Scott A Boitano

Scott A Boitano

Associate Research Scientist, Respiratory Sciences
Professor, BIO5 Institute
Professor, Cellular and Molecular Medicine
Professor, Physiological Sciences - GIDP
Professor, Physiology
Primary Department
Department Affiliations
Contact
(520) 626-2105

Research Interest

Research Interest
Dr. Scott Boitano Ph.D., is a Professor of Physiology, Cellular and Molecular Medicine, the BIO5 Institute and Associate Research Scientist of the Arizona Respiratory Center. Dr. Boitano received a B.S. in Plant Biology from University of California; Berkeley and a Ph.D. in Genetics & Cell Biology from Washington State University. Dr. Boitano’s primary research interest is in cell respiration. This encompasses the analysis and observation of cell physiology, cell-cell communications and cell-pathogen interactions. Dr. Boitano’s research pertains to the upper airway epithelium is an active cellular layer with ciliary movement to clear materials, the ability to secrete inflammatory effectors, and a biological barrier function that helps protect against pathogenic microorganisms, foreign insults and injury. Although much is known concerning the microbial genetics and microbial signaling of infection by Bordetella, relatively little is known about host cell pathology after exposure to Bordetella. Individuals have a primary tissue culture system that serves as an in vitro model of airway cell signaling and communication, and a battery of B. bronchiseptica strains, some of which are mutant in key factors shown to inhibit their ability to establish infection in animal models. His research goal is to define specific pathogen factors that alter host cell physiology to initiate or overcome host cell defense. The Boitano lab also analyzes the layers of the alveoli of the distal mammalian lung. Minimal information is known about this subject and Dr. Boitano believes that this model system for alveolar intercellular communication could expedite the formulating and testing of new medical treatments for dysfunctional alveolar cell physiology that underlies specific airway conditions following disease, insult and injury in the lung.

Publications

Boitano, S., Hoffman, J., Tillu, D. V., Asiedu, M. N., Zhang, Z., Sherwood, C. L., Wang, Y., Dong, X., Price, T. J., & Vagner, J. (2014). Development and evaluation of small peptidomimetic ligands to protease-activated receptor-2 (PAR2) through the use of lipid tethering. PloS one, 9(6), e99140.

Protease-activated receptor-2 (PAR2) is a G-Protein Coupled Receptor (GPCR) activated by proteolytic cleavage to expose an attached, tethered ligand (SLIGRL). We evaluated the ability for lipid-tethered-peptidomimetics to activate PAR2 with in vitro physiological and Ca2+ signaling assays to determine minimal components necessary for potent, specific and full PAR2 activation. A known PAR2 activating compound containing a hexadecyl (Hdc) lipid via three polyethylene glycol (PEG) linkers (2at-LIGRL-PEG3-Hdc) provided a potent agonist starting point (physiological EC50 = 1.4 nM; 95% CI: 1.2-2.3 nM). In a set of truncated analogs, 2at-LIGR-PEG3-Hdc retained potency (EC50 = 2.1 nM; 1.3-3.4 nM) with improved selectivity for PAR2 over Mas1 related G-protein coupled receptor type C11, a GPCR that can be activated by the PAR2 peptide agonist, SLIGRL-NH2. 2at-LIG-PEG3-Hdc was the smallest full PAR2 agonist, albeit with a reduced EC50 (46 nM; 20-100 nM). 2at-LI-PEG3-Hdc retained specific activity for PAR2 with reduced EC50 (310 nM; 260-360 nM) but displayed partial PAR2 activation in both physiological and Ca2+ signaling assays. Further truncation (2at-L-PEG3-Hdc and 2at-PEG3-Hdc) eliminated in vitro activity. When used in vivo, full and partial PAR2 in vitro agonists evoked mechanical hypersensitivity at a 15 pmole dose while 2at-L-PEG3-Hdc lacked efficacy. Minimum peptidomimetic PAR2 agonists were developed with known heterocycle substitutes for Ser1 (isoxazole or aminothiazoyl) and cyclohexylalanine (Cha) as a substitute for Leu2. Both heterocycle-tetrapeptide and heterocycle-dipeptides displayed PAR2 specificity, however, only the heterocycle-tetrapeptides displayed full PAR2 agonism. Using the lipid-tethered-peptidomimetic approach we have developed novel structure activity relationships for PAR2 that allows for selective probing of PAR2 function across a broad range of physiological systems.

Maura, C. L., Boitano, S. A., Vagner, J., & Burt, J. M. (2017). Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca2+-wave propagation. American Journal of Physiology: Cell Physiology, In Revision.

Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap 27: SRPTEKTIFII, and Gap36: KRDPCHQVDCFLSRPTEK) have been used as reversible, Cx-specific inhibitors of gap junction intercellular communication. Typically, these peptides require relatively high concentrations (i.e., 100–200 μM with IC50s ~20–30 μM) to achieve inhibition. We hypothesized that lipidation of Cx mimetic peptides would improve their ability to concentrate in the plasma membrane and thus more effectively interfere with intercellular signaling. A hexadecyl (Hdc) lipid tail was added to the conserved SRPTEKT peptide sequence of Gap27 (SRPTEKT-Hdc) and its efficacy in inhibiting mechanically-induced intercellular Ca2+-wave propagation in confluent cultures of both Madin-Darby canine kidney epithelial cells expressing Cx43 (MDCK43) and primary-cultured rabbit tracheal epithelial cells (RTECs) assessed. SRPTEKT-Hdc reversibly inhibited in a time- and concentration- dependent manner intercellular Ca2+-wave propagation in both cell types with an IC50 in the picomolar range (i.e., 31.8–138.9 pM), five orders of magnitude lower than the non-lipidated Gap27 peptide. SRPTEKT-Hdc was less effective at inhibiting dye coupling. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca2+-wave propagation. Immunoblot analysis revealed no change in Cx43 expression, but a small reduction in plaque-associated Cx43 following SRPTEKT-Hdc incubation. Our results suggest SRPTEKT-Hdc potently inhibits, in a channel conformation-specific manner, intercellular IP3 signaling at concentrations that minimally affect dye (electrical) coupling. In summary, lipidation of mimetic peptides represents a paradigm for development of highly potent, efficacious and selective inhibitors of gap junction-mediated intercellular signaling.

Kurzius-Spencer, M., Foster, K., Littau, S., Richey, K. J., Clark, B. M., Sherrill, D., Goodman, R. B., Boitano, S., & Burgess, J. L. (2015). Tracheobronchial markers of lung injury in smoke inhalation victims. Journal of burn care & research : official publication of the American Burn Association, 29(2), 311-8.
BIO5 Collaborators
Scott A Boitano, Jefferey L Burgess

Although smoke inhalation injury victims frequently develop severe hypoxemia and are at increased risk of acute respiratory distress syndrome (ARDS), no early prognostic tests are currently available. The objectives were to determine early longitudinal changes in tracheobronchial fluid inflammatory markers and assess the value of initial concentrations as predictors of subsequent lung injury. Partial pressure of arterial oxygen (Pao2) and the fraction of inspired oxygen (Fio2) were recorded approximately every 6 hours from intubated smoke inhalation victims admitted to a regional burn center. Tracheobronchial suction fluid was collected every 2 hours and assayed for interleukins (IL-1beta, -8, and -10), tumor necrosis factor-alpha, transforming growth factor-beta1, soluble Fas ligand (sFasL), and complement factor 5a. Temporal trends in marker concentrations during 36 hours and the relations between initial concentrations and lowest Pao2/Fio2 or ARDS within 72 hours were assessed using random coefficients modeling and cross-sectional analysis. In 21 subjects with tracheobronchial samples collected within 6.5 hours of intubation, 14 (66.7%) developed acute hypoxemia (Pao2/Fio2 200 (P = .042) during 72 hours. In smoke inhalation victims, tracheobronchial IL-1beta and IL-8 increase rapidly and high initial IL-8 may predict improved oxygenation.

Flynn, A. N., Tillu, D. V., Asiedu, M. N., Hoffman, J., Vagner, J., Price, T. J., & Boitano, S. (2011). The protease-activated receptor-2-specific agonists 2-aminothiazol-4-yl-LIGRL-NH2 and 6-aminonicotinyl-LIGRL-NH2 stimulate multiple signaling pathways to induce physiological responses in vitro and in vivo. The Journal of biological chemistry, 286(21), 19076-88.

Protease-activated receptor-2 (PAR(2)) is one of four protease-activated G-protein-coupled receptors. PAR(2) is expressed on multiple cell types where it contributes to cellular responses to endogenous and exogenous proteases. Proteolytic cleavage of PAR(2) reveals a tethered ligand that activates PAR(2) and two major downstream signaling pathways: mitogen-activated protein kinase (MAPK) and intracellular Ca(2+) signaling. Peptides or peptidomimetics can mimic binding of the tethered ligand to stimulate signaling without the nonspecific effects of proteases. The most commonly used peptide activators of PAR(2) (e.g. SLIGRL-NH(2) and SLIGKV-NH(2)) lack potency at the receptor. However, although the potency of 2-furoyl-LIGRLO-NH(2) (2-f-LIGRLO-NH(2)) underscores the use of peptidomimetic PAR(2) ligands as a mechanism to enhance pharmacological action at PAR(2), 2-f-LIGRLO-NH(2) has not been thoroughly evaluated. We evaluated the known agonist 2-f-LIGRLO-NH(2) and two recently described pentapeptidomimetic PAR(2)-specific agonists, 2-aminothiazol-4-yl-LIGRL-NH(2) (2-at-LIGRL-NH(2)) and 6-aminonicotinyl-LIGRL-NH(2) (6-an-LIGRL-NH(2)). All peptidomimetic agonists stimulated PAR(2)-dependent in vitro physiological responses, MAPK signaling, and Ca(2+) signaling with an overall rank order of potency of 2-f-LIGRLO-NH(2) ≈ 2-at-LIGRL-NH(2) > 6-an-LIGRL-NH(2) ≫ SLIGRL-NH(2). Because PAR(2) plays a major role in pathological pain conditions and to test potency of the peptidomimetic agonists in vivo, we evaluated these agonists in models relevant to nociception. All three agonists activated Ca(2+) signaling in nociceptors in vitro, and both 2-at-LIGRL-NH(2) and 2-f-LIGRLO-NH(2) stimulated PAR(2)-dependent thermal hyperalgesia in vivo. We have characterized three high potency ligands that can be used to explore the physiological role of PAR(2) in a variety of systems and pathologies.

Yeee, M. C., Nichols, H. L., Polley, D., Pal, K., Lee, K., Daines, M. O., Boitano, S. A., Hollenberg, M. D., & DeFea, K. A. (2017). Protease-activated receptor 2-induced signaling through β-arrestin-2 mediates Alternaria serine protease-induced airway inflammation. PLoS One, In Revision.

Protease-Activated Receptor-2 (PAR2) is a G-protein-coupled receptor that is activated by a variety of trypsin-like serine proteases, including those found in common allergens such as Alternaria fungi, which are causative in asthma-associated morbidity and mortality worldwide. PAR2 signals through both G-protein and β-arrestin-dependent pathways and β-arrestin-2 mediates PAR2-induced leukocyte chemotaxis into the airway. Previous studies showed that an unidentified serine protease activity in Alternaria alternata can activate PAR2 to trigger an inflammatory cell pathology in murine lung. We hypothesized that a serine-protease-mediated PAR2/β-arrestin signaling axis plays a key role in Alternaria-induced lung inflammation. Here we isolate, from Alternaria extracts, the single serine protease responsible for PAR2 activation, AASP (Alternaria Alkaline Serine Protease). Both extracts and isolated AASP promote PAR2 β-arrestin-dependent signaling in cultured cells. Intranasal administration of Alternaria extract in wild-type (but not β-arrestin-2-/- or PAR2-/- mice) increases airway epithelial damage, mucin production, and recruitment of eosinophils, CD4+T-cells and macrophages and this response is abolished by administration of soybean trypsin inhibitor (SBTI). Administration of AASP alone is sufficient to induce eosinophil and lymphocyte recruitment. Our data demonstrate that a single serine protease in an aeroallergen like Alternaria alternata can activate PAR2 signaling through β-arrestin-2 to cause an inflammatory asthma phenotype. Thus, β-arrestin-biased PAR2 antagonists represent attractive therapeutic targets for treating aeroallergen-induced asthma.