Robert M. Petrovich

The Low Affinity IgE Receptor (CD23) Is Cleaved by the Metalloproteinase ADAM10

The low affinity IgE receptor, FcϵRII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity of ADAM (a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.

Selective Mutations in Estrogen Receptor α D-domain Alters Nuclear Translocation and Non-estrogen Response Element Gene Regulatory Mechanisms

The three main mechanisms of ERα action are: 1) nuclear, genomic, direct DNA binding, 2) nuclear, genomic, “tethered”-mediated, protein-protein interactions, and 3) non-nuclear, non-genomic, rapid action responses. Reports suggest the D-domain or hinge region of ERα plays an important role in mechanisms 1 and 2 above. Studies demonstrating the functionality of the ERα hinge region have resected the full D-domain; therefore, site directed mutations were made to attribute precise sequence functionality to this domain. This study focuses on the characterization and properties of three novel site directed ERα- D-domain mutants. The Hinge 1 (H1) ERα mutant has disrupted nuclear localization, can no longer perform tethered mediated responses and has lost interaction with c-Jun, but retains estrogen response element (ERE)-mediated functions as demonstrated by confocal microscopy, reporter assays, endogenous gene expression and co-immunoprecipitation. The H2 ERα mutant is non-nuclear, but translocates to the nucleus with estradiol (E2) treatment and maintains ERE-mediated functionality. The H2+NES ERα mutant does not maintain nuclear translocation with hormone binding, no longer activates ERE-target genes, functions in ERE- or tethered-mediated luciferase assays, but does retain the non-genomic, non-nuclear, rapid action response. These studies reveal the sequence(s) in the ERα hinge region that are involved in tethered-mediated actions as well as nuclear localization and attribute important functionality to this region of the receptor. In addition, the properties of these ERα mutants will allow future studies to further dissect and characterize the three main ERα mechanisms of action and determine the mechanistic role each action has in estrogen hormone regulation.

ADAM9 Inhibition Increases Membrane Activity of ADAM10 and Controls α-Secretase Processing of Amyloid Precursor Protein

Background: Raising ADAM10 α-secretase activity has been considered as an attractive therapeutic option in Alzheimer disease. Results: Administration of the prodomain of ADAM9 (proA9) prevents ADAM10 shedding and increases membrane α-secretase activity in a neuronal cell line. Conclusion: Use of proA9 is a means to modulate cellular ADAM10 activity. Significance: proA9 can be used in vivo to increase ADAM10 α-secretase activity. Prodomains of A disintegrin and metalloproteinase (ADAM) metallopeptidases can act as highly specific intra- and intermolecular inhibitors of ADAM catalytic activity. The mouse ADAM9 prodomain (proA9; amino acids 24–204), expressed and characterized from Escherichia coli, is a competitive inhibitor of human ADAM9 catalytic/disintegrin domain with an overall inhibition constant of 280 ± 34 nm and high specificity toward ADAM9. In SY5Y neuroblastoma cells overexpressing amyloid precursor protein, proA9 treatment reduces the amount of endogenous ADAM10 enzyme in the medium while increasing membrane-bound ADAM10, as shown both by Western and activity assays with selective fluorescent peptide substrates using proteolytic activity matrix analysis. An increase in membrane-bound ADAM10 generates higher levels of soluble amyloid precursor protein α in the medium, whereas soluble amyloid precursor protein β levels are decreased, demonstrating that inhibition of ADAM9 increases α-secretase activity on the cell membrane. Quantification of physiological ADAM10 substrates by a proteomic approach revealed that substrates, such as epidermal growth factor (EGF), HER2, osteoactivin, and CD40-ligand, are increased in the medium of BT474 breast tumor cells that were incubated with proA9, demonstrating that the regulation of ADAM10 by ADAM9 applies for many ADAM10 substrates. Taken together, our results demonstrate that ADAM10 activity is regulated by inhibition of ADAM9, and this regulation may be used to control shedding of amyloid precursor protein by enhancing α-secretase activity, a key regulatory step in the etiology of Alzheimer disease.

Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta.

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain.

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Phosphorylation at Serines 216 and 221 Is Important for Drosophila HeT-A Gag Protein Stability

Telomeres from Drosophila appear to be very different from those of other organisms – in size and the mechanism of their maintenance. In the absence of the enzyme telomerase, Drosophila telomeres are maintained by retrotransposition of three elements, HeT-A, TART, and TAHRE, but details of their transposition mechanisms are not known. Here we characterized some biochemical characteristics of the HeT-A Gag protein encoded by the HeT-A element to understand this mechanism. The HeT-A Gag protein when overexpressed in S2 cells was localized to the nucleus but was resistant to high salt, detergents and nuclease extraction treatments. Analysis of the HeT-A Gag protein by tandem mass spectrophotometry revealed that serines 216 and 221 are phosphorylated. Substituting these serines with alanine or aspartic acid by site-directed mutagenesis did not result in any changes in HeT-A Gag translocation across the nucleus, suggesting that phosphorylation of these sites is not associated with HeT-A Gag translocation, but time course experiments showed that these phosphorylation sites are important for Gag-protein stability.