Type I photosensitization of Ubiquitin leads to fluorescent adduct formation, cleavage and dimerization

ERRA-BALSELLS, ROSA; THOMAS, ANDRÉS H.; REID, LARA O.; LHIAUBET-VALLET,VIRGINIE; DANTOLA, MARIA LAURA LAURA; PETROSELLI, GABRIELA; MIRANDA, MIGUEL A.

Viña del Mar

Congreso; XIV Encuentro Latinoamericano de Fotoquimica y Fotobiologia; 2019

Universidad Andres Bello

Different compounds are able to inducephotosensitivity as a result from exposure to certain molecules and light;these includes phototoxic and photoallergic reactions. The photoallergy normallyinvolves a covalent binding between proteins and photosensitizer agents leadingto the formation of a complete photoantigen, which may trigger ahypersensitivity reaction due to a cell-mediated immune response.Pterins belong to a family of heterocycliccompounds present in a wide range of living systems and play different roles ranging from pigments to enzymatic cofactorsfor numerous redox and one-carbon transfer reactions. Inpathological conditions, such as vitiligo, a skin disorder characterized by the acquiredloss of constitutional pigmentation, pterins accumulate in the affected tissues at concentrations which are significantlyhigher than those reported for healthy cells.1 Under UV- radiation, this compounds are able to photosensitize damage in proteins, DNA andtheir components by Type I (electron ?transfer) and Type II (singlet oxygen)mechanisms.1,2  Recently, it has been reportedthat, in the absence of molecular oxygen, pterin (Ptr) the parent and unsubstituted compound of oxidized pterins, is able toreact with thymine (Thy) yielding an adduct Ptr-Thy in which the photosensitizeris covalently linked to the Thy moiety.4Therefore, given thebiological and medical relevance of the photosensitizing properties of pterins,the aim of this work isto study if Ptr, is able to generate photoadducts with proteins and establishits photoallergic potencial. For this study, aqueous acidic solution ofUbiquitin (Ub) and Ptr were irradiated (lex=350 nm) at room temperature and in differentatmospheres. Ub was used as a model protein given that is a small (8.5 kDa) regulatoryprotein, which has only one Tyrosine (Tyr) residue and none Tryptophan residue.The irradiated solution were analyzed by UV/visible spectrophotometry,HPLC, fluorescence spectroscopy, SDS-PAGE and mass spectrometry.Under UV-A radiation Ptr is able to form an adduct with Ub, and thisreaction is much more efficient in the absence of O2. Thespectroscopic analysis reveals that the emission and the excitation spectrumare similar to those corresponding to Ptr, as well as the fluorescencelifetime. On the other hand, as a consequence of the photosensitized process,the protein suffers oligomerization mediated by Tyr dimers, and also afragmentation can occur, which is dependent of the oxygen concentration in theatmosphere.References1- Schallreuter K. U., et al, J.Invest. Dermatol. 116, 167,2001. 2- Lorente C., Thomas A. H., Acc. Chem. Res., 39, 395, 2006.3- Reid L. O., etal, Biochemistry, 55(34), 4777,2016.4-EstebanezS., et al, Chemphyschem, 19, 300, 2018.