ffect of FKBP12-Derived Intracellular Peptides on Rapamycin-Induced FKBP-FRB Interaction and Autophag

Author(s):Show less -

Parada, Carolina A. ^[1] ; de Oliveira, Ivan Pires ^{[1, 2]} ; Gewehr, Mayara C. F. ^[1] ; Machado-Neto, Joao Agostinho ^[1] ; Lima, Keli ^[1] ; Eichler, Rosangela A. S. ^[1] ; Lopes, Lucia R. ^[1] ; Bechara, Luiz R. G. ^[3] ; Ferreira, Julio C. B. ^[3] ; Festuccia, William T. ^[4] ; Censoni, Luciano ^[5] ; Tersariol, Ivarne Luis S. ^[6] ; Ferro, Emer S. ^[1]

Affiliation

^[1] Univ Sao Paulo, Biomed Sci Inst, Dept Pharmacol, BR-05508900 Sao Paulo, SP - Brazil

^[2] Univ Fed Minas Gerais, Inst Agr Sci, BR-39400149 Montes Claros, MG - Brazil

^[3] Univ Sao Paulo, Biomed Sci Inst, Dept Anat, BR-05508900 Sao Paulo, SP - Brazil

^[4] Univ Sao Paulo, Biomed Sci Inst, Dept Physiol, BR-05508900 Sao Paulo, SP - Brazil

^[5] Univ Estadual Campinas, Inst Chem, Dept Phys Chem, BR-13083970 Campinas, SP - Brazil

^[6] Sao Paulo Fed Univ UNIFESP, Paulista Med Sch, Dept Biochem, BR-04021001 Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: ELL; v. 11, n. 3 FEB 2022.
• Web of Science Citations: 0

Abstract

Intracellular peptides (InPeps) generated by proteasomes were previously suggested as putative natural regulators of protein-protein interactions (PPI). Here, the main aim was to investigate the intracellular effects of intracellular peptide VFDVELL (VFD7) and related peptides on PPI. The internalization of the peptides was achieved using a C-terminus covalently bound cell-penetrating peptide (cpp; YGRKKRRQRRR). The possible inhibition of PPI was investigated using a NanoBiT(R) luciferase structural complementation reporter system, with a pair of plasmids vectors each encoding, simultaneously, either FK506-binding protein (FKBP) or FKBP-binding domain (FRB) of mechanistic target of rapamycin complex 1 (mTORC1). The interaction of FKBP-FRB within cells occurs under rapamycin induction. Results shown that rapamycin-induced interaction between FKBP-FRB within human embryonic kidney 293 (HEK293) cells was inhibited by VFD7-cpp (10-500 nM) and FDVELLYGRKKRRQRRR (VFD6-cpp; 1-500 nM); additional VFD7-cpp derivatives were either less or not effective in inhibiting FKBP-FRB interaction induced by rapamycin. Molecular dynamics simulations suggested that selected peptides, such as VFD7-cpp, VFD6-cpp, VFAVELLYGRKKKRRQRRR (VFA7-cpp), and VFEVELLYGRKKKRRQRRR (VFA7-cpp), bind to FKBP and to FRB protein surfaces. However, only VFD7-cpp and VFD6-cpp induced changes on FKBP structure, which could help with understanding their mechanism of PPI inhibition. InPeps extracted from HEK293 cells were found mainly associated with macromolecular components (i.e., proteins and/or nucleic acids), contributing to understanding InPeps' intracellular proteolytic stability and mechanism of action-inhibiting PPI within cells. In a model of cell death induced by hypoxia-reoxygenation, VFD6-cpp (1 mu M) increased the viability of mouse embryonic fibroblasts cells (MEF) expressing mTORC1-regulated autophagy-related gene 5 (Atg5), but not in autophagy-deficient MEF cells lacking the expression of Atg5. These data suggest that VFD6-cpp could have therapeutic applications reducing undesired side effects of rapamycin long-term treatments. In summary, the present report provides further evidence that InPeps have biological significance and could be valuable tools for the rational design of therapeutic molecules targeting intracellular PPI. (AU)

• FAPESP's process: 19/25049-9 - Mitochondrial formyl peptides as signaling molecules in cardiac Ischemia-reperfusion injury
• Grantee: Julio Cesar Batista Ferreira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 19/27149-0 - Knockout mice for thimet oligopeptidase as a model for the study of Obesity and Obesity-associated diseases
• Grantee: Mayara Calegaro Ferrari Gewehr
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 17/02201-4 - Study of the inhibition of the accumulation of cyclic nucleotide by pyridopyrimidine derivatives using molecular dynamics simulations
• Grantee: Ivan Pires de Oliveira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 16/04000-3 - Pharmacology of oligopeptidases and intracellular peptides
• Grantee: Emer Suavinho Ferro
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/07332-5 - Structural basis of intracellular peptides-protein interactions
• Grantee: Carolina Angelica da Silva Parada
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 19/23864-7 - Comprehensive analysis of genomic data for identification and validation of novel therapeutic targets involved in cellular cytoskeleton regulation in acute Leukemia
• Grantee: João Agostinho Machado Neto
• Support Opportunities: Regular Research Grants