role of the modulating complementary subunit, the flexibility of the binding site and options for changing ligand properties. We analyzed anti-smoking compounds and used this model system for identification of novel ligands.

Co-workers

Aarsman, Ivette

Ivette Aarsman, MSc

Ph.D. student

Experience

I graduated from the VU Amsterdam in August 2018 with majors in Molecular Cell Biology and Biochemistry. I started my PhD in September 2018 at the NKI under supervisor of Titia Sixma.

The focus of my project is the deubiquitinating enzyme USP48. USP48 counteracts the ubiquitinating activity of BRCA1 on the H2A histone tail. The aims of my project are to obtain the structure of USP48 and/or related proteins complexes, to determine the molecular mechanism of the site-specific activity of USP48 on a minimal substrate and finally to generate separation-of-function mutants deficient for specific aspects of USP48 regulation.

 

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Baas, Roy

Roy Baas

Postdoctoral Fellow

Personal details

Groups

Experience

I obtained my PhD from Utrecht University in 2017 in the field of transcription and epigenetics where I applied confocal microscopy, affinity purification and mass spectrometry to study protein localization and protein complex composition.

I now apply these techniques to study DUBs and their respective complexes. The expertise of obtaining protein structural information at the Sixma lab allows me to combine this information to gain insight and better understanding of these DUBs in their protein complex environment.

 

 

 

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Bruekner

Susanne Bruekner

Ph.D. student

Experience

I studied Biochemistry at the University of Tuebingen in Germany. As a PhD student in Titia Sixma's group I work on DNA mismatch repair, an essential process that corrects replication errors. With the help of cryo EM and other biophysical methods I try to elucidate the structure and function of the proteins that form the repair machinery. This knowledge can help finding new treatments for a colon cancer (Lynch syndrome) that is associated with defects in the mismatch repair proteins.

 

 

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Dharadhar, Shreya

Shreya Dharadhar

Ph.D. student

Experience

I received my undergraduate training atthe University of Mumbai and VU Amsterdam. My work in Titia Sixma's lab ismainly focused on understanding  themechanism of Ubiquitination and Deubiquitination of PCNA which is involved invarious DNA damage repair pathways.

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Alex Fish

Postdoctoral Fellow

Experience

Major scientific project DNA mismatch repair.

Major scientific expertise biophysical characterization of bimolecular interactions using different techniques such as SPR, ITC, MALLS and spectroscopic methods.

Experience in protein purification, X-ray crystallography, protein mass spectrometry.

Operator of biophysical instruments and mass spectrometer. Provides support for biophysical experiments. Expertise: Surface Plasmon Resonance (SPR); Fluorescence Polarisation (FP); Isothermal Titration Calorimetry (ITC); mass spectrometry.

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Guo, Xiaohu

Xiaohu Guo

Postdoctoral Fellow

Experience

I graduated from Uppsala University, Sweden 2015 as a structural biologist. My current interest in NKI is to study ubiquitin in DNA regulation. If the process is malfunctioning, this can lead to cancer. I use X-ray crystallography, Cryo-EM or other methods to determine the molecular model of the complexes involved in the process, which will help us understand the detail of the mechanisms involved. This valuable knowledge could in turn be helpful for drug design.

 

 

 

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Keijzer, Niels

Niels Keijzer

Ph.D. student

Experience

I studied Biomolecular sciences at VU Amsterdam and I started as a PhD student in November 2018 at Titia Sixma's group. 

My project here is focused on PCNA, a protein with several important roles in replication and DNA damage repair. The focus of my project is on ubiquitination and deubiquitination of PCNA, which is important after DNA damage by UV and allows translesion synthesis to take place.
In order to gain new insights into this process, I will try to solve structures of PCNA ubiquitination and deubiquitination complexes.

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Doreth Kok

Ph.D. student

Experience

I finished my master Bio-molecular sciences at the VU
University in August 2014 and started as a PhD student in September 2014 in the
group of Titia Sixma. I am studying DNA Mismatch repair, a repair mechanism for
mismatches that are incorporated in our DNA after DNA replication. I am
interested in the structure of the activation states of this machinery and we
are aiming to study this with a variation of structural approaches.

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Martinelli, L

Luca Martinelli

Postdoctoral Fellow

Experience

My project focuses on the biophysical and structural characterization of a highly target-specific E3 ligase involved in cholesterol homeostasis.

Scientific Experience:

06/2015 to present NKI PostDoc, (Dr. T. Sixma)

07/2014 to 05/2015 IBMB-CSIC (Barcelona, Spain), PostDoc

09/2008 to 06/2014 IRB and IBMB-CSIC (Barcelona, Spain), PhD student, Structural Biology

09/2005 to 07/2007 University of Bologna (Bologna, Italy), MSc, Bioinformatics

09/2002 to 09/2005 University of Modena and Reggio Emilia (Modena, Italy), BSc, Medical Biotechnology

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Andrea Murachelli

Postdoctoral Fellow

Experience

I am a structural biologist: I use bioinformatics, biochemistry and protein crystallography to determine the structure of protein complexes. This allows me to understand in detail how these complexes function in a healthy cell and how they fail in case of disease.

During my graduate studies, I investigated protein complexes involved in endocytosis, splicing and apoptosis.

In 2013 I started my postdoc in Titia Sixma's lab, working on the structure of complexes involved in ubiquitin conjugation and removal during DNA repair. From these structures I hope to better understand how ubiquitin signaling affects DNA repair and, possibly, cancer development.

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Priyanka, Anu

Anu Priyanka, Ph.D.

Postdoctoral Fellow

Experience

I graduated from CSIR-IMTECH, India in 2017 with Ph.D. in Structural Biochemistry. I joined NKI as a postdoc in the group of Titia Sixma in October 2018. My current interest at NKI is structural and biochemical characterizations of USPs. USPs have been implicated to play important roles in cell signaling pathways and have been associated with several diseases like neurological disorders, cancer etc. The outcome of our research will provide valuable insights toward generating specific high-affinity inhibitors against USPs of interest. This will help in unraveling complex biological problems relating to health and disease.

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Stijf, Yvette

Yvette Stijf-Bultsma

Lab Manager, Technician

Experience

I started working at the NKI in the department of Cellular Biochemistry in 2002. Since then I have spent 15 years working on nuclear phosphoinositides, focusing in particular on the role of PIP4K and phosphatidylinositol 5-phosphate in transcription. Along the way I moved to the UK and experienced a different research environment in both an independent research institute and university. Nine years and many experiments later I returned to the NKI in 2016 to take up the role of lab manager at the department of Biochemistry. In the group of Titia Sixma I'm using my skills in cell biology and biochemistry to understand the role of BRCA1-A and BRISC complexes using a proteomics approach.

 

 

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Van Dijk, Pim

Pim van Dijk

Technician

Experience

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Key publications View All Publications

  • MutS/MutL crystal structure reveals that the MutS sliding clamp loads MutL onto DNA

    (2015) Elife. Jul 11

    Groothuizen FS, Winkler I, Cristóvão M, Fish A, Winterwerp HH, Reumer A, Marx AD, Hermans N, Nicholls RA, Murshudov GN, Lebbink JH, Friedhoff et al.

    Link to PubMed
  • Mechanism of UCH-L5 activation and inhibition by DEUBAD domains in RPN13 and INO80G

    (2015) Mol Cell. Mar 5;57(5):887-900. Epub 2015 Feb 19

    Sahtoe DD, van Dijk WJ, El Oualid F, Ekkebus R, Ovaa H, Sixma TK.

    Link to PubMed
 
 

Recent publications View All Publications

  • Active site alanine mutations convert deubiquitinases into high-affinity ubiquitin-binding proteins

    (2018) EMBO Rep. Oct;19(10). pii: e45680

    Morrow ME, Morgan MT, Clerici M, Growkova K, Yan M, Komander D, Sixma TK, Simicek M, Wolberger C.

    Link to PubMed
  • ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability

    (2018) Mol Cell. Apr 5;70(1):165-174.e6.

    Haahr P, Borgermann N, Guo X, Typas D, Achuthankutty D, Hoffmann S, Shearer R, Sixma TK, Mailand N.

    Link to PubMed
 

Contact

  • Office manager

    Mirna Ekelschot - van Diermen

  • E-mail

    m.v.diermen@nki.nl

  • Telephone Number

    +31 20 512 9127

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