The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins.

Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized onto immobilized artificial membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC (U87MG) column, and the binding affinities (Kd) determined were 1.08±0.49 and 0.0086±0.0006µM, respectively, consistent with previously reported values. Furthermore, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX, and rotenone. In addition, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC (U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy.

The orphan tyrosine kinase receptor, ROR2, mediates Wnt5A signaling in metastatic melanoma.

Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we show, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays, we show that ROR2 is expressed predominantly in metastatic melanoma. As ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our earlier data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis assays, we show that ROR2 is necessary for the Wnt5A-mediated metastasis of melanoma cells. These data imply that ROR2 may represent a novel target for melanoma therapy.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage.

The Werner syndrome protein (WRN) and chromatin assembly factor 1 (CAF-1) are both involved in the maintenance of genome stability. In response to DNA-damaging signals, both of these proteins relocate to sites where DNA synthesis occurs. However, the interaction between WRN and CAF-1 has not yet been investigated. In this report, we show that WRN interacts physically with the largest subunit of CAF-1, hp150, in vitro and in vivo. Although hp150 does not alter WRN catalytic activities in vitro, and the chromatin assembly activity of CAF-1 is not affected in the absence of WRN in vivo, this interaction may have an important role during the cellular response to DNA replication fork blockage and/or DNA damage signals. In hp150 RNA-mediated interference (RNAi) knockdown cells, WRN partially formed foci following hydroxyurea (HU) treatment. However, in the absence of WRN, hp150 did not relocate to form foci following exposure to HU and ultraviolet light. Thus, our results demonstrate that WRN responds to DNA damage before CAF-1 and suggest that WRN may recruit CAF-1, via interaction with hp150, to DNA damage sites during DNA synthesis.

Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion.

The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.

Analysis of the tetraspanin CD9-integrin alphaIIbbeta3 (GPIIb-IIIa) complex in platelet membranes and transfected cells.

The platelet integrin, alphaIIbbeta3 (GPIIb-IIIa), and the tetraspanin, CD9, are integral membrane proteins that are abundant in platelet membranes. We have identified several proteins, including CD9, which were co-precipitated by anti-alphaIIbbeta3 antibody from untreated, resting platelets that were solubilized with the poly(oxyethylene) non-ionic detergent, Brij-35. Immunoblot and quantitative immunoprecipitation showed that the association of alphaIIbbeta3 with CD9 is specific and stoichiometric. The interaction between CD9 and alphaIIbbeta3 is probably hydrophobic, as Triton X-100 and hydrophobic detergents of the Brij series completely dissociated the CD9-alphaIIbbeta3 complex. Recombinant CD9 and alphaIIbbeta3 can associate after transfection into Chinese hamster ovary cells, as seen by co-immunoprecipitation and co-localization in the periphery of spreading cells and in the lamellipodia of cells plated on fibrinogen. This co-localization is absent from focal adhesions. Furthermore, anti-CD9-coated latex beads clustered alphaIIbbeta3 with CD9. This work indicates that the tetraspanin, CD9, is associated with beta3 integrins in resting platelets and transfected cells.

Calpain cleavage of the cytoplasmic domain of the integrin beta 3 subunit.

The cytoplasmic domains of integrin beta subunits are involved in bidirectional transmembrane signaling. We report that the cytoplasmic domain of the integrin beta 3 subunit undergoes limited proteolysis by calpain, an intracellular calcium-dependent protease. Calpain cleavage occurs during platelet aggregation induced by agonists such as thrombin. Five cleavage sites have been identified. Four of these sites (C-terminal to Thr741, Tyr747, Phe754, and Tyr759) are utilized in intact platelets and flank two NXXY motifs (Asn744-Pro-Leu-Tyr747 and Asn756-Ile-Thr-Tyr759). The fifth site (Ala735) is accessible to calpain after EDTA treatment of the alpha IIb beta 3 heterodimer. The NXXY motif is critical to the bidirectional signaling functions of beta 3 integrins and their association with the cytoskeleton. Thus, calpain cleavage of the beta 3 cytoplasmic domain may provide a means to regulate integrin signaling functions.

Hydrolysis of thymic humoral factor gamma 2 by neutral endopeptidase (EC 3.4.24.11).

A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.

Neutral endopeptidase (EC 3.4.24.11) is highly expressed on osteoblastic cells and other marrow stromal cell types.

A series of cultured mouse marrow stromal cell lines of different phenotype characteristics were examined for their neutral endopeptidase (NEP) activity using the highly selective chromophoric substrate of enzyme, 3-carboxypropanoyl-alanyl-alanyl-leucine 4-nitroanilide. All the cell lines tested contained appreciable amounts of NEP activity, but the cells expressing an osteoblastic phenotype, MBA-15, showed the high levels. Other non-stromal osteoblastic cell lines, MC-3T3-E1, ROS 17/2.8 cells derived from mouse long bone explants, were also rich in NEP. A four-fold stimulation of NEP activity was observed when certain MBA-15 clones were cultured in the presence of 1 alpha,25-dihydroxyvitamin D3. Since NEP is an effective cell-surface endopeptidase, it may play a role in the dynamics of bone formation, via interaction with biologically active polypeptides.

Endothelins are more sensitive than sarafotoxins to neutral endopeptidase: possible physiological significance.

Incubation of endothelins (ETs) with bovine kidney neutral endopeptidase (NEP) resulted in a selective two-step degradation with loss of biochemical activity. The Km of the enzyme indicated high-affinity binding, and hydrolysis was completely inhibited by phosphoramidon. The first step was nicking of the Ser5-Leu6 bond, followed by cleavage at the amino side of Ile19. The nicked peptide exhibited biochemical activities comparable to those of the intact peptide--i.e., binding to the ET receptor, induction of inositol phospholipid hydrolysis, and toxicity. The twice-cleaved product was inactive. The sarafotoxins (SRTXs) were more resistant than the ETs to NEP: for example, the half-time for ET-1 was approximately 1 hr, while it was approximately 4 hr for SRTX-b and even higher for SRTX-c. These in vitro findings may indicate a regulatory role of NEP (or similar enzymes) in the physiological inactivation of ETs. They might also help to explain why under certain physiological conditions ETs may be less toxic than SRTXs.

Investigation of neutral endopeptidases (EC 3.4.24.11) and of neutral proteinases (EC 3.4.24.4) using a new sensitive two-stage enzymatic reaction.

A sensitive two-stage enzymatic reaction for mammalian and bacterial metalloendopeptidases has been developed using the substrate 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide supplemented with Streptomyces griseus amino-peptidase. Neutral endopeptidase EC 3.4.24.11 from bovine kidney hydrolyzes the substrate (pH 7.5, 25 degrees C) with a catalytic efficiency (kcat = 1.2 x 10(2) s-1, Km = 0.15 mM) of the highest ever reported for the enzyme acting on synthetic chromophoric and fluorogenic substrates. Thermolysin hydrolyzes the substrate at a faster rate (kcat = 1.2 x 10(3) s-1) but the overall efficiency is diminished by a higher Km (4.2 mM). Suspensions of human neutrophil cells and culture filtrates of Bacillus cereus have been assayed sensitively for their neutral endopeptidases and neutral proteinase activities, respectively. The assay provides a convenient tool for the kinetic investigation of neutral endopeptidases and neutral proteinases and for assessing their function in biological systems.