Electronic Circular Dichroism Detects Conformational Changes Associated with Proteasome Gating Confirmed Using AFM Imaging.

Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and ß-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and ß-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.

Universal Markers Unveil Metastatic Cancerous Cross-Sections at Nanoscale.

The characterization of cancer histological sections as metastatic, M, or not-metastatic, NM, at the cellular size level is important for early diagnosis and treatment. We present timely warning markers of metastasis, not identified by existing protocols and used methods. Digitized atomic force microscopy images of human histological cross-sections of M and NM colorectal cancer cells were analyzed by multifractal detrended fluctuation analysis and the generalized moments method analysis. Findings emphasize the multifractal character of all samples and accentuate room for the differentiation of M from NM cross-sections. Two universal markers emphatically achieve this goal performing very well: (a) the ratio of the singularity parameters (left/right), which are defined relative to weak/strong fluctuations in the multifractal spectrum, is always greater than 0.8 for NM tissues; and (b) the index of multifractality, used to classify universal multifractals, points to log-normal distribution for NM and to log-Cauchy for M tissues. An immediate large-scale screening of cancerous sections is doable based on these findings.

Formulation matters! A spectroscopic and molecular dynamics investigation on the peptide CIGB552 as itself and in its therapeutical formulation.

Synthetic therapeutic peptides (STP) are intensively studied as new-generation drugs, characterized by high purity, biocompatibility, selectivity and stereochemical control. However, most of the studies are focussed on the bioactivity of STP without considering how the formulation actually used for therapy administration could alter the physico-chemical properties of the active principle. The aggregation properties of a 20-mer STP (Ac-His-Ala-Arg-Ile-Lys-D-Pro-Thr-Phe-Arg-Arg-D-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe-Trp-NH2 ), showing antitumor activity, were investigated by optical spectroscopy and atomic force microscopy imaging, as itself (CIGB552) and in its therapeutic formulation (CIGB552TF). It has found that the therapeutic formulation deeply affects the aggregation properties of the investigated peptide and the morphology of the aggregates formed on mica by deposition of CIGB552 and CIGB552TF millimolar solutions. Molecular dynamics simulations studied the first steps of CIGB552 aggregation under physiological ionic strength conditions (NaCl 150 mM), showing that peptide oligomers, from dimers to tetramers, are preferentially formed in this environment. Interestingly, cell viability assays performed on H-460 cell lines indicate a major antiproliferative activity of the peptide in its therapeutic formulation with respect to the peptide aqueous solution.

Stepwise rapid tracking strategy to identify active molecules from Ixeris sonchifolia Hance based on "'affinity mass spectrometry-atomic force microscopy imaging'" technology.

In this study, a novel stepwise rapid tracking strategy was reported to identify the active molecules from Ixeris sonchifolia Hance (IsH) in the treatment of coronary heart disease (CHD) based on "affinity mass spectrometry (MS)-atomic force microscopy (AFM) imaging" technology. First, vascular endothelial growth factor receptor 2 (VEGFR2) of the vascular endothelial growth factor (VEGF) signal transduction pathway located on the cell membrane was revealed to be the core target protein in CHD treatment through network pharmacology and bioinformatics. In addition, affinity MS screening based on VEGFR2 identified isochlorogenic acid A and luteolin-7-O-glucuronide as having stronger affinity with VEGFR2. Then, the active molecule was elucidated based on the observation that its actions accompanied the molecular morphological changes by AFM imaging and it could act on the binding pocket of VEGFR2 through molecular docking which further demonstrated the analysis and inference of AFM imaging. The methyl thiazolyl tetrazolium (MTT) assay finally confirmed that the active molecules specifically combined with the potential core target protein to protect the viability of cardiomyocytes, which identified the main potential active molecules in IsH for the treatment of CHD and provided a possible mechanism for the protective role of the drug. The technology established in this study could facilitate the rapid tracing of potential active molecules in traditional Chinese medicine (TCM), which would provide further a reference for research on quality, molecular mechanisms and new drugs.

Nitrosative stress affects the interaction of integrin alphaIIbbeta3 with its ligands.

Binding of integrin alphaIIbbeta3 (αiibß3) to its ligands is a highly restricted and regulated mechanism. Any modification of the protein structure yields a dysfunctional role, especially in a redox environment. Here, we examine the effect of nitrosative stress on the αiibß3 reconstituted into nanodiscs. Using single molecule force spectroscopy, we measured the interaction between αiibß3 and its ligand RGD and found that in the presence of exogenous nitric oxide (NO) two force regimes are generated: a low force regime of ~100pN indicating the presence of integrin in a normal status, and a broad spectrum of high force regime (~210-450pN) suggesting the protein modification/aggregation. By high resolution atomic force microscopy imaging, we demonstrate that both NO and nitrite (a stable product formed from NO) are involved in destabilizing the transmembrane protein complex leading to release of αiibß3 from the lipid bilayer and protein aggregation. Our experimental setup opens new ways for testing in a membrane environment the effect of radical species on integrins under clinically relevant conditions.