Focus on DNA Glycosylases-A Set of Tightly Regulated Enzymes with a High Potential as Anticancer Drug Targets.

Cancer is the second leading cause of death with tens of millions of people diagnosed with cancer every year around the world. Most radio- and chemotherapies aim to eliminate cancer cells, notably by causing severe damage to the DNA. However, efficient repair of such damage represents a common mechanism of resistance to initially effective cytotoxic agents. Thus, development of new generation anticancer drugs that target DNA repair pathways, and more particularly the base excision repair (BER) pathway that is responsible for removal of damaged bases, is of growing interest. The BER pathway is initiated by a set of enzymes known as DNA glycosylases. Unlike several downstream BER enzymes, DNA glycosylases have so far received little attention and the development of specific inhibitors of these enzymes has been lagging. Yet, dysregulation of DNA glycosylases is also known to play a central role in numerous cancers and at different stages of the disease, and thus inhibiting DNA glycosylases is now considered a valid strategy to eliminate cancer cells. This review provides a detailed overview of the activities of DNA glycosylases in normal and cancer cells, their modes of regulation, and their potential as anticancer drug targets.

Förster Resonance Energy Transfer Based Biosensor for Targeting the hNTH1-YB1 Interface as a Potential Anticancer Drug Target.

The Y-box binding protein 1 (YB1) is an established metastatic marker: high expression and nuclear localization of YB1 correlate with tumor aggressiveness, drug resistance, and poor patient survival in various tumors. In the nucleus, YB1 interacts with and regulates the activities of several nuclear proteins, including the DNA glycosylase, human endonuclease III (hNTH1). In the present study, we used Förster resonance energy transfer (FRET) and AlphaLISA technologies to further characterize this interaction and define the minimal regions of hNTH1 and YB1 required for complex formation. This work led us to design an original and cost-effective FRET-based biosensor for the rapid in vitro high-throughput screening for potential inhibitors of the hNTH1-YB1 complex. Two pilot screens were carried out, allowing the selection of several promising compounds exhibiting IC50 values in the low micromolar range. Interestingly, two of these compounds bind to YB1 and sensitize drug-resistant breast tumor cells to the chemotherapeutic agent, cisplatin. Taken together, these findings demonstrate that the hNTH1-YB1 interface is a druggable target for the development of new therapeutic strategies for the treatment of drug-resistant tumors. Moreover, beyond this study, the simple design of our biosensor defines an innovative and efficient strategy for the screening of inhibitors of therapeutically relevant protein-protein interfaces.

A small C-terminal sequence of Aurora B is responsible for localization and function.

Aurora B, a protein kinase required in mitosis, localizes to inner centromeres at metaphase and the spindle midzone in anaphase and is required for proper chromosome segregation and cytokinesis. Aurora A, a paralogue of Aurora B, localizes instead to centrosomes and spindle microtubules. Except for distinct N termini, Aurora B and Aurora A have highly similar sequences. We have combined small interfering RNA (siRNA) ablation of Aurora B with overexpression of truncation mutants to investigate the role of Aurora B sequence in its function. Reintroduction of Aurora B during siRNA treatment restored its localization and function. This permitted a restoration of function test to determine the sequence requirements for Aurora B targeting and function. Using this rescue protocol, neither N-terminal truncation of Aurora B unique sequence nor substitution with Aurora A N-terminal sequence affected Aurora B localization or function. Truncation of unique Aurora B C-terminal sequence from terminal residue 344 to residue 333 was without effect, but truncation to 326 abolished localization and function. Deletion of residues 326-333 completely abolished localization and blocked cells at prometaphase, establishing this sequence as critical to Aurora B function. Our findings thus establish a small sequence as essential for the distinct localization and function of Aurora B.

Erratum to: Characterizing Intact Macromolecular Complexes Using Native Mass Spectrometry.

The chapter author provided the below additional text to be added in the acknowledgement section. This has now been updated in the revised version of the book.

Characterizing Intact Macromolecular Complexes Using Native Mass Spectrometry.

Native mass spectrometry (MS) enables the characterization of macromolecular assemblies with high sensitivity. It can reveal the stoichiometry of subunits as well as their two-dimensional interaction network and provide information regarding the dynamic behavior of macromolecular complexes. Here, we describe the workflow to perform native MS experiments. In addition, we illustrate the quality control analysis of proteins using MS in denaturing conditions.

Fusion to a homo-oligomeric scaffold allows cryo-EM analysis of a small protein.

Recent technical advances have revolutionized the field of cryo-electron microscopy (cryo-EM). However, most monomeric proteins remain too small (<100 kDa) for cryo-EM analysis. To overcome this limitation, we explored a strategy whereby a monomeric target protein is genetically fused to a homo-oligomeric scaffold protein and the junction optimized to allow the target to adopt the scaffold symmetry, thereby generating a chimeric particle suitable for cryo-EM. To demonstrate the concept, we fused maltose-binding protein (MBP), a 40 kDa monomer, to glutamine synthetase, a dodecamer formed by two hexameric rings. Chimeric constructs with different junction lengths were screened by biophysical analysis and negative-stain EM. The optimal construct yielded a cryo-EM reconstruction that revealed the MBP structure at sub-nanometre resolution. These findings illustrate the feasibility of using homo-oligomeric scaffolds to enable cryo-EM analysis of monomeric proteins, paving the way for applying this strategy to challenging structures resistant to crystallographic and NMR analysis.

Centromeric histone variant CENP-A represses acetylation-dependent chromatin transcription that is relieved by histone chaperone NPM1.

Mammalian centromeric histone H3 variant, CENP-A, is involved in maintaining the functional integrity and epigenetic inheritance of the centromere. CENP-A causes transcriptional repression of centromeric chromatin through an unknown mechanism. Here, we report that reconstituted CENP-A nucleosomes are amenable to ATP-dependent SWI/SNF-mediated remodelling but are less permissive to acetylation and acetylation-dependent in vitro chromatin transcription. Remarkably, the transcriptional repression of the CENP-A chromatinized template could be relieved by the ectopic addition of histone chaperone, nucleophosmin.

Molecular distinctions between Aurora A and B: a single residue change transforms Aurora A into correctly localized and functional Aurora B.

Aurora A and Aurora B, paralogue mitotic kinases, share highly similar primary sequence. Both are important to mitotic progression, but their localizations and functions are distinct. We have combined shRNA suppression with overexpression of Aurora mutants to address the cause of the distinction between Aurora A and Aurora B. Aurora A residue glycine 198 (G198), mutated to asparagine to mimic the aligned asparagine 142 (N142) of Aurora B, causes Aurora A to bind the Aurora B binding partner INCENP but not the Aurora A binding partner TPX2. The mutant Aurora A rescues Aurora B mitotic function. We conclude that binding to INCENP is alone critical to the distinct function of Aurora B. Although G198 of Aurora A is required for TPX2 binding, N142G Aurora B retains INCENP binding and Aurora B function. Thus, although a single residue change transforms Aurora A, the reciprocal mutation of Aurora B does not create Aurora A function. An Aurora A-Delta120 N-terminal truncation construct reinforces Aurora A similarity to Aurora B, because it does not associate with centrosomes but instead associates with kinetochores.

Localization of aurora A and aurora B kinases during interphase: role of the N-terminal domain.

Aurora kinases possess a conserved catalytic domain (CD) and a N-terminal domain (ND) that varies in size and sequence. We have previously reported that the N-terminal domain of AuroraA (AurA) participates in the localization of the kinase to the centrosome in interphase. AuroraB (AurB) is a chromosome passenger protein and its N-terminal domain is not necessary for its localization or function during mitosis. Using various combinations of GFP-AurA and AurB protein domains we show that AurB N-terminal domain is required for nuclear localization in Xenopus XL2 cells in interphase. In human cells, however, we found both AurA and AurB kinases in the nucleus, AurA being mainly cytoplasmic and AurB mainly nuclear. Both proteins are actively excluded from the nucleus by a CRM1 dependent pathway. Interestingly, at a functional level, in interphase, every combination of Aurora kinase domains (ND-CD) rescues histone H3 Serine10 phosphorylation defect induced by AurB knockdown. This clearly indicates the presence of a functional AurA in the nucleus. However, the chimera ND-AurA/CD-AurB was much more efficient than the ND-AurB/ CD-AurA to rescue multinucleation also induced by AurB knockdown. This indicates that the catalytic domain of AurB is required to fulfill specific functions during mitosis that cannot be fulfilled by the catalytic domain of AurA, probably for localization reasons during mitosis.

Nucleolin is a histone chaperone with FACT-like activity and assists remodeling of nucleosomes.

Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A-H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A-H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription.

The histone variant macroH2A interferes with transcription factor binding and SWI/SNF nucleosome remodeling.

The unusual histone variant macroH2A (mH2A) has been associated with repression of transcription, but the molecular mechanisms by which it exerts this function are unknown. Here we have identified a mechanism by which the different domains of mH2A may be involved in the repression of transcription. Evidence is presented that the presence of mH2A in a positioned nucleosome interferes with the binding of the transcription factor NF-kappaB. The nonhistone region of mH2A was identified to be associated with this interference. Importantly, the presence of macroH2A was found to severely impede SWI/SNF nucleosome remodeling and movement to neighboring DNA segments. This property of mH2A was demonstrated to reside only in its H2A-like domain. A hypothesis explaining the role of histone variants in transcriptional regulation is proposed.

SWI/SNF remodeling and p300-dependent transcription of histone variant H2ABbd nucleosomal arrays.

A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd-H2B dimer to a tetrameric histone H3-H4 particle. In addition, the p300- and Gal4-VP16-activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed.

The histone octamer is invisible when NF-kappaB binds to the nucleosome.

The transcription factor NF-kappaB is involved in the transcriptional control of more than 150 genes, but the way it acts at the level of nucleosomal templates is not known. Here we report on a study examining the interaction of NF-kappaB p50 with its DNA recognition sequence in a positioned nucleosome. We demonstrate that NF-kappaB p50 was able to bind to the nucleosome with an apparent association constant close to that for free DNA. In agreement with this, the affinity of NF-kappaB p50 binding does not depend on the localization of its recognition sequence relative to the nucleosome dyad axis. In addition, the binding of NF-kappaB p50 does not induce eviction of histones and does not perturb the overall structure of the nucleosome. The NF-kappaB p50-nucleosome complex exhibits, however, local structural alterations within the NF-kappaB p50 recognition site. Importantly, these alterations were very similar to those found in the NF-kappaB p50-DNA complex. Our data suggest that NF-kappaB p50 can accommodate the distorted, bent DNA within the nucleosome. This peculiar property of NF-kappaB p50 might have evolved to meet the requirements for its function as a central switch for stress responses.