The C-Terminus of Transmembrane Helix 2 (TM2) of the Escherichia coli Tar Chemorecptor Determines Signal Output and Ligand Sensitivity

Methyl-accepting chemotaxis proteins MCPs can bind one or more receptor- specific ligands. In the case of the Tar MCP of Escherichia coli (TarEc), a primary attractant ligand is aspartate. Its binding to the periplasmic domain of Tar generates a conformational change that is transmitted via helix 4 transmembrane helix 2 (TM2). An inward movement of TM2 initiates a transmembrane signal to the cytoplasmic HAMP (histidine kinases, adenyl cyclases, methyl-accepting proteins, phosphatases) domain. Baseline CheA kinase-stimulating activity and ligand-induced responses are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). The cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in TarEc, is of particular importance. These residues are not highly conserved among transmembrane receptors having a HAMP domain, although there are almost always some aromatic residues in this region. The question thus becomes what properties of this aromatic anchor are necessary for proper signal transduction. In this dissertation, I studied the effect on TarEc function by substituting all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. This library of TarEc variants allowed the direct assessment of the effect of the residue composition of the aromatic anchor and led to a model of how the wild-type anchor maintains the base-line signaling state in TarEc. Additional receptor variants containing double aromatic tandems and Ala substitutions for the periplasmic Trp residue were created, and the aromatic residues were also shifted in position within the six residues 207-212. Trp, Tyr, and Phe, in that order, had the greatest effect on function when they were moved to novel positions. It was also discovered that Gly-211 plays a critical role in maintaining receptor function. A model was generated that proposes that Gly-211 plays a role in maintaining the flexibility of the TM2-HAMP domain connector. The results suggest that the signaling properties of the transmembrane sensor kinases of two-component systems can be predicted by the nature of their TM2-HAMP connections. It may also be possible to modulate their activity in a controlled way by manipulating the amino acid sequences that comprise those connections

Repellent Taxis in Response to Nickel Ion Requires Neither Ni2+ Transport nor the Periplasmic NikA Binding Protein

Ni(2+) and Co(2+) are sensed as repellents by the Escherichia coli Tar chemoreceptor. The periplasmic Ni(2+) binding protein, NikA, has been suggested to sense Ni(2+). We show here that neither NikA nor the membrane-bound NikB and NikC proteins of the Ni(2+) transport system are required for repellent taxis in response to Ni(2+)

Activation of Parathyroid Hormone 2 Receptor Induces Decorin Expression and Promotes Wound Repair.

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair

Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment

The Parathyroid Hormone Second Receptor PTH2R and its Ligand Tuberoinfundibular Peptide of 39 Residues TIP39 Regulate Intracellular Calcium and Influence Keratinocyte Differentiation

Genes related to the parathyroid hormone (PTH) influence cutaneous immune defense and development, but the full functions of the PTH family in cutaneous biology remain incompletely understood. In this study, we examined the expression and potential functions of the PTH second receptor (PTH2R) and its ligand, the tuberoinfundibular peptide of 39 residues (TIP39), in the skin. TIP39 and PTH2R mRNA and protein were detectable in both human and mouse skin, and in cultured keratinocytes and adipocytes. TIP39 was observed in the basal layer of human skin, whereas PTH2R was detected in the spinous to granular layer. The subcellular localization of TIP39 in keratinocytes changed during calcium-induced differentiation and shifted to colocalize with PTH2R at the membrane. The addition of recombinant TIP39 to normal human keratinocytes in culture induced an increase in intercellular calcium and triggered aspects of terminal differentiation including decreased keratin-14 and increased involucrin expression. Consistent with these observations, PTH2R(-/-) mice were observed to have increased epidermal thickness. In summary, identification of TIP39 and its receptor in the epidermis reveals an additional PTH family member that is expressed in the skin and may influence keratinocyte function

IL-1 Receptor–Knockout Mice Develop Epidermal Cysts and Show an Altered Innate Immune Response after Exposure to UVB Radiation

In this study, we observed that mice lacking the IL-1 receptor (IL-1R) (IL1r-/-) or deficient in IL1-β developed multiple epidermal cysts after chronic UVB exposure. Cysts that developed in IL1r-/- mice were characterized by the presence of the hair follicle marker Sox 9, keratins 10 and 14, and normal melanocyte distribution and retinoid X receptor-α expression. The increased incidence of cysts in IL1r-/- mice was associated with less skin inflammation as characterized by decreased recruitment of macrophages, and their skin also maintained epidermal barrier function compared with wild-type mice. Transcriptional analysis of the skin of IL1r-/- mice after UVB exposure showed decreased gene expression of proinflammatory cytokines such as tumor necrosis factor-α and IL-6. In vitro, primary keratinocytes derived from IL1r-/- mice were more resistant to UVB-triggered cell death compared with wild-type cells, and tumor necrosis factor-α release was completely blocked in the absence of IL-1R. These observations illustrate an unexpected yet prominent phenotype associated with the lack of IL-1R signaling in mice and support further investigation into the role of IL-1 ligands in epidermal repair and innate immune response after damaging UVB exposure

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway

Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment