Kerstin Freidel

Concurrent knock‐out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae

The hexose transporter family of Saccharomyces cerevisiae comprises 18 proteins (Hxt1–17, Gal2). Here, we demonstrate that all these proteins, except Hxt12, and additionally three members of the maltose transporter family (Agt1, Ydl247, Yjr160) are able to transport hexoses. In a yeast strain deleted for HXT1–17, GAL2, AGT1, YDL247w and YJR160c, glucose consumption and transport activity were completely abolished. However, as additional deletion of the glucose sensor gene SNF3 partially restored growth on hexoses, our data indicate the existence of even more proteins able to transport hexoses in yeast.

Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae

SummaryWe have analysed functional properties of putative proteins encoded by the yeast transposable element, Ty1, by overexpression of TY genes. High-level expression was achieved by appropriate fusion of a Ty sequence, TY9C, to the yeast ADH1 promoter and transformation of yeast cells with this construction. As shown recently by others (Garfinkel et al. 1985; Mellor et al. 1985c) TY over-expression leads to an increase in particle-bound reverse transcriptase activity and to an intracellular accumulation of virus-like particles (Ty-VLPs). We have used a number of deletions in the second open reading frame (TYB) to identify functional domains required for processing and assembly of Ty proteins. Deletions in the TYB region with homology to acid proteases result in overproduction of an unprocessed form of the TYA protein (pro-TYA) which represents the major protein of Ty-VLPs. One particular mutant construction, TY9C-Δ36, led to the accumulation of a particle-bound, 160 kDa protein which cross-reacted with a mouse antiserum raised against purified pro-TYA protein. This supports the hypothesis that TYB is expressed as a TYA/TYB fusion protein which is processed by a TYB-encoded protease activity. Ty-VLPs are formed in the absence of protein processing and even when the TYB gene is not expressed. Thus, we assume that the assembly of Ty particles occurs prior to processing of Ty proteins.

Characterization of trans-acting mutations affecting Ty and Ty-mediated transcription in Saccharomyces cerevisiae.

SummaryBy recessive mutations, we have identified five genes, TYE1-TYE5, that are required for Ty-mediated expression of ADH2. These tye mutations not only suppress transcription of ADH2 when associated with a Ty element but are also defective in transcription of all Ty1 and Ty2 elements. Moreover, some of these mutations cause growth defects on non-fermentable carbon sources as well as sporulation defects. tye mutations also strongly suppress ADH2 expression when controlled by a polyA/T insertion mutation. Genetic analysis revealed that genes TYE3 and TYE4 are allelic to the previously identified genes SNF2 and SNF5 which code for transcription factors. These findings suggest that TYE gene products influence transcription of many genes rather than specifically Ty and Ty-mediated transcrption. We have also found that null alleles of certain STE genes (ste7, ste11 and ste12), known to affect cell-type specific gene expression and expression of some Ty-adjacent genes, have a clear effect on Ty-controlled ADH2 expression depending on the carbon source. On the basis of ADH2 transcript levels in glucose-grown cells, all three ste alleles cause of five-fold reduction of ADH2 expression/transcription. In ethanol-grown cells, ste11 and ste12 mutations caused an almost complete loss of Ty-mediated ADH2 activation while ste7 has only a rather moderate effect. Surprisingly, ste11 and ste12 mutations lead to a significant increase in total Ty transcript levels. This would indicate that the STE12 protein, which is known to bind specifically to Ty1 sequences and thereby serve as an activator of a Ty-adjacent gene, can negatively modulate Ty transcription. The STE7 and STE11 genes which encode protein kinases apparently act in a different manner in both Ty and Ty-mediated transcription.

Cyclooxygenase COX-2a, a novel COX-2 mRNA variant, in platelets from patients after coronary artery bypass grafting.

There are two principal cyclooxygenase isoforms referred to as COX-1 and COX-2. Recently, COX-3 has been identified. We have demonstrated the expression of COX-2 in platelets from patients after coronary artery bypass grafting (CABG). Careful biochemical analysis revealed that, when compared to recombinant COX-2, platelet COX-2 had a slightly higher electrophoretic mobility. Two COX-2 sequences (approximately 1.8 kb, approximately 1.7 kb) were cloned from platelet mRNA. The approximately 1.7 kb sequence, designated COX-2a, differed from the human COX-2 sequence only in a deletion from position +458 to +567. Similar to the human COX-3, there is a frame shift in the COX-2a sequence resulting in a TAA stop codon at position +490. Thus, the expression of a COX-2a protein corresponding to the 67 kDa COX-2 protein is not clear. However, the marked shifting from COX-2 to COX-2a in platelets from some patients after CABG is a striking finding.

Redox regulation of human protease-activated receptor-2 by activated factor X

Activated factor X (FXa) exerts coagulation-independent actions such as proliferation of vascular smooth muscle cells (SMCs) through the protease-activated receptors PAR-1 and PAR-2. Both receptors are upregulated upon vascular injury but the underlying mechanisms have not been defined. We examined if FXa regulates PAR-1 and PAR-2 in human vascular SMCs. FXa increased PAR-2 mRNA, protein, and cell-surface expression and augmented PAR-2-mediated mitogenesis. PAR-1 was not influenced. The regulatory action of FXa on PAR-2 was concentration-dependent and mimicked by a PAR-2-selective activating peptide. PAR-2 regulation was not influenced by the thrombin inhibitor argatroban or PAR-1 siRNA. FXa increased dichlorofluorescein diacetate fluorescence and 8-isoprostane formation and induced expression of the NADPH oxidase subunit NOX-1. NOX-1 siRNA prevented FXa-stimulated PAR-2 regulation, as did ebselen and cell-permeative and impermeative forms of catalase. Exogenous H(2)O(2) increased PAR-2 expression and mitogenic activity. FXa promoted nuclear translocation and PAR-2/DNA binding of nuclear factor κB (NF-κB); NF-κB inhibition prevented PAR-2 regulation by FXa. FXa also promoted PAR-2 mRNA stabilization through increased human antigen R (HuR)/PAR-2 mRNA binding and cytoplasmic shuttling. HuR siRNA abolished FXa-stimulated PAR-2 expression. Thus FXa induces functional expression of PAR-2 but not of PAR-1 in human SMCs, independent of thrombin formation, via a mechanism involving NOX-1-containing NADPH oxidase, H(2)O(2), NF-κB, and HuR.

Complete Downmodulation of P-Selectin Glycoprotein Ligand in Monocytes Undergoing Apoptosis

Objectives—Apoptotic monocytes release membrane microparticles which may play a major role in thrombogenicity through a P-selectin glycoprotein ligand (PGSL-1)–mediated mechanism. We have studied systematically the regulation of PSGL-1 expression and function in apoptotic monocytic cells. Methods and Results—PSGL-1 expression (flow cytometry, immunofluorescence microscopy, immunoblot) was virtually abolished in apoptotic monocytes by proteolytic shedding. This was accompanied by a complete loss of PSGL-1–mediated platelet–leukocyte (flow cytometry) and leukocyte–endothelial cell (parallel plate flow chamber) interactions. Systematic screening of protease inhibitors combined with knock-out and siRNA experiments characterized the PSGL-1-cleaving enzyme as an N-ethylmaleimide-inhibitable metalloproteinase of the ADAM family. Conclusions—Downmodulation of PGSL-1 in apoptotic monocytes may prevent ectopic cell clearance in the peripheral vasculature to reduce local inflammatory and proliferative responses. Depletion of PSGL-1 expression on apoptotic microparticles may also act as a molecular switch to modulate their thrombogenic activity.