Simone König

The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Kaposis sarcoma–associated herpesvirus (KSHV) is the causative agent of Kaposis sarcoma, a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies. A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells. We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA2 and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry. Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposis sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposis sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.

Follicular Fluid High Density Lipoprotein-associated Sphingosine 1-Phosphate Is a Novel Mediator of Ovarian Angiogenesis

Angiogenesis plays an important role in the development of the ovarian follicle and its subsequent transition into the corpus luteum. Accordingly, follicular fluid is a rich source of mitogenic and angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor secreted by granulosa cells. In the present study, we show that follicular fluid deprived of basic fibroblast growth factor or vascular endothelial growth factor by means of thermal denaturation or antibody neutralization retains its capacity to stimulate endothelial proliferation and angiogenesis. Mass spectrometric analysis of chromatographic fractions stimulating endothelial growth obtained from follicular fluid revealed that the heat-stable mitogenic activity is identical with the subfraction α of high density lipoproteins purified from follicular fluid (FF-HDL). Further investigations demonstrated that sphingosine 1-phosphate (S1P), one of the lysophospholipids associated with HDL, accounts for the capacity of this lipoprotein to stimulate endothelial growth and the formation of new vessels. Activation of mitogen-activated protein kinase (p42/44ERK1/2), protein kinase C, and protein kinase Akt represent signaling pathways utilized by FF-HDL and S1P to induce endothelial proliferation and angiogenesis. We conclude that FF-HDL represents a novel mitogenic and angiogenic factor present in follicular fluid and that S1P is one of the FF-HDL lipid components accounting for this activity.

Small colony variants of Staphylococcus aureus reveal distinct protein profiles

Small‐colony variants (SCVs) of Staphylococcus aureus represent a slow‐growing subpopulation causing chronic and relapsing infections due to their physiological adaptation on an intracellular lifestyle. In this first proteomic study on physiological changes associated with a natural, clinically derived SCV, its proteomic profile was investigated in comparison to corresponding isogenic strains displaying normal (clinical wild‐type strain, complemented hemB mutant and spontaneous revertant of the clinical SCV) and SCV phenotypes (hemB mutant and gentamicin‐induced SCV). Applying an ultra‐high resolution chromatography and high mass accuracy MSE‐based label‐free relative and absolute protein quantification approach, the whole cytoplasmic proteome of this strain sextet was investigated in a growth phase‐controlled manner covering early‐exponential, late‐exponential and stationary phases. Of 1019 cytoplasmic proteins identified, 154 were found to be differently regulated between strains. All SCV phenotypes showed down‐regulation of the tricarboxylic acid (TCA) cycle‐related proteins and of a protein cluster involved in purine/pyrimidine and folate metabolism. In contrast to hemB mutant and gentamicin‐induced SCVs, the clinically derived SCVs showed no prominent up‐regulation of glycolytic proteins. The spontaneous switch into the normal phenotype resulted in up‐regulation of TCA cycle‐related parts, while oxidative stress‐related proteins were down‐regulated. However, the natural revertant from the clinical SCV retained also dominant protein features of the clinical SCV phenotype. In conclusion, physiological changes between normal and SCV S. aureus phenotypes are more complex than reflected by defined electron transport chain‐interrupting mutants and their complemented counterparts.

Both Estrogen Receptor Subtypes, α and β, Attenuate Cardiovascular Remodeling in Aldosterone Salt–Treated Rats

Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ER&agr; and ER&bgr;. Therefore, we determined whether selective activation of ER&agr; or of ER&bgr; would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt–treated rats. The ER&agr; agonist 16&agr;-LE2, the ER&bgr; agonist 8&bgr;-VE2, and the nonselective estrogen receptor agonist 17&bgr;-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16&agr;-LE2 and 17&bgr;-estradiol but not by 8&bgr;-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ER&agr; or ER&bgr; protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ER&bgr; agonists such as 8&bgr;-VE2 have a therapeutic potential in the treatment of hypertensive heart disease.

Evidence for perforin-like activity associated with earthworm leukocytes

Earthworm (Eisenia fetida) coelomic fluid contains several leukocytes (coelomocytes): basophils, acidophils and neutrophils as well as chloragocytes. Small coelomocytes and coelomocyte lysate are cytotoxic for the tumor cell target K562. The expression of a lytic factor was investigated by immunocytochemistry using light and transmission electron microscopy. A rat-anti-mouse-perforin-mAb labeled mainly small coelomocytes (nearly 20%) as visualized by light microscopy. TEM analysis using immunogold showed a homogenous labeling in the cytoplasm of small coelomocytes. The highest number of immunogold particles was estimated in coelomocytes with many small cytoplasmic granules. Coelomocytes with large lysosomal granules were also labeled but less intensely. No antibody binding was observed for chloragocytes either in light or electron microscopy. This suggests that the perforin-like activity is associated with only one cell type and that chloragocytes are responsible for other lytic activities. MALDI-MS revealed calreticulin usually associated with perforin in mammalian cells that mediate lysis (e.g. NK, CTL). Together, results strongly suggest the presence of putative perforin in earthworms. This in turn supports the hypothesis that perforin is a conserved component important in immune defense during evolution.

Activation Mechanism of Pro-astacin: Role of the Pro-peptide, Tryptic and Autoproteolytic Cleavage and Importance of Precise Amino-terminal Processing

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family.

Formation and Decomposition of Water Clusters as Observed in a Triple Quadrupole Mass Spectrometer

Water clustering up to mass 4000 has been observed using the Finnigan TSQ-700 electrospray mass spectrometer operating in either the normal or discharge modes with the capillary just above room temperature. The mechanism of cluster formation and structure has been studied by changing instrumental parameters including capillary temperature, auxiliary gas flow, and tube lens (skimmer) voltage. As expected, and in agreement with earlier work, enhanced abundances were observed at clusters of n = 21 and 28 molecules of water. Abundances of these same clusters are enhanced after fragmentation of higher mass clusters by MS/MS. Existing models including the clathrate structure are examined and it is suggested that with the exception of the structures at n = 21 and 28, the clusters are based on ice Ic. Also in agreement with previous work, water clusters were found to contain both protons and ammonium ions and the presence of the latter was proved by accurate mass measurement. In one case, these ions also attached to decomposition products formed by the discharge from traces of residual polyethylene glycol.

Capillary electrophoresis and off-line capillary electrophoresis-electrospray ionization quadrupole time-of-flight tandem mass spectrometry of carbohydrates

The use of off-line high-performance capillary electrophoresis in connection with nanospray electrospray ionization quadrupole time-of-flight tandem mass spectrometry for identification of complex carbohydrates of biological origin is presented. The method was applied to the identification of O-glycosylated amino acids and -glycopeptides from the urine of patients suffering from a hereditary disease - N-acetylhexosaminidase deficiency. Structural elements typical for O-glycosylation of proteins, like expression of core 1 and 2 type O-glycans with different numbers of N-acetyllactosaminyl repeats and different degrees of sialylation, can be directly detected.

Identification of poly(ADP-ribose)polymerase-1 and Ku70/Ku80 as transcriptional regulators of S100A9 gene expression

BackgroundS100 proteins, a multigenic family of non-ubiquitous cytoplasmic Ca2+-binding proteins, have been linked to human pathologies in recent years. Dysregulated expression of S100 proteins, including S100A9, has been reported in the epidermis as a response to stress and in association with neoplastic disorders. Recently, we characterized a regulatory element within the S100A9 promotor, referred to as MRE that drives the S100A9 gene expression in a cell type-specific, activation- and differentiation-dependent manner (Kerkhoff et al. (2002) J. Biol. Chem. 277, 41879–41887).ResultsIn the present study, we investigated transcription factors that bind to MRE. Using the MRE motif for a pull-down assay, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80 were identified by mass spectrometry and confirmed by chromatin immunoprecipitation. Furthermore, TPA-induced S100A9 gene expression in HaCaT keratinocytes was blocked after the pharmacologic inhibition of PARP-1 with 1,5-isoquinolinediol (DiQ).ConclusionThe candidates, poly(ADP-ribose)polymerase-1 (PARP-1) and the heterodimeric complex Ku70/Ku80, are known to participate in inflammatory disorders as well as tumorgenesis. The latter may indicate a possible link between S100 and inflammation-associated cancer.

Monitoring neuropeptide‐specific proteases: processing of the proopiomelanocortin peptides adrenocorticotropin and α‐melanocyte‐stimulating hormone in the skin

Abstract:  The neuroendocrine precursor protein proopiomelanocortin (POMC) and its derived neuropeptides are involved in a number of important regulatory processes in the central nervous system as well as in peripheral tissues. Despite its important role in controlling the local activation of melanocortin (MC) receptors, the extracellular proteolytic processing of POMC peptides has received little attention. The mechanisms relevant for controlling the bioavailability of adrenocorticotropin and melanocyte‐stimulating hormones for the corresponding MC receptors in the skin by specific peptidases such as neprilysin (neutral endopeptidase; NEP) or angiotensin‐converting enzyme (ACE) have been addressed in a number of recent investigations. This review summarizes the current body of knowledge concerning the qualitative and quantitative POMC peptide processing with respect to the action and specificity of NEP and ACE and discusses relevant recent analytical methodologies.