A Small Molecule, UAB126, Reverses Diet-Induced Obesity and its Associated Metabolic Disorders.

Targeting retinoid X receptor (RXR) has been proposed as one of the therapeutic strategies to treat individuals with metabolic syndrome, as RXR heterodimerizes with multiple nuclear receptors that regulate genes involved in metabolism. Despite numerous efforts, RXR ligands (rexinoids) have not been approved for clinical trials to treat metabolic syndrome due to the serious side effects such as hypertriglyceridemia and altered thyroid hormone axis. In this study, we demonstrate a novel rexinoid-like small molecule, UAB126, which has positive effects on metabolic syndrome without the known side effects of potent rexinoids.

Topoisomerases and cancer chemotherapy: recent advances and unanswered questions.

DNA topoisomerases are enzymes that catalyze changes in the torsional and flexural strain of DNA molecules. Earlier studies implicated these enzymes in a variety of processes in both prokaryotes and eukaryotes, including DNA replication, transcription, recombination, and chromosome segregation. Studies performed over the past 3 years have provided new insight into the roles of various topoisomerases in maintaining eukaryotic chromosome structure and facilitating the decatenation of daughter chromosomes at cell division.

UBC9 Mutant Reveals the Impact of Protein Dynamics on Substrate Selectivity and SUMO Chain Linkages.

SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs.

DNA topoisomerase-targeting chemotherapeutics: what's new?

To resolve the topological problems that threaten the function and structural integrity of nuclear and mitochondrial genomes and RNA molecules, human cells encode six different DNA topoisomerases including type IB enzymes (TOP1 and TOP1mt), type IIA enzymes (TOP2α and TOP2β) and type IA enzymes (TOP3α and TOP3β). DNA entanglements and the supercoiling of DNA molecules are regulated by topoisomerases through the introduction of transient enzyme-linked DNA breaks. The covalent topoisomerase-DNA complexes are the cellular targets of a diverse group of cancer chemotherapeutics, which reversibly stabilize these reaction intermediates.

Molecular Mechanism of DNA Topoisomerase I-Dependent rDNA Silencing: Sir2p Recruitment at Ribosomal Genes.

Saccharomyces cerevisiae sir2Δ or top1Δ mutants exhibit similar phenotypes involving ribosomal DNA, including (i) loss of transcriptional silencing, resulting in non-coding RNA hyperproduction from cryptic RNA polymerase II promoters; (ii) alterations in recombination; and (iii) a general increase in histone acetylation. Given the distinct enzymatic activities of Sir2 and Top1 proteins, a histone deacetylase and a DNA topoisomerase, respectively, we investigated whether genetic and/or physical interactions between the two proteins could explain the shared ribosomal RNA genes (rDNA) phenotypes. We employed an approach of complementing top1Δ cells with yeast, human, truncated, and chimeric yeast/human TOP1 constructs and of assessing the extent of non-coding RNA silencing and histone H4K16 deacetylation.

Acoustic Droplet Ejection Technology and Its Application in High-Throughput RNA Interference Screening.

The development of acoustic droplet ejection (ADE) technology has resulted in many positive changes associated with the operations in a high-throughput screening (HTS) laboratory. Originally, this liquid transfer technology was used to simply transfer DMSO solutions of primarily compounds. With the introduction of Labcyte's Echo 555, which has aqueous dispense capability, the application of this technology has been expanded beyond its original use.

Point mutations of the mTOR-RHEB pathway in renal cell carcinoma.

Aberrations in the mTOR (mechanistic target of rapamycin) axis are frequently reported in cancer. Using publicly available tumor genome sequencing data, we identified several point mutations in MTOR and its upstream regulator RHEB (Ras homolog enriched in brain) in patients with clear cell renal cell carcinoma (ccRCC), the most common histology of kidney cancer. Interestingly, we found a prominent cluster of hyperactivating mutations in the FAT (FRAP-ATM-TTRAP) domain of mTOR in renal cell carcinoma that led to an increase in both mTORC1 and mTORC2 activities and led to an increased proliferation of cells.

DNA topoisomerase I domain interactions impact enzyme activity and sensitivity to camptothecin.

During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain.

Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg.

Development and histopathological characterization of tumorgraft models of pancreatic ductal adenocarcinoma.

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.

4-Hydroxytamoxifen induces autophagic death through K-Ras degradation.

Tamoxifen is widely used to treat estrogen receptor-positive breast cancer. Recent findings that tamoxifen and its derivative 4-hydroxytamoxifen (OHT) can exert estrogen receptor-independent cytotoxic effects have prompted the initiation of clinical trials to evaluate its use in estrogen receptor-negative malignancies. For example, tamoxifen and OHT exert cytotoxic effects in malignant peripheral nerve sheath tumors (MPNST) where estrogen is not involved.

High-throughput RNA interference screening: tricks of the trade.

The process of validating an assay for high-throughput screening (HTS) involves identifying sources of variability and developing procedures that minimize the variability at each step in the protocol. The goal is to produce a robust and reproducible assay with good metrics. In all good cell-based assays, this means coefficient of variation (CV) values of less than 10% and a signal window of fivefold or greater.

Cellular strategies for regulating DNA supercoiling: a single-molecule perspective.

Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA.

Disulfide cross-links reveal conserved features of DNA topoisomerase I architecture and a role for the N terminus in clamp closure.

In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions.

Mutation of Gly721 alters DNA topoisomerase I active site architecture and sensitivity to camptothecin.

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended alpha-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket.

Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.

TOR signaling is a determinant of cell survival in response to DNA damage.

The conserved TOR (target of rapamycin) kinase is part of a TORC1 complex that regulates cellular responses to environmental stress, such as amino acid starvation and hypoxia. Dysregulation of Akt-TOR signaling has also been linked to the genesis of cancer, and thus, this pathway presents potential targets for cancer chemotherapeutics. Here we report that rapamycin-sensitive TORC1 signaling is required for the S-phase progression and viability of yeast cells in response to genotoxic stress.

Antitumour drugs impede DNA uncoiling by topoisomerase I.

Increasing the ability of chemotherapeutic drugs to kill cancer cells is often hampered by a limited understanding of their mechanism of action. Camptothecins, such as topotecan, induce cell death by poisoning DNA topoisomerase I, an enzyme capable of removing DNA supercoils. Topotecan is thought to stabilize a covalent topoisomerase-DNA complex, rendering it an obstacle to DNA replication forks.

Structure of a SUMO-binding-motif mimic bound to Smt3p-Ubc9p: conservation of a non-covalent ubiquitin-like protein-E2 complex as a platform for selective interactions within a SUMO pathway.

The SUMO ubiquitin-like proteins play regulatory roles in cell division, transcription, DNA repair, and protein subcellular localization. Paralleling other ubiquitin-like proteins, SUMO proteins are proteolytically processed to maturity, conjugated to targets by E1-E2-E3 cascades, and subsequently recognized by specific downstream effectors containing a SUMO-binding motif (SBM). SUMO and its E2 from the budding yeast Saccharomyces cerevisiae, Smt3p and Ubc9p, are encoded by essential genes.

Inhibition of topoisomerase I cleavage activity by thiol-reactive compounds: importance of vicinal cysteines 504 and 505.

DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species.

Alterations in linker flexibility suppress DNA topoisomerase I mutant-induced cell lethality.

Eukaryotic DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of a covalent enzyme-DNA intermediate, which is reversibly stabilized by the anticancer agent camptothecin (CPT). Crystallographic studies of the 70-kDa C terminus of human Top1p bound to duplex DNA describe a monomeric protein clamp circumscribing the DNA helix. The structures, which lack the N-terminal domain, comprise the conserved clamp, an extended linker domain, and the conserved C-terminal active site Tyr domain.

Distinct functional domains of Ubc9 dictate cell survival and resistance to genotoxic stress.

Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P(123)L mutant was viable at 36 degrees C yet exhibited enhanced sensitivity to DNA damage.

Trapping of DNA topoisomerase I on nick-containing DNA in cell free extracts of Saccharomyces cerevisiae.

The aim of the present study was to identify proteins that bind nicked DNA intermediates formed in the course of base excision repair (BER) in cell free extracts of Saccharomyces cerevisiae. In mammalian cells, nicks in DNA are targets of proteins such as PARP-1 or XRCC1 that have no homologues in yeast. One of the most promising methodologies to trap proteins that interact with damaged DNA lies in using a photocrosslinking technique with photoactivable dNTP analogues such as exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2'-deoxycytidine-5'-triphosphate (FAP-dCTP) for enzymatic synthesis of DNA probes with a photoreactive dNMP residue at the 3'-margin of a nick.

Defects in SUMO (small ubiquitin-related modifier) conjugation and deconjugation alter cell sensitivity to DNA topoisomerase I-induced DNA damage.

Eukaryotic DNA topoisomerase I (Top1p) has important functions in DNA replication, transcription, and recombination. This enzyme also constitutes the cellular target of camptothecin (CPT), which induces S-phase-dependent cytotoxicity. To define cellular pathways that regulate cell sensitivity to Top1p-induced DNA lesions, we described a yeast genetic screen for conditional tah (top1T722A-hypersensitive) mutants with enhanced sensitivity to low levels of the CPT mimetic mutant top1T722A (Reid, R.

Enhanced antitumor activity of irofulven in combination with irinotecan in pediatric solid tumor xenograft models.

Purpose: Irofulven, a novel chemotherapeutic agent with a broad spectrum of activity, is effective against preclinical models of pediatric tumors. The cytotoxic activity of irofulven is augmented when combined with agents that interact with DNA topoisomerase I; however, none of the reported studies have used the protracted dosing schedule found to be active clinically in treatment of childhood cancers. The objective of this study was to evaluate the antitumor activity of irofulven in combination with irinotecan administered on a protracted schedule in a panel of pediatric solid tumor xenografts.