Histone deacetylase 6 inhibition restores autophagic flux to promote functional recovery after spinal cord injury.

After spinal cord injury (SCI), the inhibitory molecules derived from scars at the lesion sites and the limited regenerative capacity of neuronal axons pose difficulties for the recovery after SCI. Remodeling of cytoskeleton structures including microtubule assembly and tubulin post-translational modification are widely accepted to play a crucial role in initiation of growth cone and regrowth of injured axon. Although increasing studies have focused on the association between tubulin acetylation and autophagy due to the role of tubulin acetylation in organelles and substances transport, there are no studies exploring the effect of tubulin acetylation on autophagy after spinal cord injury (SCI). Here, we found that histone deacetylase 6 (HDAC6) was significantly up-regulated after SCI, while inhibition of HDAC6 by Tubastatin A induced functional recovery after SCI. In view of enzyme-dependent and -independent mechanisms of HDAC6 to adjust diverse cellular processes, such as autophagy, the ubiquitin proteasome system and post-translational modification of tubulin, we mainly focused on the significance of HDAC6 in axonal regeneration and autophagy after SCI. Western blotting, Co-immunoprecipitation and immunofluorescence staining were conducted to showed that Tubastatin A treatment in nocodazole-treated cells and mice suffering from SCI prompted acetylation and stabilization of microtubules and thus restored transport function, which may contribute to restored autophagic flux and increased axonal length. Whereas inhibition of degradation of autolysosomes by bafilomycin A1 (Baf-A1) reversed functional recovery caused by Tubastatin A, revealing the association between tubulin acetylation and autophagy, which supports HDAC6 inhibition as a potential target for SCI treatment.

A Multi-Biochemical and In Silico Study on Anti-Enzymatic Actions of Pyroglutamic Acid against PDE-5, ACE, and Urease Using Various Analytical Techniques: Unexplored Pharmacological Properties and Cytotoxicity Evaluation.

In the current study, pyroglutamic acid (pGlu), a natural amino acid derivative, has efficiently inhibited the catalytic activities of three important enzymes, namely: Human recombinant phosphodiesterase-5A1 (PDE5A1), human angiotensin-converting enzyme (ACE), and urease. These enzymes were reported to be associated with several important clinical conditions in humans. Radioactivity-based assay, spectrophotometric-based assay, and an Electrospray Ionization-Mass Spectrometry-based method were employed to ascertain the inhibitory actions of pGlu against PDE5A1, ACE, and urease, respectively. The results unveiled that pGlu potently suppressed the activity of PDE5A1 (half-maximal inhibitory concentration; IC50 = 5.23 µM) compared with that of standard drug sildenafil citrate (IC50 = 7.14 µM). Moreover, pGlu at a concentration of 20 µg/mL was found to efficiently inhibit human ACE with 98.2% inhibition compared with that of standard captopril (99.6%; 20 µg/mL). The urease-catalyzed reaction was also remarkably inactivated by pGlu and standard acetohydroxamic acid with IC50 values of 1.8 and 3.9 µM, respectively. Remarkably, the outcome of in vitro cytotoxicity assay did not reveal any significant cytotoxic properties of pGlu against human cervical carcinoma cells and normal human fetal lung fibroblast cells. In addition to in vitro assays, molecular docking analyses were performed to corroborate the outcomes of in vitro results with predicted structure-activity relationships. In conclusion, pGlu could be presented as a natural and multifunctional agent with promising applications in the treatment of some ailments connected with the above-mentioned anti-enzymatic properties.

Tubastatin A inhibits HDAC and Sirtuin activity rather than being a HDAC6-specific inhibitor in mouse oocytes.

Tubastatin A (TubA) is a highly selective histone deacetylase 6 (HDAC6) inhibitor. As expected, mouse germinal vesicle oocytes fail to extrude the first polar body following TubA treatment. However, a previous study demonstrated that homozygous Hdac6 knockout (KO) mice can be viable and fertile. Therefore, we asked whether TubA is indeed a specific inhibitor of HDAC6 activity. RNA-sequencing and in silico analysis demonstrated that the TubA-treated group presented significant changes in the expression of Hdac subfamily genes such as Hdac6, 10, and 11, and Sirtuin 2, 5, 6, and 7. Additionally, gene expression related to the p53, MAPK, Wnt, and Notch signaling pathways in the TubA-treated group were increased significantly; in contrast, gene expression related to metabolism, DNA replication, and oxidative phosphorylation was decreased significantly. Furthermore, gene expression related to cell cycle, cell structure, pyrimidine metabolism, pentose phosphate pathway, mitochondrial activation, proteasome pathway, RNA polymerase, DNA replication, cyclin-dependent kinase, nucleolar activity, and MI arrest were significantly decreased, indicating that TubA-induced abnormal meiotic maturation and oocyte senescence may be due to the combined effects of HDAC and Sirtuin inhibition, and not HDAC6 inhibition alone. Thus, we believed that this system could provide a model for monitoring the effects of TubA on mouse oocytes.

Warhead biosynthesis and the origin of structural diversity in hydroxamate metalloproteinase inhibitors.

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are  potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining.

Chlamydia spp. development is differentially altered by treatment with the LpxC inhibitor LPC-011.

BACKGROUND: Chlamydia species are obligate intracellular bacteria that infect a broad range of mammalian hosts. Members of related genera are pathogens of a variety of vertebrate and invertebrate species. Despite the diversity of Chlamydia, all species contain an outer membrane lipooligosaccharide (LOS) that is comprised of a genus-conserved, and genus-defining, trisaccharide 3-deoxy-D-manno-oct-2-ulosonic acid Kdo region. Recent studies with lipopolysaccharide inhibitors demonstrate that LOS is important for the C. trachomatis developmental cycle during RB- > EB differentiation. Here, we explore the effects of one of these inhibitors, LPC-011, on the developmental cycle of five chlamydial species. RESULTS: Sensitivity to the drug varied in some of the species and was conserved between others. We observed that inhibition of LOS biosynthesis in some chlamydial species induced formation of aberrant reticulate bodies, while in other species, no change was observed to the reticulate body. However, loss of LOS production prevented completion of the chlamydial reproductive cycle in all species tested. In previous studies we found that C. trachomatis and C. caviae infection enhances MHC class I antigen presentation of a model self-peptide. We find that treatment with LPC-011 prevents enhanced host-peptide presentation induced by infection with all chlamydial-species tested. CONCLUSIONS: The data demonstrate that LOS synthesis is necessary for production of infectious progeny and inhibition of LOS synthesis induces aberrancy in certain chlamydial species, which has important implications for the use of LOS synthesis inhibitors as potential antibiotics.

Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V).

Cultures of Shewanella putrefaciens grown in medium containing 10mM 1,4-diamino-2-butanone (DBO) as an inhibitor of ornithine decarboxylase and 10mM 1,5-diaminopentane (cadaverine) showed the simultaneous biosynthesis of the macrocyclic dihydroxamic acids: putrebactin (pbH2), avaroferrin (avH2) and bisucaberin (bsH2). The level of DBO did not completely repress the production of endogenous 1,4-diaminobutane (putrescine) as the native diamine substrate of pbH2. The relative concentration of pbH2:avH2:bsH2 was 1:2:1, which correlated with the substrate selection of putrescine:cadaverine in a ratio of 1:1. The macrocycles were characterised using LC-MS as free ligands and as 1:1 complexes with Fe(III) of the form [Fe(pb)]+, [Fe(av)]+ or [Fe(bs)]+, with labile ancillary ligands in six-coordinate complexes displaced during ESI-MS acquisition; or with Mo(VI) of the form [Mo(O)2(pb)], [Mo(O)2(av)] or [Mo(O)2(bs)]. Chromium(V) complexes of the form [CrO(pb)]+ were detected from solutions of Cr(VI) and pbH2 in DMF using X-band EPR spectroscopy. Supplementation of S. putrefaciens medium with DBO and 1,3-diaminopropane, 1,6-diaminohexane or 1,4-diamino-2(Z)-butene (Z-DBE) resulted only in the biosynthesis of pbH2. The work has identified a native system for the simultaneous biosynthesis of a suite of three macrocyclic dihydroxamic acid siderophores and highlights both the utility of precursor-directed biosynthesis for expanding the structural diversity of siderophores, and the breadth of their coordination chemistry.

Inhibition of Histone Deacetylase Activity Aggravates Coxsackievirus B3-Induced Myocarditis by Promoting Viral Replication and Myocardial Apoptosis.

UNLABELLED: Viral myocarditis, which is most prevalently caused by coxsackievirus B3 (CVB3), is a serious clinical condition characterized by excessive myocardial inflammation. Recent studies suggest that regulation of protein acetylation levels by inhibiting histone deacetylase (HDAC) activity modulates inflammatory response and shows promise as a therapy for several inflammatory diseases. However, the role of HDAC activity in viral myocarditis is still not fully understood. Here, we aim to investigate the role of HDAC activity in viral myocarditis and its underlying mechanism. CVB3-infected BALB/c mice were treated with the HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA). We found inhibition of HDAC activity aggravated rather than ameliorated the severity of CVB3-induced myocarditis, which was contrary to our expectations. The aggravated myocarditis by HDACI treatment seemed not to be caused by an elevated inflammatory response but by the increased CVB3 replication. Further, it was revealed that the increased CVB3 replication was closely associated with the HDACI-enhanced autophagosome formation. Inhibition of autophagosome formation by wortmannin or ATG5 short hairpin RNA dramatically suppressed the HDACI-increased CVB3 replication. The increased viral replication subsequently elevated CVB3-induced myocardial apoptosis. Conversely, inhibition of CVB3 replication and ensuing myocardial apoptosis by the antiviral drug ribavirin significantly reversed the HDACI-aggravated viral myocarditis. In conclusion, we elucidate that the inhibition of HDAC activity increases CVB3 replication and ensuing myocardial apoptosis, resulting in aggravated viral myocarditis. Possible adverse consequences of administering HDACI should be considered in patients infected (or coinfected) with CVB3. IMPORTANCE: Viral myocarditis, which is most prevalently caused by CVB3, is characterized by excessive myocardial inflammation. Inhibition of HDAC activity was originally identified as a powerful anti-cancer therapeutic strategy and was recently found to be implicated in the regulation of inflammatory response. HDACI has been demonstrated to be efficacious in animal models of several inflammatory diseases. Thus, we hypothesize that inhibition of HDAC activity also protects against CVB3-induced viral myocarditis. Surprisingly, we found inhibition of HDAC activity enhanced myocardial autophagosome formation, which led to the elevated CVB3 viral replication and ensuing increased myocardial apoptosis. Viral myocarditis was eventually aggravated rather than ameliorated by HDAC inhibition. In conclusion, we elucidate the role of HDAC activity in viral myocarditis. Moreover, given the importance of HDACI in preclinical and clinical treatments, the possible unfavorable effect of HDACI should be carefully evaluated in patients infected with viruses, including CVB3.

Trichostatin A specifically stimulates gonadotropin FSHß gene expression in gonadotroph LßT2 cells.

Trichostatin A (TSA) is a selective inhibitor of mammalian histone deacetylase. In the present study, TSA was found to selectively increase gene expression of the pituitary gonadotropin ß-subunit of follicle-stimulating hormone (FSH). Stimulation of mouse pituitary gonadotroph cell lines, LßT2, with TSA for 24 h resulted in no change in mRNA expression of the α- and LHß-subunit. On the other hand, FSHß-subunit mRNA expression was significantly increased in a dose-dependent fashion. Similarly, specific induction of the FSHß-subunit gene with TSA stimulation was observed in primary cultures of rat pituitary cells. Histone acetylation in whole cell lysates of LßT2 cells was significantly increased after TSA treatment, but not gonadotropin-releasing hormone (GnRH) treatment. The effect of TSA on FSHß mRNA expression was prominent compared to that of GnRH; however, TSA-stimulated FSHß mRNA expression was significantly reduced with combined TSA and GnRH treatment. TSA caused a slight increase in extracellular signal-regulated kinase (ERK) phosphorylation, while GnRH-increased ERK phosphorylation was potentiated in the presence of TSA. In addition, TSA, but not GnRH, significantly stimulated gene expression of retinaldehyde dehydrogenase 1 (RALDH1), a retinoic acid (RA) synthesizing enzyme involved in cell differentiation. These findings demonstrate that TSA specifically increases FSHß subunit gene expression with a concomitant increase in whole cell histone acetylation. Moreover, although GnRH is a stimulator of FSHß gene expression, it interfered with the stimulatory effect of TSA on FSHß mRNA expression, without modification of TSA-increased whole cell histone acetylation. This suggests that the mechanisms of TSA and GnRH-induced gonadotropin subunit gene expression are entirely distinct.

Induction of the liver cancer-down-regulated long noncoding RNA uc002mbe.2 mediates trichostatin-induced apoptosis of liver cancer cells.

Differential expression of long non-coding RNAs (lncRNAs) plays critical roles in hepatocarcinogenesis. Considerable attention has focused on the antitumor effect of histone deacetylase inhibitor (Trichostatin A, TSA) as well as the coding gene expression-induced apoptosis of cancer cells. However, it is not known whether lncRNA has a role in TSA-induced apoptosis of human hepatocellular carcinoma (HCC) cells. The global expression of lncRNAs and coding genes was analyzed with the Human LncRNA Array V2.0 after 24 h treatment. Expression was verified in cell lines and tissues by quantitative real-time PCR. The data showed that 4.8% (959) of lncRNA and 6.1% (1849) of protein coding gene were significantly differentially expressed. The differential expressions of lncRNA and protein coding genes had distinguishable hierarchical clustering expression profiling pattern. Among these differentially expressed lncRNAs, the greatest change was noted for uc002mbe.2, which had more than 300 folds induction upon TSA treatment. TSA selectively induced uc002mbe.2 in four studied HCC cell lines. Compared with normal human hepatocytes and adjacent noncancerous tissues, uc002mbe.2 expression level was significantly lower in the HCC cell lines and liver cancer tissues. The TSA-induced uc002mbe.2 expression was positively correlated with the apoptotic effect of TSA in HCC cells. In addition, knockdown the expression of uc002mbe.2 significantly reduced TSA-induced apoptosis of Huh7cells. Therefore, TSA-induced apoptosis of HCC cells is uc002mbe.2 dependent and reduced expression of uc002mbe.2 may be associated with liver carcinogenesis.

Okadaic acid inhibits the trichostatin A-mediated increase of human CYP46A1 neuronal expression in a ERK1/2-Sp3-dependent pathway.

The CYP46A1 gene codes for the cholesterol 24-hydroxylase, a cytochrome P450 specifically expressed in neurons and responsible for the majority of cholesterol turnover in the central nervous system. Previously, we have demonstrated the critical participation of Sp transcription factors in the CYP46A1 response to histone deacetylase (HDAC) inhibitors, and in this study we investigated the involvement of intracellular signaling pathways in the trichostatin A (TSA) effect. Our results show that pretreatment of neuroblastoma cells with chemical inhibitors of mitogen-activated kinase kinase (MEK)1 significantly potentiates the TSA-dependent induction of cholesterol 24-hydroxylase, whereas inhibition of protein phosphatases by okadaic acid (OA) or overexpression of MEK1 partially impairs the TSA effect without affecting histone hyperacetylation at the promoter. Immunoblotting revealed that TSA treatment decreases ERK1/2 phosphorylation concomitantly with a decrease in Sp3 binding activity, which are both reversed by pretreatment with OA. Chromatin immunoprecipitation analysis demonstrated that TSA induces the release of p-ERK1/2 from the CYP46A1 proximal promoter, whereas pretreatment with OA restores the co-occupancy of Sp3-ERK1/2 in the same promoter fragments. We demonstrate for the first time the participation of MEK-ERK1/2 signaling pathway in HDAC inhibitor-dependent induction of cytochrome P450 gene expression, underlying the importance of this regulatory signaling mechanism in the control of brain cholesterol elimination.

Binding of 5-phospho-D-arabinonohydroxamate and 5-phospho-D-arabinonate inhibitors to zinc phosphomannose isomerase from Candida albicans studied by polarizable molecular mechanics and quantum mechanics.

Type I phosphomannose isomerase (PMI) is a Zn-dependent metalloenzyme involved in the isomerization of D-fructose 6-phosphate to D-mannose 6-phosphate. One of our laboratories has recently designed and synthesized 5-phospho-D-arabinonohydroxamate (5PAH), an inhibitor endowed with a nanomolar affinity for PMI (Roux et al., Biochemistry 2004, 43, 2926). By contrast, the 5-phospho-D-arabinonate (5PAA), in which the hydroxamate moiety is replaced by a carboxylate one, is devoid of inhibitory potency. Subsequent biochemical studies showed that in its PMI complex, 5PAH binds Zn(II) through its hydroxamate moiety rather than through its phosphate. These results have stimulated the present theoretical investigation in which we resort to the SIBFA polarizable molecular mechanics procedure to unravel the structural and energetical aspects of 5PAH and 5PAA binding to a 164-residue model of PMI. Consistent with the experimental results, our theoretical studies indicate that the complexation of PMI by 5PAH is much more favorable than by 5PAA, and that in the 5PAH complex, Zn(II) ligation by hydroxamate is much more favorable than by phosphate. Validations by parallel quantum-chemical computations on model of the recognition site extracted from the PMI-inhibitor complexes, and totaling up to 140 atoms, showed the values of the SIBFA intermolecular interaction energies in such models to be able to reproduce the quantum-chemistry ones with relative errors < 3%. On the basis of the PMI-5PAH SIBFA energy-minimized structure, we report the first hypothesis of a detailed view of the active site of the zinc PMI complexed to the high-energy intermediate analogue inhibitor, which allows us to identify active site residues likely involved in the proton transfer between the two adjacent carbons of the substrates.

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453.

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.

Substrate and inhibitor specificity of class 1 and class 2 histone deacetylases.

Histone deacetylases (HDACs) are key enzymes in the transcriptional regulation of gene expression in eukaryotic cells. In recent years HDACs have attracted considerable attention as promising new targets in anticancer therapy. Currently, different histone deacetylase subtypes are divided into four groups denoted as classes 1-4. Here, we compare in more detail representatives of class 1 HDACs and FB188 HDAH as a close bacterial homologue of class 2 HDAC6, in regard of substrate and inhibitor specificity. Structure comparison is used to identify candidate regions responsible for observed specificity differences. Knowledge of these structural elements expedite studies on the biochemical role of different HDAC subtypes as well as the development of highly selective HDAC inhibitors as antitumor agents.

Histone deacetylase inhibitor-mediated radiosensitization of human cancer cells: class differences and the potential influence of p53.

Histone deacetylase inhibitors (HDI) are emerging as potentially useful components of the anticancer armamentarium and as useful tools to dissect mechanistic pathways. HDIs that globally inhibit histone deacetylases (HDAC) have radiosensitizing effects, but the relative contribution of specific HDAC classes remains unclear. Newly characterized HDIs are now available that preferentially inhibit specific HDAC classes, including SK7041 (inhibits class I HDACs) and splitomicin (inhibits class III HDACs). We investigated in human cancer cells the relative radiosensitizations that result from blocking specific HDAC classes. We found that trichostatin A (TSA; inhibitor of both class I and II HDACs) was the most effective radiosensitizer, followed by the class I inhibitor SK7041, whereas splitomicin (inhibitor of class III) had least effect. Interestingly, radiosensitization by TSA in cell lines expressing p53 was more pronounced than in isogenic lines lacking p53. Radiosensitization of cells expressing p53 by TSA was reduced by pifithrin-alpha, a small-molecule inhibitor of p53. In contrast, the radiosensitization by TSA of cells expressing low levels of p53 was enhanced by transfection of wild-type p53-expressing vector or pretreatment with leptomycin B, an inhibitor of nuclear export that increased intracellular levels of p53. These effects on radiosensitization were respectively muted or not seen in cells treated with SK7041 or splitomicin. To our knowledge, this may be among the first systematic investigations of the comparative anticancer effects of inhibiting specific classes of HDACs, with results suggesting differences in the degrees of radiosensitization, which in some cell lines may be influenced by p53 expression.

A novel series of histone deacetylase inhibitors incorporating hetero aromatic ring systems as connection units.

A series of structurally novel HDAC inhibitors, in which a hetero aromatic ring connects the spacer with the hydrophobic group, has been designed and synthesized. These new inhibitors are very potent in in vitro enzymatic assays and display antiproliferation activity against two human cancer cell lines.

Reduction of experimental laser-induced choroidal neovascularization by orally administered BPHA, a selective metalloproteinase inhibitor.

BACKGROUND: N-Biphenyl sulfonyl-phenylalanine hydroxamic acid (BPHA), a synthetic, selective matrix metalloproteinase (MMP)-2, -9, -14 inhibitor, has been reported to show significant antiangiogenic activity without unpleasant adverse effects. After film in situ zymography (FIZ) and conventional zymography were performed to detect MMP in experimental choroidal neovascularizations (CNVs), we studied the reducible effect of BPHA on CNVs. METHODS: Using FIZ, the gelatinolytic activity of MMPand BPHA-reduction on gelatinolysis were examined in diode-laser-induced CNV lesions in a total of 22 male Brown Norway rats. The MMP subtypes were studied in the CNV lesions of three rats using conventional zymography. Vehicle solution only or 25-, 50-, or 100 mg/kg-body-weight of BPHA was administered orally twice daily for 14 days after the laser photocoagulation in 18 rats, respectively. Fluorescein angiograms were taken, and the late hyperfluorescence of CNVs was given scores by three researchers using four grades. The thickness of CNV lesions was studied histologically. RESULTS: In laser-induced CNVs, the gelatinolytic activity of MMP and reduction of gelatinolysis by BPHA were observed on FIZ, and MMP-2 and proMMP-2 were identified by conventional zymography. The scores given to the late dye leakage and staining on angiograms were lower in the BPHA-treated groups ( p<0.01) than in the controls, and the effect appeared to be dose-dependent. Similarly, the CNV lesions in the BPHA-treated groups were less thick than in the controls ( p<0.01). CONCLUSIONS: MMP-2 played a role in laser-induced CNV development, and administration of BPHA reduced the experimental CNVs.

Chemical genetic modifier screens: small molecule trichostatin suppressors as probes of intracellular histone and tubulin acetylation.

Histone deacetylase (HDAC) inhibitors are being developed as new clinical agents in cancer therapy, in part because they interrupt cell cycle progression in transformed cell lines. To examine cell cycle arrest induced by HDAC inhibitor trichostatin A (TSA), a cytoblot cell-based screen was used to identify small molecule suppressors of this process. TSA suppressors (ITSAs) counteract TSA-induced cell cycle arrest, histone acetylation, and transcriptional activation. Hydroxamic acid-based HDAC inhibitors like TSA and suberoylanilide hydroxamic acid (SAHA) promote acetylation of cytoplasmic alpha-tubulin as well as histones, a modification also suppressed by ITSAs. Although tubulin acetylation appears irrelevant to cell cycle progression and transcription, it may play a role in other cellular processes. Small molecule suppressors such as the ITSAs, available from chemical genetic suppressor screens, may prove to be valuable probes of many biological processes.

Multiple metalloproteinases process protransforming growth factor-alpha (proTGF-alpha).

Shedding of TNF-alpha requires a single cleavage event, whereas the ectodomain of proTGF-alpha is cleaved at N-proximal (N-terminal) and membrane proximal (C-terminal) sites to release mature TGF-alpha. Tumor necrosis factor-alpha converting enzyme (TACE) was shown to have a central role in the shedding of both factors. Here we show that cleavage of the proTGF-alpha C-terminal site, required for release of mature growth factor, is less sensitive to a panel of hydroxamates than TNF-alpha processing. Recombinant TACE cleaves TNF-alpha and N-terminal TGF-alpha peptides 50-fold more efficiently than the C-terminal TGF-alpha peptide. Moreover, fractionation of rat liver epithelial cell membranes yields two populations: one contains TACE and cleaves peptides corresponding to TNF-alpha and both proTGF-alpha processing sites, while the other lacks detectable TACE and cleaves only the C-terminal proTGF-alpha processing site. Activities in both fractions are inhibited by hydroxamates and EDTA but not by cysteine, aspartate, or serine protease inhibitors. Both membrane fractions also contain ADAM 10. ADAM 10 correctly cleaves peptides and a soluble form of precursor TGF-alpha (proTGFecto) at the N-terminal site but not the C-terminal site. However, the kinetics of N-terminal peptide cleavage by ADAM 10 are 90-fold less efficient than TACE. Our findings indicate that while TACE is an efficient proTGF-alpha N-terminal convertase, a different activity, distinguishable from TACE, exists that can process proTGF-alpha at the C-terminal site. A model that accounts for these findings and the requirement for TACE in TGF-alpha shedding is proposed.

Small molecule modulation of the human chromatid decatenation checkpoint.

After chromosome replication, the intertwined sister chromatids are disentangled by topoisomerases. The integrity of this process is monitored by the chromatid decatenation checkpoint. Here, we describe small molecule modulators of the human chromatid decatenation checkpoint identified using a cell-based, chemical genetic modifier screen. Similar to 1,2,7-trimethylyxanthine (caffeine), these small molecules suppress the G(2)-phase arrest caused by ICRF-193, a small molecule inhibitor of the enzymatic activity of topoisomerase II. Analysis of specific suppressors, here named suptopins for suppressor of Topoisomerase II inhibition, revealed distinct effects on cell cycle progression, microtubule stability, nucleocytoplasmic transport of cyclin B1, and no effect on the chromatin deacetylation checkpoint induced by trichostatin A. The suptopins provide new molecular tools for dissecting the role of topoisomerases in maintaining genomic stability and determining whether inhibiting the chromatid decatenation checkpoint sensitizes tumor cells to chemotherapeutics.

Trichostatin A, lead compound for development of antifibrogenic drugs.

Eukaryotic gene expression has mainly been studied in the context of trans-acting transcription factors and their interaction with regulatory cis-elements. Evidence is accumulating, that the higher order structure of chromatin also plays an essential role in eukaryotic gene expression. Hepatic stellate cells are the major cellular source of extracellular matrix synthesis in chronic liver diseases leading to fibrosis. We explored the antifibrogenic effect of the histone deacetylase inhibitor trichostatin A (TSA) on hepatic stellate cells in vitro. Primary hepatic stellate cells as well as activated, subcultured stellate cells were exposed to 10(-7) M-10(-9) M TSA. Collagens type I and III, and smooth muscle alpha-actin (alpha-SMA), a marker for transdifferentiation, were investigated at the protein and mRNA level by performing Northern hybridisation and quantitative immunoprecipitation. The antiproliferative effect was examined by 3H-thymidine incorporation and cell counting. Hyperacetylation of histone H4 was demonstrated by acid urea Triton-X-100 (AUT) polyacrylamide gel electrophoresis. TSA at 10(-7) M retarded the morphological changes characteristics for activation of primary stellate cells. Synthesis of collagens type I and III, and alpha-SMA was strongly inhibited at both protein and mRNA level. The proliferation rate of primary hepatic stellate cells was strongly suppressed by 10(-7) M TSA. Hyperacetylation of histone H4 showed to be maximal at 10(-7) M TSA. Primary hepatic stellate cells were more affected by TSA than subcultured stellate cells.