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C‐terminal domain is responsible for a Kunitz‐type inhibitor uptake by tumor cells
Author(s) -
ChudzinskiTavassi Ana Marisa,
Morais Katia Luciano Pereira,
Servent Denis,
Sciani Juliana Mozer,
Iqbal Asif
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb188
Subject(s) - circular dichroism , confocal microscopy , microbiology and biotechnology , alexa fluor , intracellular , cell culture , biology , cell penetrating peptide , cytotoxicity , chemistry , peptide , biochemistry , in vitro , fluorescence , genetics , physics , quantum mechanics
Amblyomin‐X is a recombinant Kunitz‐type FXa inhibitor that was identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth and reduction of metastasis. In this regard, recently arose question about it structure‐fuction. Thereby, we investigated the effects of two truncated (synthetic) regions of Amblyomin‐X, which we termed Kunitz and C‐terminal domains. Here, using MTT and FXa chromogenic activity assay, we found that both domains have no cytotoxicity in human tumor cells (SK‐MEL‐ 28 and MIA PaCa‐2) and only Kunitz domain inhibit FXa slightly. Also, through confocal microscopy and high content analysis (ImageXpress Micro Confocal High‐Content, Molecular Devices), we observed C‐terminal domains (Alexa Fluor 488 conjugated) uptake by tumor cells previously than Amblyomin‐X (whole protein), but with similar intracellular trafficking and negligible nuclear translocation. Besides, circular dichroism analysis shown that Kunitz domain has quite a structure of α‐helix and β‐sheet, whereas the C‐terminal domain has the great part of random structure. Taken all results together, we can suggest that the C‐terminal domain is responsible for Amblyomin‐X uptake and could be an unusual cell penetrating peptide. Support or Funding Information Supported by BNDES, Fapesp and União Química Farmacêutica This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .