Photonic Activation of Plasminogen Induced by Low Dose UVB

Abstract

Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm). Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å). Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760–765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the elimination of blood clots.

EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

“…The presence of oxidative conditions induced by light can lead to the oxidation of the aromatic residues in proteins [14,15,19,31,41]. UVB excitation of aromatic residues in proteins leads to the disruption of SS bridges [14–17,19] and to the formation of photoproducts, such as N-formylkynurenine (NFK), kynurenine (Kyn) [25,42] and dityrosine (DT) [26].…”

“…Furthermore, UV excitation of the side chains of aromatic residues induces the disruption of disulphide (SS) bonds, mediated by an electron transfer process, leading to the formation of a transient disulphide electron adduct and to changes in the fluorescence quantum yield of proteins [16,17]. The effect of UV light on the structure and function of key medically relevant proteins, such as Epidermal Growth Factor Receptor (EGFR) [19], insulin [14] and plasminogen [15] has been reported.…”

Biophysical, photochemical and biochemical characterization of a protease from Aspergillus tamarii URM4634

“…The peptide backbone, Trp, Tyr, Phe, and cystine residues are the primary targets of photodegradation in proteins [37]. UVB excitation of aromatic residues in proteins leads to the disruption of SS bridges and to the formation of photoproducts [38], such as N-formylkynurenine (NFK), kynurenine (Kyn), dityrosine (DT), isodityrosine, trityrosine and pulcherosine [39]. Light at 320 nm excites both NFK, Kyn and DT, and at 360 nm excites NFK and Kyn but does not excite DT [16].…”

Expression and ultrastructural localization of plasmin(ogen) in the terminally differentiated layers of normal human epidermis

“…The proteases of the plasminogen system in the epidermis are activated by inflammatory conditions, such as UV light exposure , or by proinflammatory cytokines, such as IL‐1β, IL‐8 and TNFα . Plasmin is a relatively unspecific serine protease.…”