Copper-Triggered Aggregation of Ubiquitin

Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80∶20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing β-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates

Probing the interaction between cisplatin and the therapeutic monoclonal antibody trastuzumab

Trastuzumab (Herceptin) is a fully humanized monoclonal immunoglobulin γ-1 (IgG1) antibody directed against the HER2/neu receptor, which is up-regulated in about 20–25% of gastric and breast cancer patients. The combination of trastuzumab with cisplatin results in a favourable toxicity profile in the case of advanced HER2-positive gastric and gastroesophageal cancers, gaining the approval by the U.S. FDA in 2010. However, the current literature reveals no atomic details about platinum drug–trastuzumab interactions, probably because of size limitations. Our research aim has been to probe the interaction between the monoclonal antibody trastuzumab and cisplatin by means of electrospray mass spectrometry analysis within the “divide and conquer” approach and 1H, 15N-HSQC NMR spectroscopy (using 15N-labeled cisplatin). We demonstrate that trastuzumab is able to bind platinum and that there is no involvement of the residues belonging to the antigen-binding region so that the receptor recognition is not affected

Probing the Interaction of Cisplatin with the Human Copper Chaperone Atox1 by Solution and In-Cell NMR Spectroscopy

Among anticancer therapeutics, platinum-based drugs have a prominent role. They carry out their antitumor activity by forming stable adducts with DNA, thus interfering with replication and transcription processes. Cellular uptake of these drugs is tightly connected to copper transport. The major Cu(I) influx transporter Ctr1 has been found to mediate transport of cisplatin and its analogues. Evidence also suggests that ATP7A and ATP7B mediate cisplatin sequestration and efflux from cells, thus influencing drug resistance. The copper-chaperone Atox1, which normally binds Cu(I) via two cysteines and delivers the metal to ATP7A/B, has also been reported to interact with cisplatin in in vitro experiments. In the present investigation we apply a combined approach, using solution and in-cell NMR spectroscopy methods, to probe intracellular drug delivery and interaction of cisplatin with Atox1. The intracellular environment provides itself the suitable conditions for the preservation of the protein in its active form. Initially a {Pt(NH₃)₂}-Atox1 adduct is formed. At longer reaction time we observed protein dimerization and loss of the ammines. Such a process is reminiscent of the copper-promoted formation of Atox1 dimers which have been proposed to be able to cross the nuclear membrane and act as a transcription factor. We also show that overexpression of Atox1 in <i>E. coli</i> reduces the amount of DNA platination and, consequently, the degree of cell filamentation

Synthesis, Characterization, and in Vitro Evaluation of a New TSPO-Selective Bifunctional Chelate Ligand

The 18-kDa translocator protein (TSPO) is overexpressed in many types of cancers and is also abundant in activated microglial cells occurring in inflammatory neurodegenerative diseases. Thus, TSPO has become an extremely attractive subcellular target not only for imaging disease states overexpressing this protein, but also for a selective mitochondrial drug delivery. In this work we report the synthesis, the characterization, and the in vitro evaluation of a new TSPO-selective ligand, 2-(8-(2-(bis­(pyridin-2-yl)­methyl)­amino)­acetamido)-2-(4-chlorophenyl)<i>H</i>-imidazo­[1,2-<i>a</i>]­pyridin-3-yl)-<i>N</i>,<i>N</i>-dipropylacetamide (<b>CB256</b>), which fulfils the requirements for a bifunctional chelate approach. The goal was to provide a new TSPO ligand that could be used further to prepare coordination complexes of a metallo drug to be used in diagnosis and therapy. However, the ligand itself proved to be a potent tumor cell growth inhibitor and DNA double-strand breaker

C60@Lysozyme: Direct observation by nuclear magnetic resonance of a 1:1 fullerene protein adduct

Integrating carbon nanoparticles (CNPs) with proteins to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science. The comprehension of CNP-protein interactions requires the still-missing identification and characterization of the 'binding pocket' for the CNPs. Here, using Lysozyme and C-60 as model systems and NMR chemical shift perturbation analysis, a protein-CNP binding pocket is identified unambiguously in solution and the effect of the binding, at the level of the single amino acid, is characterized by a variety of experimental and computational approaches. Lysozyme forms a stoichiometric 1:1 adduct with C-60 that is dispersed monomolecularly in water. Lysozyme maintains its tridimensional structure upon interaction with C-60 and only a few identified residues are perturbed. The C-60 recognition is highly specific and localized in a well-defined pocket

E-consensus on telemedicine in proctology: A RAND/UCLA-modified study

Background: Coronavirus disease 2019 is revolutionizing healthcare delivery. The aim of this study was to reach a consensus among experts as to the possible applications of telemedicine in the proctologic field. Methods: A group of 55 clinical practice recommendations was developed by a clinical guidance group based on coalescence of evidence and expert opinion. The Telemedicine in Proctology Italian Working Group included 47 Italian Society of Colorectal Surgery nominated experts evaluating the appropriateness of each clinical practice recommendations based on published RAND/UCLA methodology in 2 rounds. Results: Stakeholder median age was 53 years (interquartile range limits 40-60), and 38 (81%) were men. Nine (19%) panelists reported no experience with telemedicine before the pandemic. Agreement was obtained on a minimum of 3 to 5 years of practice in the proctologic field before starting teleconsultations, which should be regularly paid, with advice and prescriptions incorporated into a formal report sent to the patient by e-mail along with a receipt. Of the panelists, 35 of 47 (74%) agreed that teleconsultation carries the risk of misdiagnosis of cancer, thus recommending an in-person assessment before scheduling any surgery. Fifteen additional clinical practice recommendations were re-elaborated in the second round and assessed by 44 of 47 (93.6%) panelists. The application of telemedicine for the diagnosis of common proctologic conditions (eg, hemorrhoidal disease, anal abscess and fistula, anal condylomas, and anal fissure) and functional pelvic floor disorders was generally considered inappropriate. Teleconsultation was instead deemed appropriate for the diagnosis and management of pilonidal disease. Conclusion: This e-consensus revealed the boundaries of telemedicine in Italy. Standardization of infrastructures, logistics, and legality remain to be better elucidated