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.

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.

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.

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.

Hepatoma-derived growth factor-related protein-3 interacts with microtubules and promotes neurite outgrowth in mouse cortical neurons.

Hepatoma-derived growth factor-related proteins (HRP) comprise a family of 6 members, which the biological functions are still largely unclear. Here we show that during embryogenesis HRP-3 is strongly expressed in the developing nervous system. At early stages of development HRP-3 is located in the cytoplasm and neurites of cortical neurons. Upon maturation HRP-3 relocalizes continuously to the nuclei and in the majority of neurons of adult mice it is located exclusively in the nucleus. This redistribution from neurites to nuclei is also found in embryonic cortical neurons maturing in cell culture. We show that HRP-3 is necessary for proper neurite outgrowth in primary cortical neurons. To identify possible mechanisms of how HRP-3 modulate neuritogenesis we isolated HRP-3 interaction partners and demonstrate that it binds tubulin through the N-terminal so called HATH region, which is strongly conserved among members of the HRP family. It promotes tubulin polymerization, stabilizes and bundles microtubules. This activity depends on the extranuclear localization of HRP-3. HRP-3 thus could play an important role during neuronal development by its modulation of the neuronal cytoskeleton.

Hepatoma-derived growth factor (HDGF) is dispensable for normal mouse development.

Hepatoma-derived growth factor (HDGF) is suggested to be involved in organ development and exhibits proliferative, angiogenic, and neurotrophic activity. The in vivo functions are, however, so far unknown. In this study, we generated HDGF-deficient mice, in which parts of the HDGF gene were replaced by a gene encoding green fluorescent protein (eGFP). HDGF-/- mice are viable with no apparent morphological abnormalities. Cultured HDGF-deficient dermal fibroblasts show unaltered proliferation rates and cell-cycle distributions. In contrast to previous studies, our data demonstrate that signal pathways involved in the response to extracellular HDGF do not depend on the presence of intracellular HDGF. Contrary to the reported role of HDGF as a modulator of apoptosis, similar apoptotic rates were found between wild-type and HDGF-deficient fibroblasts following tumor necrosis factor alpha (TNFalpha) -induced apoptosis or cellular stress. The lack of obvious biochemical and morphological phenotypes in HDGF-deficient mice demonstrates that in vivo HDGF is dispensable for normal development in mice.

HDGF is dephosphorylated during the early steps of endothelial cell apoptosis in a caspase-dependent way.

We were looking by a proteomic approach for new phospho-proteins involved during the early steps of the TNF + cycloheximide (CHX)-induced apoptosis-preceding mitochondrial membrane permeabilization-of endothelial cells (BAEC). In the present study, we observed on the autoradiography from 2D gel of (32)P-labeled samples a string of proteins undergoing a complete dephosphorylation after 1 h of stimulation with TNF + CHX-while mitochondrial membrane permeabilization was observed after 3 h-identified the different spots by mass spectrometry as one and only protein, HDGF, and confirmed the identity by western blot. The intensity of the 2D phosphorylation pattern of HDGF was correlated with the amount of apoptosis induced by TNF + CHX and TNF or CHX alone and this event was inhibited by the Caspase specific inhibitor zVADfmk. Moreover the TNF + CHX-treatment did not affect the nuclear localization of GFP-HDGF. Taken together, our data suggest an involvement of HDGF during the initiation phase of the apoptotic process downstream from an initiator Caspase and a regulation of this protein by phosphorylation in the nucleus.

SUMOylation of the hepatoma-derived growth factor negatively influences its binding to chromatin.

Hepatoma-derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma-derived growth factor, its routes of secretion and internalization or post-translational modifications. In the present study, we show for the first time that hepatoma-derived growth factor is modified by the covalent attachment of small ubiquitin-related modifier 1 (SUMO-1), a post-translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI-TOF-MS based peptide analysis, we identified the lysine residue SUMOylated located in the N-terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma-derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma-derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma-derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma-derived growth factor.

Expression of hepatoma-derived growth factor family members in the adult central nervous system.

BACKGROUND: Hepatoma-derived growth factor (HDGF) belongs to a polypeptide family containing five additional members called HDGF related proteins 1-4 (HRP-1 to -4) and Lens epithelial derived growth factor. Whereas some family members such as HDGF and HRP-2 are expressed in a wide range of tissues, the expression of others is very restricted. HRP-1 and -4 are only expressed in testis, HRP-3 only in the nervous system. Here we investigated the expression of HDGF, HRP-2 and HRP-3 in the central nervous system of adult rats on the cellular level by immunohistochemistry. In addition we performed Western blot analysis of various brain regions as well as neuronal and glial cell cultures. RESULTS: HDGF was rather evenly expressed throughout all brain regions tested with the lowest expression in the substantia nigra. HRP-2 was strongly expressed in the thalamus, prefrontal and parietal cortex, neurohypophysis, and the cerebellum, HRP-3 in the bulbus olfactorius, piriform cortex and amygdala complex. HDGF and HRP-2 were found to be expressed by neurons, astrocytes and oligodendrocytes. In contrast, strong expression of HRP-3 in the adult nervous system is restricted to neurons, except for very weak expression in oligodendrocytes in the brain stem. Although the majority of neurons are HRP-3 positive, some like cerebellar granule cells are negative. CONCLUSION: The coexpression of HDGF and HRP-2 in glia and neurons as well as the coexpression of all three proteins in many neurons suggests different functions of members of the HDGF protein family in cells of the central nervous system that might include proliferation as well as cell survival. In addition the restricted expression of HRP-3 point to a special function of this family member for neuronal cells.

Hepatoma-derived growth factor. Significance of amino acid residues 81-100 in cell surface interaction and proliferative activity.

Hepatoma-derived growth factor (HDGF) has proliferative, angiogenic, and neurotrophic activity. It plays a putative role in the development and progression of cancer. When expressed in cells, the mitogenic activity of HDGF depends on its nuclear localization, but it also stimulates proliferation when added to the cell culture medium. A cell surface receptor for HDGF has not been identified so far. We investigated the interaction of various purified recombinant HDGF fusion proteins with the cell surface of NIH 3T3 fibroblasts. We showed that binding of a HDGF-beta-galactosidase fusion protein to the cell surface of NIH 3T3 fibroblasts was saturable, occurred with high affinity (K(D) = 14 nm), and had a proliferative effect. We identified a peptide comprising amino acid residues 81-100 within the amino-terminal part of HDGF that bound to the cell surface of NIH 3T3 cells with saturation and affinity values similar to those of HDGF. When added to primary human fibroblasts, this peptide stimulated proliferation. Substitution of a single amino acid (K96A) within this peptide was sufficient to abolish its binding to the cell surface and its proliferative activity. In contrast, when expressed transiently in NIH 3T3 cells, a HDGF-beta-galactosidase fusion protein in which amino acid residues 81-100 were deleted still had proliferative activity, whereas a fusion protein containing only the 81-100 peptide did not. Our results suggest the existence of a plasma membrane-located HDGF receptor for which signaling depends on amino acid residues 81-100 of HDGF. This region differs from the one that has been recently identified to be essential for mitogenic activity depending on the nuclear localization of HDGF. Thus, HDGF exerts its proliferative activity via two different pathways.