Exploring the Anticancer Activity of Tamoxifen-Based Metal Complexes Targeting Mitochondria

Two new 'hybrid' metallodrugs of Au(III)(AuTAML)and Cu(II) (CuTAML) were designed featuring a tamoxifen-derived pharmacophoreto ideally synergize the anticancer activity of both the metal centerand the organic ligand. The compounds have antiproliferative effectsagainst human MCF-7 and MDA-MB 231 breast cancer cells. Moleculardynamics studies suggest that the compounds retain the binding activityto estrogen receptor (ER & alpha;). In vitro and in silico studies showed that the Au(III) derivative isan inhibitor of the seleno-enzyme thioredoxin reductase, while theCu(II) complex may act as an oxidant of different intracellular thiols.In breast cancer cells treated with the compounds, a redox imbalancecharacterized by a decrease in total thiols and increased reactiveoxygen species production was detected. Despite their different reactivitiesand cytotoxic potencies, a great capacity of the metal complexes toinduce mitochondrial damage was observed as shown by their effectson mitochondrial respiration, membrane potential, and morphology

De novo design of coiled-coil protein switch

Narava je tekom evolucije uporabila le del konformacijskega prostora, ki je na voljo polipeptidnim molekulam. Z načrtovanjem sintetičnih proteinov je mogoče ta nepreizkušen prostor načrtno preiskati in v njem odkriti popolnoma nova proteinska zvitja z novimi, v naravi neopaženimi lastnostmi. Eden izmed trenutnih izzivov na področju dizajniranja proteinov je načrtovanje proteinov, ki ob spremembi okoljskih pogojev spremenijo svoje konformacijsko stanje. Konformacijske spremembe strukture proteinov v odziv na kemijski ali fizikalni signal so osnovni del številnih regulatornih, transportnih in drugih mehanizmov v bioloških sistemih. Zmožnost načrtovanja proteinov, katerih konformacijsko stanje je mogoče natančno in reverzibilno nadzorovati, bi omogočila razvoj naprednih biomaterialov ali molekulskih strojev, prikrojenih za specifične aplikacije. V doktorskem delu smo z načrtovanjem vezavnih mest za kovinske ione razvili konformacijska stikala na osnovi motiva ovite vijačnice. Ovite vijačnice so pogost strukturni motiv v naravnih proteinih, z njimi pa je mogoče zgraditi tudi sintetične proteinske nanostrukture. V prvem delu smo načrtovali peptid poimenovan SwitCCh, ki je v prisotnosti Zn(II) ionov ali pri nizkem pH tvoril paralelen homodimer ovite vijačnice, drugače pa je v raztopini zavzel strukturo naključnega klobčiča. Dodatek Zn(II) ionov je povzročil tvorbo paralelnega homodimera, ob čemer se je temperaturna stabilnost peptida povišala za več kot 30 °C. Prehod med ovito vijačnico in razvitim stanjem je bil reverzibilen in ponovljiv. Peptid SwitCCh je bil ortogonalen glede na predhodno načrtovani set dimerov ovite vijačnice, kar pomeni, da bi ga bilo mogoče uporabiti kot kontrolni element za nadzorovanje zlaganja nanostruktur in materialov na osnovi ovite vijačnice. V drugem delu smo s pomočjo načrtovanja vezavnih mest za kovinske ione v predhodno načrtovani ortogonalen set razvili set Zn(II)-odzivnih ovitih vijačnic. Spektroskopija cirkularnega dikroizma in velikostno izključitvena kromatografija sklopljena s statičnim sipanjem svetlobe pri različnih kotih sta potrdili, da so se peptidi povezali v heterodimer ovite vijačnice le v prisotnosti Zn(II) ionov. Poleg tega so načrtovani peptidi delovali tudi kot pH stikala, saj so nizke vrednosti pH preprečile koordinacijo Zn(II) ionov, kar je vodilo do razvitja ovitih vijačnic. Na osnovi načrtovanega seta ovitih vijačnic smo uspeli pripraviti proteinski trikotnik, katerega zvitje je bilo pod kontrolo Zn (II) ionov. To kaže, da je načrtovani set Zn(II)-odzivnih ovitih vijačnic mogoče uporabiti za razvoj proteinskih kletk na osnovi ovitih vijačnic, katerih zvijanje in razvijanje je mogoče enostavno nadzorovati.De novo protein design represents an exciting opportunity to explore the conformational space unsampled by nature and develop novel protein folds and functionality. One of the current challenges in the protein design field is the design of proteins that change their conformation in response to environmental cues. Conformational change of proteins in response to chemical or physical signals is the underlying principle of many regulatory and transport mechanisms in biological systems. The ability to design proteins whose conformational state can be precisely and reversibly controlled would facilitate the development of smart bio-inspired materials or molecular machines tailored for specific applications. We explored metal-binding site design to engineer peptide-based conformational switches that assemble into a dimeric coiled-coil in response to the addition of Zn(II) ions. Coiled-coil dimers are present in many natural proteins and have been used to construct synthetic protein nanostructures. Firstly, we designed a peptide called SwitCCh that formed a parallel homodimeric coiled-coil in the presence of Zn(II) or low pH. The addition of Zn(II) promoted formation of a parallel homodimer with an increase in thermal stability by more than 30 °C. The peptide could be reversibly cycled between the coiled-coil and random conformation. Furthermore, the SwitCCh peptide was orthogonal to the previously developed coiled-coil dimer set, indicating it could be used for regulated self-assembly of coiled-coil based nanostructures and materials. We further advanced our work by utilizing metal-binding site design to render a previously designed orthogonal set of coiled-coil heterodimers Zn(II)-responsive. Circular dichroism spectroscopy and size exclusion chromatography coupled to multi-angle light scattering confirmed the designed peptides assembled into coiled-coil heterodimers only in the presence of Zn(II). Additionally, designed peptides also acted as pH switches, since low pH prevented coordination of Zn(II) and lead to disassembling of coiled-coils. Our results showed the incorporation of a metal binding site not only preserved orthogonality, but that it is also a viable strategy for increasing the size of orthogonal sets. The designed Zn(II)-responsive coiled-coils were used for the construction of a triangular fold, whose assembly and disassembly was under the control of Zn(II) ions, demonstrating the designed set could facilitate the development of coiled-coil protein cages with easily controllable folding and unfolding

Modelling data for article "Intrinsically disordered ectodomain modulates ion permeation through a metal transporter"

Full-length hCtr1 models used to perform classical molecular dynamics (MD) simulations described in the article "Intrinsically disordered ectodomain modulates ion permeation through a metal transporter" by Aupič et al. The zipped file contains three folders named alpha, beta and unfolded. Each contains the full-length all-atom model of the human copper transporter 1 (hCtr1) with the N-terminal domains in the alpha, beta or the unfolded conformational state. We provide both the initial model (initial-model.pdb) created with MODELLER (version 10.0) homology modelling software (B. Webb, A. Sali, Comparative Protein Structure Modeling Using MODELLER. Curr. Protoc. Bioinforma. 54, 5–6 (2016)) and the model structure embedded in the lipid bilayer as obtained after a 1 μs MD simulation (after-md.pdb)

Intrinsically disordered ectodomain modulates ion permeation through a metal transporter

The function of many channels and transporters is enriched by the conformational plasticity of intrinsically disordered regions (IDRs). Copper transporter 1 (Ctr1) is the main entry point for Cu(I) ions in eukaryotes and contains IDRs both at its N-terminal (Nterm) and C-terminal ends. The former delivers copper ions from the extracellular matrix to the selectivity filter in the Ctr1 lumen. However, the molecular mechanism of this process remains elusive due to Nterm’s disordered nature. Here, we combine advanced molecular dynamics simulations and circular dichroism experiments to show that Cu(I) ions and a lipidic environment drive the insertion of the Nterm into the Ctr1 selectivity filter, causing its opening. Through a lipid-aided conformational switch of one of the transmembrane helices, the conformational change of the selectivity filter propagates down to the cytosolic gate of Ctr1. Taken together, our results elucidate how conformational variability of IDRs modulates ion transport

The conformational plasticity of the selectivity filter methionines controls the in-cell Cu(I) uptake through the CTR1 transporter

Copper is a trace element vital to many cellular functions. Yet its abnormal levels are toxic to cells, provoking a variety of severe diseases. The high affinity copper transporter 1 (CTR1), being the main in-cell copper [Cu(I)] entry route, tightly regulates its cellular uptake via a still elusive mechanism. Here, all-atoms simulations unlock the molecular terms of Cu(I) transport in eukaryotes disclosing that the two methionine (Met) triads, forming the selectivity filter, play an unprecedented dual role both enabling selective Cu(I) transport and regulating its uptake rate thanks to an intimate coupling between the conformational plasticity of their bulky side chains and the number of bound Cu(I) ions. Namely, the Met residues act as a gate reducing the Cu(I) import rate when two ions simultaneously bind to CTR1. This may represent an elegant autoregulatory mechanism through which CTR1 protects the cells from excessively high, and hence toxic, in-cell Cu(I) levels. Overall, our outcomes resolve fundamental questions in CTR1 biology and open new windows of opportunity to tackle diseases associated with an imbalanced copper uptake