The mission of the Protein Microcharacterization Core Facility (PMCF) is to provide state-of-the-art mass spectrometric support, including education, to the Division of Intramural Research at the NIEHS. A brief summary of the projects completed in 2006/2007 or are on-going include:? ADAM10 Project: The low affinity IgE receptor, CD23, is a regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species 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 ADAM10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in CD23. 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 proteinase-deficient ADAM10 partially inhibited the production of soluble CD23. 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. The specific contribution of the PMCF to this project was to characterize the ADAM10 cleavage sites in synthetic CD23 peptides. ? Amado Collaboration: The PMCF in collaboration with the Tomer Mass Spectrometry Research Group served as a host to Dr. Rui Vitorino. The results of the proteomic analyses performed in collaboration with Dr. Vitorino resulted in two manuscripts: Vitorino, R., Lobo, M.J.C., Williams, J.G., Ferrer-Correia, A.J., Tomer, K.B., Duarte, J., Pedro M. Domingues, P.M., and Amado, F.M.L. in press Biomed. Chrom. Peptidomic Analysis of Human Acquired Enamel Pellicle and Vitorino, R., Ferreira, R., Neuparth, M., Guedes, S., Williams, J., Tomer, K.B., Domingues, P.M., Appell, H.J., Duarte, J.A., Amado, F.M.L. (2007) Anal. Biochem. 366(2), 156-169. Subcellular Proteomics of Mice Gastrocnemius and Soleus Muscles? Nrf2 Project: Nrf2 is a key transcription factor in the cellular response to oxidative stress. In this study we identify two phosphorylated forms of endogenous human Nrf2 after chemically induced oxidative stress and provide evidence that protein kinase CK2-mediated sequential phosphorylation plays potential roles in Nrf2 activation and degradation. Human Nrf2 has a predicted molecular mass of 66 kDa. However, immunoblots showed that two bands at 98 and 118 kDa, which are identified as phosphorylated forms, are increased in response to Nrf2 inducers. In addition, human Nrf2 was found to be a substrate for CK2 which mediated two steps of phosphorylation, resulting in two forms of Nrf2 migrating with differing M(r) at 98 kDa (Nrf2-98) and 118 kDa (Nrf2-118). Our results support a role in which calmodulin binding regulates CK2 activity. Gel shift assays showed that the Nrf2-118 does not bind to the antioxidant response element, indicating that Nrf2-98 has transcriptional activity. In contrast, Nrf2-118 is more susceptible to degradation. These results provide evidence for phosphorylation by CK2 as a critical controlling factor in Nrf2-mediated cellular antioxidant response. The specific contribution of the PMCF to these studies was the mass spectrometric confirmation of Nrf2 in the SDS-PAGE gels.? Penaeidin project: In collaboration with the Tomer research group and Dr. Perry Blackshear, the PMCF was involved in the mass spectrometric characterization of the primary sequence of a penaeidin, litset 4, using electron capture dissociation and mass spectrometry. This technique proved to be very powerful in that it allowed for the sequence determination the N-terminus of the intact pinaeidin.? PPInsP5 Kinase Project: Mammalian cells utilize multiple signaling mechanisms to protect against the osmotic stress that accompanies plasma membrane ion-transport, solute uptake, and turnover of protein and carbohydrates. Recently, osmotic stress was found to increase synthesis of bisdiphosphoinositol tetrakisphosphate, a high-energy inositol pyrophosphate. We have now described the purification from rat brain of a diphosphoinositol pentakisphosphate kinase (PPIP5K) that synthesizes PP2-InsP4. Partial amino-acid sequence, obtained by mass spectrometry, matched the sequence of a 160 kDa rat protein containing a putative ATP-grasp kinase domain. Recombinant human PPIP5K1, expressed in E. coli, was found to phosphorylate PP-InsP5 to PP2-InsP4. Over-expression in HEK cells of either PPIP5K1 or PPIP5K2 substantially increased levels of PP2-InsP4, whereas over-expression of a catalytically-dead PPIP5K1D332A mutant had no effect. Immunopurification of over-expressed PPIP5K1 from osmotically-stressed HEK cells revealed a persistent, 4-fold activation compared to control cells. PPIP5Ks are likely to be important signaling enzymes. The PMCF was instrumental in this project. Dr. Shears provided partially purified samples from rodent brain extracts and the core facility identified all the proteins within the limits of detection. Dr. Shears then systematically examined the identified proteins for PPInsP5 kinase activity. Serendipitously, the top scoring protein in the samples was found to be the PPInsP5 kinase.? VKOR Project: More than 21 million prescriptions for warfarin are written yearly in the U.S. Despite its importance, warfarin's target, vitamin K epoxide reductase (VKOR), has resisted purification since its identification in 1972. We have now reported its purification and reconstitution. It has been reported that VKOR is a multisubunit enzyme. Our results, however, suggest that a single peptide can accomplish both the conversion of vitamin K epoxide to vitamin K and vitamin K to reduced vitamin K. This purification will allow further characterization of VKOR in relation to other components of the vitamin K cycle and should facilitate its structural determination. The contribution of the PMCF in this collaboration with the Tomer research group and Dr. Darrel Stafford was to identify the low level contaminating proteins in the VKOR preparation used in these experiments. Recent results suggest that perhaps undetectable levels of contaminating proteins may have lead to incorrect conclusions. In conjunction with the Stafford laboratory, the PMCF is attempting to address these questions.? Phosphorylopathies: We have found that a common polymorphism in the human ERG1 potassium channel gene, which is known to increase the risk of fatal cardiac arrhythmias, creates a recognition site on the channel for the Akt protein kinase that reverses the effects of hormonal signaling through PI3Kinase on channel activity. A systematic bioinformatics search of genes encoding channel proteins reveals fourteen additional candidate """"""""phosphorylopathies,"""""""" disease-associated SNPs that are predicted to alter channel phosphorylation. Changes in protein phosphorylation might represent a general mechanism for the effects of genetic variation on human health and its interaction with the environment. The PMCF discovered the phosphorylation on the residue, K897T, that is generated by the SNP.? Phosphorylation of GR: The PMCF in collaboration with Dr. Cidlowski is assessing the occupation of conserved predicted sites of phosphorylation on the glucocorticoid receptor in the presence and absence of dexamethasone.? Protein Identification: The PMCF is involved in a number of in depth collaborations (Drs. Olden, Tennant, and Wilson) that all have resulted in manuscripts being submitted. Many additional protein identification and protein characterization projects are also on-going.
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