Daniel F. Cimino

Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Individual Carboxyl-Terminal Serine and Threonine Residues

Adaptation, defined as the diminution of receptor signaling in the presence of continued or repeated stimulation, is critical to cellular function. G protein-coupled receptors (GPCRs) undergo multiple adaptive processes, including desensitization and internalization, through phosphorylation of cytoplasmic serine and threonine residues. However, the relative importance of individual and combined serine and threonine residues to these processes is not well understood. We examined this mechanism in the context of the N-formyl peptide receptor (FPR), a well-characterized member of the chemoattractant/chemokine family of GPCRs critical to neutrophil function. To evaluate the contributions of individual and combinatorial serine and threonine residues to internalization, desensitization, and arrestin2 binding, 30 mutant forms of the FPR, expressed in the human promyelocytic U937 cell line, were characterized. We found that residues Ser328, Ser332, and Ser338 are individually critical, and indeed sufficient, for internalization, desensitization, and arrestin2 binding, but that the presence of neighboring threonine residues can inhibit these processes. Additionally, we observed no absolute correlation between arrestin binding and either internalization or desensitization, suggesting the existence of arrestin-independent mechanisms for these processes. Our results suggest C-terminal serine and threonine residues of the FPR represent a combinatorial code, capable of both positively and negatively regulating signaling and trafficking. This study is among the first detailed analyses of a complex regulatory site in a GPCR, and provides insight into GPCR regulatory mechanisms.

Evidence for Developmental Hypopituitarism in Ill Preterm Infants

OBJECTIVE: The relationship between the concentrations of cortisol and T4 with outcome in the preterm infants has not been well studied.STUDY DESIGN: Mean cortisol (days 2, 4, and 6) and T4 values were correlated to gestational age, illness, and outcome in 210 infants using ANOVA.RESULTS: Cortisol significantly decreased and T4 increased across gestational age. For both hormones, higher values were found in infants on low ventilatory settings. Combined lower cortisol (mean <5 μg/dl (138 nmol/l)) and T4 concentrations (<4 μg/dl) were found in 20/210 (9.5%) infants; 11/20 in a high-acuity group (22% of total) including 48% (12/25) of the deaths. Lower cortisol values were found in infants who died (p<0.005) in contrast to a lack of relationship with T4.CONCLUSIONS: Lower cortisol values in infants who died are consistent with the role for cortisol in survival. Combined lower cortisol and T4 concentrations in infants who failed to improve clinical status may suggest that these hormones are markers of a poor physiological state. In contrast, we suggest that these results reflect a developmental hypopituitarism, a necessary role for cortisone and T4 in successful early neonatal transition. Treatment of hypothyroidism in the setting of coexistent low cortisol concentrations (central dysfunction of the hypothalamic–pituitary axes) is known to precipitate cortisol crisis in older populations. Therefore, we caution against treatment of low neonatal thyroid concentrations until more is known about the relationship between cortisol and T4 preterm infant population.

Glutathione-S-transferase-green fluorescent protein fusion protein reveals slow dissociation from high site density beads and measures free GSH

Glutathione, a ubiquitous tripeptide, is an important cellular constituent, and measurement of reduced and oxidized glutathione is a measure of the redox state of cells. Glutathione‐S‐transferase (GST) fusion proteins bind naturally to beads derivatized with glutathione, and elution of such bead‐bound fusion proteins with buffer containing millimolar glutathione is a commonly used method of protein purification. Many protein–protein interactions have been established by using GST fusion proteins and measuring binding of fusion protein binding partners by GST pulldown assays, usually monitored by Western blot methodology.

Regulation of N-formyl peptide-mediated degranulation by receptor phosphorylation.

One of the major functions of the N-formyl peptide receptor (FPR) is to mediate leukocyte degranulation. Phosphorylation of the C-terminal domain of the FPR is required for receptor internalization and desensitization. Although arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of novel signaling cascades for a number of G protein-coupled receptors, their roles in FPR regulation and signaling remain unclear. CXCR1-mediated degranulation of RBL-2H3 cells is promoted by arrestin binding. To determine whether receptor phosphorylation or arrestin binding is required to promote FPR-mediated degranulation, we used RBL-2H3 cells stably transfected with either the wild-type FPR or a mutant form, ΔST, which is incapable of undergoing ligand-stimulated phosphorylation. We observed that stimulation of wild-type FPR resulted in very low levels of degranulation compared with that mediated by cross-linking of the FcεRI receptor. Stimulation of the ΔST mutant, however, resulted in levels of degranulation comparable to those of the FcεRI receptor, demonstrating that neither receptor phosphorylation nor arrestin binding was necessary to initiate FPR-mediated degranulation. Degranulation initiated by the ΔST mutant was proportional to the level of active cell surface receptor, suggesting that either receptor internalization or desensitization may be responsible for terminating degranulation of the wild-type FPR. To distinguish between these possibilities, we used a partially phosphorylation-deficient mutant of the FPR that can undergo internalization, but not desensitization. Degranulation by this mutant FPR was indistinguishable from that of the ΔST mutant, indicating that FPR phosphorylation or binding of arrestin but not internalization terminates the degranulation response.

Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer

Background: EphA5 is a functional target in lung cancer, the most common cause of tumor-related death in mankind. Results: EphA5 regulates cell cycle checkpoints and DNA damage repair induced by ionizing radiation. Conclusion: EphA5 is a novel regulator of DNA damage repair with clinical implications. Significance: EphA5 may serve as a novel biomarker of radioresistance and a candidate target for therapeutic intervention in human lung cancer. Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

Abstract 3543: Ligand-directed and transcription-based molecular imaging and treatment of cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Inflammatory breast cancer (IBC) is a rare and aggressive disease that progresses rapidly, often in a matter of weeks or months. In the United States, IBC accounts for 1 to 5 percent of all breast cancers diagnosed per year. Due to the lack of reliable and effective detection methods, IBC can be difficult to diagnose and thus, it is more likely to have metastasized by the time it is identified. Specific delivery of drugs and/or imaging agents to tumors takes advantage of dynamic molecular addresses on the surface of tumor cells and the tumor vasculature through inherent receptor/ligand interactions. By using a combinatorial phage display library in vivo (consisting of more than 1×109unique CX(7/8)C amino acid residues) we identified an unique peptide sequence - WIFPWIQL - that mediates targeting to breast cancer metastasis. We further validated that the WIFPWIQL peptide specifically binds to the stress-response 78 kDa glucose-regulated protein (GRP78), expressed at the cell surface of IBC tumors. GRP78 expression is elevated with breast cancer progression, and correlates with metastatic disease in IBC. Using near infrared (NIR) fluorescent-tagged phage variants and/or antibody fragments, we demonstrate WIFPIWIQL specifically targets breast tumors in vivo. As a therapeutic approach, we coupled a toxic apoptotic moiety D(KLAKLAK)2 - to the WIFPWIQL peptide to create BMTP-78. BMTP-78 is cytotoxic to cultured IBC cells in vitro. Furthermore, BMTP-78 prevents the growth of IBC tumor xenografts, reduces tumor burden and mitigates metastasis in breast cancer pre-clinical models. Taking into consideration that phage particles can be adapted to transduce mammalian cells by targeting a specific receptor, we developed a unique phage-based targeted vector by combining genomic elements from adeno-associated virus (AAV) and an M13-derived phage (P) - termed AAVP. AAVP chimeras are able to deliver reporter genes that can be used to image, diagnose and treat pre-clinical breast cancer. In our initial in vivo assessment, AAVP particles harboring the tumor targeting peptide, WIFPWIQL, delivered the Herpes Simplex Virus thymidine kinase gene (HSVtk) driven by the CMV promoter, to EF43.fgf-4 mouse mammary tumors after a single intravenous injection. Positron emission tomography (PET) studies demonstrated WIFPIWIQL-targeted AAVP-HSKtk tumors could be non-invasively imaged. More importantly, transcriptional targeting of AAVP vectors was achieved by replacing the CMV promoter with a human GRP78 promoter that selectively controls transgene expression and allows molecular imaging of breast tumors in real time. Taken together, our data presents a GRP78 receptor/ligand system that can be used to specifically image and target breast tumors. Our findings infer potential clinical applications of AAVP for targeted detection and eradication of IBC tumors via a GRP78-targeted mechanism in a disease for which no effective treatment currently exists. Citation Format: Andrey S. Dobroff, Bedrich L. Eckhardt, Carolina C. Salmeron, Daniel F. Cimino, Wadih Arap, Renata Pasqualini. Ligand-directed and transcription-based molecular imaging and treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3543. doi:10.1158/1538-7445.AM2015-3543