St. John's wort extract Ze 117 alters the membrane fluidity of C6 glioma cells by influencing cellular cholesterol metabolism.

Chronic stress is associated with major depressive disorder (MDD). Increased glucocorticoid levels caused by uncontrolled release through the hypothalamic‒pituitary‒adrenal (HPA) axis can cause changes in the lipid content of the cellular plasma membrane. These changes are suspected to be involved in the development of depressive disorders. St. John's wort extract (SJW) Ze 117 has long been used as an alternative to synthetic antidepressants. Part of its effect may be due to an effect on the cellular lipid composition and thus on the properties of plasma membranes and receptor systems embedded therein. In this study, we investigated the effect of Ze 117 on that of dexamethasone and simvastatin. Dexamethasone increases the fluidity of C6 cell plasma membranes. This effect is counteracted by administration of Ze 117. Here we demonstrate that this is not due to a change in C16:1/16:0 and C18:1/18:0 ratios in C6 cell fatty acids. On the other hand, Ze 117 increased the cellular cholesterol content by 42.5%, whereas dexamethasone reduced cholesterol levels similarly to simvastatin. Lowering cholesterol levels by dexamethasone or simvastatin resulted in decreased ß-arrestin 2 recruitment to the 5-HT1a receptor. This effect was counterbalanced by Ze 117, whereas the SJW extract had little effect on ß-arrestin 2 recruitment in non-stressed cells. Taken together, in C6 cells, Ze 117 induces changes in membrane fluidity through its effect on cellular cholesterol metabolism rather than by affecting fatty acid saturation. This effect is reflected in an altered signal transduction of the 5-HT1a receptor under Ze 117 administration. The current in vitro results support the hypothesis that Ze 117 addresses relevant parts of the cellular lipid metabolism, possibly explaining some of the antidepressant actions of Ze 117.

Ivy Leaf Dry Extract EA 575® Has an Inhibitory Effect on the Signalling Cascade of Adenosine Receptor A2B.

Ivy leaf dry extract EA 575® is used to improve complaints of chronic inflammatory bronchial diseases and acute inflammation of the respiratory tract accompanied by coughing. Its mechanism of action has so far been explained by influencing ß2-adrenergic signal transduction. In the present study, we investigated a possible influence on adenosine receptor A2B (A2BAR) signalling, as it has been described to play a significant and detrimental role in chronic inflammatory airway diseases. The influence of EA 575® on A2BAR signalling was assessed with measurements of dynamic mass redistribution. Subsequently, the effects on A2BAR-mediated second messenger cAMP levels, ß-arrestin 2 recruitment, and cAMP response element (CRE) activation were examined using luciferase-based HEK293 reporter cell lines. Lastly, the impact on A2BAR-mediated IL-6 release in Calu-3 epithelial lung cells was investigated via the Lumit™ Immunoassay. Additionally, the adenosine receptor subtype mediating these effects was specified, and A2BAR was found to be responsible. The present study demonstrates an inhibitory influence of EA 575® on A2BAR-mediated general cellular response, cAMP levels, ß-arrestin 2 recruitment, CRE activation, and IL-6 release. Since these EA 575®-mediated effects occur within a time frame of several hours of incubation, its mode of action can be described as indirect. The present data are the first to describe an inhibitory effect of EA 575® on A2BAR signalling. This may offer an explanation for the beneficial clinical effects of the extract in adjuvant asthma therapy.

Investigation of adenosine A1 receptor-mediated ß-arrestin 2 recruitment using a split-luciferase assay.

Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep-wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated ß-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of ß-arrestin 2. Stimulation of A1AR results in the recruitment of ß-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of ß-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1AR-mediated recruitment of ß-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A1AR-mediated ß-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.

Impact of St. John's wort extract Ze 117 on stress induced changes in the lipidome of PBMC.

BACKGROUND: Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. METHODS: Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John's wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 µM] PBMCs with Ze 117 [10-50 µg/ml] were analyzed by mass spectrometry. RESULTS: Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 µM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. CONCLUSION: The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

Effect of St. John's wort extract Ze 117 on the lateral mobility of ß1-adrenergic receptors in C6 cells.

Depression has been associated with altered signal transduction of serotonergic, dopaminergic and adrenergic neurotransmitter systems in the brain. Signaling relies on receptor-ligand interactions and subsequent regulatory processes, but also on lateral receptor mobility. The aim of this study was to investigate the effect of the St. John's wort extract Ze 117 on the lateral mobility of SNAP-tagged ß1-adrenergic receptors (ß1AR) in the plasma membrane of C6 cells under both, non-stimulating and isoprenaline-stimulating conditions. Single particle tracking (SPT) was used, whereby the registered trajectories were evaluated by variational Bayesian treatment of a hidden Markov model (vbSPT) and packing coefficient (Pc) analysis with respect to diffusion coefficients, receptor state occupancies and confinement. Three different diffusion states were identified, differing in their diffusion coefficients. Treatment with Ze 117 [25 µg/ml] decreased the mobility of the ß1AR, which was manifested by a relative increase in the slow-diffusing state S1 (0.21-0.30) compared to control and by an increase in receptor confinement (79.4-68.1 nm). After isoprenaline stimulation of control cells, the slow-diffusing state was more pronounced, whereas confinement was not affected. In summary, SPT has been shown to be a powerful method to analyze lateral receptor mobility. Furthermore, the present study identified a correlation between Ze 117 treatment and ß1AR mobility.

Ivy leaves dry extract EA 575® mediates biased ß2-adrenergic receptor signaling.

BACKGROUND: ß2-adrenergic receptor (ß2-AR) stimulation activates the G protein/cAMP pathway, which is opposed by the GRK2/ß-arrestin 2 pathway. The latter is undesirable in the treatment of respiratory diseases. HYPOTHESIS/PURPOSE: EA 575® is capable of mediating a biased ß2-adrenergic signaling pathway. METHODS: The impact of the ivy leaves dry extract EA 575® on ß2-adrenergic signaling was tested in a dynamic mass redistribution assay in HEK wild-type and in HEK ß-arrestin knock-out cells. cAMP formation and recruitment of ß-arrestin 2 were investigated using GloSensor™ and PathHunter® assays, respectively. NFκB transcriptional activity was determined in both HEK wild-type as well as HEK ß-arrestin knock-out cells. RESULTS: EA 575® inhibits the recruitment of ß-arrestin 2 and thereby enhances G protein/cAMP signaling under ß2-stimulating conditions, as evidenced by a corresponding increase in cAMP formation. While ß2-AR-mediated inhibition of NFκB transcriptional activity is ß-arrestin-dependent, EA 575® leads to significant inhibition of NFκB transcriptional activity in ß-arrestin knock-out cells and thus via a ß-arrestin-independent signaling pathway. CONCLUSION: EA 575® is the first active phytopharmaceutical ingredient for which biased ß2-adrenergic activation has been described. This shift towards G protein/cAMP signaling provides the molecular basis for the clinically proven efficacy of EA 575® in the treatment of lower respiratory tract diseases. In this light, EA 575® could potentially reduce ß-arrestin-mediated adverse effects in new combinatorial therapeutic approaches.

A comparative study on the influence of an ivy preparation and an ivy/thyme combination on the ß2-adrenergic signal transduction.

The ß2-adrenergic receptor (ß2AR) is relevant for surfactant formation in alveolar type 2 cells and reduction of intracellular calcium concentration in bronchial muscle cells and thus for secretolytic and bronchospasmolytic effects. Herbal medicinal products that affect the ß2AR system are used to treat common cold and bronchitis accompanied with mucus covered and narrowed airways. The present work compares the influence of an ivy preparation and an ivy/thyme combination on the ß2-adrenergic signal transduction. For receptor binding studies and characterization of the lateral mobility of ß2AR we have used single molecule detection by fluorescence correlation spectroscopy and single particle tracking. For the determination of both the second messenger cAMP and the internalization of ß2AR we have generated luciferase based reporter cell lines, which produce a cAMP-dependent luciferase in the cytosol and express ß2AR with extracellular luciferase moiety in the plasma membrane. While both preparations increased the ß2AR binding, a significant increase of the cAMP level was observed only for the ivy preparation, which can be explained by the inhibited internalization of HiBiT-tagged ß2AR under stimulating conditions. In contrast, isoprenaline-mediated internalization of HiBiT-tagged ß2AR of ivy/thyme combination pre-treated cells was not inhibited. Cells comparatively pre-treated with a thyme preparation did not show inhibition of ß2AR internalization either. Furthermore, SNAP-tagged ß2AR of ivy preparation pre-treated cells, which were not internalized after isoprenaline stimulation, showed a redistribution from fast-to-slowly diffusing ß2AR. A corresponding redistribution of these receptors was not observed after pre-treatment with both the ivy/thyme combination and the thyme preparation. Comparable to the ivy/thyme combination, no decrease in the intratrack transitioning probability ratio (p23/p32) for fast and slow diffusing ß2AR was found for the thyme preparation, which, however, significantly decreased for control cells and for pre-treatment with the ivy preparation under stimulating conditions. It can therefore be concluded that the thyme fluid extract fraction in the ivy/thyme combination may have in part a negative effect on the ß2-adrenergic signal transduction.

St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells.

BACKGROUND: Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. METHODS: Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of ß1-adrenoceptors and changes in cAMP formation were measured by ELISA. RESULTS: Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of ß1-adrenoceptors as well as reduced cAMP formation after stimulation with the ß1-adrenoceptor agonist dobutamine. CONCLUSION: Obviously, the signaling of ß1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.

Anti-inflammatory effects of ivy leaves dry extract: influence on transcriptional activity of NFκB.

EA 575® is an ivy leaves dry extract (DER 5-7.5:1, 30% ethanol) used against diseases of the lower respiratory tract associated with productive cough. EA 575® improves symptoms associated with chronic inflammatory bronchial conditions. Compared to its bronchospasmolytic and secretolytic properties, the anti-inflammatory effects of EA 575® are mostly untried. Therefore, we addressed the question of whether the anti-inflammatory effect of EA 575® is due to an impact on the NFκB pathway. NFκB nuclear translocation was visualized by immunofluorescence in J774.2 as well as HEK293 cells. In the latter, a luciferase-based reporter was used to monitor NFκB transcriptional activity. Phosphorylation of RelA and its inhibitor IκB was measured by Western blot analysis. Additionally, changes in the stability of NFκB:IκB complex were shown by protein fragment complementation. Decreased transcriptional activity of NFκB under treatment with EA 575® was also shown for a human monocytic as well as a human lung epithelial cell line. EA 575® is able to inhibit NFκB transcriptional activity by partially inhibiting its translocation to the nucleus after stimulation with TNFα. Furthermore, phosphorylation of IκBα is reduced while phosphorylation of RelA is enhanced after pre-incubation with EA 575®, leading to an enhanced stability of NFκB:IκBα complex. EA 575® has an regulatory impact on the NFκB pathway, possibly by switching specificity of IKK from IκBα to RelA, resulting in enhanced stability of NFκB:IκBα complex and reduced RelA translocation into the nucleus.

Splice variants of perlucin from Haliotis laevigata modulate the crystallisation of CaCO3.

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO3 and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.

Targeting cancer with a bi-functional peptide: in vitro and in vivo results.

BACKGROUND: Current therapies to treat cancer, although successful for some patients, have significant side-effects and a high number of patients have disease that is either non-responsive or which develops resistance. Our goal was to design a small peptide that possesses similar functions to an antibody drug conjugate with regard to targeting and killing cancer cells, but that overcomes size restrictions. MATERIALS AND METHODS: We designed a novel cancer-specific killer peptide created by fusion of the toxic peptide (KLAKLAK)2 with the cancer recognition peptide LTVSPWY. RESULTS: This bi-functional peptide showed toxicity to breast cancer, prostate cancer, and neuroblastoma cell lines. Only low toxicity to non-cancer cells, colon cancer, lung cancer, and lymphoma cell lines was observed. In vivo injections of the bi-functional peptide caused tumor growth retardation compared to mice treated with control peptides. The bi-functional peptide caused retardation of MDA-MB-435S tumors in vivo and increased survival to 80% at day 100 after tumor implantation, whereas all control animals died at day 70. Previous reports showed that the recognition moiety LTVSPWY targets the tumor-associated antigen HER2. Here we found that our new peptide TP-Tox also excerts toxic effects on HER2-negative cell lines. Therefore, we searched for the molecular target of the bi-specific peptide using immunoprecipitation and mass spectrometry. Our data suggest a possible interaction with RAS GTPase-activating protein binding protein 1 (G3BP1). CONCLUSION: We designed a bi-functional peptide of 23 amino acids and demonstrated its ability to bind and kill several cancer cell lines in vitro and to strongly increase survival in breast cancer bearing mice in vivo. This novel toxin could be used in future cancer therapies and warrants further pre-clinical and clinical exploration.

Hepatoma-derived growth factor and nucleolin exist in the same ribonucleoprotein complex.

BACKGROUND: Hepatoma-derived growth factor (HDGF) is a protein which is highly expressed in a variety of tumours. HDGF has mitogenic, angiogenic, neurotrophic and antiapoptotic activity but the molecular mechanisms by which it exerts these activities are largely unknown nor has its biological function in tumours been elucidated. Mass spectrometry was performed to analyse the HDGFStrep-tag interactome. By Pull-down-experiments using different protein and nucleic acid constructs the interaction of HDGF and nucleolin was investigated further. RESULTS: A number of HDGFStrep-tag copurifying proteins were identified which interact with RNA or are involved in the cellular DNA repair machinery. The most abundant protein, however, copurifying with HDGF in this approach was nucleolin. Therefore we focus on the characterization of the interaction of HDGF and nucleolin in this study. We show that expression of a cytosolic variant of HDGF causes a redistribution of nucleolin into the cytoplasm. Furthermore, formation of HDGF/nucleolin complexes depends on bcl-2 mRNA. Overexpression of full length bcl-2 mRNA increases the number of HDGF/nucleolin complexes whereas expression of only the bcl-2 coding sequence abolishes interaction completely. Further examination reveals that the coding sequence of bcl-2 mRNA together with either the 5' or 3' UTR is sufficient for formation of HDGF/nucleolin complexes. When bcl-2 coding sequence within the full length cDNA is replaced by a sequence coding for secretory alkaline phosphatase complex formation is not enhanced. CONCLUSION: The results provide evidence for the existence of HDGF and nucleolin containing nucleoprotein complexes which formation depends on the presence of specific mRNAs. The nature of these RNAs and other components of the complexes should be investigated in future.

Lysosomal di-N-acetylchitobiase-deficient mouse tissues accumulate Man2GlcNAc2 and Man3GlcNAc2.

Most lysosomal storage diseases are caused by defects in genes encoding for acidic hydrolases. Deficiency of an enzyme involved in the catabolic pathway of N-linked glycans leads to the accumulation of the respective substrate and consequently to the onset of a specific storage disorder. Di-N-acetylchitobiase and core specific α1-6mannosidase represent the only exception. In fact, to date no lysosomal disease has been correlated to the deficiency of these enzymes. We generated di-N-acetylchitobiase-deficient mice by gene targeting of the Ctbs gene in murine embryonic stem cells. Accumulation of Man2GlcNAc2 and Man3GlcNAc2 was evaluated in all analyzed tissues and the tetrasaccharide was detected in urines. Multilamellar inclusion bodies reminiscent of polar lipids were present in epithelia of a scattered subset of proximal tubules in the kidney. Less constantly, enlarged Kupffer cells were observed in liver, filled with phagocytic material resembling partly digested red blood cells. These findings confirm an important role for lysosomal di-N-acetylchitobiase in glycans degradation and suggest that its deficiency could be the cause of a not yet described lysosomal storage disease.

γ-Interferon-regulated chaperone governs human lymphocyte antigen class II expression.

Antigen presentation by human lymphocyte antigen (HLA) class II peptide receptors alerts the immune system to infections. In antigen-presenting cells (APCs), HLA class II, HLA-DM, and associated invariant chain-encoding genes are exclusively regulated by the interferon γ (IFNγ)-inducible class II transactivator (CIITA). Control of CIITA expression could therefore govern expression of class II peptide receptors in the diverse group of APCs. We discovered that elevation of the HLA class III region encoded B-associated transcript 3 (BAT3) increases and depletion of BAT3 decreases expression of HLA class II, HLA-DM, and invariant chain. IFNγ strongly elevates BAT3 transcription in various tumor cell lines and in primary macrophages. BAT3 chaperones the simultaneously IFNγ-induced CIITA. Following IFNγ-treatment, both CIITA and BAT3 translocate from the cytosol to the nucleus. The nuclear import of CIITA mediated by IFNγ controls activation of HLA class II genes. BAT3 is a novel key regulator of components of the HLA class II processing pathway. We present a mechanism explaining how parallel IFNγ-mediated regulation of CIITA and of its chaperone BAT3 controls the level of components of the HLA class II processing pathway.

Overexpression of hepatoma-derived growth factor in melanocytes does not lead to oncogenic transformation.

BACKGROUND: HDGF is a growth factor which is overexpressed in a wide range of tumors. Importantly, expression levels were identified as a prognostic marker in some types of cancer such as melanoma. METHODS: To investigate the presumed oncogenic/transforming capacity of HDGF, we generated transgenic mice overexpressing HDGF in melanocytes. These mice were bred with mice heterozygous for a defective copy of the Ink4a tumor suppressor gene and were exposed to UV light to increase the risk for tumor development both genetically and physiochemically. Mice were analyzed by immunohistochemistry and Western blotting. Furthermore, primary melanocytes were isolated from different strains created. RESULTS: Transgenic animals overexpressed HDGF in hair follicle melanocytes. Interestingly, primary melanocytes isolated from transgenic animals were not able to differentiate in vitro whereas cells isolated from wild type and HDGF-deficient animals were. Although, HDGF-/-/Ink4a+/- mice displayed an increased number of epidermoid cysts after exposure to UV light, no melanomas or premelanocytic alterations could be detected in this mouse model. CONCLUSIONS: The results therefore provide no evidence that HDGF has a transforming capacity in tumor development. Our results in combination with previous findings point to a possible role in cell differentiation and suggest that HDGF promotes tumor progression after secondary upregulation and may represent another protein fitting into the concept of non-oncogene addiction of tumor tissue.

Identification of specific nuclear structural protein alterations in human breast cancer.

Breast cancer is the most commonly diagnosed type of cancer and a major cause of death in women. Reliable biomarkers are urgently needed to improve early detection or to provide evidence of the prognosis for each individual patient through expression levels in tumor tissue or body fluids. This proteomic analysis focused on the nuclear structure of human breast cancer tissue, which has been shown to be a promising tool for cancer biomarker development. The nuclear matrix composition of human breast cancer (n = 14), benign controls (n = 2), and healthy controls (n = 2) was analyzed by high-resolution two-dimensional gel electrophoresis and mass spectrometry. Validation studies were performed in an individual sample set consisting of additional breast cancer tissues (n = 3) and additional healthy control tissues (n = 2) by one-dimensional immunoblot. In this setting, we identified five proteins that were upregulated in human breast cancer tissue, but absent in the healthy and benign controls (P < 0.001). These spots were also present in the investigated human breast cancer cell lines, but absent in the MCF10a cell line, which represents normal human epithelial breast cells. Two of the breast cancer-specific proteins have been confirmed to be calponin h2 and calmodulin-like protein 5 by one-dimensional immunoblot. This is the first study demonstrating the expression of both proteins in human breast cancer tissue. Further studies are required to investigate the potential role of these proteins as biomarkers for early diagnosis or prognosis in human breast cancer.

Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease.

Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

Hepatoma-derived growth factor-related protein-3: a new neurotrophic and neurite outgrowth-promoting factor for cortical neurons.

Hepatoma-derived growth factor-related proteins (HRPs) make up a family of six members. Hepatoma-derived growth factor-related protein-3 (HRP-3) is the only family member whose expression is almost restricted to nervous tissue. Here we show that soluble HRP-3 acts as a novel neurotrophic factor for cultured primary cortical neurons. Antibody-mediated neutralization of HRP-3 function results in neuronal degeneration. In contrast, HRP-3 as the only addition to a culture medium not supporting neuronal survival rescues neurons to an extent comparable to the addition of FCS. Besides this neuroprotective capability, the protein exerts a neurite outgrowth-promoting effect when it is presented as a coated substrate but not as a soluble factor. This study points to an important role of HRP-3 during the development of the nervous system.

The role of protein quality control in mitochondrial protein homeostasis under oxidative stress.

Mitochondria contribute significantly to the cellular production of ROS. The deleterious effects of increased ROS levels have been implicated in a wide variety of pathological reactions. Apart from a direct detoxification of ROS molecules, protein quality control mechanisms are thought to protect protein functions in the presence of elevated ROS levels. The reactivities of molecular chaperones and proteases remove damaged polypeptides, maintaining enzyme activities, thereby contributing to cellular survival both under normal and stress conditions. We characterized the impact of oxidative stress on mitochondrial protein homeostasis by performing a proteomic analysis of isolated yeast mitochondria, determining the changes in protein abundance after ROS treatments. We identified a set of mitochondrial proteins as substrates of ROS-dependent proteolysis. Enzymes containing oxidation-sensitive prosthetic groups like iron/sulfur clusters represented major targets of stress-dependent degradation. We found that several proteins involved in ROS detoxification were also affected. We identified the ATP-dependent protease Pim1/LON as a major factor in the degradation of ROS-modified soluble polypeptides localized in the matrix compartment. As Pim1/LON expression was induced significantly under ROS treatment, we propose that this protease system performs a crucial protective function under oxidative stress conditions.

Transport of a hyaluronan-binding protein in brain tissue.

Hyaluronan is an unsulfated linear glycosaminoglycan with the ability to nucleate extracellular matrices by the formation of aggregates with lecticans. These matrices are essential during development of the central nervous system. In the prospective white matter of the developing brain hyaluronan is organized into fiber-like structures according to confocal microscopy of fixed slices which may guide the migration of neural precursor cells [Baier, C., S.L. Baader, J. Jankowski, V. Gieselmann, K. Schilling, U. Rauch, and J. Kappler. 2007. Hyaluronan is organized into fiber-like structures along migratory pathways in the developing mouse cerebellum. Matrix Biol. 26: 348-58]. By using plasmon surface resonance, microinjection into brain slices and fluorescence correlation spectroscopy, we show that the brain-specific lecticans bind to, but also dissociate rather rapidly from hyaluronan. After microinjection into native cerebellar slices a GFP-tagged hyaluronan-binding neurocan fragment was enriched at binding sites in the prospective white matter, which had a directional orientation and formed local stationary concentration gradients in areas where binding sites are abundant. Fluorescence correlation spectroscopy measurements at fixed brain slices revealed that fiber-bound neurocan-GFP was mobile with D(fiber(neurocan-GFP))=4x10(-10)cm(2)/s. Therefore, we propose that hyaluronan-rich fibers in the prospective white matter of the developing mouse cerebellum can guide the diffusion of lecticans. Since lecticans bind a variety of growth and mobility factors, their guided diffusion may contribute to the transport of these polypeptides and to the formation of concentration gradients. This mechanism could serve to encode positional information during development.