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Biomedical subjects

Jun O Liu

Publications and source records attributed to Jun O Liu.

34 records · Page 2Linked to original sources
PubMed2005

3-Aryl beta-carbolin-1-ones as a new class of potent inhibitors of tumor cell proliferation: synthesis and biological evaluation.

A novel three-step synthesis of 3-aryl beta-carbolin-1-ones from non-indole starting materials has been developed. The two nitrogen atoms in beta-carbolin-1-one were introduced efficiently by Michael addition of ethyl acetamidocyanoacetate to chalcone. The desired pyridone and indole rings were assembled by an intramolecular ketone-nitrile annulation mediated by aqueous HCl-HOAc and a Cu(I)-catalyzed intramolecular N-arylation of the amide, respectively. The target compounds were found to possess significant activity against tumor cell proliferation.

Antineoplastic Agents↗
PubMed2005

Mechanism of recruitment of class II histone deacetylases by myocyte enhancer factor-2.

Class II histone deacetylases (HDACs) bind myocyte enhancer factor-2 (MEF2) and repress specific gene expression in a calcium-dependent manner. Despite their significant physiological functions in muscle, immune and neuronal cells, the mechanism of recruitment of class II HDACs by MEF2 is not well understood. Here, we have characterized the complex between the MEF2-binding motif of class II HDACs and the MADS-box/MEF2S domain of MEF2B by structural and biochemical methods. The crystal structure of a HDAC9/MEF2/DNA complex reveals that HDAC9 binds to a hydrophobic groove of the MEF2 dimer. The overall binding mode is similar to that seen in the Cabin1/MEF2/DNA complex. The detailed binding interactions at the HDAC9/MEF2 interface, however, show marked differences from those at the Cabin1/MEF2 interface. Our studies suggest a general mechanism by which class II HDACs and possibly other transcriptional co-regulators are recruited by MEF2. On the other hand, the differential binding between MEF2 and its various partners may confer specific regulatory and functional properties to MEF2 in distinct cellular processes. Such specificity provides a basis for selectively disrupting a particular MEF2/co-regulator complex by mutations or small molecules.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The yins of T cell activation.

Transcription factors activated in response to T cell receptor (TCR) signaling include nuclear factor of activated T cells (NFAT) family, which is highly phosphorylated and thereby maintained in the cytoplasm of resting T cells, the nuclear factor NF-kappaB, which is kept in the cytoplasm of resting cells through its association with the inhibitor protein IkappaB, and activating protein-1 (AP-1), which is only transcribed after TCR stimulation. Negative regulators of TCR signaling can be divided into two groups: Class 1 regulators help maintain the quiescent state of unstimulated T cells, whereas class 2 regulators are themselves transcriptionally induced in response to TCR signaling and serve to limit and terminate the activating signal. Class 1 regulators include the autoinhibitory domain of the phosphatase calcineurin; IkappaB and its transcriptional activators Foxj1 and Foxo3a; and various transcriptional coregulators that inhibit interleukin-2 (IL-2) production. Class 2 regulators include the calcipressins, which, like NFATp and NFAT4 are feedback inhibitors of calcineurin-NFAT signaling, IkappaB, and the mitogen-activated protein kinase (MAPK) phosphatases, which inhibit MAPK signaling and thus the nuclear localization of AP-1 components.

Animals↗
PubMed2004

The C. elegans methionine aminopeptidase 2 analog map-2 is required for germ cell proliferation.

We have investigated the physiological function of type 2 methionine aminopeptidases (MetAP2) using Caenorhabditis elegans as a model system. A homolog of human MetAP2 was found in the C. elegans genome, which we termed MAP-2. MAP-2 protein displayed methionine aminopeptidase activity and was sensitive to inhibition by fumagillin. Downregulation of map-2 expression by RNAi led to sterility, resulting from a defect in germ cell proliferation. These observations suggest that MAP-2 is essential for germ cell development in C. elegans and that this ubiquitous enzyme may play important roles in a tissue specific manner.

Amino Acid Sequence↗
PubMed2004

Evidence for separate binding and scaffolding domains in the immunosuppressive and antitumor marine natural product, pateamine a: design, synthesis, and activity studies leading to a potent simplified derivative.

Pateamine A (PatA), a marine metabolite from Mycale sp., is a potent inhibitor of the intracellular signal transduction pathway emanating from the T-cell receptor leading to the transcription of cytokines such as interleukin-2 (IL-2). On the basis of the structure of PatA and initial biological results, a hypothesis was developed regarding the presence of distinct binding and scaffolding domains in the PatA structure with respect to interactions with its putative cellular receptor(s). Employing a highly convergent approach involving a Hantzsch coupling strategy, we probed this hypothesis by preparing a simplified PatA derivative (desmethyl, desamino PatA, DMDAPatA, 3). This derivative was prepared in 10 fewer synthetic steps relative to PatA and was indeed found to exhibit equal to greater potency (IC50 0.81 +/- 0.27 nM) in inhibition of IL-2 production relative to PatA (IC50 4.01 +/- 0.94 nM) thus providing support for the binding/scaffolding domain hypothesis. In addition, as a means to find more stable derivatives and gain further insights into structure-activity relationships, several PatA derivatives were synthesized and assayed in the IL-2 reporter gene assay. Several of these derivatives displayed lower potency but marked stability relative to the natural product and provide further insights into the nature of the binding domain required for activity.

Antineoplastic Agents↗
PubMed2004

Myocyte enhancer factor 2 mediates calcium-dependent transcription of the interleukin-2 gene in T lymphocytes: a calcium signaling module that is distinct from but collaborates with the nuclear factor of activated T cells (NFAT).

The second messenger calcium plays an essential role in the T cell receptor-mediated signal transduction pathways leading to transcription of the interleukin-2 gene. A key mechanism of calcium signaling has been shown to be mediated by calcineurin and NFAT. We report herein that the transcription factor myocyte enhancer factor (MEF)-2 is another calcium signal transducer involved in the regulation of the interleukin (IL)-2 promoter. A MEF2-binding site was identified in proximity to the TATA box of the IL-2 promoter. This site was shown to be bound by MEF2 in both resting and activated T cells. Overexpression of MEF2 enhanced, while overexpression of a dominant negative form of MEF2 or the MEF2-specific transcriptional corepressors Cabin1 and histone deacetylase 4 inhibited, the T cell receptor-dependent activation of an IL-2 reporter gene. Down-regulation of MEF2 by RNA interference in primary human T cells led to the inhibition of endogenous IL-2 transcription. These results suggest that MEF2 is required for the transcriptional activation of IL-2 and likely other cytokine genes in response to calcium signaling and may serve as a novel target for development of immunosuppressants.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Nur77 activated by hypoxia-inducible factor-1alpha overproduces proopiomelanocortin in von Hippel-Lindau-mutated renal cell carcinoma.

Mutation in the von Hippel-Lindau (VHL) protein associated with renal cell carcinoma causes hypoxia-inducible factor (HIF) to stabilize and consequently to induce various HIF-targeting proteins. In this study, we found that proopiomelanocortin (POMC), an adrenocorticotropic hormone precursor, is up-regulated constitutively in VHL-mutated renal cell carcinoma. A critical transcription factor responsible for POMC overproduction was identified as Nur77, a member of the orphan steroid receptor superfamily. Little is known about how VHL mutation leads to activation of Nur77. We report that Nur77 is directly regulated by HIF. We show that HIF-1alpha, but not HIF-2alpha, binds to a putative HIF responsive element in the Nur77 promoter, activating the expression of Nur77. Mutation or deletion of the HIF binding site in the Nur77 promoter abrogates activation of a luciferase reporter gene under the control of Nur77 promoter by HIF-1alpha. The treatment of Nur77 antisense oligonucleotide reduces POMC transcription under hypoxic conditions. We confirmed that Nur77 and POMC are up-regulated in VHL-mutated renal cell carcinoma. In this study, we provide the first molecular evidence that Nur77 activated by HIF under hypoxic conditions regulates production of the peptide hormone precursor POMC.

Amino Acid Substitution↗
PubMed2003

Endogenous protein inhibitors of calcineurin.

A growing family of endogenous inhibitors of calcineurin has been identified in recent years. These endogenous calcineurin inhibitors are throwing new light on the function and regulation of calcineurin in a wide variety of cellular processes and cell types.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Menin, a tumor suppressor, represses JunD-mediated transcriptional activity by association with an mSin3A-histone deacetylase complex.

Menin, a gene product of multiple endocrine neoplasia type I (MEN1), is known to act as a tumor suppressor to repress JunD transcription factor. However, the mechanism by which Menin represses JunD transcriptional activity was still unclear. In this study, we found that Menin is a corepressor against JunD transcriptional activity via recruitment of histone deacetylases in an mSin3A-dependent manner. The amino acid search revealed that central domain of Menin includes a alpha-helical mSin3-interacting domain [SID (371-387)]. The SID mutation of Menin (L381P/A385P) abolished the interaction between mSin3A and paired amphipathic helix 2 domain of Menin and reduced its ability to repress JunD transcriptional activity, implicating that SID of Menin is important for recruiting an mSin3A-histone deacetylase complex to repress JunD transcriptional activity.

Amino Acid Sequence↗
PubMed2003

Fumagalone, a reversible inhibitor of type 2 methionine aminopeptidase and angiogenesis.

Fumagillin and ovalicin constitute a family of structurally related natural products that possess antiangiogenic activity. We report the synthesis of a new fumagillin analogue, fumagalone, in which the spiroepoxide group is replaced with an aldehyde. Fumagalone inhibits type 2 methionine aminopeptidase (MetAP2) with IC(50) = 8 microM and endothelial cell proliferation with IC(50) = 52 nM. With dialysis and competition assays, it was unambiguously demonstrated that binding of fumagalone to MetAP2 is reversible.

Aminopeptidases↗
PubMed2003

Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B.

The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes. Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form. In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes. Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C. We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues. Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications. Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form. The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120. Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway. These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions.

Amino Acid Sequence↗
PubMed2003

Sequence-specific recruitment of transcriptional co-repressor Cabin1 by myocyte enhancer factor-2.

The myocyte enhancer factor-2 (MEF2) family of transcription factors has important roles in the development and function of T cells, neuronal cells and muscle cells. MEF2 is capable of repressing or activating transcription by association with a variety of co-repressors or co-activators in a calcium-dependent manner. Transcriptional repression by MEF2 has attracted particular attention because of its potential role in hypertrophic responses of cardiomyocytes. Several MEF2 co-repressors, such as Cabin1/Cain and class II histone deacetylases (HDACs), have been identified. However, the molecular mechanism of their recruitment to specific promoters by MEF2 remains largely unknown. Here we report a crystal structure of the MADS-box/MEF2S domain of human MEF2B bound to a motif of the transcriptional co-repressor Cabin1 and DNA at 2.2 A resolution. The crystal structure reveals a stably folded MEF2S domain on the surface of the MADS box. Cabin1 adopts an amphipathic alpha-helix to bind a hydrophobic groove on the MEF2S domain, forming a triple-helical interaction. Our studies of the ternary Cabin1/MEF2/DNA complex show a general mechanism by which MEF2 recruits transcriptional co-repressor Cabin1 and class II HDACs to specific DNA sites.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes.

Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the common target for two immunophilin-immunosuppressant complexes, cyclophilin A-cyclosporin A (CyPA-CsA) and FKBP-FK506. How the two structurally distinct immunophilin-drug complexes bind the same target has remained unknown. We report the crystal structure of calcineurin (CN) in complex with CyPA-CsA at 2.8-A resolution. The CyPA-CsA complex binds to a composite surface formed by the catalytic and regulatory subunits of CN, where the complex of FK506 and its binding protein FKBP also binds. While the majority of the CN residues involved in the binding are common for both immunophilin-immunosuppressant complexes, a significant number of the residues are distinct. Unlike FKBP-FK506, CyPA-CsA interacts with Arg-122 at the active site of CN, implying direct involvement of CyPA-CsA in the regulation of CN catalysis. The simultaneous interaction of CyPA with both the composite surface and the active site of CN suggests that the composite surface may serve as a substrate recognition site responsible for the narrow substrate specificity of CN. The comparison of CyPA-CsA-CN with FKBP-FK506-CN significantly contributes to understanding the molecular basis of regulation of CN activity by the immunophilin-immunosuppressant.

Calcineurin↗
PubMed2002

Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death.

Cain/cabin1 is an endogenous inhibitor of calcineurin (Cn), a calcium-dependent serine/threonine phosphatase involved in various cellular functions including apoptosis. We show here that during apoptosis cain/cabin1 is cleaved by calpain at the carboxyl terminus to generate a cleavage product with a molecular mass of 32 kDa as a necessary step leading to Cn-mediated cell death. Mouse cain/cabin1 was identified from a thymus cDNA library by an in vitro substrate-screening assay with calpain. Exposure of Jurkat cells to the calcium ionophore, induced cain/cabin1 cleavage and cell death, accompanied by activation of calpain and Cn. The calpain inhibitors, calpeptin and zLLY, suppressed both -induced cain/cabin1 cleavage and Cn activation, indicating that Cn activation and cain/cabin1 cleavage are calpain-dependent. Expression of cain/cabin1 or a catalytically inactive Cn mutant [CnA beta(2)(1-401/H160N)] and treatment with FK506 reduced -induced cell death. In vitro calpain cleavage and immunoprecipitation assays with deletion mutants of cain/cabin1 showed that cleavage occurred in the Cn-binding domain of cain/cabin1, indicating that the cleavage at its C terminus by calpain prevented cain/cabin1 from binding to Cn. In addition, in vitro binding assays showed that cain/cabin1 bound to the Cn B-binding domain of Cn A. Taken together, these results indicate that calpain cleaves the calcineurin-binding domain of cain/cabin1 to activate Cn and elicit calcium-triggered cell death.

Adaptor Proteins, Signal Transducing↗