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1.
Toxins (Basel) ; 13(6)2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203879

RESUMO

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.


Assuntos
Antineoplásicos Hormonais/farmacologia , Toxina Shiga I/metabolismo , Toxina Shiga II/metabolismo , Tamoxifeno/farmacologia , Endossomos/efeitos dos fármacos , Células HeLa , Humanos , Transporte Proteico/efeitos dos fármacos
2.
Chem Sci ; 12(11): 3984-3998, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-34163669

RESUMO

SfmD is a heme-dependent enzyme in the biosynthetic pathway of saframycin A. Here, we present a 1.78 Šresolution de novo crystal structure of SfmD, which unveils a novel heme cofactor attached to the protein with an unusual Hx n HxxxC motif (n ∼ 38). This heme cofactor is unique in two respects. It contains a single thioether bond in a cysteine-vinyl link with Cys317, and the ferric heme has two axial protein ligands, i.e., His274 and His313. We demonstrated that SfmD heme is catalytically active and can utilize dioxygen and ascorbate for a single-oxygen insertion into 3-methyl-l-tyrosine. Catalytic assays using ascorbate derivatives revealed the functional groups of ascorbate essential to its function as a cosubstrate. Abolishing the thioether linkage through mutation of Cys317 resulted in catalytically inactive SfmD variants. EPR and optical data revealed that the heme center undergoes a substantial conformational change with one axial histidine ligand dissociating from the iron ion in response to substrate 3-methyl-l-tyrosine binding or chemical reduction by a reducing agent, such as the cosubstrate ascorbate. The labile axial ligand was identified as His274 through redox-linked structural determinations. Together, identifying an unusual heme cofactor with a previously unknown heme-binding motif for a monooxygenase activity and the structural similarity of SfmD to the members of the heme-based tryptophan dioxygenase superfamily will broaden understanding of heme chemistry.

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