Holger Rehmann

University Medical Center Utrecht, Utrecht, Utrecht, Netherlands

Are you Holger Rehmann?

Claim your profile

Publications (70)613.35 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: CDC25 homology domain (CDC25-HD) containing Guanine Nucleotide Exchange Factors (GEFs) initiate signalling by small G-proteins of the Ras-family. Each GEF acts on a small subset of the G-proteins only, thus providing signalling selectivity. Rlf is a GEF with selectivity for the G-proteins RalA and RalB. Here the crystal structure of Rlf in complex with Ral is determined. The Rlf•Ral complex crystallised into two different crystal forms, which represent different steps of the exchange reaction. Thereby general insight in the CDC25-HD catalysed nucleotide exchange is obtained. In addition, the basis for the selectivity of the interaction is investigated. The exchange activity is monitored by the use of recombinant proteins. Selectivity determinants in the binding interface are identified and confirmed by a mutational study.
    No preview · Article · Dec 2015 · Journal of Structural Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Modulation of protein function is used to intervene in cellular processes but is often done indirectly by means of introducing DNA or mRNA encoding the effector protein. Thus far, direct intracellular delivery of proteins has remained challenging. We developed a method termed iTOP, for induced transduction by osmocytosis and propanebetaine, in which a combination of NaCl hypertonicity-induced macropinocytosis and a transduction compound (propanebetaine) induces the highly efficient transduction of proteins into a wide variety of primary cells. We demonstrate that iTOP is a useful tool in systems in which transient cell manipulation drives permanent cellular changes. As an example, we demonstrate that iTOP can mediate the delivery of recombinant Cas9 protein and short guide RNA, driving efficient gene targeting in a non-integrative manner. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Apr 2015 · Cell
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.
    Preview · Article · Mar 2015 · Proceedings of the National Academy of Sciences
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Author Summary cAMP is a small molecule produced by cells that activates proteins involved in a wide range of biological processes, including olfaction, pacemaker activity, regulation of gene expression, insulin secretion, and many others. In the case of insulin secretion, cAMP seems to impinge on different stages of the signalling cascade to regulate secretory activity in pancreatic β-cells. Here we have developed a chemically modified version of cAMP that specifically only activates Epac2, one of the cAMP-responsive proteins in this cascade. Furthermore, our cAMP analogue activates Epac2 more potently than cAMP itself does. We have determined several crystal structures of Epac2 in complex with cAMP analogues to help us explain the molecular basis of the observed selectivity and the strong activation potential. In addition, we were able to show that the analogue is able to potentiate glucose-induced secretion of insulin from human pancreatic islets. The principal challenge during this study was identifying and understanding small differences in the cAMP-binding domains of cAMP-regulated proteins and matching these differences with suitable modifications of the cAMP molecule.
    Full-text · Article · Jan 2015 · PLoS Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.
    Full-text · Article · Aug 2014 · Biochemical and Biophysical Research Communications
  • [Show abstract] [Hide abstract]
    ABSTRACT: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a central role in the regulation of cardiac and neuronal firing rate, and these channels can be dually activated by membrane hyperpolarization and by binding of cyclic nucleotides. cAMP has been shown to directly bind HCN channels and modulate their activity. Despite this, while there are selective inhibitors that block the activation potential of the HCN channels, regulation by cAMP analogs has not been well investigated. A comprehensive screen of 47 cyclic nucleotides with modifications in the nucleobase, ribose moiety, and cyclic phosphate was tested on the three isoforms HCN1, HCN2, and HCN4. 7-CH-cAMP was identified to be a high affinity binder for HCN channels and crosschecked for its ability to act on other cAMP receptor proteins. While 7-CH-cAMP is a general activator for cAMP- and cGMP-dependent protein kinases as well as for the guanine nucleotide exchange factors Epac1 and Epac2, it displays the highest affinity to HCN channels. The molecular basis of the high affinity was investigated by determining the crystal structure of 7-CH-cAMP in complex with the cyclic nucleotide binding domain of HCN4. Electrophysiological studies demonstrate a strong activation potential of 7-CH-cAMP for the HCN4 channel in vivo. So, this makes 7-CH-cAMP a promising activator of the HCN channels in vitro whose functionality can be translated in living cells.
    No preview · Article · Mar 2014 · ACS Chemical Biology
  • Source
    Holger Rehmann
    [Show abstract] [Hide abstract]
    ABSTRACT: cAMP is a universal second messenger. Its signalling is mediated by protein kinase A, Epac and certain types of ion channels in mammalians. cAMP signalling is involved in many physiological processes ranging from vision to the control of insulin secretion, pacemaker activity and gene transcription and therefore selective pharmacological interference is of medical interest. Whereas selective inhibitors of PKA and selective activators of Epac are well established, no inhibitors of Epac were available until recently. Here the action of four of the novel Epac inhibitors was analysed by biophysical means. ESI-05 is confirmed as a selective inhibitor of Epac2. No direct action of Brefeldin A on Epac could be demonstrated. ESI-09 and HJC0197 were found to act as chemicals with general protein denaturing properties and do not act on Epac selectively.
    Preview · Article · Oct 2013 · Scientific Reports
  • [Show abstract] [Hide abstract]
    ABSTRACT: Rlf is a Guanine Nucleotide Exchange Factor for the small G-proteins RalA and RalB and couples Ras- to Ral-signalling. Here the crystal structure of the catalytic module of Rlf consisting of a REM- and a CDC25-homology domain is determined. The structure is distinguished by an extended three stranded β-sheet called the flagpole. The flagpole is a conserved element in the RalGDS family of Guanine Nucleotide Exchange Factors and stabilises the orientation of the REM-domain relative to the CDC25-homology domain. A proline-rich sequence in the flagpole is unique to Rlf and several proteins that interact with this sequence by SH3 domains are identified. Conformational pre-selection results in a gain of affinity and contributes to the establishment of SH3 domain selectivity.
    No preview · Article · Jul 2013 · Journal of Structural Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Forkhead box O (FOXO) transcription factors are tumor suppressors and increase lifespan of model organisms. Cellular stress, in particular oxidative stress due to an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionary conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF, which is in complex with JIP1 and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4 and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C.elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.
    Preview · Article · Jun 2013 · Journal of Biological Chemistry
  • Milica Popovic · Marije Rensen-de Leeuw · Holger Rehmann
    [Show abstract] [Hide abstract]
    ABSTRACT: The Ras-family of small G-proteins plays an essential role in the regulation of a variety of signal transduction processes, ranging from cell cycle control to the regulation of exocytosis. Signalling by the Ras G-proteins is initiated by the CDC25-homology domain (CDC25-HD) containing Guanidine Nucleotide Exchange Factors (GEFs); each GEF, with its specific selectivity profile towards G-proteins, commonly acts on only a small subset of the Ras-family members. Thus, GEFs play a pivotal part in establishing the activation of the downstream signalling routes. The structural basis for the establishment of selectivity in the GEF-G-protein interaction is only partially understood, and several controversies on the selectivity of GEFs are discussed in the literature. In the present study, we undertook a systematic approach to determine the selectivity of CDC25-HD for members of the Ras-family. We generated a dataset of 126 pairs using a standardised in vitro approach encompassing purified recombinant proteins, and a comprehensive mutational study analysed the basis of the selectivity. Together, these data highlight the distinct selectivity of various GEFs and allow for predictions of untested combinations of GEFs and G-proteins.
    No preview · Article · May 2013 · Journal of Molecular Biology
  • Source
    Dataset: Montreal

    Full-text · Dataset · May 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2.
    Preview · Article · Feb 2013 · PLoS ONE
  • Source
    Dataset: 0310083

    Full-text · Dataset · Jan 2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Epac proteins are activated by binding of the second messenger cAMP and then act as guanine nucleotide exchange factors for Rap proteins. The Epac proteins are involved in the regulation of cell adhesion and insulin secretion. Here we have determined the structure of Epac2 in complex with a cAMP analogue (Sp-cAMPS) and RAP1B by X-ray crystallography and single particle electron microscopy. The structure represents the cAMP activated state of the Epac2 protein with the RAP1B protein trapped in the course of the exchange reaction. Comparison with the inactive conformation reveals that cAMP binding causes conformational changes that allow the cyclic nucleotide binding domain to swing from a position blocking the Rap binding site towards a docking site at the Ras exchange motif domain.
    Full-text · Dataset · Dec 2012
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Proper regulation of the formation and stabilization of epithelial cell-cell adhesion is crucial in embryonic morphogenesis and tissue repair processes. Defects in this process lead to organ malformation and defective epithelial barrier function. A combination of chemical and mechanical cues is used by cells to drive this process. We have investigated the role of the actomyosin cytoskeleton and its connection to cell-cell junction complexes in the formation of an epithelial barrier in MDCK cells. We find that the E-cadherin complex is sufficient to mediate a functional link between cell-cell contacts and the actomyosin cytoskeleton. This link involves the actin binding capacity of α-catenin and the recruitment of the mechanosensitive protein Vinculin to tensile, punctate cell-cell junctions that connect to radial F-actin bundles, which we name Focal Adherens Junctions (FAJ). When cell-cell adhesions mature, these FAJs disappear and linear junctions are formed that do not contain Vinculin. The rapid phase of barrier establishment (as measured by Trans Epithelial Electrical Resistance (TER)) correlates with the presence of FAJs. Moreover, the rate of barrier establishment is delayed when actomyosin contraction is blocked or when Vinculin recruitment to the Cadherin complex is prevented. Enhanced presence of Vinculin increases the rate of barrier formation. We conclude that E-cadherin-based FAJs connect forming cell-cell adhesions to the contractile actomyosin cytoskeleton. These specialized junctions are sites of Cadherin mechanosensing, which, through the recruitment of Vinculin, is a driving force in epithelial barrier formation.
    Full-text · Article · Nov 2012 · Biology Open
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To remodel endothelial cell-cell adhesion, inflammatory cytokine- and angiogenic growth factor-induced signals impinge on the vascular endothelial cadherin (VE-cadherin) complex, the central component of endothelial adherens junctions. This study demonstrates that junction remodeling takes place at a molecularly and phenotypically distinct subset of VE-cadherin adhesions, defined here as focal adherens junctions (FAJs). FAJs are attached to radial F-actin bundles and marked by the mechanosensory protein Vinculin. We show that endothelial hormones vascular endothelial growth factor, tumor necrosis factor α, and most prominently thrombin induced the transformation of stable junctions into FAJs. The actin cytoskeleton generated pulling forces specifically on FAJs, and inhibition of Rho-Rock-actomyosin contractility prevented the formation of FAJs and junction remodeling. FAJs formed normally in cells expressing a Vinculin binding-deficient mutant of α-catenin, showing that Vinculin recruitment is not required for adherens junction formation. Comparing Vinculin-devoid FAJs to wild-type FAJs revealed that Vinculin protects VE-cadherin junctions from opening during their force-dependent remodeling. These findings implicate Vinculin-dependent cadherin mechanosensing in endothelial processes such as leukocyte extravasation and angiogenesis.
    Preview · Article · Mar 2012 · The Journal of Cell Biology
  • [Show abstract] [Hide abstract]
    ABSTRACT: Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.
    No preview · Article · Feb 2012 · Cell
  • Source
    Holger Rehmann
    [Show abstract] [Hide abstract]
    ABSTRACT: Sulfonylureas are widely used oral drugs in the treatment of diabetes mellitus. They function by the inhibition of ATP-sensitive K+ channels in pancreatic β-cells, which are thus considered the 'classical' sulfonylurea receptor. Next to the ATP-sensitive K+ channels, additional sulfonylurea-interacting proteins were identified, which might contribute to the physiological effects of this drug family. Most recently, Epac2 (exchange protein directly activated by cAMP 2) was added to the list of sulfonylurea receptors. However, this finding caused controversy in the literature. The critical discussion of the present paper comes to the conclusion that sulfonylureas are not able to activate Epac2 directly and are unlikely to bind to Epac2. Increased blood glucose levels after food intake result in the secretion of insulin from pancreatic β-cells. Glucose levels are detected 'indirectly' by β-cells: owing to increased glycolysis rates, the ratio of cellular ATP/ADP increases and causes the closure of ATP-sensitive K+ channels. In consequence, cells depolarize and voltage-dependent Ca2+ channels open to cause an increase in the cellular Ca2+ concentration. Finally, Ca2+ induces the fusion of insulin-containing granules with the plasma membrane. Sulfonylureas, such as tolbutamide, glibenclamide or acetohexamide, form a class of orally applicable drugs used in the treatment of non-insulin-dependent diabetes mellitus.
    Preview · Article · Feb 2012 · Biochemical Society Transactions
  • [Show abstract] [Hide abstract]
    ABSTRACT: Photodynamic therapy has great potential as nephron sparing therapy for small renal masses. Using mTHPC [meso-tetra(hydroxyphenyl)chlorin] (BioLitec Pharma, Dublin, Ireland), a photosensitizer that targets vasculature and tissue, we determined whether renal tumors could be ablated using mTHPC mediated photodynamic therapy in a translational renal carcinoma mouse model. We administered mTHPC intravenously in kidney tumor bearing mice. Tumor diameter was about 7 mm. At several drug-light intervals a cylindrical laser fiber was placed intratumorally for interstitial illumination using a wavelength of 652 nm. We determined mTHPC biodistribution up to 48 hours after administration and tumor destruction after mTHPC mediated photodynamic therapy. In vitro mTHPC uptake and photodynamic therapy induced cytotoxicity were studied in human endothelial, renal and renal cell carcinoma cell lines. Ablated regions with a maximum diameter of 9.3 mm and complete loss of cell viability were observed at a drug-light interval of 4 hours, when mTHPC was increased in blood and tissue. Viable renal tissue remained detectable outside the illuminated area. In endothelial cells mTHPC uptake and sensitivity to photodynamic therapy were increased compared to those in renal cell carcinoma and renal cells. mTHPC mediated photodynamic therapy is a nephron sparing therapy. The extent of renal tumor destruction is adequate for clinical translation. Localization of mTHPC in tumor vasculature and tissue produces a strong combined effect. Our findings justify further preclinical studies of the applicability of photodynamic therapy for renal cell carcinoma before photodynamic therapy can become a valuable addition to current minimally invasive treatments of small renal masses.
    No preview · Article · Nov 2011 · The Journal of urology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In vertebrates, left-right (LR) axis specification is determined by a ciliated structure in the posterior region of the embryo. Fluid flow in this ciliated structure is responsible for the induction of unilateral left-sided Nodal activity in the lateral plate mesoderm, which in turn regulates organ laterality. Bmp signalling activity has been implied in repressing Nodal expression on the right side, however its mechanism of action has been controversial. In a forward genetic screen for mutations that affect LR patterning, we identified the zebrafish linkspoot (lin) mutant, characterized by cardiac laterality and mild dorsoventral patterning defects. Mapping of the lin mutation revealed an inactivating missense mutation in the Bmp receptor 1aa (bmpr1aa) gene. Embryos with a mutation in lin/bmpr1aa and a novel mutation in its paralogue, bmpr1ab, displayed a variety of dorsoventral and LR patterning defects with increasing severity corresponding with a decrease in bmpr1a dosage. In Bmpr1a-deficient embryos we observed bilateral expression of the Nodal-related gene, spaw, coupled with reduced expression of the Nodal-antagonist lefty1 in the midline. Using genetic models to induce or repress Bmp activity in combination with Nodal inhibition or activation, we found that Bmp and Nodal regulate lefty1 expression in the midline independently of each other. Furthermore, we observed that the regulation of lefty1 by Bmp signalling is required for its observed downregulation of Nodal activity in the LPM providing a novel explanation for this phenomenon. From these results we propose a two-step model in which Bmp regulates LR patterning. Prior to the onset of nodal flow and Nodal activation, Bmp is required to induce lefty1 expression in the midline. When nodal flow has been established and Nodal activity is apparent, both Nodal and Bmp independently are required for lefty1 expression to assure unilateral Nodal activation and correct LR patterning.
    Full-text · Article · Sep 2011 · PLoS Genetics

Publication Stats

4k Citations
613.35 Total Impact Points

Institutions

  • 2000-2015
    • University Medical Center Utrecht
      • Department of Physiological Chemistry
      Utrecht, Utrecht, Netherlands
  • 2011
    • Medizinische Universität Wien
      • Zentrum für Hirnforschung
      Vienna, Vienna, Austria
  • 2006-2008
    • Universiteit Utrecht
      • • Bijvoet Institute for Biomolecular Research
      • • Department of Physiological Chemistry
      Utrecht, Utrecht, Netherlands
  • 2003
    • Max Planck Institute of Molecular Physiology
      Dortmund, North Rhine-Westphalia, Germany