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Utilising rabbit corneal endothelial cells (CEC) in three different paradigms, two human FGF1 derivatives (TTHX1001 and TTHX1114), engineered to exhibit greater stability, were tested as proliferative agents. Primary CECs and mouse NIH 3T3 cells treated with the two FGF1 derivatives showed equivalent EC

Purpose: Corneal endothelial dystrophies are characterized by endothelial cell loss and dysfunction. Recent evidence suggests that corneal endothelial cells (CECs) can regenerate although they do not do so under normal conditions. This work sought to test whether CECs can be stimulated to proliferate in organ culture by wounding and/or by treatment with the engineered human fibroblast growth factor 1 (FGF1) derivative TTHX1114. Methods: Human donor corneas obtained from eye banks were maintained in organ culture in the presence or absence of TTHX1114. Wounds in the corneas were created by quartering the corneas. The CEC monolayer was identified as a regular layer by Hoechst staining of the nuclear DNA with cell outlines delineated by immunohistochemical identification of ZO-1. Nuclei and nuclei incorporating 5-ethynyl-2′-deoxyuridine (EdU) were counted using ImageJ. Results: CECs in normal corneas in undisturbed monolayers had low, but measurable, rates of proliferation. CECs at the edge of a wound had higher rates of proliferation, probably due to the release of contact inhibition. TTHX1114 increased proliferation at wound edges. After 7 days of culture, proliferating CECs formed contiguous groups of labeled cells that did not migrate away from one another. TTHX1114-treated cells, including the EdU labeled proliferating cells, retained normal morphology, including cell/cell junction ZO-1 staining. Conclusions: Proliferation of CECs in organ-cultured corneas is low, but can be stimulated by wounding or by the administration of TTHX1114 with the effects of each being additive. The CEC monolayer appears to have a population of progenitor cells that are susceptible to stimulation.

Despite progress in genomic and proteomic technology and applications, the validation of cancer biomarkers of use as clinical early detection diagnostics has remained elusive. As described in this brief viewpoint, there are now recognized to be many types of clinical biomarkers and proteomic analyses, particularly when combined with other 'omic analyses, have been effective in many such biomarker identifications. However, in the area of early diagnosis of cancers, the problems associated with the conversion from identification to diagnostic have largely not been overcome. Notably, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI), has been particularly successful in refining the analytical steps needed to tackle this challenging issue and has provided positive insight into how to solve many of the underlying problems. The potential for developing clinical diagnostics for early detection of highly lethal cancers and possible new therapeutic strategies through proteomic analyses, as seen through these CPTAC successes, is more promising than ever.

Purpose Caveolin (Cav) regulates various aspect of endothelial cell signaling and cell-permeable peptides (CPPs) fused to domains of Cav can reduce retinal damage and inflammation in vivo. Thus, the goal of the present study was to identify a novel CPP that improves delivery of a truncated Cav modulator in vitro and in vivo. Methods Phage display technology was used to identify a small peptide (RRPPR) that was internalized into endothelial cells. Fusions of Cav with the peptide were compared to existing molecules in three distinct assays, vascular endothelial growth factor-A (VEGF) induced nitric oxide (NO) release, VEGF induced vascular leakage, and in a model of immune mediated uveitis. Results RRPPR was internalized efficiently and was potent in blocking NO release. Fusing RRPPR with a minimal Cav inhibitory domain (CVX51401) dose-dependently blocked NO release, VEGF induced permeability, and retinal damage in a model of uveitis. Conclusions CVX51401 is a novel Cav modulator that reduces VEGF and immune mediated inflammation. Translational relevance CVX51401 is an optimized Cav modulator that reduces vascular permeability and ocular inflammation that is poised for clinical development.

The Centre for Cellular and Molecular Biology, Hyderabad, India, was host for an international forum, or “brainstorming meeting,” on proteomics held in November 2014, which provided the opportunity to showcase proteomic science in India and to allow discussions between Indian scientists and students and several international visitors. This provided an amalgamation of speakers and participants whose interests lay mainly in developing and using mass-spectrometry-based proteomics to advance their research work. A week-long workshop with hands-on training in proteomic methodology followed the meeting.

Nerve growth factor (NGF) and its precursor (proNGF) are primarily considered as regulators of neuronal function that induce their responses via the tyrosine kinase receptor TrkA and the pan-neurotrophin receptor p75NTR. It has been generally held that NGF exerts its effects primarily through TrkA, inducing a cascade of tyrosine kinase-initiated responses, while proNGF binds more strongly to p75NTR. When this latter entity interacts with a third receptor, sortilin, apoptotic responses are induced in contrast to the survival/differentiation associated with the other two. Recent studies have outlined portions of the downstream phosphoproteome of TrkA in the neuronal PC12 cells and have clarified the contribution of individual docking sites in the TrkA endodomain. The patterns observed showed a similarity with the profile induced by the epidermal growth factor receptor, which is extensively associated with oncogenesis. Indeed, as with other neurotrophic factors, the distribution of TrkA and p75NTR is not limited to neuronal tissue, thus providing an array of targets outside the nervous systems. One such source is breast cancer cells, in which NGF and proNGF stimulate breast cancer cell survival/growth and enhance cell invasion, respectively. This latter activity is exerted via TrkA (as opposed to p75NTR) in conjunction with sortilin. Another tissue overexpressing proNGF is prostate cancer and here the ability of cancer cells to induce neuritogenesis has been implicated in cancer progression. These studies show that the non-neuronal functions of proNGF/NGF are likely integrated with their neuronal activities and point to the clinical utility of these growth factors and their receptors as biomarkers and therapeutic targets for metastasis and cancer pain.

Nerve growth factor (NGF) is a protein whose importance to research and its elucidation of fundamental mechanisms in cell and neurobiology far outstrips its basic physiological roles. It was the first of a broad class of cell regulators, largely acting through autocrine and paracrine interactions which will be described herein. It was of similar significance in establishing the identity and unique roles of neurotrophic factors in the development and maintenance of the peripheral and central nervous systems. Finally, it contributed to many advances in the elaboration of cell surface receptor mechanisms and intracellular cell signaling. As such, it can be considered to be a "molecular Rosetta Stone". In this brief review, the highlights of these various studies are summarized, particularly as illustrated by their coverage in the 13 NGF international meetings that have been held since 1986.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing data sets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous assumptions/perceptions about the specificity and purposes of NME are not entirely correct. Although NME, through the universal enzymatic specificity of the methionine aminopeptidases, results in the removal of the initiator Met in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val, the comparative genomic analyses suggest that this specificity may vary modestly in some organisms. In addition, the functional role of NME may be primarily to expose Ala and Ser rather than all seven of these residues. Although any of this group provide “stabilizing” N termini in the N-end rule, and de facto leave the remaining 13 amino acid types that are classed as “destabilizing” (in higher eukaryotes) protected by the initiator Met, the conservation of NME-substrate proteins through evolution suggests that the other five are not crucially important for proteins with these residues in the second position. They are apparently merely inconsequential players (their function is not affected by NME) that become exposed because their side chains are smaller or comparable to those of Ala and Ser. The importance of exposing mainly two amino acids at the N terminus, i.e. Ala and Ser, is unclear but may be related to NTA or other post-translational modifications. In this regard, these analyses also reveal that NTA is more prevalent in some prokaryotes than previously appreciated.

Receptor tyrosine kinases (RTKs) function through protein kinase entities located in the intracellular domain of each protomer. Following activation by ligand binding, they selectively form phosphotyrosine residues by autocatalytic modification. Some of these sites are involved in maintaining the active conformation of the kinase, while others become docking sites for various adaptor/effector/scaffold proteins, which, after complexing with the receptor, then initiate further responses through cascades of post-translational modifications and the generation of lipid second messengers. Although there is substantial overlap in the pathways and activities stimulated by this superfamily, the molecular features of the endodomains of the sub-families and the moieties that they interact with to perpetrate their signals are surprisingly distinct, which may play a significant role in the regulation and responses of the individual RTK types. Some use large scaffold proteins as the basis for most, if not all, of their signal-generating interactions, while others have numerous receptor endodomain phosphotyrosine sites that are quite overlapping in specificity. The members of the Trk family of receptors each have several tyrosine residues that are phosphorylated following stimulation, including those in the kinase activation loop, but there are only two established sites (Y490 and Y785 on TrkA) that are known to be directly involved in signal propagation. Taking advantage of this limited repertoire of docking sites, we have applied phosphoproteomic methods to dissect the signaling responses of both the native protein and derivatives that have had these two sites modified. Interestingly, a clear subset that was not dependent on either docking site was identified. A comparison with a similar set of data for EGFR indicates a considerable degree of similarity in the downstream signaling profile between these two RTKs.

Stably transfected PC12 cells expressing a chimeric receptor composed of the extracellular domain of the platelet-derived growth factor receptor BB and the transmembrane and intracellular domains of TrkA, the nerve growth factor receptor, were stimulated for 20 min with platelet-derived growth factor and the resulting phosphoproteome was determined from affinity purified tryptic peptides identified by tandem MS (MS/MS) analyses. The changes in the levels of individual phosphorylation sites in stimulated cells versus control were ascertained by the stable isotope labeling of amino acids in cell culture technique. A total of 2035 peptides (806 proteins) were indentified and quantified in both data sets. Of these, 424 phosphopeptides on 259 proteins were found to be up-regulated and 392 sites on 206 proteins were down-regulated (1.8-fold or more). Protein kinases and phosphatases, as well as sites in many proteins involved in G-protein signaling, were prominently represented in the up-regulated group and more than half of the kinase up-regulated phosphosites could be clustered into three sequence motifs; a similar distribution was also found for the down-regulated sites. A comparison of the up-regulated motif profile observed to that calculated from a previous study of the EGFR-induced phosphoproteome in human HeLa cells at the same time point showed a considerable amount of similarity, supporting the view that RTK signal transduction pathways and downstream modifications are likely to be extensively overlapping.

International audience; Given the essential role of proteomics in understanding the biology of plants, we are establishing a global plant proteomics organization to properly organize, preserve and disseminate collected information on plant proteomics. We call this organization 'International Plant Proteomics Organization (INPPO; http://www.inppo.com).' Ten initiatives of INPPO are outlined along with how to address them in multiple phases. As our vision is global, we sincerely hope the scientific communities around the world will come together to support and join INPPO.

The RTKs are one of the most important families mediating transmembrane signaling and they participate and are instrumental in regulating a broad range of physiological activities. Indeed, tyrosine kinases in general, and the processes that they control and/or stimulate, provide a rich source of drug targets, particularly in growth related disorders such as cancer (Zwick et al., 2002; Krause and Van Etten, 2005). However, there remain many questions regarding their activation and downstream signaling and the application of proteomic analyses promises to answer many of them. There have been relatively few detailed studies of this type to date and it will require considerably more of them to better define the pathways with respect to both the major and minor PTMs that, along with the protein-protein interactions, are the means to direct the flow of the signals generated. It will take such approaches to define the specificity that characterize the individual families, even appreciating that to some degree all are capable of activating many, if not all, of the principal pathways. It will also be necessary to understand, in the highly complex networks of intracellular phosphorylation (that contain thousands of sites of modification and clearly have not yet been fully determined in any paradigm), exactly which kinases modify which substrates, and to work out the inter-relationships with other modifications such as O-GlcNAcylation and acetylation. Only then will it be possible to determine which modifications are physiologically significant and which are simply background. Along theway, these studies should continue to provide potential drug targets and perhaps improve the current lackluster biomarker discovery track record.

On February 28, when Richard “Dick” Hanson succumbed, after a protracted battle, to cancer at the age of 78, the world lost a truly remarkable scientist, artist, educator, and leader, and I lost a great friend and kindred spirit. His impressive list of accomplishments and the well-deserved recognition he received in his more-than-50-year career do not do justice to the tremendous impact he had on students, colleagues, and friends alike, and the positive effects of his legacy will continue to be felt by many succeeding generations for a long time to come.

Purpose Organ cultures of rabbit corneas have been used to ascertain the effectiveness of a human fibroblast growth factor (FGF)-1 derivative (TTHX1114), lacking cysteine residues, to protect against and/or repair epithelial lesions following exposure to nitrogen mustard (NM). Methods Rabbit corneas were exposed to NM and cultured for up to 14 days, with or without drug (TTHX1114). At specified times, tissue was examined by histopathology and graded by a novel composite scale. Proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and the expression of native FGF-1 and ADAM-17 after NM exposure was determined by immunofluorescence. Results Rabbit corneas, exposed to a single dose of NM, showed a nearly complete loss of epithelial cells by day 6 but were significantly regenerated by day 14. When treated continuously with TTHX1114 following vesicant exposure, the losses remained at day 2 levels. The loss of keratocytes in the stroma was not affected by TTHX1114. EdU incorporation over the same time course showed a steady increase in tissue that had not been treated with TTHX1114, while corneas that were treated with the drug showed a higher percent incorporation initially, which then decreased, indicating the strong proliferative response to TTHX1114. ADAM-17 was not significantly altered by TTHX1114 treatment. Corneal epithelial FGF-1 disappeared after only 1 day following exposure to NM. Conclusions TTHX1114 is protective against NM-induced damage of the corneal epithelium, possibly by supplying an NM-resistant source of trophic support and by stimulating regeneration of new epithelial cells. These responses underscore the potential value of TTHX1114 as an anti-vesicant therapeutic.

The octapeptide, Ser-Ser-Thr-His-Pro-Val-Phe-His, corresponding to the first eight amino-terminal residues in the sequence of mouse nerve growth factor, has been synthesized by the manual solid-phase technique. After cleavage of the peptide material from the peptide-resin by HF, extensive purification produced the expected product in good yield (27% of potential starting sites; 58% of the released product). The peptide was characterized by chromatographic techniques, sequence analysis, enzymatic digestion, CD and ORD spectral analyses, and biological and pharmacological assays. Detailed analysis of the reaction by-products, isolated during the purification of the principal product, provided technical information regarding possible synthetic side reactions and further evidence for dynamic solvation of the polymer matrix as a dominant factor in solid phase synthesis.

Editorial Author’s Choice © 2015 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org On Credibility, Clarity, and Compliance Al Burlingame, Steven A. Carr, Ralph A. Bradshaw, and Robert J. Chalkley Since its inception, MCP has recognized the promise of proteomics and its underlying technologies to significantly move the boundaries of knowledge in biology and medicine. As has been noted (1), proteomics represented a paradigm shift in how experiments were designed and executed and how the resultant data were interpreted and stored. But the journal has also recognized that the value of the proteomic approach and the data generated were only as good as the quality and reliability of that data—a corollary of the compu- tational maxim, “garbage in, garbage out.” Thus, in its very earliest stages, the editorial staff of MCP, particularly Steve Carr, Ruedi Aebersold, and Al Burlingame, began earnest discussions about how a journal should evaluate data arising from large scale, often called “shotgun,” mass spectrometric experiments, which ultimately lead to the first set of guidelines (2). Initially, these questions focused on correctly identifying peptide sequences (as surrogates for the proteins they were derived from) but quickly expanded to the identification and localization of post-translational modifications (PTMs) and mass-spectrometric-based quantification as well (3, 4). In the main, it was decided that manuscripts submitted to the jour- nal should provide sufficient information to allow an appropri- ate assessment by first reviewers and then readers. At first, the requested information consisted of the methods used for collection and interpretation (basically search parameters) of the data as well as the identifications themselves. As the guidelines evolved, tutorials and checklists were introduced to aid the submission process. The latter were eventually expanded to include checklists for papers of clinical relevance and those reporting glycomic analyses (5, 6). Finally, after first introducing the guidelines simply as recommendations, the journal began enforcing them by subjecting all submitted articles to a review to determine if they were compliant. The compliance check is not, and was never meant to be, a part of the evaluative peer review process, as these checks only determine whether the necessary information has been sup- plied and do not assess in any way its quality or interpretation. One of the most challenging aspects of the documentation process as it developed was the requirement that authors provide “annotated” spectra for all MS/MS protein identifica- Received June 3, 2015, and in revised form, June 3, 2015 Published, MCP Papers in Press, June 3, 2015, DOI 10.1074/ mcp.E115.052506 Author’s Choice—Final version free via Creative Commons CC-BY license. Author contributions: A.L.B., S.A.C., R.A.B., and R.J.C. wrote the paper. Molecular & Cellular Proteomics 14.7 tions based on only a single unique peptide (including the spectra used in peptide mapping fingerprints (PMFs)) and all PTM assignments. Annotated spectra means the labeling of the m/z for all significant peaks in the spectra as well as their fragment ion designations (e.g. y, b, etc. if spectra are from an MS/MS experiment) relative to the sequence being reported. Given the variations in software supporting different instru- ments and in the outputs of the many individual search en- gines, this single requirement became an increasingly difficult one to manage. In keeping with the historical “maintenance of the public record” role, which basically requires journals to report all the data that are germane to the claims and con- clusions of an article, MCP at the outset considered that these spectra needed to be submitted with the article and be a part of the journal. Electronic publishing of biological journals, which began with the Journal of Biological Chemistry in 1995 (7), made this possible, at least at first. However, when very large-scale identifications of PTMs became commonplace, the problem was substantially exacerbated, and it became clear that, at least in some cases, it was impractical to insist that these spectra be submitted as supplemental material. In addition, many authors were meeting this compliance require- ment through the production of one large pdf file of static screenshots of spectra, a format that made finding relevant spectra and more than cursory examination of the assignment difficult and reanalysis of data impossible. At the same time, the journal was also wrestling with the issue of raw data and whether it should require authors to also make this information available. It is germane to note that these were two separate issues: The spectra were considered to be a part of the manuscript, while the raw data were not. However, they were certainly interrelated. Thus, as a compro- mise, and in recognition of the importance of supporting repositories that store data in a secure form while allowing full public access, MCP waived the requirement that spectra be submitted with the article, allowing deposition in an accepta- ble public repository in lieu of inclusion in the manuscript as an alternative. At the same time, it announced that it would require all raw data to be deposited in a similar public site. Unfortunately this led, in many cases, to the often incorrect conclusion that simply depositing the raw data met both requirements without appreciating the different goals of the two submissions: one providing the ability to easily assess the authors’ interpretation of their data, the other allowing reanal- ysis and reuse of acquired data. This well-meaning decision to require the deposition of raw data subsequently hit “a serious snag” when one of the major sites available at the time began

Infiltration of the tumor microenvironment by nerve fibers is an understudied aspect of breast carcinogenesis. In this study, the presence of nerve fibers was investigated in a cohort of 369 primary breast cancers (ductal carcinomas in situ, invasive ductal and lobular carcinomas) by immunohistochemistry for the neuronal marker PGP9.5. Isolated nerve fibers (axons) were detected in 28% of invasive ductal carcinomas as compared to only 12% of invasive lobular carcinomas and 8% of ductal carcinomas in situ (p = 0.0003). In invasive breast cancers, the presence of nerve fibers was observed in 15% of lymph node negative tumors and 28% of lymph node positive tumors (p = 0.0031), indicating a relationship with the metastatic potential. In addition, there was an association between the presence of nerve fibers and the expression of nerve growth factor (NGF) in cancer cells (p = 0.0001). In vitro, breast cancer cells were able to induce neurite outgrowth in PC12 cells, and this neurotrophic activity was partially inhibited by anti-NGF blocking antibodies. In conclusion, infiltration by nerve fibers is a feature of the tumor microenvironment that is associated with aggressiveness and involves NGF production by cancer cells. The potential participation of nerve fibers in breast cancer progression needs to be further considered.

Nerve growth factor (NGF) is the ligand for two unrelated cellular receptors, TrkA and p75(NTR), and acts as a mediator in the development and maintenance of the mammalian nervous system. Signaling through TrkA kinase domains promotes neuronal survival, whereas activation of the p75(NTR) "death domains" induces apoptosis under correct physiological conditions. However, co-expression of these receptors leads to enhanced neuronal survival upon NGF stimulation, possibly through a ternary p75(NTR) x NGF x TrkA complex. We have expressed human p75(NTR) ligand binding domain as a secreted glycosylated protein in Trichoplusia ni cells. Following assembly and purification of soluble p75(NTR) x NGF complexes, mass spectrometry, analytical ultracentrifugation, and solution x-ray scattering measurements are indicative of 2:2 stoichiometry, which implies a symmetric complex. Molecular models of the 2:2 p75(NTR) x NGF complex based on these data are not consistent with the further assembly of either symmetric (2:2:2) or asymmetric (2:2:1) ternary p75(NTR) x NGF x TrkA complexes.

To assess whether nerve growth factor (NGF) expression would respond to booster dosing with the inducing agent ponasterone A, human embryonic kidney cells (HEK-293) were transfected with human NGF cDNA. Cells were cultured for 5 days in media with or without ponasterone A. On day 5, controls received a ponasterone A media replacement, whereas experimental groups received ponasterone A booster media replacement. NGF protein expression bioactivity was assessed using a PC-12 cell bioassay and the concentration of secreted NGF was quantified using NGF enzyme-linked immunosorbent assay. Cells with and without ponasterone A were left for 5 days without changing the medium. On day 5, the supernatants were collected and flash-frozen for enzyme-linked immunosorbent assay. The ponasterone A-positive and -negative booster medium was replaced in the appropriate wells. Supernatants were collected from the wells at 2, 4, and 6 days after the booster dose and removal of original supernatant. The medium was flash-frozen for enzyme-linked immunosorbent assay (1.5 ml), and the remaining 500 mul was transferred to PC-12 cells seeded onto 12-well plates to determine NGF bioactivity. All experiments were performed in quadruplicate. NGF production was measured daily by enzyme-linked immunosorbent assay over a 6-day period after the ponasterone A booster to a maximal release of 1233 +/- 130 pg/ml at day 6 (11 days after original induction). Maximal NGF production per 10(3) cells was 2.5 +/- 0.61 pg at day 6. Bioactivity was determined by percentage differentiation (per 100 cells counted) at 26, 52, and 98 percent for ponasterone A-treated wells on 2, 4, and 6 days after booster dosing (7, 9, and 11 days after induction), respectively. PC-12 cell differentiation was not visualized in the ponasterone A-negative control wells. Human NGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent ponasterone A. Furthermore, repeated bioactive NGF expression peaks beyond that previously demonstrated can be achieved using induction agent booster dosing, indicating the ability to regulate the system over an extended period.

Tissue-engineering strategies offer hope to patients facing functional impairment after nerve injury. We have previously demonstrated that HEK-293 cells can release nerve growth factor (NGF) in vitro, using an inducible system of expression. In this study, our objective was to assess the efficacy of the NGF delivery system in vivo, using nude rats. HEK-293 cells were transfected with human NGF cDNA. Ponasterone A (PonA) was used as the inducing agent. NGF collection chambers were implanted subcutaneously in nude rats. Sealed chambers were filled with one of the following: (1) DMEM, (2) untransfected 293 cells (EcR-293) plus PonA, (3) untransfected EcR-293 without PonA, (4) transfected 293 cells (hNGF-EcR-293) plus PonA, or (5) transfected hNGF-EcR-293 without PonA. Chambers were aspirated 24, 48, and 120 h postimplantation. NGF secretion was analyzed in the following ways: (1) NGF protein expression bioactivity was assessed in a PC-12 cell bioassay, and (2) the concentration of secreted NGF was quantified by NGF ELISA. NGF quantification by ELISA reached a maximal release of 12.9 +/- 3.57 ng/mL at 120 h. PC-12 cells exposed to media from induced transfected HEK-293 cell chambers demonstrated higher levels of differentiation compared with controls. We conclude that hNGF-EcR-293 cells can inducibly secrete bioactive NGF when exposed to the induction agent PonA. This regulated delivery system can secrete bioactive NGF for up to 5 days in vivo. We believe this regulated delivery system will be useful for tissue-engineered nerve constructs.

Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction.

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 +/- 2.6 ng/ml at day 9. Maximal NGF production was 8.46 +/- 2.1 pg/10(3) cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p

SummaryThrombin is the product of the hemostatic response essential to the conversion of fibrinogen to fibrin. In addition, it is also responsible for the aggregation of blood platelets in the formation of the “platelet plug” as well as the activation of factor VIII, factor V, factor XI, factor XIII and protein C. The action of thrombin is not confined to the hemostatic response as it also has a critical function in the wound healing process by stimulating ‘mitogenic’ events through interaction with cell surface receptors. In this review, we consider the various biological activities of thrombin as they relate to current therapeutic use. While there has been considerable interest in the development of fibrin sealant products, there has been considerably less interest in documenting the continuing use of thrombin as a therapeutic. The use of thrombin for topical hemostasis and the treatment of pseudoaneurysms will be discussed in detail. It is concluded that the use of thrombin as a drug will not only continue but also will significantly increase. However, the availability of a safe human thrombin preparation will be critical for the continued use of thrombin as a therapeutic.

Methionine, used to initiate protein synthesis in eukaryotes, is largely recovered as a co-translational hydrolysis event catalysed by two related enzymes, methionine aminopeptidase (MetAP)1 and 2. One of these, MetAP2, is covalently modified, and thus catalytically inactivated, by a class of drugs related to the fungal polyene antibiotic, fumagillin. This irreversible inhibition results in cell cycle arrest in endothelial cells (as well as a number of other cells) and prevents vascular growth. TNP-470, a member of this group, has been examined in clinical trials. Thus, these and other inhibitors of MetAP2, are a part of the growing class of antiangiogenic drugs with potential for treating cancer and other diseases related to neovascularisation. A large class of non-peptidic reversible inhibitors, as well as antisense and dominant-negative inhibitors, have been developed toward this end.

The Trk family is composed of three principal members (A, B and C), also termed neurotrophin tyrosine kinase receptors (NTRK1, NTRK2, NTRK3), with several additional splice variants, which in humans are located on three different chromosomes (1, 9, 15). The Trk proteins are receptors for the neurotrophin family of growth factors (NGF, BDNF, NT-3, and NT4/5); they also bind and respond more weakly to the proforms of these neurotrophins. Both the neurotrophins and pro-neurotrophins utilize other receptors (p75NTR, sortilin) that can interact with the Trks and affect their activity. The Trks are predominantly expressed in nervous tissues, where they are essential for the growth, development, and maintenance of select types of both peripheral and central neurons and are involved in neurodegenerative diseases. The Trks are also found in some nonneuronal tissues and in a wide variety of tumors, where they can play an active role in tumor progression and metastasis. The activated Trk receptors primarily bind and signal through Shc, FRS2, and PLCγ, which in turn activate PI3K, the Erks, and the hydrolysis of inositol phospholipids, among other events, leading to the modulation of gene transcription.

The initiator methionine residue of proteins is removed during synthesis by a specific and ubiquitous enzyme, methionine aminopeptidase (MetAP). Prokaryotes have a single gene, while eukaryotes have two isoforms. This family of metalloenzymes generally cleaves substrates in which the penultimate residue is one of the seven smaller amino acids (glycine, alanine, serine, threonine, proline, cysteine and valine). One of the eukaryotic isoforms (MetAP2) has an additional non-proteolytic function and is the principle target of a family of anti-angiogenic drugs that are related to fumagillin. The resulting covalent modification inhibits the protease activity of MetAP2 and blocks cell-cycle function in endothelial and some cancer cells. The role of MetAP2 in the mitogenic activity of these cells is unknown.

To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.

We previously identified the urokinase plasminogen activator receptor (UPAR) as a gene induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF), in PC12 cells (Farias-Eisner et al. [2000] J. Neurosci. 20:230–239). Antisense oligonucletides for the UPAR mRNA or an antibody directed against UPAR protein, added simultaneously with NGF, block NGF-induced morphological and biochemical differentiation of PC12 cells. In this report, we show that anti-UPAR antibody blocks morphological differentiation and the expression of two NGF-specific secondary response genes, collagenase-1 and transin, in PC12 cells only during the first 2 hr following NGF exposure. These data suggest that induced UPAR expression is required only over a short period of time following exposure to NGF for the differentiation program in PC12 cells to proceed. For two models of “primed” PC12 cells, we found that UPAR expression and function are not required for NGF-induced differentiation. UPAR and the secondary response genes collagenase-1 and transin are not induced in “primed” PC12 cells in response to NGF, and anti-UPAR antibody does not block morphological differentiation in these cells. Our data suggests that UPAR is required only transiently during the “priming” of PC12 cells in NGF-induced PC12 cell differentiation. J. Neurosci. Res. 63:341–346, 2001. © 2001 Wiley-Liss, Inc.

Analysis of protein post-translational modifications using mass spectrometry is an intensive area of proteomic research. This perspective discusses the current state of the field with respect to what can be achieved, the challenges encountered, most notably with modification site assignment, the reliability of the published results, consequences of unreliable data and what is needed to be done to improve the situation.

Nerve growth factor binds to the TrkA and p75(NTR) (p75) and generates signals leading to neuronal cell survival, differentiation, and programmed cell death. Here we describe a series of experiments involving selective activation of either TrkA or p75 in which distinct cell-signaling intermediates promote different cellular consequences. We analyzed pheochromocytoma 12 (PC12) cells stably expressing chimeras consisting of the extracellular domain of PDGF receptor (PDGFR) fused to the transmembrane and cytoplasmic segments of p75 or TrkA. Because PC12 cells lack endogenous PDGFR, addition of PDGF to these cell lines permits selective activation of the p75 or TrkA responses without stimulating endogenous receptors. Although both p75 and TrkA activated nuclear factor-kappaB (NF-kappaB), we show that distinct proximal-signaling intermediates are used by each receptor. A dominant-negative mutant of TRAF6 blocked p75- but not TrkA-mediated induction of NF-kappaB. Conversely a dominant-negative mutant of Shc inhibited TrkA but not p75 activation of NF-kappaB. Both of these distinct signaling pathways subsequently converge, leading to activation of the IkappaB kinase complex. Moreover, the activation of NF-kappaB by these distinct pathways after stimulation of either TrkA or p75 leads to different physiological consequences. Blocking p75-mediated activation of NF-kappaB by ecdysone-inducible expression of a nondegradable mutant of IkappaBalpha significantly enhanced apoptosis. In contrast, blocking NF-kappaB induction via TrkA significantly inhibited neurite process formation in PC12 cells. Together these findings indicate that, although both of these receptors lead to the activation of NF-kappaB, they proceed via distinct proximal-signaling intermediates and contribute to different cellular outcomes.