Glioblastoma (GBM), characterized by fast growth and invasion into adjacent tissue, is the most aggressive cancer of brain origin. Current protocols, which include cytotoxic chemotherapeutic agents, effectively treat localized disease; however, these aggressive therapies present side effects due to the high doses administered. Therefore, more efficient ways of drug delivery have been studied to reduce the therapeutic exposure of the patients. We have isolated and fully characterized small extracellular vesicles (EVs) from seven patient-derived GBM cell lines. After loading them with two different drugs, Temozolomide (TMZ) and EPZ015666, we observed a reduction in the total amount of drugs needed to trigger an effect on tumor cells. Moreover, we observed that GBM-derived small EVs, although with lower target specificity, can induce an effect on pancreatic cancer cell death. These results suggest that GBM-derived small EVs represent a promising drug delivery tool for further preclinical studies and potentially for the clinical development of GBM treatments.

PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1–5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.

RYBP (Ring1 and YY 1 binding protein) is a multifunctional, intrinsically disordered protein (IDP), best described as a transcriptional regulator. It exhibits a ubiquitin-binding functionality, binds to other transcription factors, and has a key role during embryonic development. RYBP, which folds upon binding to DNA, has a Zn-finger domain at its N-terminal region. By contrast, PADI4 is a well-folded protein and it is one the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline. As both proteins intervene in signaling pathways related to cancer development and are found in the same localizations within the cell, we hypothesized they may interact. We observed their association in the nucleus and cytosol in several cancer cell lines, by using immunofluorescence (IF) and proximity ligation assays (PLAs). Binding also occurred in vitro, as measured by isothermal titration calorimetry (ITC) and fluorescence, with a low micromolar affinity (~1 μM). AlphaFold2-multimer (AF2) results indicate that PADI4's catalytic domain interacts with the Arg53 of RYBP docking into its active site. As RYBP sensitizes cells to PARP (Poly (ADP-ribose) polymerase) inhibitors, we applied them in combination with an enzymatic inhibitor of PADI4 observing a change in cell proliferation, and the hampering of the interaction of both proteins. This study unveils for the first time the possible citrullination of an IDP, and suggests that this new interaction, whether it involves or not citrullination of RYBP, might have implications in cancer development and progression.

Cancer cells need to implement and maintain molecular mechanisms relying on an energy trade-off between resistance and key functions to survive. Among them, changes in lipid metabolism are crucial. Phosphatidylserine (PS), phosphatidylcholine (PC) and phosphatidic acid (PA) are some of the lipids forming cell membranes and having key functions in lipid metabolism. PADI4 is an enzyme implicated in the conversion of arginine to citrulline (citrullination), that has been related with the development of several types of cancers. In this work, we report the lipid binding properties of PADI4. Such binding was assayed in vitro against zwitterionic (PC) and anionic (PA and PS) lipids, and it was monitored by several biophysical techniques. Furthermore, results in silico showed that the binding of PADI4 with PA, PC and PS occurred at the active site of the enzyme. This binding was confirmed in cellulo by using PS as the target lipid, employing immunofluorescence (IF) and proximity ligation assay (PLA) in different cancer cell lines. The use in cellulo of a specific enzymatic inhibitor of PADI4, GSK484, abolished the binding between PADI4 and PS in cancer cells, further indicating that their interaction occurred at the protein active site. Altogether, this work shows that PADI4 was capable of binding to lipids, and opens the venue to study the role that it could be playing in deimination processes and cancer development. Moreover, this study lays the foundation for developing novel cancer therapies from new perspectives, based on the interaction of lipids with citrullinating enzymes.

El método de espectroscopía de absorción atómica (AA) de llama para la determinación de cadmio (Cd) en almendra de cacao (Theobroma cacao) utilizado por Agrocalidad es tóxico para el ser humano y el ambiente; por ello, se pretende utilizar el método de espectroscopia de absorción atómica con horno de grafito (GFAAS) por ser más confiable y seguro. Así, se realizó la verificación de cuatro parámetros de desempeño del método GFAAS para cuantificar Cd en almendra de cacao utilizando material de referencia certificado (MRC) y muestras provenientes de cuatro fincas (A, B, C, D) ubicadas en la zona cacaotera de Ecuador, cantón Flavio Alfaro, provincia de Manabí. Se realizó una prueba inter-laboratorios y finalmente se elaboró el protocolo (PEE/B/14). Sobre el MRC (Cód. 07206B y 07167A) se verificó: linealidad, precisión, veracidad e incertidumbre de acuerdo con la Guía Eurachem de Eurolab España y Morillas (2016), y con el estándar IRAM 35050 (2001) se encontró linealidad entre 0 y 8 ppb con R2=0.9988; desviación estándar de 0.0005 y 0.0022 respectivamente; sesgo en 0.007 y porcentaje de recuperación de 109.75; la incertidumbre estándar de 0.00013 y 0.00082. El contenido de Cd en las muestras de la finca A con 0.54 ppm, las Fincas B-D con 0.26 ppm y 0.15 ppm en la finca C. En la prueba inter-laboratorios se estableció la misma concentración de cadmio para la muestra C3 y, de acuerdo con lo estipulado por la Unión Europea, el cacao de las cuatro fincas podría ser exportado sin restricciones.