Medical Biochemistry (research)

The research discipline of medical biochemistry studies the molecular mechanisms underlying a multitude of diseases. These include infectious disease, metabolic disorders, storage disorders and cardiovascular disease.

Vascular Cell Biology, prof. de Vries (Head of Department) & dr V. de Waard

The Vascular Cell Biology group is part of the Department of Medical Biochemistry and is composed of technicians, postdocs, PhD-students and students who work as a team applying a plethora of molecular/cellular techniques and animal models. We collaborate with many research groups within the AMC, throughout the country and abroad.

Our research aims to delineate crucial regulatory mechanisms that determine initiation and progression of vascular disease, among which atherosclerosis, (in-stent) restenosis and aneurysm formation. To understand these vascular pathologies we study activation of cultured primary smooth muscle cells (SMCs), endothelial cells, monocytes and macrophages and their subsequent response. These studies involve the analysis of cellular functions such as differentiation, proliferation, inflammation and apoptosis. The underlying signaling mechanisms and the crucial regulatory factors directing these processes are studied applying up to date biochemical and molecular biological techniques. To substantiate functional involvement of specific signaling pathways and their regulatory proteins in the diseased vessel wall, we verify their role in dedicated (transgenic/atherosclerotic) animal models.

Publications de Vries (PubMed)

Publications de Waard (PubMed)

Our strong embedding in the AMC, involving among others a longstanding collaboration with the Department of Cardiology, provides ideal opportunities to translate basic research into clinical applications.

Vascular Immunopathology, prof. Lutgens

Our research focuses on the function of macrophages in atherogenesis. Macrophages play a crucial role in regulating the development of atherosclerotic lesions and plaque stability. Not only by the accumulation of lipids leading to foam cell formation but most importantly by the production of inflammatory mediators that determine the growth and stability of the plaques. Using both in vitro and in vivo approaches, we try to establish the regulatory mechanisms that affect macrophage function in atherosclerosis. We study how lipids affect the macrophages, which signaling pathways and transcription factors are crucial in atherosclerosis and assess novel opportunities to modulate macrophage function and thereby prevent progression of atherosclerotic disease.

  • Macrophages in Atherosclerosis, prof. de Winther

Our research focuses on the function of macrophages in atherogenesis. Macrophages play a crucial role in regulating the development of atherosclerotic lesions and plaque stability. Not only by the accumulation of lipids leading to foam cell formation but most importantly by the production of inflammatory mediators that determine the growth and stability of the plaques. Using both in vitro and in vivo approaches, we try to establish the regulatory mechanisms that affect macrophage function in atherosclerosis. We study how lipids affect the macrophages, which signaling pathways and transcription factors are crucial in atherosclerosis and assess novel opportunities to modulate macrophage function and thereby prevent progression of atherosclerotic disease.

Transcriptional Regulation of Metabolism, prof. Zelcer

Contribution of the LXR-IDOL-LDLR axis to cholesterol metabolism. Identification and characterization of novel metabolic genes. Studying the contribution of the UPS system to metabolism.

www.zelcerlab.eu

Vascular Microenvironment & Integrity, dr Huveneers

  • Investigating vascular integrity in inflammation and cardiovascular disease.
  • Elucidating cytoskeletal-mediated cell-cell junction regulation.
  • Understanding how mechanical forces control endothelial adhesions.

Endothelial mechanotransduction in inflammation and vascular disease

We aim to understand the molecular basis of vascular diseases that entail endothelial permeability and inflammation. Within blood vessels, endothelial cell-cell and cell-matrix adhesions are crucial to preserve barrier function. These adhesions are tightly controlled during vascular development, angiogenesis and migration of immune or tumor cells. Interestingly, endothelial cells respond to mechanical changes in the vasculature by a complex array of intracellular biochemical and structural changes. This mechanotransduction response include signals that promote, but also signals that protect against, monolayer permeability. Excessive vascular extracellular matrix (ECM) stiffening during aging, or after cancer therapies, disturbs this delicate balance and underlies pathological permeability and inflammation. Surprisingly, the underlying molecular events that control stiffness-induced mechanotransduction remain elusive. We recently discovered that differences in wall stiffness of human arterial and venous vessels alter endothelial morphology, and we are investigating the impact of this phenomenon in vascular biology. To achieve this we study the endothelium in the physiological setting of the vascular wall. See also our Circulation Research 2015 and ATVB 2014 publications.

Regulation of the VE-cadherin complex and endothelial integrity

The integrity of endothelial cell-cell adhesions is regulated in space and time during angiogenesis and inflammation. To achieve this, VE-cadherin, the crucial transmembrane receptor of endothelial cell-cell adhesions, is targeted by many major vascular signaling pathways. However, the molecular effects of angiogenic or inflammatory signals on the VE-cadherin complex, that may explain the tight spatiotemporal control of cell-cell adhesion, remain largely unclear. It is known that local organization of the actin cytoskeleton plays an important role in regulating the VE-cadherin complex. See our Journal of Cell Biology 2012 and Journal of Cell Science 2013 publications for details. Identifying the molecular changes at the VE-cadherin-based adhesion that occur upon cytoskeletal-dependent remodeling, and unveiling their dynamics at cell-cell junctions in response to signals will be key to understand the control of endothelial cell-cell adhesion during angiogenesis and inflammation. Our previous work showed that permeability agonists such as VEGF, histamine or thrombin, alter the organization and molecular composition of endothelial cell-cell adhesions. The mechanotransduction protein vinculin and other proteins specifically associate with the active subset of VE-cadherin-based adhesions that is linked to, and dependent on, the contractile actin cytoskeleton. We are elucidating the molecular regulation of the VE-cadherin complex in the context of inflammation and vascular diseases, such as atherosclerosis.

Internships

We facilitate a limited number of longterm internships for undergraduate students that are incorporated in our research projects. Key approaches include live fluorescence imaging, cell biology and biochemistry. If you're interested, please contact Stephan Huveneers to discuss the possibilities.

  • Functional Genomics of Eukaryotes, dr Distel

  • Regulation of protein function by cysteine ubiquitination.
  • Molecular mechanism of cysteine ubiquitination.
  • Understanding the molecular and cellular basis of Angelman Syndrome.
  • The function of BAR proteins in cell membrane remodelling.

Molecular mechanism of cysteine ubiquitination

Peroxisomes are single membrane bound organelles present in nearly all eukaryotes. They contribute to cellular metabolism in various ways depending on species, but a consistent feature is the presence of enzymes to degrade fatty acids. Defects in the biogenesis of these organelles cause severe, often lethal, human disorders known as peroxisome biogenesis disorders (PBDs). Our understanding of the genetic defects that underlie PBDs has greatly advanced by analyzing the peroxisome biogenesis process in simple model eukaryotes such as yeast.

The import of proteins into peroxisomes is mediated by cycling receptors, which bind the peroxisomal targeting signal-containing proteins in the cytosol, transport them to the peroxisomal membrane, release them into the peroxisomal matrix and then (re-) cycle back to the cytosol for another round of import. The cycling of these import receptors is regulated by ubiquitination, a posttranslational modification, where the 7kDa protein ubiquitin is covalently linked to a target protein. In the model yeast Saccharomyces cerevisiae, we established that Pex5p, the major peroxisomal import receptor in all eukaryotes, undergoes a novel type of ubiquitination that targets cysteine residues. The components of the peroxisomal ubiquitination machinery have been identified and future work will be aimed at unraveling the mechanistic details of cysteine ubiquitination. For this we have established in the group in vitro and in vivo ubiquitination assays, and for structural analysis of proteins involved in cysteine ubiquitination we collaborate with the group of Prof Wilmanns (EMBL, Hamburg). As cysteine ubiquitination is not restricted to yeast but also occurs in humans, we plan to extend our studies on ubiquitination to mammalian cells.

Understanding the molecular and cellular basis of Angelman Syndrome

Angelman syndrome (AS) is a severe neurological disorder, affecting 1:10,000-15,000 children. It is characterized by severe mental retardation, epilepsy, motor dysfunction and absence of speech. The disease is caused by the loss of a functional copy of the maternal UBE3A gene. Since the paternal UBE3A gene is silenced in the brain, such a mutation results in the complete absence of neuronal UBE3A expression. The UBE3A gene encodes the ubiquitin protein ligase E6AP, however since its identification 16 years ago, little progress has been made that provides insight in its role in neuronal function.

To gain insight into the molecular mechanisms underlying Angelman syndrome we have teamed up with the neurobiological laboratory of Prof Elgersma (ErasmusMC, Rotterdam) specialized in synaptic plasticity using mouse models. We have applied multi-disciplinary approaches to find candidate targets of the ubiquitin protein ligase E6AP and are in the process of validating these targets using in vitro and in vivo ubiquitination assays. The next step will be to validate these E6AP targets in mouse models. Understanding the molecular defects in Angelman Syndrome is a first step towards treatment of this severe neurological disorder.

The function of BAR proteins in cell membrane remodelling

Membrane dynamics is an essential process for cell locomotion, cytokinesis, vesicular transport and organelle morphogenesis. Formation of tubes or buds from a nearly flat membrane is a widely spread feature of the membrane shape changes. The BAR (Bin/Amphiphysin/Rvs-homology) domain dimers exhibit long extended curved shapes and appear to be outstandingly suitable modules for this purpose.

We have identified novel BAR proteins in two yeast species: C. albicans and S. pombe. The function of these novel BAR proteins are studied using a variety of in vitro and in vivo approaches such as yeast two-hybrid, liposome-binding and -vesiculation assays, GFP-tagging and gene knock out.

Nanomedicine, prof. Mulder

AMC Protein Technology Lab (AMC-PTL), dr Speijer & dr Bleijlevens

The department of Medical Biochemistry hosts the AMC-Protein Technology Laboratory (AMC-PTL). This facility provides technology and expertise in a broad range of protein research involving protein chemistry & analysis, ligand binding and protein-protein interactions.

Available technology in the AMC-PTL

  • Mass spectrometry, nanoLC-MS/MS (Synapt G2, Waters) for proteome analysis, interaction studies after immunoprecipitation or tag-based purification, characterization of post-translational modifications (PTMs).
  • Production and purification of recombinant proteins: advanced FPLC system (NGC, BioRad) for multi-dimensional purification in a thermostated cold cabinet.
  • Real-time characterization of protein-protein interactions, biolayer interferometry (BLItz, Pall ForteBio).
  • Multifunctional fluorescence spectrophotometer for protein-ligand interaction studies; fluorescence anisotropy (ligand binding/conformational changes), steady-state fluorescence, temperature ramping (via Peltier element) for stability studies (LS55, Perkin-Elmer).
  • Typhoon FLA 9500 imaging system (GE Healthcare), fluorescence scanner.
  • Ultracentrifuges.
  • Protein modeling: (1) homology modeling (multiple platforms) and structure analysis. (2) ligand binding simulations, virtual screening (autodock vina), prediction of binding modes of ‘small molecules’ (ligands) to proteins.

News

  • Dave Speijer's publication on evolution of peroxisomes was highlighted in BioEssays. A video abstract of his paper can be seen here.

    • Anthony Alioui (Zelcer group) published his work on the role of LXRs in constraining prostate cancer progression in Nature Communications.
    • A review on the immune modulation of brown(ing) of adipose tissue in obesity by Susan van den Berg (Lutgens & de Winther groups) and colleagues from the LUMC made the cover of Endocrine Reviews and was selected as Editor's Choice.
    • Jan Van den Bossche and his colleagues (de Winther group) published their work on macrophage immunometabolism in Cell Reports.

Researchers.

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