Proximity Mapping of CCP6 Reveals Its Association with Centrosome Organization and Cilium Assembly.

The cytosolic carboxypeptidase 6 (CCP6) catalyzes the deglutamylation of polyglutamate side chains, a post-translational modification that affects proteins such as tubulins or nucleosome assembly proteins. CCP6 is involved in several cell processes, such as spermatogenesis, antiviral activity, embryonic development, and pathologies like renal adenocarcinoma. In the present work, the cellular role of CCP6 has been assessed by BioID, a proximity labeling approach for mapping physiologically relevant protein-protein interactions (PPIs) and bait proximal proteins by mass spectrometry. We used HEK 293 cells stably expressing CCP6-BirA* to identify 37 putative interactors of this enzyme. This list of CCP6 proximal proteins displayed enrichment of proteins associated with the centrosome and centriolar satellites, indicating that CCP6 could be present in the pericentriolar material. In addition, we identified cilium assembly-related proteins as putative interactors of CCP6. In addition, the CCP6 proximal partner list included five proteins associated with the Joubert syndrome, a ciliopathy linked to defects in polyglutamylation. Using the proximity ligation assay (PLA), we show that PCM1, PIBF1, and NudC are true CCP6 physical interactors. Therefore, the BioID methodology confirms the location and possible functional role of CCP6 in centrosomes and centrioles, as well as in the formation and maintenance of primary cilia.

Synthesis and In Vitro Studies of Photoactivatable Semisquaraine-type Pt(II) Complexes.

The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.

Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.

A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.

Proximity-based activation of AURORA A by MPS1 potentiates error correction.

Faithfull cell division relies on mitotic chromosomes becoming bioriented with each pair of sister kinetochores bound to microtubules oriented toward opposing spindle poles. Erroneous kinetochore-microtubule attachments often form during early mitosis, but are destabilized through the phosphorylation of outer kinetochore proteins by centromeric AURORA B kinase (ABK) and centrosomal AURORA A kinase (AAK), thus allowing for re-establishment of attachments until biorientation is achieved. MPS1-mediated phosphorylation of NDC80 has also been shown to directly weaken the kinetochore-microtubule interface in yeast. In human cells, MPS1 has been proposed to transiently accumulate at end-on attached kinetochores and phosphorylate SKA3 to promote microtubule release. Whether MPS1 directly targets NDC80 and/or promotes the activity of AURORA kinases in metazoans remains unclear. Here, we report a novel mechanism involving communication between kinetochores and centrosomes, wherein MPS1 acts upstream of AAK to promote error correction. MPS1 on pole-proximal kinetochores phosphorylates the C-lobe of AAK thereby increasing its activation at centrosomes. This proximity-based activation ensures the establishment of a robust AAK activity gradient that locally destabilizes mal-oriented kinetochores near spindle poles. Accordingly, MPS1 depletion from Drosophila cells causes severe chromosome misalignment and erroneous kinetochore-microtubule attachments, which can be rescued by tethering either MPS1 or constitutively active AAK mutants to centrosomes. Proximity-based activation of AAK by MPS1 also occurs in human cells to promote AAK-mediated phosphorylation of the NDC80 N-terminal tail. These findings uncover an MPS1-AAK cross-talk that is required for efficient error correction, showcasing the ability of kinetochores to modulate centrosome outputs to ensure proper chromosome segregation.

SiR-DNA/SiR-Hoechst-induced chromosome entanglement generates severe anaphase bridges and DNA damage.

SiR-DNA/SiR-Hoechst is a far-red fluorescent DNA probe that is routinely used for live-cell imaging of cell nuclei in interphase and chromosomes during mitosis. Despite being reported to induce DNA damage, SiR-DNA has been used in more than 300 research articles, covering topics like mitosis, chromatin biology, cancer research, cytoskeletal research, and DNA damage response. Here, we used live-cell imaging to perform a comprehensive analysis of the effects of SiR-DNA on mitosis of four human cell lines (RPE-1, DLD-1, HeLa, and U2OS). We report a dose-, time-, and light-dependent effect of SiR-DNA on chromosome segregation. We found that, upon the exposure to light during imaging, nanomolar concentrations of SiR-DNA induce non-centromeric chromosome entanglement that severely impairs sister chromatid segregation and spindle elongation during anaphase. This causes DNA damage that is passed forward to the following cell cycle, thereby having a detrimental effect on genome integrity. Our findings highlight the drawbacks in using SiR-DNA for investigation of late mitotic events and DNA damage-related topics and urge the use of alternative labeling strategies to study these processes.

Cell-penetrating peptide-conjugated copper complexes for redox-mediated anticancer therapy.

Metal-based chemotherapeutics like cisplatin are widely employed in cancer treatment. In the last years, the design of redox-active (transition) metal complexes, such as of copper (Cu), has attracted high interest as alternatives to overcome platinum-induced side-effects. However, several challenges are still faced, including optimal aqueous solubility and efficient intracellular delivery, and strategies like the use of cell-penetrating peptides have been encouraging. In this context, we previously designed a Cu(II) scaffold that exhibited significant reactive oxygen species (ROS)-mediated cytotoxicity. Herein, we build upon the promising Cu(II) redox-active metallic core and aim to potentiate its anticancer activity by rationally tailoring it with solubility- and uptake-enhancing functionalizations that do not alter the ROS-generating Cu(II) center. To this end, sulfonate, arginine and arginine-rich cell-penetrating peptide (CPP) derivatives have been prepared and characterized, and all the resulting complexes preserved the parent Cu(II) coordination core, thereby maintaining its reported redox capabilities. Comparative in vitro assays in several cancer cell lines reveal that while specific solubility-targeting derivatizations (i.e., sulfonate or arginine) did not translate into an improved cytotoxicity, increased intracellular copper delivery via CPP-conjugation promoted an enhanced anticancer activity, already detectable at short treatment times. Additionally, immunofluorescence assays show that the Cu(II) peptide-conjugate distributed throughout the cytosol without lysosomal colocalization, suggesting potential avoidance of endosomal entrapment. Overall, the systematic exploration of the tailored modifications enables us to provide further understanding on structure-activity relationships of redox-active metal-based (Cu(II)) cytotoxic complexes, which contributes to rationalize and improve the design of more efficient redox-mediated metal-based anticancer therapy.

Study and Preparation of Multifunctional Poly(L-Lysine)@Hyaluronic Acid Nanopolyplexes for the Effective Delivery of Tumor Suppressive MiR-34a into Triple-Negative Breast Cancer Cells.

Non-viral gene delivery using exogenous microRNAs is a potential strategy for fighting cancers with poor prognosis and which lack specific therapies, such as triple-negative breast cancer (TNBC). Herein we report the synthesis of six nontoxic electrostatic polymeric nanocapsules (P1 to P6) for microRNA delivery in TNBC cells. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Scanning Electron Microscopy (SEM) were used to characterize the nanopolyplexes, synthesized with Poly(L-Lysine) and hyaluronic acid (Ha). Studies on the activity of the ternary HA/PLI/miRNA-34 nanopolyplexes towards TNBC cell line MDA-MB-231 were conducted. The nanopolyplexes mediated intracellular restoration of tumor suppressor miR34a was evaluated by using Western blotting to quantify the expression level of the Bcl-2 protein. The results suggest that the P5, with a ratio PLI/Ha of 0.05, was the most promising for the delivery of miR-34a into TNBC cells; the P5 nanocapsules were able to reduce Bcl-2 expression at a protein level, and had an effect in the overall cell viability after 24 h treatment.

Iridium(III) coordination of N(6) modified adenine derivatives with aminoacid chains.

In this manuscript we report the preparation of three N6-aminoacid-adenine-derivatives: N-(7H-purin-6-yl)glycine·0.5H2O (N6-GlyAde), N-(7H-purin-6-yl)-ß-alanine·1.5H2O (N6-ß-AlaAde) and N-(7H-purin-6-yl)-γ-aminobutyric·2H2O (N6-GabaAde) and the synthesis and X-ray characterization of three Ir(III) NAMI-A derivatives (NAMI-A is [imidazoleH][trans-RuIIICl4(DMSO-κS)(imidazole)]) [trans-IrIIICl4(DMSO-κS)(N3-H)-(7H-purin-6-yl)glycine-κN9] (1), [trans-IrIIICl4(DMSO-κS)(N3-H)-(7H-purin-6-yl)-ß-alanine-κN9] hydrate (2) and [trans-IrIIICl4(DMSO-κS)(N3-H)-(7H-purin-6-yl)-γ-aminobutyryl-κN9] (3). In all complexes the metal center shows octahedral geometry with coordination to four chlorido ligands and one S coordinated dimethylsulfoxide (DMSO-κS). The coordination sphere of the metal is completed by the modified adenine molecule which is bound via N(9) and protonated at N(3). In two complexes the importance of lone pair (lp)-π interactions involving the adenine ring have been studied using density functional theory (DFT) calculations and the Bader's theory of atoms in molecules. Furthermore, the ability of complexes (1-3) to affect the cell viability was evaluated against three different cancer cell lines: human lung carcinoma cells (A549), human cervical carcinoma cells (HeLa) and human breast cancer cells (MCF7). We have also analyzed their ability to cleave the DNA experimentally and their affinity for two models of DNA has been studied using molecular docking simulations.

New Cyclams and Their Copper(II) and Iron(III) Complexes: Synthesis and Potential Application as Anticancer Agents.

New cyclam derivatives (HOCH2 CH2 CH2 )2 (PhCH2 )2 Cyclam and (HOCH2 CH2 CH2 )2 ( 4-CF3 PhCH2 )2 Cyclam, as well as their CuII and FeIII complexes, were synthesized and characterized and their stability in cellular media was assessed. The cytotoxic effect of all compounds was examined on human cervical cancer (HeLa) cells, revealing strong anticancer activity. After 24 h, only complexes with the (HOCH2 CH2 CH2 )2 ( 4-CF3 PhCH2 )2 Cyclam ligand are cytotoxic, whereas after incubation for 72 h all compounds show significant antiproliferative effects. Notably, compounds containing 4-CF3 PhCH2 pendant arms on the cyclam ring revealed the most activity, with cytotoxicity values up to 12 times higher than those of cisplatin. All metal complexes seem to induce cell death through the formation of reactive oxygen species.

Studying the reactivity of "old" Cu(II) complexes for "novel" anticancer purposes.

Reactive oxygen species (ROS) formation appears as one of the most promising pathways to induce cell death. The interesting Cu(II)/Cu(I) redox pair has been reported to biologically generate ROS and induce cell damage. Simple metal complexes, such as cisplatin, sometimes offer even better properties than others highly accurately synthesized, which imply considerable time and economical efforts. This work relies on the synthesis and characterisation of four existing Cu(II) complexes bearing N-donor ligands, previously used for a totally different intend, but tested now for anticancer purposes. Furthermore, a relationship between their coordinating features, i.e. their redox behaviour, with their biological activity have been inferred to further understand the medicinal role of the Cu(II)/Cu(I) redox pair. Cytotoxicity studies and interactions towards DNA have been assessed, studying both covalent and non-covalent modes of binding via mass spectrometry (MS), UV-Vis and fluorescence, evaluating the cleaving properties of the assayed compounds, as well as their capacity to generate ROS inside the cells. The role of the ligand for one of the complexes has been evaluated by a computational approach. The idea of using "old" complexes for "novel" anticancer purposes can offer promising results in the future, being a simple but interesting approach to study, as we demonstrate here for most of the complexes analysed, showing a non-expected "new" and beneficial role.