Protein-DNA/RNA Interactions: An Overview of Investigation Methods in the -Omics Era

The fields of application of functional proteomics are not limited to the study of protein-protein interactions; they also extend to those involving protein complexes that bind DNA or RNA. These interactions affect fundamental processes such as replication, transcription, and repair in the case of DNA, as well as transport, translation, splicing, and silencing in the case of RNA. Analytical or preparative experimental approaches, both in vivo and in vitro, have been developed to isolate and identify DNA/RNA binding proteins by exploiting the advantage of the affinity shown by these proteins toward a specific oligonucleotide sequence. The present review proposes an overview of the approaches most commonly employed in proteomics applications for the identification of nucleic acid-binding proteins, such as affinity purification (AP) protocols, EMSA, chromatin purification methods, and CRISPR-based chromatin affinity purification, which are generally associated with mass spectrometry methodologies for the unbiased protein identification

The cell cycle checkpoint inhibitors in the treatment of leukemias

open3noThe study was funded by the University of Bologna and by the Italian Association for Cancer Research (AIRC).The inhibition of the DNA damage response (DDR) pathway in the treatment of cancers has recently reached an exciting stage with several cell cycle checkpoint inhibitors that are now being tested in several clinical trials in cancer patients. Although the great amount of pre-clinical and clinical data are from the solid tumor experience, only few studies have been done on leukemias using specific cell cycle checkpoint inhibitors. This review aims to summarize the most recent data found on the biological mechanisms of the response to DNA damages highlighting the role of the different elements of the DDR pathway in normal and cancer cells and focusing on the main genetic alteration or aberrant gene expression that has been found on acute and chronic leukemias. This review, for the first time, outlines the most important pre-clinical and clinical data available on the efficacy of cell cycle checkpoint inhibitors in single agent and in combination with different agents normally used for the treatment of acute and chronic leukemias.openGhelli Luserna di Rora', A; Iacobucci, I; Martinelli, GGhelli Luserna di Rora', A; Iacobucci, I; Martinelli,

Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A-Treated Stem Cells

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications

Characterisation of AMS H35 HV-CMOS monolithic active pixel sensor prototypes for HEP applications

Monolithic active pixel sensors produced in High Voltage CMOS (HV-CMOS) technology are being considered for High Energy Physics applications due to the ease of production and the reduced costs. Such technology is especially appealing when large areas to be covered and material budget are concerned. This is the case of the outermost pixel layers of the future ATLAS tracking detector for the HL-LHC. For experiments at hadron colliders, radiation hardness is a key requirement which is not fulfilled by standard CMOS sensor designs that collect charge by diffusion. This issue has been addressed by depleted active pixel sensors in which electronics are embedded into a large deep implantation ensuring uniform charge collection by drift. Very first small prototypes of hybrid depleted active pixel sensors have already shown a radiation hardness compatible with the ATLAS requirements. Nevertheless, to compete with the present hybrid solutions a further reduction in costs achievable by a fully monolithic design is desirable. The H35DEMO is a large electrode full reticle demonstrator chip produced in AMS 350 nm HV-CMOS technology by the collaboration of Karlsruher Institut f\"ur Technologie (KIT), Institut de F\'isica d'Altes Energies (IFAE), University of Liverpool and University of Geneva. It includes two large monolithic pixel matrices which can be operated standalone. One of these two matrices has been characterised at beam test before and after irradiation with protons and neutrons. Results demonstrated the feasibility of producing radiation hard large area fully monolithic pixel sensors in HV-CMOS technology. H35DEMO chips with a substrate resistivity of 200$\Omega$ cm irradiated with neutrons showed a radiation hardness up to a fluence of $10^{15}$n$_{eq}$cm$^{-2}$ with a hit efficiency of about 99% and a noise occupancy lower than $10^{-6}$ hits in a LHC bunch crossing of 25ns at 150V

Nilotinib: a novel encouraging therapeutic option for chronic myeloid leukemia patients with imatinib resistance or intolerance

Although high rates of complete hematologic and cytogenetic remission have been observed in patients with chronic phase chronic myeloid leukemia (CML) treated with imatinib, a short duration of response with eventual emergence of imatinib resistance has also been reported in a subset of CML patients. The most frequent clinically relevant mechanisms that change imatinib sensitivity in BCR-ABL-transformed cells are mutations within the Abl kinase domain, affecting several of its properties. Crystal structure analysis of the Abl-imatinib complex has proven helpful in identifying potential critical residues that hinder interactions of imatinib with mutated Abl. This has led to the development of a second generation of targeted therapies such as nilotinib and dasatinib, already in phase II clinical trials or SKI-606 and MK-0457 in phase I trials. In this review, we discuss the activity of nilotinib, developed by Novartis using a rational drug design strategy in which imatinib served as the lead compound. Preliminary studies demonstrated that nilotinib has more efficacy than imatinib in inhibiting proliferation of BCR-ABL-dependent cells, a relatively safety profile and clinical efficacy in all phases of CML

Structural determinants in ApoA-I amyloidogenic variants explain improved cholesterol metabolism despite low HDL levels.

Twenty Apolipoprotein A-I (ApoA-I) variants are responsible for a systemic hereditary amyloidosis in which protein fibrils can accumulate in different organs, leading to their failure. Several ApoA-I amyloidogenic mutations are also associated with hypoalphalipoproteinemia, low ApoA-I and high-density lipoprotein (HDL)-cholesterol plasma levels; however, subjects affected by ApoA-I-related amyloidosis do not show a higher risk of cardiovascular diseases (CVD). The structural features, the lipid binding properties and the functionality of four ApoA-I amyloidogenic variants were therefore inspected in order to clarify the paradox observed in the clinical phenotype of the affected subjects. Our results show that ApoA-I amyloidogenic variants are characterized by a different oligomerization pattern and that the position of the mutation in the ApoA-I sequence affects the molecular structure of the formed HDL particles. Although lipidation increases ApoA-I proteins stability, all the amyloidogenic variants analyzed show a lower affinity for lipids, both in vitro and in ex vivo mouse serum. Interestingly, the lower efficiency at forming HDL particles is compensated by a higher efficiency at catalysing cholesterol efflux from macrophages. The decreased affinity of ApoA-I amyloidogenic variants for lipids, together with the increased efficiency in the cholesterol efflux process, could explain why, despite the unfavourable lipid profile, patients affected by ApoA-I related amyloidosis do not show a higher CVD risk

CDKN2A-independent role of BMI1 in promoting growth and survival of Ph+ acute lymphoblastic leukemia

BMI1 is a key component of the PRC1 (polycomb repressive complex-1) complex required for maintenance of normal and cancer stem cells. Its aberrant expression is detected in chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia (ALL), but no data exist on BMI1 requirement in ALL cells. We show here that BMI1 expression is important for proliferation and survival of Ph+ ALL cells and for leukemogenesis of Ph+ cells in vivo. Levels of BIM, interferon-α (IFNα)-regulated genes and E2F7 were upregulated in BMI1-silenced cells, suggesting that repressing their expression is important for BMI1 biological effects. Consistent with this hypothesis, we found that: (i) downregulation of BIM or E2F7 abrogated apoptosis or rescued, in part, the reduced proliferation and colony formation of BMI1 silenced BV173 cells; (ii) BIM/E2F7 double silencing further enhanced colony formation and in vivo leukemogenesis of BMI1-silenced cells; (iii) overexpression of BIM and E2F7 mimicked the effect of BMI1 silencing in BV173 and SUP-B15 cells; and (iv) treatment with IFNα suppressed proliferation and colony formation of Ph+ ALL cells. These studies indicate that the growth-promoting effects of BMI1 in Ph+ ALL cells depend on suppression of multiple pathways and support the use of IFNα in the therapy of Ph+ ALL

Prototyping of an HV-CMOS demonstrator for the High Luminosity-LHC upgrade

HV-CMOS sensors can offer important advantages in terms of material budget, granularity and cost for large area tracking systems in high energy physics experiments. This article presents the design and simulated results of an HV-CMOS pixel demonstrator for the High Luminosity-LHC. The pixel demonstrator has been designed in the 0.35 μm HV-CMOS process from ams AG and submitted for fabrication through an engineering run. To improve the response of the sensor, different wafers with moderate to high substrate resistivities are used to fabricate the design. The prototype consists of four large analog and standalone matrices with several pixel flavours, which are all compatible for readout with the FE-I4 ASIC. Details about the matrices and the pixel flavours are provided in this article

Modulation of amyloidogenic peptide aggregation by photoactivatable co-releasing ruthenium(II) complexes

Three Ru(II)-based CO-releasing molecules featuring bidentate benzimidazole and terpyridine derivatives as ligands were investigated for their ability to modulate the aggregation process of the second helix of the C-terminal domain of nucleophosmin 1, namely nucleophosmin 1 (NPM1)264–277, a model amyloidogenic system, before and after irradiation at 365 nm. Thioflavin T (ThT) binding assays and UV/Vis absorption spectra indicate that binding of the compounds to the peptide inhibits its aggregation and that the inhibitory effect increases upon irradiation (half maximal effective concentration (EC50) values in the high micromolar range). Electrospray ionization mass spectrometry data of the peptide in the presence of one of these compounds confirm that the modulation of amyloid aggregation relies on the formation of adducts obtained when the Ru compounds react with the peptide upon releasing of labile ligands, like chloride and carbon monoxide. This mechanism of action explains the subtle different behavior of the three compounds observed in ThT experiments. Overall, data support the hypothesis that metal-based CO releasing molecules can be used to develop metal-based drugs with potential application as anti-amyloidogenic agents

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples