Structural analysis of the Sulfolobus solfataricus MCM protein N-terminal domain†

The Mini-Chromosome Maintenance (MCM) proteins are candidates of replicative DNA helicase in eukarya and archaea. Here we report a 2.8 Å crystal structure of the N-terminal domain (residues 1–268) of the Sulfolobus solfataricus MCM (Sso MCM) protein. The structure reveals single-hexameric ring-like architecture, at variance from the protein of Methanothermobacter thermoautotrophicus (Mth). Moreover, the central channel in Sso MCM seems significantly narrower than the Mth counterpart, which appears to more favorably accommodate single-stranded DNA than double-stranded DNA, as supported by DNA-binding assays. Structural analysis also highlights the essential role played by the zinc-binding domain in the interaction with nucleic acids and allows us to speculate that the Sso MCM N-ter domain may function as a molecular clamp to grasp the single-stranded DNA passing through the central channel. On this basis possible DNA unwinding mechanisms are discussed

Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling.

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2–286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action

A CDC6-like factor from the archaea Sulfolobus solfataricus promotes binding of the mini-chromosome maintenance complex to DNA

The archaeal replication apparatus appears to be a simplified version of the eukaryotic one with fewer polypeptides and simpler protein complexes. Herein, we report evidence that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities. These findings may provide important clues to understanding how the DNA replication initiation process has evolved in the more complex eukaryotic organisms

Biochemical characterization of a CDC6-like protein from the crenarchaeon sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function

Biotin-targeted Pluronic® P123/F127 mixed micelles delivering niclosamide: A repositioning strategy to treat drug-resistant lung cancer cells

With the aim to develop alternative therapeutic tools for the treatment of resistant cancers, here we propose targeted Pluronic1 P123/F127 mixed micelles (PMM) delivering niclosamide (NCL) as a repositioning strategy to treat multidrug resistant non-small lung cancer cell lines. To build multifunctional PMM for targeting and imaging, Pluronic1 F127 was conjugated with biotin, while Pluronic1 P123 was fluorescently tagged with rhodamine B, in both cases at one of the two hydroxyl end groups. This design intended to avoid any interference of rhodamine B on biotin exposition on PMM surface, which is a key fundamental for cell trafficking studies. Biotin-decorated PMM were internalized more efficiently than non-targeted PMM in A549 lung cancer cells, while very low internalization was found in NHI3T3 normal fibroblasts. Biotin-decorated PMM entrapped NCL with good efficiency, displayed sustained drug release in protein-rich media and improved cytotoxicity in A549 cells as compared to free NCL (P < 0.01). To go in depth into the actual therapeutic potential of NCL-loaded PMM, a cisplatin-resistant A549 lung cancer cell line (CPr-A549) was developed and its multidrug resistance tested against common chemotherapeutics. Free NCL was able to overcome chemoresistance showing cytotoxic effects in this cell line ascribable to nucleolar stress, which was associated to a significant increase of the ribosomal protein rpL3 and consequent up-regulation of p21. It is noteworthy that biotin- decorated PMM carrying NCL at low doses demonstrated a significantly higher cytotoxicity than free NCL in CPr-A549. These results point at NCL-based regimen with targeted PMM as a possible second-line chemotherapy for lung cancer showing cisplatin or multidrug resistance

Relazioni struttura funzione della DNA elicasi MCM del crenarchaeon Sulfolobus solfataricus

Il progetto scientifico sviluppato durante il triennio del Dottorato di ricerca ha riguardato lo studio delle relazioni struttura-funzione della DNA elicasi dall’archaeon iper-termofilo Sulfolobus solfataricus (SsoMCM). La replicazione del DNA negli eucarioti è caratterizzata dall’interazione di molti fattori proteici che coordinano in maniera altamente precisa una serie di eventi culminanti con la duplicazione del materiale genetico prima del completamento della divisione cellulare. Il completo sequenziamento del genoma di molte specie archeobatteriche ed i primi studi effettuati sul sistema replicativo di tali organismi indicano l’esistenza di una forte similitudine con il sistema eucariotico. Infatti, i complessi molecolari degli Archaea coinvolti nella replicazione del DNA sono costituiti da fattori proteici più simili a quelli eucariotici che non batterici e questo fa degli Archaea un ottimo modello di studio per comprendere i meccanismi biologici che regolano il complicato macchinario replicativo eucariotico. Lo studio dell’ipotetica DNA elicasi replicativa SsoMCM è un perfetto esempio in tal senso. Infatti, nel genoma di Sulfolobus solfataricus è presente un unico omologo delle proteine che compongono il complesso proteico MCM 2-7 eucariotico (Mini-Chromosome Maintenance). Studi preliminari hanno evidenziato che SsoMCM è un omo-esamero ed appartiene alla super-famiglia delle ATPasi con varie Attività cellulari Associate (AAA+ super-family), è in grado di legare differenti tipi di molecole di DNA e mostra un’attività DNA elicasica, ATPasi-dipendente, con polarità 3’->5’. Studi strutturali mediante microscopia elettronica ad elevata risoluzione e cristallografia ai raggi-X della proteina MCM dell’archaeon Methanothermobacter thermoautotrophicum (MthMCM) hanno dimostrato che essa è costituita da due esameri giustapposti, ciascuno dei quali delimita un canale centrale carico positivamente che si ritiene leghi il DNA. Grazie all’allineamento tra le sequenze altamente simili di MthMCM e di SsoMCM, perfezionato sulla base della struttura cristallografica recentemente risolta di un frammento di circa 300 residui di MthMCM, è stato possibile identificare aminoacidi con carica positiva di SsoMCM putativamente localizzati nel canale centrale e coinvolti nel legame al DNA. Tali aminoacidi (Lisine 129, 134, 194 ed Istidina 146) sono stati sostituiti con residui di Alanina mediante mutagenesi sito-diretta. Si è dimostrato che le proteine mutanti formano correttamente omo-esameri in soluzione e conservano una attività ATPasica paragonabile a quella della proteina wild type. Mediante analisi per EMSA e saggi enzimatici è stato possibile dimostrare che sono critici per il legame al DNA e/o l’attività DNA elicasica, mentre la sostituzione del residuo aminoacidico K134 con Alanina ha effetto solo sul legame al DNA a doppio filamento e non sul legame al DNA a singolo filamento ne sull’attività DNA elicasica dell’enzima. Grazie alla analisi suddetta, sono stati individuati altri residui basici (Lisina 246 ed Arginine 247, 250) situati in β-hairpin fingers ipoteticamente orientati verso il canale centrale in SsoMCM. La loro sostituzione con Alanina determina una sostanziale riduzione sia del legame al DNA che dell’attivita enzimatica. Inoltre, mediante tecniche di spettroscopia di fluorescenza sono stati monitorati cambi conformazionali di tali β-hairpin fingers promossi dal legame di SsoMCM a vari nucleotidi (ATP, ADP, ADP-AlF4-). L’effetto più importante si è osservato quando gli spettri sono stati registrati in presenza di ADP o ADP-AlF4-, un analogo dello stato di transizione della reazione di idrolisi dell’ATP. Questi risultati suggeriscono che l’idrolisi, piuttosto che il legame, dell’ATP determina il movimento di tali elementi di struttura necessario per la funzione di remodeling del DNA da parte di SsoMCM

Integrated proteomics and metabolomics analysis reveals new insight into the synergistic antitumor effect of valproic acid plus simvastatin in prostate cancer xenograft models associated with downmodulation of YPA/TAZ signaling

&lt;p&gt;Combined&nbsp;proteomic and metabolomic/lipidomic approach&nbsp;with the aim to characterize the molecular mechanism of valproic acid (VPA)/simvastatin (SIM) combination in tissue tumor samples derived from 22Rv1 mCRPC cells xenografted mice, with respect to untreated samples.&lt;/p&gt

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus▿

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adhTt) was heterologously overexpressed in Escherichia coli, and the protein (ADHTt) was purified to homogeneity and characterized. ADHTt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ∼73°C and a 30-min half-inactivation temperature of ∼90°C, as well as good tolerance to common organic solvents. ADHTt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and α-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, α-tetralone, and α-methyl and α-ethyl benzoylformates to (S)-(−)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-α-(trifluoromethyl)benzyl alcohol (93% ee), (S)-α-tetralol (>99% ee), methyl (R)-(−)-mandelate (92% ee), and ethyl (R)-(−)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily

Epigenetic Approaches to Overcome Fluoropyrimidines Resistance in Solid Tumors

Although fluoropyrimidines were introduced as anticancer agents over 60 years ago, they are still the backbone of many combination chemotherapy regimens for the treatment of solid cancers. Like other chemotherapeutic agents, the therapeutic efficacy of fluoropyrimidines can be affected by drug resistance and severe toxicities; thus, novel therapeutic approaches are required to potentiate their efficacy and overcome drug resistance. In the last 20 years, the deregulation of epigenetic mechanisms has been shown to contribute to cancer hallmarks. Histone modifications play an important role in directing the transcriptional machinery and therefore represent interesting druggable targets. In this review, we focused on histone deacetylase inhibitors (HDACis) that can increase antitumor efficacy and overcome resistance to fluoropyrimidines by targeting specific genes or proteins. Our preclinical data showed a strong synergistic interaction between HDACi and fluoropyrimidines in different cancer models, but the clinical studies did not seem to confirm these observations. Most likely, the introduction of increasingly complex preclinical models, both in vitro and in vivo, cannot recapitulate human complexity; however, our analysis of clinical studies revealed that most of them were designed without a mechanistic approach and, importantly, without careful patient selection

Tissue transglutaminase (TG2) is involved in the resistance of cancer cells to the histone deacetylase (HDAC) inhibitor vorinostat

Vorinostat demonstrated preclinical and clinical efficacy in human cancers and is the first histone deacetylase inhibitor (HDACi) approved for cancer treatment. Tissue transglutaminase (TG2) is a multifunctional enzyme that catalyzes a Ca2+ dependent transamidating reaction resulting in covalent cross-links between proteins. TG2 acts also as G-protein in trans-membrane signaling and as a cell surface adhesion mediator. TG2 up-regulation has been demonstrated in several cancers and its expression levels correlate with resistance to chemotherapy and metastatic potential. We demonstrated that the anti-proliferative effect of the HDACi vorinostat is paralleled by the induction of TG2 mRNA and protein expression in cancer cells but not in ex vivo treated peripheral blood lymphocytes. This effect was also shared by other pan-HDACi and resulted in increased TG2 transamidating activity. Notably, high TG2 basal levels in a panel of cancer cell lines correlated with lower vorinostat antiproliferative activity. Notably, in TG2-knockdown cancer cells vorinostat anti-proliferative and pro-apoptotic effects were enhanced, whereas in TG2-full-length transfected cells were impaired, suggesting that TG2 could represent a mechanism of intrinsic or acquired resistance to vorinostat. In fact, co-treatment of tumor cells with inhibitors of TG2 transamidating activity potentiated the antitumor effect of vorinostat. Moreover, vorinostat-resistant MCF7 cells selected by stepwise increasing concentrations of the drug, significantly overexpressed TG2 protein compared to parental cells, and co-treatment of these cells with TG2 inhibitors reversed vorinostat-resistance. Taken together, our data demonstrated that TG2 is involved in the resistance of cancer cells to vorinostat, as well as to other HDACi