Effect of mutations in TrkA gene found in insensitivity to congenital pain on cell signaling pathways

Author(s)

Beatriz Caroline de Moraes

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2020-09-10

Examining board members

Deborah Schechtman; Nicolas Carlos Hoch; Fábio Papes; Vanessa Olzon Zambelli

• Advisor: Deborah Schechtman

Abstract

Mutations that cause loss of function of the tropomyosin kinase A receptor (TrkA) are among the main factors that lead to the disease. TrkA is a high affinity receptor for NGF, and the formation of the NGF / TrkA complex plays a role in neuronal development, as well as in sensitization to perceived temperature increases and mechanical stimulation, which results in hyperalgesia. Many mutations compose the syndrome spectrum of CIPA, amongst them the ones found in the kinase domain are object of the present study, because they can alter this domain\'s activity and thus interfere in important signaling pathways mediated by TrkA (PLCγ, Akt, and ERK). With the objective of understanding how signaling pathways are altered by the mutations in CIPA, we searched several published articles on PubMed and identified the description of more than 131 mutations in this gene. Missense mutations were in the three-dimensional structure and classified in three kinds: (1) mutations close to the ATP binding site (catalytic site), (2) mutations in the substrate binding site (SBS), and (3) mutations in other structured regions of TrkA, that can prevent correct protein folding. We validated in vitro one of the CIPA mutations (R654C), both as a mutation at the ATP binding site and in the SBS region. As predicted in the modeling, this mutation presented total inactivation of TrkA. The nonsense and frameshift mutations would also significantly affect TrkA structure, although some of them such as Q770*, Q782*, V777Cfs*91 and L784Sfs*79, would lead to theexpression of a truncated protein, specifically effecting PLC anchoring, their presence triggered the phenotype of CIPA in patients, indicating that the activation of PLCγ would be an important route for the occurrence of pain. Based on these results, a peptide was developed that inhibits an interaction of TrkA and PLCγ, called QYP. In HEK 293 cells transfected with TrkA, this peptide was capable of inhibiting PLCγ activation mediated by NGF. In addition, in in vivo behavioral trials of acute and CFA-induced inflammatory pain, QYP was able to inhibit mechanical hypersensitivity and partially caused by inflammation for up to 6 hours. QYP also proved to be more efficient than commercial inhibitors (GNF, TrkA inhibitor and U73347, PLC inhibitor) in reversing painful sensitivity. Based on our results, we validate that the PLCγ pathway is essential for the development of mechanical pain, which is a potential target for the development of new analgesics. In addition, the QYP peptide has been shown to be effective in reversing acute and chronic mechanical painful sensitivity in in vivo models of inflammatory pain. (AU)

• FAPESP's process: 18/07201-5 - Effect of mutations in TrkA gene found in insensitivity to congenital pain on cell signaling pathways
• Grantee: Beatriz Caroline de Moraes
• Support Opportunities: Scholarships in Brazil - Master