Light-Activatable Photocaged UNC2025 for Triggering TAM Kinase Inhibition in Bladder Cancer.

Photopharmacology is an emerging field that utilizes photo-responsive molecules to enable control over the activity of a drug using light. The aim is to limit the therapeutic action of a drug at the level of diseased tissues and organs. Considering the well-known implications of protein kinases in cancer and the therapeutic issues associated with protein kinase inhibitors, the photopharmacology is seen as an innovative and alternative solution with great potential in oncology.

Type I arginine methyltransferases are intervention points to unveil the oncogenic Epstein-Barr virus to the immune system.

The oncogenic Epstein-Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is also highly antigenic. Hence, EBV seemingly evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 limits the translation of its own mRNA to the minimal level to ensure its essential function, thereby, at the same time, minimizing immune recognition.

Protein Dimerization via Tyr Residues: Highlight of a Slow Process with Co-Existence of Numerous Intermediates and Final Products.

Protein dimerization via tyrosine residues is a crucial process in response to an oxidative attack, which has been identified in many ageing-related pathologies. Recently, it has been found that for isolated tyrosine amino acid, dimerization occurs through three types of tyrosine-tyrosine crosslinks and leads to at least four final products. Herein, considering two protected tyrosine residues, tyrosine-containing peptides and finally proteins, we investigate the dimerization behavior of tyrosine when embedded in a peptidic sequence.

BrdU immuno-tagged G-quadruplex ligands: a new ligand-guided immunofluorescence approach for tracking G-quadruplexes in cells.

G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag.

Acridine-O-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity.

O-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O-methylguanine residues in DNA. MGMT substrate analogues, such as O-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine).

Oxidative radicals (HO or N) induce several di-tyrosine bridge isomers at the protein scale.

Among protein oxidative damages, di-tyrosine bridges formation has been evidenced in many neuropathological diseases. Combining oxidative radical production by gamma radiolysis with very performant chromatographic separation coupled to mass spectrometry detection, we brought into light new insights of tyrosine dimerization. Hydroxyl and azide radical tyrosine oxidation leading to di-tyrosine bridges formation was studied for different biological compounds: a full-length protein (Δ25-centrin 2), a five amino acid peptide (KTSLY) and free tyrosine.

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD.

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells.

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1.

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination. In mitosis, BRCA2 is phosphorylated by Polo-like kinase 1 (PLK1). Here we describe how this phosphorylation contributes to the control of mitosis.

Nucleolin Discriminates Drastically between Long-Loop and Short-Loop Quadruplexes.

We investigate herein the interaction between nucleolin (NCL) and a set of G4 sequences derived from the CEB25 human minisatellite that adopt a parallel topology while differing in the length of the central loop (from nine nucleotides to one nucleotide). It is revealed that NCL strongly binds to long-loop (five to nine nucleotides) G4 while interacting weakly with the shorter variants (loop with fewer than three nucleotides). Photo-cross-linking experiments using 5-bromo-2'-deoxyuridine (BrU)-modified sequences further confirmed the loop-length dependency, thereby indicating that the WT-CEB25-L191 (nine-nucleotide loop) is the best G4 substrate.

How protein structure affects redox reactivity: example of Human centrin 2.

Electron transfer inside proteins plays a central role in their reactivity and biological functions. Herein, we developed a combined approach by gamma radiolysis and electrochemistry which allowed a deep insight into the reactivity of Human centrin 2, a protein very sensitive to oxidative stress and involved in several key biological processes. This protein bears a single terminal tyrosine and was observed to be extremely sensitive to ionizing radiation sources, leading to a tyrosine dimer.

"On-the-fly" kinetics of enzymatic racemization using deuterium NMR in DNA-based chiral oriented media.

We report the in situ and real-time monitoring of the interconversion of L- and D-alanine-d3 by alanine racemase from Bacillus stearothermophilus directly observed by (2)H NMR spectroscopy in anisotropic phase. The enantiomers are distinguished by the difference of their (2)H quadrupolar splittings in a chiral liquid crystal containing short DNA fragments. The proof-of-principle, the reliability, and the robustness of this new method is demonstrated by the determination of the turnover rates of the enzyme using the Michaelis-Menten model.

Oxidative stress induces mainly human centrin 2 polymerisation.

Purpose: To determine the human centrin 2 (Hscen 2) protein response to oxidising radicals in vitro and to evaluate the consequences on its biological functions.

Materials And Methods: Hscen 2 was submitted to hydroxyl and azide radicals produced by radiolysis in the absence of oxygen. The resulting products were characterised by biochemical, spectroscopic and mass spectrometry techniques.

The carboxy-terminal domain of xeroderma pigmentosum complementation group C protein, involved in TFIIH and centrin binding, is highly disordered.

Xeroderma pigmentousum group C protein (XPC) is involved in the first step of nucleotide excision repair, with multiple functional roles including DNA damage recognition and recruitment of the repair machinery. This human protein of 940 residues forms a strong heterotrimeric complex with Rad23B and centrin 2. The structure of XPC is actually not known, and lack of significant sequence homology with proteins from structural data bases precludes any relevant prediction.

Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein.

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction.

High sensitivity of human centrin 2 toward radiolytical oxidation: C-terminal tyrosinyl residue as the main target.

Centrins are calcium-binding proteins that play a significant role in the maintenance of the centrosomal organization, mainly in the continuity between centrosome and microtubular network. Recent data showed that centrosome duplication abnormalities, like overduplication for example, could be due to hydrogen peroxide, suggesting an important impact of oxidative stress. To challenge this hypothesis, we performed one-electron oxidation experiments with human centrin 2, starting from azide radicals.

Slow backbone dynamics of the C-terminal fragment of human centrin 2 in complex with a target peptide probed by cross-correlated relaxation in multiple-quantum NMR spectroscopy.

The C-terminal domain of human centrin 2 (C-HsCen2) strongly binds to P1-XPC, a peptide comprising 17 amino acids with a NWKLLAKGLLIRERLKR sequence. This peptide corresponds to residues N847-R863 of XPC, a protein involved in the recognition of damaged DNA during the initial step of the nucleotide excision repair pathway. The slow internal dynamics of the protein backbone in the C-HsCen-P1-XPC complex was studied by measuring the relaxation rates of zero- and double-quantum coherences involving neighboring pairs of carbonyl 13C and amide 15N nuclei.

Binding of human centrin 2 to the centrosomal protein hSfi1.

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat.

Crystallization and preliminary X-ray diffraction data of the complex between human centrin 2 and a peptide from the protein XPC.

Centrins are highly conserved calcium-binding proteins involved in the nucleotide-excision repair pathway as a subunit of the heterotrimer including the XPC and hHR23B proteins. A complex formed by a Ca2+-bound human centrin 2 construct (the wild type lacking the first 25 amino acids) with a 17-mer peptide derived from the XPC sequence (residues Asn847-Arg863) was crystallized. Data were collected to 1.

Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair.

Human centrin 2 is a component of the nucleotide excision repair system, as a subunit of the heterotrimer including xeroderma pigmentosum group C protein (XPC) and hHR23B. The C-terminal domain of centrin (C-HsCen2) binds strongly a peptide from the XPC protein (P1-XPC: N(847)-R(863)). Here, we characterize the solution Ca(2+)-dependent structural and molecular features of the C-HsCen2 in complex with P1-XPC, mainly using NMR spectroscopy and molecular modeling.

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly.

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2.

Calcium and magnesium binding to human centrin 3 and interaction with target peptides.

There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties. We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2. This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography.

Disentangling ligand migration and heme pocket relaxation in cytochrome P450cam.

In this work we show that ligand migration and active site conformational relaxation can occur independently of each other in hemoproteins. The complicated kinetics of carbon monoxide rebinding with cytochrome P450cam display up to five distinct processes between 77 K and 300 K. They were disentangled by using a combination of three approaches: 1), the competition of the ligand with xenon for the occupation of internal protein cavities; 2), the modulation of the amount of distal steric hindrance within the heme pocket by varying the nature of the substrate; and 3), molecular mechanics calculations to support the proposed heme-substrate relaxation mechanism and to seek internal cavities.

Calcium-dependent self-assembly of human centrin 2.

Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca(2+)-sensitive fibers and their precise role in centrosome duplication are not known.

Xeroderma pigmentosum group C protein possesses a high affinity binding site to human centrin 2 and calmodulin.

Human centrin 2 (HsCen2), a member of the EF-hand superfamily of Ca2+-binding proteins, is commonly associated with centrosome-related structures. The protein is organized in two domains, each containing two EF-hand motifs, but only the C-terminal half exhibits Ca2+ sensor properties. A significant fraction of HsCen2 is localized in the nucleus, where it was recently found associated with the xeroderma pigmentosum group C protein (XPC), a component of the nuclear excision repair pathway.

C-terminal half of human centrin 2 behaves like a regulatory EF-hand domain.

Human centrin 2 (HsCen2) is an EF-hand protein that plays a critical role in the centrosome duplication and separation during cell division. We studied the structural and Ca(2+)-binding properties of two C-terminal fragments of this protein: SC-HsCen2 (T94-Y172), covering two EF-hands, and LC-HsCen2 (M84-Y172), having 10 additional residues. Both fragments are highly disordered in the apo state but become better structured (although not conformationally homogeneous) in the presence of Ca(2+) and depending on the nature of the cations (K(+) or Na(+)) in the buffer.