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Papers by Willm-Thomas Heyken

Yeast, 2003

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinat... more Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are coordinately regulated by a UAS element, designated ICRE (inositol/choline-responsive element). Opi1 is a negative regulator responsible for repression of ICRE-dependent genes in the presence of an excess of inositol and choline. Gene regulation by phospholipid precursors has been also reported for the pathogenic yeast Candida albicans. Screening of a data base containing raw sequences of the C. albicans genome project allowed us to identify an open reading frame exhibiting weak similarity to Opi1. Expression of the putative CaOPI1 in an opi1 mutant of S. cerevisiae could restore repression of an ICRE-dependent reporter gene. Similar to OPI1, overexpression of CaOPI1 strongly inhibited derepression of ICRE-driven genes leading to inositol-requiring transformants. Previous work has shown that Opi1 mediates gene repression by interaction with the pleiotropic repressor Sin3. The genome of C. albicans also encodes a protein similar to Sin3 (CaSin3). By two-hybrid analyses and in vitro studies for protein-protein interaction we were able to show that CaOpi1 binds to ScSin3. ScOpi1 could also interact with CaSin3, while CaOpi1 failed to bind to CaSin3. Despite of some conservation of regulatory mechanisms between both yeasts, these results suggest that repression of phospholipid biosynthetic genes in C. albicans is mediated by a mechanism which does not involve recruitment of CaSin3 by CaOpi1.

Nephrology Dialysis Transplantation, 2007

Molecular Microbiology, 2003

In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated ... more In the yeast Saccharomyces cerevisiae, genes involved in phospholipid biosynthesis are activated by ICRE (inositol/choline-responsive element) up-stream motifs and the corresponding heterodimeric binding factor, Ino2 + Ino4. Both Ino2 and Ino4 contain basic helix-loop-helix (bHLH) domains required for ICRE binding, whereas transcriptional activation is mediated exclusively by Ino2. In this work, we describe a molecular analysis of functional minimal domains responsible for specific DNA recognition and transcriptional activation (TAD1 and TAD2). We also define the importance of individual amino acids within the more important activation domain TAD1. Random mutagenesis at five amino acid positions showed the importance of acidic as well as hydrophobic residues within this minimal TAD. We also investigated the contribution of known general transcription factors and co-activators for Ino2-dependent gene activation. Although an ada5 single mutant and a gal11 paf1 double mutant were severely affected, a partial reduction in activation was found for gcn5 and srb2. Ino2 interacts physically with the basal transcription factor Sua7 (TFIIB of yeast). Interestingly, interaction is mediated by the HLH dimerization domain of Ino2 and by two non-overlapping domains within Sua7. Thus, Sua7 may compete with Ino4 for binding to the Ino2 activator, creating the possibility of positive and negative influence of Sua7 on ICRE-dependent gene expression.

Molecular Microbiology, 2005

Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring... more Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118-135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1-dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICREdependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RIDbinding activator interaction domain (AID, aa 321-380) at the C-terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID-AID interaction as the crucial step of regulated expression of ICRE-dependent genes.

Circulation Research, 2006

The endothelium plays a key role in the control of vascular tone and alteration in endothelial ce... more The endothelium plays a key role in the control of vascular tone and alteration in endothelial cell function contributes to several cardiovascular disease states. Endothelium-dependent dilation is mediated by NO, prostacyclin, and an endothelium-derived hyperpolarizing factor (EDHF). EDHF signaling is thought to be initiated by activation of endothelial Ca 2ϩ -activated K ϩ channels (K Ca ), leading to hyperpolarization of the endothelium and subsequently to hyperpolarization and relaxation of vascular smooth muscle. In the present study, we tested the functional role of the endothelial intermediateconductance K Ca (IK Ca /K Ca 3.1) in endothelial hyperpolarization, in EDHF-mediated dilation, and in the control of arterial pressure by targeted deletion of K Ca 3.1. K Ca 3.1-deficient mice (K Ca 3.1 Ϫ/Ϫ ) were generated by conventional gene-targeting strategies. Endothelial K Ca currents and EDHF-mediated dilations were characterized by patch-clamp analysis, myography and intravital microscopy. Disruption of the K Ca 3.1 gene abolished endothelial K Ca 3.1 currents and significantly diminished overall current through K Ca channels. As a consequence, endothelial and smooth muscle hyperpolarization in response to acetylcholine was reduced in K Ca 3.1 Ϫ/Ϫ mice. Acetylcholine-induced dilations were impaired in the carotid artery and in resistance vessels because of a substantial reduction of EDHF-mediated dilation in K Ca 3.1 Ϫ/Ϫ mice. Moreover, the loss of K Ca 3.1 led to a significant increase in arterial blood pressure and to mild left ventricular hypertrophy. These results indicate that the endothelial K Ca 3.1 is a fundamental determinant of endothelial hyperpolarization and EDHF signaling and, thereby, a crucial determinant in the control of vascular tone and overall circulatory regulation. (Circ Res. 2006;99:537-544.)

Cardiovascular Research, 2008

British Journal of Pharmacology, 2009

Arteriosclerosis, Thrombosis, and Vascular Biology, 2006

Objective-Ca 2ϩ -influx through transient receptor potential (TRP) channels was proposed to be im... more Objective-Ca 2ϩ -influx through transient receptor potential (TRP) channels was proposed to be important in endothelial function, although the precise role of specific TRP channels is unknown. Here, we investigated the role of the putatively mechanosensitive TRPV4 channel in the mechanisms of endothelium-dependent vasodilatation. Methods and Results-Expression and function of TRPV4 was investigated in rat carotid artery endothelial cells (RCAECs) by using in situ patch-clamp techniques, single-cell RT-PCR, Ca 2ϩ measurements, and pressure myography in carotid artery (CA) and Arteria gracilis. In RCAECs in situ, TRPV4 currents were activated by the selective TRPV4 opener 4␣-phorbol-12,13-didecanoate (4␣PDD), arachidonic acid, moderate warmth, and mechanically by hypotonic cell swelling. Single-cell RT-PCR in endothelial cells demonstrated mRNA expression of TRPV4. In FURA-2 Ca 2ϩ measurements, 4␣PDD increased [Ca 2ϩ ] i by Ϸ140 nmol/L above basal levels. In pressure myograph experiments in CAs and A gracilis, 4␣PDD caused robust endothelium-dependent and strictly endothelium-dependent vasodilatations by Ϸ80% (K D 0.3 mol/L), which were suppressed by the TRPV4 blocker ruthenium red (RuR). Shear stress-induced vasodilatation was similarly blocked by RuR and also by the phospholipase A 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3 ). 4␣PDD produced endothelium-derived hyperpolarizing factor (EDHF)-type responses in A gracilis but not in rat carotid artery. Shear stress did not produce EDHF-type vasodilatation in either vessel type. Conclusions-Ca 2ϩ entry through endothelial TRPV4 channels triggers NO-and EDHF-dependent vasodilatation.

PLoS ONE, 2007

In blood vessels, the endothelium is a crucial signal transduction interface in control of vascul... more In blood vessels, the endothelium is a crucial signal transduction interface in control of vascular tone and blood pressure to ensure energy and oxygen supply according to the organs&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; needs. In response to vasoactive factors and to shear stress elicited by blood flow, the endothelium secretes vasodilating or vasocontracting autacoids, which adjust the contractile state of the smooth muscle. In endothelial sensing of shear stress, the osmo- and mechanosensitive Ca(2+)-permeable TRPV4 channel has been proposed to be candidate mechanosensor. Using TRPV4(-/-) mice, we now investigated whether the absence of endothelial TRPV4 alters shear-stress-induced arterial vasodilation. In TRPV4(-/-) mice, loss of the TRPV4 protein was confirmed by Western blot, immunohistochemistry and by in situ-patch-clamp techniques in carotid artery endothelial cells (CAEC). Endothelium-dependent vasodilation was determined by pressure myography in carotid arteries (CA) from TRPV4(-/-) mice and wild-type littermates (WT). In WT CAEC, TRPV4 currents could be elicited by TRPV4 activators 4alpha-phorbol-12,13-didecanoate (4alphaPDD), arachidonic acid (AA), and by hypotonic cell swelling (HTS). In striking contrast, in TRPV4(-/-) mice, 4alphaPDD did not produce currents and currents elicited by AA and HTS were significantly reduced. 4alphaPDD caused a robust and endothelium-dependent vasodilation in WT mice, again conspicuously absent in TRPV4(-/-) mice. Shear stress-induced vasodilation could readily be evoked in WT, but was completely eliminated in TRPV4(-/-) mice. In addition, flow/reperfusion-induced vasodilation was significantly reduced in TRPV4(-/-) vs. WT mice. Vasodilation in response to acetylcholine, vasoconstriction in response to phenylephrine, and passive mechanical compliance did not differ between genotypes, greatly underscoring the specificity of the above trpv4-dependent phenotype for physiologically relevant shear stress. Genetically encoded loss-of-function of trpv4 results in a loss of shear stress-induced vasodilation, a response pattern critically dependent on endothelial TRPV4 expression. Thus, Ca(2+)-influx through endothelial TRPV4 channels is a molecular mechanism contributing significantly to endothelial mechanotransduction.