Biosystems and Integrative Sciences Institute

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Publications

Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1‐mediated nuclear export

Publication . Jimenez, Lucia; Mayoral‐Varo, Victor; Amenábar, Carlos; Ortega, Judit; Sequeira, João G. N.; Machuqueiro, Miguel; Mourato, Cristiana; Silvestri, Romano; Angeli, Andrea; Carta, Fabrizio; Supuran, Claudiu T.; Megías, Diego; Ferreira, Bibiana; Link, Wolfgang

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer, and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma, providing proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anticancer and antiviral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using red fluorescent protein or green fluorescent protein-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal. Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins, including PDK1, p110 alpha, STAT5A, FOXO1, 3, 4 and TRIB2, and to screen a compound collection. We show that while p110 alpha partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed the striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

2022Journal article

Open Access

Engineered metal nanoparticles in the marine environment: A review of the effects on marine fauna

Publication . Roma, Joana; Matos, Ana Rita; Vinagre, Catarina; Duarte, Bernardo

There is an increasing awareness of how damaging pollutants in the marine environment can be, however information on the effects of metal engineered nanoparticles (ENPs) on marine biota is still insufficient, despite an exponential rising in related publications in recent years. In order to provide an integrated insight on the present state of the art on metal ENP-related ecotoxicology studies on marine fauna, this review aimed to: (i) highlight the means of toxicity of metal ENPs in the marine environment, (ii) identify the principal biotic and abiotic factors that may alter metal ENP toxicity, and (iii) analyse and categorize results of these studies, including accumulation, molecular and histological biomarkers, genotoxicity and behavioural changes. Data retrieved from Scopus yielded 134 studies that met pre-established criteria. Most often, the target ENPs were titanium, zinc, copper or silver, and most studies (61.2%) focused on the phylum Mollusca. The degree of toxicity of metal ENPs was often dependent on the concentrations tested, length of exposure and the type of tissue sampled. Effects from simple tissue accumulation to DNA damage or behavioural alterations were identified, even when concentrations below environmentally available levels were used. It is proposed that other phyla besides the traditional Mollusca (and within it Bivalvia) should be used more often in this kind of studies, that exact pathways of toxicity be further explored, and lastly that co-stressors be used in order to best mimic conditions observed in nature. In this review, the current knowledge on engineered metal nanoparticles and their effects on marine fauna was summarized, highlighting present knowledge gaps. Guidelines for future studies focusing on under-developed subjects in ENP toxicology are also briefly provided.

2020-10Journal article

Restricted

Unveiling molecular details behind improved activity at neutral to alkaline pH of an engineered DyP-type peroxidase

Publication . Borges, Patrícia T.; Silva, Diogo; Silva, Tomás F.D.; Brissos, Vânia; Cañellas, Marina; Lucas, Maria Fátima; Masgrau, Laura; Melo, Eduardo; Machuqueiro, Miguel; Frazão, Carlos; Martins, Lígia O.

DyP-type peroxidases (DyPs) are microbial enzymes that catalyze the oxidation of a wide range of substrates, including synthetic dyes, lignin-derived compounds, and metals, such as Mn2+ and Fe2+, and have enormous biotechnological potential in biorefineries. However, many questions on the molecular basis of enzyme function and stability remain unanswered. In this work, high-resolution structures of PpDyP wild-type and two engineered variants (6E10 and 29E4) generated by directed evolution were obtained. The X-ray crystal structures revealed the typical ferredoxin-like folds, with three heme access pathways, two tunnels, and one cavity, limited by three long loops including catalytic residues. Variant 6E10 displays significantly increased loops' flexibility that favors function over stability: despite the considerably higher catalytic efficiency, this variant shows poorer protein stability compared to wild-type and 29E4 variants. Constant-pH MD simulations revealed a more positively charged microenvironment near the heme pocket of variant 6E10, particularly in the neutral to alkaline pH range. This microenvironment affects enzyme activity by modulating the pK(a) of essential residues in the heme vicinity and should account for variant 6E10 improved activity at pH 7-8 compared to the wild-type and 29E4 that show optimal enzymatic activity close to pH 4. Our findings shed light on the structure-function relationships of DyPs at the molecular level, including their pH-dependent conformational plasticity. These are essential for understanding and engineering the catalytic properties of DyPs for future biotechnological applications. (c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.

2022-12Journal article

Open Access