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Biomedical subjects

Jun O Liu

Publications and source records attributed to Jun O Liu.

At least 19 recordsLinked to original sources
PubMed2007

Feedback inhibition of calcineurin and Ras by a dual inhibitory protein Carabin.

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression.

Processing of the N-terminal initiator methionine is an essential cellular process conserved from prokaryotes to eukaryotes. The enzymes that remove N-terminal methionine are known as methionine aminopeptidases (MetAPs). Human MetAP2 has been shown to be required for the proliferation of endothelial cells and angiogenesis. The physiological function of MetAP1, however, has remained elusive. In this report we demonstrate that a family of inhibitors with a core structure of pyridine-2-carboxylic acid previously developed for the bacterial and yeast MetAP1 is also specific for human MetAP1 (HsMetAP1), as confirmed by both enzymatic assay and high-resolution x-ray crystallography. Treatment of tumor cell lines with the MetAP1-specific inhibitors led to an accumulation of cells in the G(2)/M phase, suggesting that HsMetAP1 may play an important role in G(2)/M phase transition. Overexpression of HsMetAP1, but not HsMetAP2, conferred resistance of cells to the inhibitors, and the inhibitors caused retention of N-terminal methionine of a known MetAP substrate, suggesting that HsMetAP1 is the cellular target for the inhibitors. In addition, when HsMetAP1 was knocked down by gene-specific siRNA, cells exhibited slower progression during G(2)/M phase, a phenotype similar to cells treated with MetAP1 inhibitors. Importantly, MetAP1 inhibitors were able to induce apoptosis of leukemia cell lines, presumably as a consequence of their interference with the G(2)/M phase checkpoint. Together, these results suggest that MetAP1 plays an important role in G(2)/M phase of the cell cycle and that it may serve as a promising target for the discovery and development of new anticancer agents.

Aminopeptidases↗
PubMed2006

Synthesis, characterization, and utility of thermoresponsive natural/unnatural product macroligands for affinity chromatography.

[Structure: see text] The synthesis and characterization of thermoresponsive, water-soluble poly-N-isopropyl acrylamide (PNIPAM) derived macroligands displaying cyclosporin A (CsA) and dexamethasone (Dex) for use as novel affinity resins are described. Characterization of these soluble macroligands, including ligand loading and integrity, was determined by 1H NMR spectroscopy. One of the CsA macroligands was used in a protein affinity experiment to capture known binding proteins of CsA, the cyclophilins, from Jurkat T-cell lysates.

Chromatography, Affinity↗
PubMed2006

Total synthesis of the cyclodepsipeptide apratoxin A and its analogues and assessment of their biological activities.

A novel total synthesis of apratoxin A is described, with key steps including the assembly of its ketide segment through a D-proline-catalyzed direct aldol reaction and Oppolzer's anti aldol reaction and the preparation of its thiazoline unit in a biomimetic synthesis. An oxazoline analogue of apratoxin A has also been elaborated by a similar approach. This compound has a potency against HeLa cell proliferation only slightly lower than that of apratoxin A, whilst a C(40)-demethylated oxazoline analogue of apratoxin A displays a much lower cytotoxicity and the C(37)-epimer and C(37) demethylation product of this new analogue are inactive. These results suggest that the two methyl groups at C(37) and C(40) and the stereochemistry at C(37) are essential for the potent cellular activity of the oxazoline analogue of apratoxin A. Further biological analysis revealed that both synthetic apratoxin A and its oxazoline analogue inhibited cell proliferation by causing cell cycle arrest in the G1 phase.

Antineoplastic Agents↗
PubMed2006

Fumarranol, a rearranged fumagillin analogue that inhibits angiogenesis in vivo.

The fumagillin family of natural products inhibits angiogenesis through the irreversible inhibition of the type 2 methionine aminopeptidase (MetAP2). Herein is reported a novel fumagillin analogue named fumarranol. It is shown that, like fumagillin, fumarranol selectively inhibits MetAP2 and endothelial cell proliferation. It is also active in a mouse model of angiogenesis in vivo. Unlike TNP-470, fumarranol does not covalently bind to MetAP2. Fumarranol may serve as a lead for a new class of angiogenesis inhibitors.

Aminopeptidases↗
PubMed2006

Inhibitors of Plasmodium falciparum methionine aminopeptidase 1b possess antimalarial activity.

With >1 million deaths annually, mostly among children in sub-Saharan Africa, malaria poses one of the most critical challenges in medicine today. Although introduction of the artemisinin class of antimalarial drugs has offered a temporary solution to the problem of drug resistance, new antimalarial drugs are needed to ensure effective control of the disease in the future. Herein, we have investigated members of the methionine aminopeptidase family as potential antimalarial targets. The Plasmodium falciparum methionine aminopeptidase 1b (PfMetAP1b), one of four MetAP proteins encoded in the P. falciparum genome, was cloned, overexpressed, purified, and used to screen a 175,000-compound library for inhibitors. A family of structurally related inhibitors containing a 2-(2-pyridinyl)-pyrimidine core was identified. Structure/activity studies led to the identification of a potent PfMetAP1b inhibitor, XC11, with an IC(50) of 112 nM. XC11 was highly selective for PfMetAP1b and did not exhibit significant cytotoxicity against primary human fibroblasts. Most importantly, XC11 inhibited the proliferation of P. falciparum strains 3D7 [chloroquine (CQ)-sensitive] and Dd2 (multidrug-resistant) in vitro and is active in mouse malaria models for both CQ-sensitive and CQ-resistant strains. These results suggest that PfMetAP1b is a promising target and XC11 is an important lead compound for the development of novel antimalarial drugs.

Amino Acid Sequence↗
PubMed2006

Eukaryotic initiation factor 2alpha-independent pathway of stress granule induction by the natural product pateamine A.

Stress granules are aggregates of small ribosomal subunits, mRNA, and numerous associated RNA-binding proteins that include several translation initiation factors. Stress granule assembly occurs in the cytoplasm of higher eukaryotic cells under a wide variety of stress conditions, including heat shock, UV irradiation, hypoxia, and exposure to arsenite. Thus far, a unifying principle of eukaryotic initiation factor 2alpha phosphorylation prior to stress granule formation has been observed from the majority of experimental evidence. Pateamine A, a natural product isolated from marine sponge, was recently reported to inhibit eukaryotic translation initiation and induce the formation of stress granules. In this report, the protein composition and fundamental progression of stress granule formation and disassembly induced by pateamine A was found to be similar to that for arsenite. However, pateamine A-induced stress granules were more stable and less prone to disassembly than those formed in the presence of arsenite. Most significantly, pateamine A induced stress granules independent of eukaryotic initiation factor 2alpha phosphorylation, suggesting an alternative mechanism of formation from that previously described for other cellular stresses. Taking into account the known inhibitory effect of pateamine A on eukaryotic translation initiation, a model is proposed to account for the induction of stress granules by pateamine A as well as other stress conditions through perturbation of any steps prior to the rejoining of the 60S ribosomal subunit during the entire translation initiation process.

Animals↗
PubMed2006

A novel function of Nur77: physical and functional association with protein kinase C.

Despite the involvement in diverse physiological process and pleiotropic expression profile, the molecular functions of Nur77 are not likely to be fully elucidated. From the effort to find a novel function of Nur77, we detected molecular interaction between Nur77 and PKC. Details of interaction revealed that C-terminal ligand binding domain (LBD) of Nur77 specifically interacted with highly conserved glycine-rich loop of PKC required for catalytic activity. This molecular interaction resulted in inhibition of catalytic activity of PKCtheta by Nur77. C-terminal LBD of Nur77 is sufficient for inhibiting the phosphorylation of substrate by PKCtheta. Ultimately, inhibition of catalytic activity by Nur77 is deeply associated with repression of PKC-mediated activation of AP-1 and NF-kappaB. Therefore, these findings demonstrate a novel function of Nur77 as a PKC inhibitor and give insights into molecular mechanisms of various Nur77-mediated physiological phenomena.

Animals↗
PubMed2006

Reading dynamic kinase activity in living cells for high-throughput screening.

Protein kinases, as crucial signaling molecules, represent an emerging class of drug targets, and the ability to assay their activities in living cells with high-throughput screening should provide exciting opportunities for drug discovery and chemical and functional genomics. Here, we describe a general method for high-throughput reading of dynamic kinase activities using ratiometric fluorescent sensors, and showcase an example of reading intracellular activities of protein kinase A (PKA) and the cyclic adenosine monophosphate (cAMP)/PKA pathway downstream of many G-protein coupled receptors (GPCRs). We further demonstrate the first compound screen based on the ability of compounds to modulate dynamic kinase activities in living cells and show that such screening of a collection of clinical compounds has successfully identified modulators of the GPCR/cAMP/PKA pathway.

Cell Line↗
PubMed2006

A clinical drug library screen identifies astemizole as an antimalarial agent.

The high cost and protracted time line of new drug discovery are major roadblocks to creating therapies for neglected diseases. To accelerate drug discovery we created a library of 2,687 existing drugs and screened for inhibitors of the human malaria parasite Plasmodium falciparum. The antihistamine astemizole and its principal human metabolite are promising new inhibitors of chloroquine-sensitive and multidrug-resistant parasites, and they show efficacy in two mouse models of malaria.

Animals↗
PubMed2006

Identification of type 1 inosine monophosphate dehydrogenase as an antiangiogenic drug target.

To rapidly discover clinically useful angiogenesis inhibitors, we created and screened a library of existing drugs for inhibition of endothelial cell proliferation. Mycophenolic acid (MPA), an immunosuppressive drug, was found to potently inhibit endothelial cell proliferation in vitro and block tumor-induced angiogenesis in vivo. Using RNA interference, we found that knockdown of one of the two known isoforms of inosine monophosphate dehydrogenase (IMPDH-1) is sufficient to cause endothelial cell cycle arrest.

Angiogenesis Inhibitors↗
PubMed2005

Inhibition of eukaryotic translation initiation by the marine natural product pateamine A.

Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.

Epoxy Compounds↗
PubMed2005

OSW saponins: facile synthesis toward a new type of structures with potent antitumor activities.

[reaction: see text] OSW saponins, featuring a 16beta,17alpha-dihydroxycholest-22-one aglycon and an acylated beta-D-xylopyranosyl-(1-->3)-alpha-L-arabinopyranosyl residue attached to the 16-hydroxyl group, have recently been discovered from a group of lily plants, which show potent antitumor activities with a novel mechanism of action. This paper describes an aldol approach to the stereoselective construction of the 16alpha,17alpha-dihydroxycholest-22-one structure from 16alpha-hydroxy-5-androsten-17-ones and propionates. Elaboration of the aldol adducts toward OSW-1, involving installation of the isoamyl ketone side chain, inversion of the 16-hydroxyl configuration, and selective protection of the C22-oxy function, has been explored and accomplished. In particular, the present route was found convenient for the synthesis of OSW saponin analogues with a C22-ester side chain. Thus, the 23-oxa-analogue of OSW-1 (40) was prepared starting from the industrial dehydroisoandrosterone (1) in a linear eight-step sequence and in 26% overall yield. Analogues with a variety of modified side chains were prepared, via aldol condensation with propionates of varying length, thiopropionate, and acetate (for preparation of 68-75) or via aminolysis of the 22,16-lactone 26 (for preparation of the 23-N-analogues). Cross metathesis (CM) reaction was also found feasible for modification at the final stage from C22-allyl ester 70. Valuable structure-activity relationships (SAR), together with the practical synthetic approach, have thus been provided to set a new stage for further studies on this new type of antitumor structures.

Antineoplastic Agents↗
PubMed2005

Structural basis for the functional differences between type I and type II human methionine aminopeptidases.

Determination of the crystal structure of human MetAP1 makes it possible, for the first time, to compare the structures of a Type I and a Type II methionine aminopeptidase (MetAP) from the same organism. Comparison of the Type I enzyme with the previously reported complex of ovalicin with Type II MetAP shows that the active site of the former is reduced in size and would incur steric clashes with the bound inhibitor. This explains why ovalicin and related anti-angiogenesis inhibitors target Type II human MetAP but not Type I. The differences in both size and shape of the active sites between MetAP1 and MetAP2 also help to explain their different substrate specificity. In the presence of excess Co(2+), a third cobalt ion binds in the active site region, explaining why metal ions in excess can be inhibitory. Also, the N-terminal region of the protein contains three distinct Pro-x-x-Pro motifs, supporting the prior suggestion that this region of the protein may participate in binding to the ribosome.

Amino Acid Sequence↗
PubMed2005

Calmodulin-dependent protein kinase IV regulates nuclear export of Cabin1 during T-cell activation.

Calcium signaling is critical for activation of T lymphocytes and has been proposed to be transduced through multiple calmodulin target proteins. Whereas the calcineurin-NFAT signaling module is critical for all mammalian T cells, the role of calmodulin-dependent kinase IV (CaMKIV) in mouse naïve CD4+ T-cell activation remains enigmatic. We have applied lentivius-mediated RNA interference of CaMKIV to human T cells and found that knockdown of CaMKIV abrogates T-cell receptor-mediated transcription of the IL-2 gene. We demonstrate that CaMKIV directly phosphorylates Cabin1, a transcriptional corepressor for myocyte enhancer factor 2, creating a docking site for 14-3-3, which causes its nuclear export. CaMKIV-mediated nuclear export of Cabin1 is likely to account for a significant part of the requirement of CaMKIV during human T-cell activation.

14-3-3 Proteins↗
PubMed2005

Identification of an SH3-binding motif in a new class of methionine aminopeptidases from Mycobacterium tuberculosis suggests a mode of interaction with the ribosome.

The crystal structure of the methionine aminopeptidase (MetAP) from Mycobacterium tuberculosis (MtMetAP1c) has been determined in the apo- and methionine-bound forms. This is the first structure of a type I MetAP with a significant extension at the amino terminus. The catalytic domain is similar to that of Escherichia coli MetAP (EcMetAP), and the additional 40-residue segment wraps around the surface with an extended but well-defined structure. There are several members of the actinomyces family of bacteria that contain MetAPs with such N-terminal extensions, and we classify these as MetAP type Ic (MetAP1c). Some members of this family of bacteria also contain a second MetAP (type Ia) similar in size to EcMetAP. The main difference between the apo- and the methionine-bound forms of MtMetAP1c is in the conformation of the metal-binding residues. The position of the methionine bound in the active site is very similar to that found in many of the known members of this family. Side chains of several residues in the S1 and S1' subsites shift as much as 1.5 A compared to EcMetAP. Residues 14-17 have the sequence Pro-Thr-Arg-Pro and adopt the conformation of a polyproline II helix. Model-building suggests that this PxxP segment can bind to an SH3 protein motif. Other type Ib and type Ic MetAPs with N-terminal extensions contain similarly located PxxP motifs. Also, several ribosomal proteins are known to include SH3 domains, one of which is located close to the tunnel from which the nascent polypeptide chain exits the ribosome. Therefore, it is proposed that the binding of MetAPs to the ribosome is mediated by a complex between a PxxP motif on the protein and an SH3 domain on the ribosome. It is also possible that zinc-finger domains, which are located at the extreme N-terminus of type I MetAPs, may participate in interactions with the ribosome.

Amino Acid Motifs↗