The kinetic mechanism of human uridine phosphorylase 1: Towards the development of enzyme inhibitors for cancer chemotherapy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cysteine proteinase inhibitors regulate human and mouse osteoclastogenesis by interfering with RANK signaling

The cysteine proteinase inhibitor cystatin C inhibited RANKL-stimulated osteoclast formation in mouse bone marrow macrophage cultures, an effect associated with decreased mRNA expression of Acp5, Calcr, Ctsk, Mmp9, Itgb3, and Atp6i, without effect on proliferation or apoptosis. The effects were concentration dependent with half-maximal inhibition at 0.3 μM. Cystatin C also inhibited osteoclast formation when RANKL-stimulated osteoclasts were cultured on bone, leading to decreased formation of resorption pits. RANKL-stimulated cells retained characteristics of phagocytotic macrophages when cotreated with cystatin C. Three other cysteine proteinase inhibitors, cystatin D, Z-RLVG-CHN2 (IC50 0.1 μM), and E-64 (IC 50 3 μM), also inhibited osteoclast formation in RANKL-stimulated macrophages. In addition, cystatin C, Z-RLVG-CHN2, and E-64 inhibited osteoclastic differentiation of RANKL-stimulated CD14+ human monocytes. The effect by cystatin C on differentiation of bone marrow macrophages was exerted at an early stage after RANKL stimulation and was associated with early (4 h) inhibition of c-Fos expression and decreased protein and nuclear translocation of c-Fos. Subsequently, p52, p65, IκBα, and Nfatc1 mRNA were decreased. Cystatin C was internalized in osteoclast progenitors, a process requiring RANKL stimulation. These data show that cystatin C inhibits osteoclast differentiation and formation by interfering intracellularly with signaling pathways downstream RANK. © FASEB.

Both XPA and DNA polymerase eta are necessary for the repair of doxorubicin-induced DNA lesions

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Natural compounds Camptothecin and Triptolide: highly specific enzyme inhibitors and tools to dissect transcriptional functions

With this work I elucidated new and unexpected mechanisms of two strong and highly specific transcription inhibitors: Triptolide and Campthotecin. Triptolide (TPL) is a diterpene epoxide derived from the Chinese plant Trypterigium Wilfoordii Hook F. TPL inhibits the ATPase activity of XPB, a subunit of the general transcription factor TFIIH. In this thesis I found that degradation of Rbp1 (the largest subunit of RNA Polymerase II) caused by TPL treatments, is preceded by an hyperphosphorylation event at serine 5 of the carboxy-terminal domain (CTD) of Rbp1. This event is concomitant with a block of RNA Polymerase II at promoters of active genes. The enzyme responsible for Ser5 hyperphosphorylation event is CDK7. Notably, CDK7 downregulation rescued both Ser5 hyperphosphorylation and Rbp1 degradation triggered by TPL. Camptothecin (CPT), derived from the plant Camptotheca acuminata, specifically inhibits topoisomerase 1 (Top1). We first found that CPT induced antisense transcription at divergent CpG islands promoter. Interestingly, by immunofluorescence experiments, CPT was found to induce a burst of R loop structures (DNA/RNA hybrids) at nucleoli and mitochondria. We then decided to investigate the role of Top1 in R loop homeostasis through a short interfering RNA approach (RNAi). Using DNA/RNA immunoprecipitation techniques coupled to NGS I found that Top1 depletion induces an increase of R loops at a genome-wide level. We found that such increase occurs on the entire gene body. At a subset of loci R loops resulted particularly stressed after Top1 depletion: some of these genes showed the formation of new R loops structures, whereas other loci showed a reduction of R loops. Interestingly we found that new peaks usually appear at tandem or divergent genes in the entire gene body, while losses of R loop peaks seems to be a feature specific of 3’ end regions of convergent genes.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.

The polarized distribution of poly(A+)-mRNA-induced functional ion channels in the Xenopus oocyte plasma membrane is prevented by anticytoskeletal drugs

Foreign mRNA was expressed in Xenopus laevis oocytes. Newly expressed ion currents localized in defined plasma membrane areas were measured using the two-electrode voltage clamp technique in combination with a specially designed chamber, that exposed only part of the surface on the oocytes to channel agonists or inhibitors. Newly expressed currents were found to be unequally distributed in the surface membrane of the oocyte. This asymmetry was most pronounced during the early phase of expression, when channels could almost exclusively be detected in the animal hemisphere of the oocyte. 4 d after injection of the mRNA, or later, channels could be found at a threefold higher density at the animal than at the vegetal pole area. The pattern of distribution was observed to be similar with various ion channels expressed from crude tissue mRNA and from cRNAs coding for rat GABAA receptor channel subunits. Electron microscopical analysis revealed very similar microvilli patterns at both oocyte pole areas. Thus, the asymmetric current distribution is not due to asymmetric surface structure. Upon incubation during the expression period in either colchicine or cytochalasin D, the current density was found to be equal in both pole areas. The inactive control substance beta-lumicolchicine had no effect on the asymmetry of distribution. Colchicine was without effect on the amplitude of the expressed whole cell current. Our measurements reveal a pathway for plasma membrane protein expression endogenous to the Xenopus oocyte, that may contribute to the formation and maintenance of polarity of this highly organized cell.

Mechanisms of transcription inhibition by 8-amino-adenosine

Nucleoside analogs are a class of chemotherapeutic agents with tremendous utility in treating viral infections and cancers. Traditional nucleoside analogs are DNA-directed. However, there is a new group of nucleoside analogs that induce cell death by a direct effect on RNA synthesis. The adenosine analog, 8-chloroadenosine, is incorporated into RNA and is currently in clinical trials. Another congener, 8-amino-adenosine has demonstrated toxicity in multiple myeloma cell lines. Like other nucleoside analogs, 8-amino-adenosine must be metabolized to its triphosphate to elicit a cytotoxic effect. Furthermore, 8-amino-adenosine causes a decline of the intracellular ATP pool and inhibits mRNA poly(A) adenylation. ^ Because of the previously known adenosine analog mechanism as well as the scope of the RNA directed nucleoside analog field, I hypothesized there are multiple mechanisms of transcription inhibition mediating 8-amino-adenosine-induced cell death. Prior to investigating these mechanisms, cell death by 8-amino-adenosine was characterized. 8-Amino-adenosine activates PARP cleavage and induces the caspase cascade. 8-Amino-adenosine increases Annexin V binding and the mitochondrial membrane permeability in wild-type MEF cells. In BAX/BAK deficient MEF cells, 8-amino-adenosine decreases the mitochondrial membrane permeability and induces autophagy. ^ Once cell death was characterized, the mechanisms of 8-amino-adenosine transcription inhibition were assessed. It was established that 8-aminoadenosine treatment causes 8-amino-ATP accumulation and decreases the intracellular ATP concentration, resulting in RNA synthesis inhibition. Several other mechanisms are identified. First, a relationship between ATP decline by 8-amino-adenosine or other known ATP synthesis inhibitors and RNA synthesis is established indicating that effects on cellular bioenergy, regardless of the mechanism of ATP decline, can decrease RNA synthesis. Second, 8-aminoadenosine treatment decreases the phosphorylation of serine residues on the RNA polymerase II C-terminal domain which regulates transcription initiation and elongation. Third, evidence is provided to demonstrate 8-amino-ATP is a substrate for RNA synthesis. Fourth, 8-amino-ATP is incorporated at the 3'-terminal position leading to chain termination. Finally, in vitro transcription assays show that 8-amino-ATP may compete with ATP to decrease de novo mRNA synthesis. Overall, this work demonstrates 8-amino-adenosine is a cytotoxic nucleoside analog that functions to inhibit RNA transcription through multiple mechanisms. ^

Hybrid Ty1/HIV-1 elements used to detect inhibitors and monitor the activity of HIV-1 reverse transcriptase

We previously demonstrated that hybrid retrotransposons composed of the yeast Ty1 element and the reverse transcriptase (RT) of HIV-1 are active in the yeast Saccharomyces cerevisiae. The RT activity of these hybrid Ty1/HIV-1 (his3AI/AIDS RT; HART) elements can be monitored by using a simple genetic assay. HART element reverse transcription depends on both the polymerase and RNase H domains of HIV-1 RT. Here we demonstrate that the HART assay is sensitive to inhibitors of HIV-1 RT. (−)-(S)-8-Chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione monohydrochloride (8 Cl-TIBO), a well characterized non-nucleoside RT inhibitor (NNRTI) of HIV-1 RT, blocks propagation of HART elements. HART elements that express NNRTI-resistant RT variants of HIV-1 are insensitive to 8 Cl-TIBO, demonstrating the specificity of inhibition in this assay. HART elements carrying NNRTI-resistant variants of HIV-1 RT can be used to identify compounds that are active against drug-resistant viruses.

Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis

The oocyte nuclear antigen of the monoclonal antibody 32-5B6 of Xenopus laevis is subject to regulated nuclear translocation during embryogenesis. It is distributed in the cytoplasm during oocyte maturation, where it remains during cleavage and blastula stages, before it gradually reaccumulates in the nuclei during gastrulation. We have now identified this antigen to be the enzyme S-adenosylhomocysteine hydrolase (SAHH). SAHH is the only enzyme that cleaves S-adenosylhomocysteine, a reaction product and an inhibitor of all S-adenosylmethionine-dependent methylation reactions. We have compared the spatial and temporal patterns of nuclear localization of SAHH and of nuclear methyltransferase activities during embryogenesis and in tissue culture cells. Nuclear localization of Xenopus SAHH did not temporally correlate with DNA methylation. However, we found that SAHH nuclear localization coincides with high rates of mRNA synthesis, a subpopulation colocalizes with RNA polymerase II, and inhibitors of SAHH reduce both methylation and synthesis of poly(A)+ RNA. We therefore propose that accumulation of SAHH in the nucleus may be required for efficient cap methylation in transcriptionally active cells. Mutation analysis revealed that the C terminus and the N terminus are both required for efficient nuclear translocation in tissue culture cells, indicating that more than one interacting domain contributes to nuclear accumulation of Xenopus SAHH.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G1

Transcription of ribosomal RNA genes by RNA polymerase (pol) I oscillates during the cell cycle, being maximal in S and G2 phase, repressed during mitosis, and gradually recovering during G1 progression. We have shown that transcription initiation factor (TIF)-IB/SL1 is inactivated during mitosis by cdc2/cyclin B-directed phosphorylation of TAFI110. In this study, we have monitored reactivation of transcription after exit from mitosis. We demonstrate that the pol I factor UBF is also inactivated by phosphorylation but recovers with different kinetics than TIF-IB/SL1. Whereas TIF-IB/SL1 activity is rapidly regained on entry into G1, UBF is reactivated later in G1, concomitant with the onset of pol I transcription. Repression of pol I transcription in mitosis and early G1 can be reproduced with either extracts from cells synchronized in M or G1 phase or with purified TIF-IB/SL1 and UBF isolated in the presence of phosphatase inhibitors. The results suggest that two basal transcription factors, e.g., TIF-IB/SL1 and UBF, are inactivated at mitosis and reactivated by dephosphorylation at the exit from mitosis and during G1 progression, respectively.

Reverse biochemistry: Use of macromolecular protease inhibitors to dissect complex biological processes and identify a membrane-type serine protease in epithelial cancer and normal tissue

Serine proteases of the chymotrypsin fold are of great interest because they provide detailed understanding of their enzymatic properties and their proposed role in a number of physiological and pathological processes. We have been developing the macromolecular inhibitor ecotin to be a “fold-specific” inhibitor that is selective for members of the chymotrypsin-fold class of proteases. Inhibition of protease activity through the use of wild-type and engineered ecotins results in inhibition of rat prostate differentiation and retardation of the growth of human PC-3 prostatic cancer tumors. In an effort to identify the proteases that may be involved in these processes, reverse transcription–PCR with PC-3 poly(A)+ mRNA was performed by using degenerate oligonucleotide primers. These primers were designed by using conserved protein sequences unique to chymotrypsin-fold serine proteases. Five proteases were identified: urokinase-type plasminogen activator, factor XII, protein C, trypsinogen IV, and a protease that we refer to as membrane-type serine protease 1 (MT-SP1). The cloning and characterization of the MT-SP1 cDNA shows that it encodes a mosaic protein that contains a transmembrane signal anchor, two CUB domains, four LDLR repeats, and a serine protease domain. Northern blotting shows broad expression of MT-SP1 in a variety of epithelial tissues with high levels of expression in the human gastrointestinal tract and the prostate. A His-tagged fusion of the MT-SP1 protease domain was expressed in Escherichia coli, purified, and autoactivated. Ecotin and variant ecotins are subnanomolar inhibitors of the MT-SP1 activated protease domain, suggesting a possible role for MT-SP1 in prostate differentiation and the growth of prostatic carcinomas.

Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level.

Electrical currents associated with nucleotide transport by the reconstituted mitochondrial ADP/ATP carrier.

The electrophoretic export of ATP against the import of ADP in mitochondria bridges the intra- versus extramitochondrial ATP potential gap. Here we report that the electrical nature of the ADP/ATP exchange by the mitochondrial ADP/ATP carrier (AAC) can be directly studied by measuring the electrical currents via capacitive coupling of AAC-containing vesicles on a planar lipid membrane. The currents were induced by the rapid liberation of ATP or ADP with UV flash photolysis from caged nucleotides. Six different transport modes of the AAC were studied: heteroexchange with either ADP or ATP inside the vesicles, initiated by photolysis of caged ATP or ADP; homoexchange with ADPex/ADPin or ATPex/ATPin; and caged ADP or ATP with unloaded vesicles. The heteroexchange produced the largest currents with the longest duration in line with the electrical charge difference ATP4- versus ADP3-. Surprisingly, also in the homoexchange and with unloaded vesicles, small currents were measured with shorter duration. In all three modes with caged ATP, a negative charge moved into the vesicles and with caged ADP it moved out of the vesicles. All currents were completely inhibited by a mixture of the inhibitors of the AAC, carboxyatractyloside and hongkrekate, which proves that the currents are exclusively due to AAC function. The observed charge movements in the heteroexchange system agree with the prediction from transport studies in mitochondria and reconstituted vesicles. The unexpected charge movements in the homoexchange or unloaded systems are interpreted to reveal transmembrane rearrangements of charged sites in the AAC when occupied with ADP or ATP. The results also indicate that not only ATP4- but also ADP3- contribute, albeit in opposite direction, to the electrical nature of the ADP/ATP exchange, which is at variance with former conclusions from biochemical transport studies. These measurements open up new avenues of studying the electrical interactions of ADP and ATP with the AAC.