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.

Identification and characterization of a small inhibitory peptide that can target DNA-PKcs autophosphorylation and increase tumor radiosensitivity.

PURPOSE: The DNA protein kinase catalytic subunit (DNA-PKcs) is one of the critical elements involved in the DNA damage repair process. Inhibition of DNA-PKcs results in hypersensitivity to ionizing radiation (IR); therefore, this approach has been explored to develop molecular targeted radiosensitizers. Here, we aimed to develop small inhibitory peptides that could specifically target DNA-PKcs autophosphorylation, a critical step for the enzymatic activation of the kinase in response to IR. METHODS AND MATERIALS: We generated several small fusion peptides consisting of 2 functional domains, 1 an internalization domain and the other a DNA-PKcs autophosphorylation inhibitory domain. We characterized the internalization, toxicity, and radiosensitization activities of the fusion peptides. Furthermore, we studied the mechanisms of the inhibitory peptides on DNA-PKcs autophosphorylation and DNA repair. RESULTS: We found that among several peptides, the biotin-labeled peptide 3 (BTW3) peptide, which targets DNA-PKcs threonine 2647 autophosphorylation, can abrogate IR-induced DNA-PKcs activation and cause prolonged γ-H2AX focus formation. We demonstrated that BTW3 exposure led to hypersensitivity to IR in DNA-PKcs-proficient cells but not in DNA-PKcs-deficient cells. CONCLUSIONS: The small inhibitory peptide BTW3 can specifically target DNA-PKcs autophosphorylation and enhance radiosensitivity; therefore, it can be further developed as a novel class of radiosensitizer.

N- and C-terminal degradation of ecdysteroid receptor isoforms, when transiently expressed in mammalian CHO cells, is regulated by the proteasome and cysteine and threonine proteases.

Transcriptional activity of nuclear receptors is the result of transactivation capability and the concentration of the receptor protein. The concentration of ecdysteroid receptor (EcR) isoforms, constitutively expressed in mammalian CHO cells, is dependent on a number of factors. As shown previously, ligand binding stabilizes receptor protein concentration. In this paper, we investigate the degradation of EcR isoforms and provide evidence that N-terminal degradation is modulated by isoform-specific ubiquitination sites present in the A/B domains of EcR-A and -B1. This was demonstrated by the increase in EcR concentration by treatment with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), an inhibitor of ubiquitin-mediated proteasomal degradation and by deletion of ubiquitination sites. In addition, EcR is degraded by the peptidyl-dipeptidase cathepsin B (CatB) and the endopeptidase cathepsin S (CatS) at the C-terminus in an isoform-specific manner, despite identical C-termini. Ubiquitin-proteasome-mediated degradation and the proteolytic action are modulated by heterodimerization with Ultraspiracle (USP). The complex regulation of receptor protein concentration offers an additional opportunity to regulate transcriptional activity in an isoform- and target cell-specific way and allows the temporal limitation of hormone action.

Monocillin II inhibits human breast cancer growth partially by inhibiting MAPK pathways and CDK2 Thr160 phosphorylation.

Twenty-two ß-resorcylic acid lactones (RALs) were evaluated for cytotoxicity against human breast cancer cells to find their structure-activity relationship (SAR). Monocillin II, a trans-enone RAL without epoxy and conjugated dienone, was found to have higher activity in inhibiting tumor cell growth in both in vitro experiment and in vivo nude xenografted mice model than its analogue radicicol, an anticancer lead compound. We demonstrated for the first time that monocillin II could arrest breast cancer cell cycle in G1 phase, which might partially be the result of its inhibition effect on the phosphorylation of the Thr160 residue of cyclin dependent kinase 2 (CDK2), a key enzyme in cell-cycle regulation. Moreover, monocillin II exhibited inhibition of heat shock protein 90 (Hsp90) and depleted its target proteins, Raf-1 and A-Raf, which are involved in Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway. Remarkably, we found that monocillin II could inhibit activation of MAPKs including ERK, JNK and p38, which might be involved in the inactivation of CDK2. These results suggest that monocillin II has potential therapeutic benefits in breast cancer prevention and intervention.

Inhibition of Thr-55 phosphorylation restores p53 nuclear localization and sensitizes cancer cells to DNA damage.

The p53 tumor suppressor induces cell growth arrest and apoptosis in response to DNA damage. Because these functions are achieved largely by the transcriptional properties of p53, nuclear localization of the protein is essential. Indeed, the tumors with aberrant cytoplasmic localization of wild-type p53 often exhibit an impaired response to DNA damage. In this study, we report that Thr-55 phosphorylation induces the association of p53 with the nuclear export factor CRM1, leading to p53 nuclear export. We further show that MDM2 also promotes the CRM1-p53 association and Thr-55 phosphorylation is required for this process. Interestingly, inhibition of Thr-55 phosphorylation by a dietary flavonoid, apigenin, specifically blocks the CRM1-p53 association, restores p53 nuclear localization, and sensitizes tumor cells with cytoplasm localized wild-type p53 to DNA damage. These data provide insights into the regulation of p53 nuclear localization by post-translational modification and suggest an avenue for targeted therapy for cancers caused by aberrant cytoplasm localization of wild-type p53.

Dual phosphorylation of phosphoinositide 3-kinase adaptor Grb2-associated binder 2 is responsible for superoxide formation synergistically stimulated by Fc gamma and formyl-methionyl-leucyl-phenylalanine receptors in differentiated THP-1 cells.

The class Ia phosphoinositide (PI) 3-kinase consisting of p110 catalytic and p85 regulatory subunits is activated by Tyr kinase-linked membrane receptors such as FcgammaRII through the association of p85 with the phosphorylated receptors or adaptors. The heterodimeric PI 3-kinase is also activated by G protein-coupled chemotactic fMLP receptors, and activation of the lipid kinase plays an important role in various immune responses, including superoxide formation in neutrophils. Although fMLP-induced superoxide formation is markedly enhanced in FcgammaRII-primed neutrophils, the molecular mechanisms remain poorly characterized. In this study, we identified two Tyr-phosphorylated proteins, c-Cbl (Casitas B-lineage lymphoma) and Grb2-associated binder 2 (Gab2), as PI 3-kinase adaptors that are Tyr phosphorylated upon the stimulation of FcgammaRII in differentiated neutrophil-like THP-1 cells. Interestingly, Gab2 was, but c-Cbl was not, further Ser/Thr phosphorylated by fMLP. Thus, the adaptor Gab2 appeared to be dually phosphorylated at the Ser/Thr and Tyr residues through the two different types of membrane receptors. The Ser/Thr phosphorylation of Gab2 required the activation of extracellular signal-regulated kinase, and fMLP receptor stimulation indeed activated extracellular signal-regulated kinase in the cells. Enhanced superoxide formation in response to Fcgamma and fMLP was markedly attenuated when the Gab2 Ser/Thr phosphorylation was inhibited. These results show the importance of the dual phosphorylation of PI 3-kinase adaptor Gab2 for the enhanced superoxide formation in neutrophil-type cells.

Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53.

Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulation of membrane excitability. To determine whether Kir2.3 is modulated by direct phosphorylation of its channel protein and identify the phosphorylation site of protein kinase C (PKC), we performed experiments using several recombinant and mutant Kir2.3 channels. Whole-cell Kir2.3 currents were inhibited by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another member in the Kir2 family that is insensitive to PMA, the chimerical channel lost its PMA sensitivity. However, substitution of the C terminus was ineffective. Four potential PKC phosphorylation sites in the N terminus were studied by comparing mutations of serine or threonine with their counterpart residues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36, and 39 had no effect on the channel sensitivity to PMA, mutation of threonine 53 completely eliminated the channel response to PMA. Interestingly, creation of this threonine residue at the corresponding position (I79T) in Kir2.1 lent the mutant channel a PMA sensitivity almost identical to the wild-type Kir2.3. These results therefore indicate that Kir2.3 is directly modulated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3.

Serine/threonine phosphatases play a role in stimulus-secretion coupling in pancreatic acinar cells.

The role of serine/threonine phosphatases in Ca2+/IP3- and cAMP- mediated stimulus-secretion coupling was investigated in isolated pancreatic acinar cells. Cyclosporine A, an inhibitor of type 2b serine/threonine phosphatases, maximally reduced CCK-8-stimulated amylase secretion by 33%. In contrast, the secretory response to secretin or PACAP-(1-27) was not significantly altered by cyclosporine A independent of the secretagogue-concentration okadaic acid significantly reduced amylase release, induced by Ca2+/IP3-mediated- (CCK-8) or cAMP-mediated agonists (secretin, PACAP-(1-27), VIP) at concentrations that primarily inactivate type 1 and 2b phosphatases. Calyculin A, another type 1 and 2a phosphatase inhibitor, had a similar inhibitory effect on CCK-8-, secretin- or PACAP-(1-27)-induced secretion. In permeabilized acini, cyclosporine A reduced calcium-induced amylase release by 20%, whereas okadaic acid and calyculin A had an inhibitory effect by 55% and 52%, respectively. The ultrastructure of CsA-incubated acinar cells was not different from vehicle-incubated control lobules. In contrast, incubation with okadaic acid for 60 min resulted in morphological alterations of the Golgi apparatus, leading to a fragmentation of Golgi cisternae into small vesicles. Our data suggest a role of type 1 and 2b phospatases in stimulus-secretion coupling of both signal-transduction pathways in pancreatic acinar cells. These phosphatases might also be important for the maintenance of pancreatic cellular ultrastructure.

Posttranslational processing of a human myeloid lysosomal protein, myeloperoxidase.

Myeloperoxidase (MPO) is a lysosomal enzyme present in the azurophilic granules of human neutrophils and monocytes and is important for optimal oxygen-dependent killing of microorganisms. The native molecule is a heterodimer composed of a pair of heavy-light protomers, each containing a 59-kDa and 13.5-kDa subunit. The intracellular processing during biosynthesis of MPO was examined in the human promyelocytic cell line HL-60. Endoglycosidase H and F digestion of immunoprecipitated pro-MPO demonstrated the presence of five N-linked--high-mannose oligosaccharide side chains and no complex mannose units. Incorporation of the threonine analogue beta-hydroxynorvaline produced species approximately 2.5 kDa and approximately 5 kDa smaller than the fully glycosylated pro-MPO, suggesting that two of the glycans were in the asparagine-X-threonine tripeptide sequence. Processing of pro-MPO occurred very rapidly, within approximately five minutes, and was best identified using glucosidase inhibitors. The presence of such inhibitors resulted in synthesis of a 92-kDa glycoprotein rather than the usually identified 89-kDa peptide. Swainsonine, a Golgi mannosidase inhibitor, did not alter the size of the earliest synthesized protein, suggesting that pro-MPO exited the endoplasmic reticulum or cis-Golgi proximal to the site of mannosidases. Intracellular transport and proteolytic maturation of MPO was retarded by weak bases (NH4Cl, chloroquine) or monensin at concentrations shown to raise intralysosomal pH. However, these agents did not qualitatively alter transport nor increase secretion. Thus, although MPO biosynthesis resembled that of other lysosomal enzymes, significant differences exist, including only limited oligosaccharide processing and intracellular transport and proteolytic maturation of pro-MPO that was only retarded by alkalinizing lysosomes without affecting the products or the fraction of pro-MPO secreted. Characterization of the determinants for targeting and of the regulatory factors in processing lysosomal enzymes in myeloid cells will provide insight into the molecular mechanisms underlying common disorders such as myeloperoxidase deficiency.

Enhanced degradation of cathepsin D synthesized in the presence of the threonine analog beta-hydroxynorvaline.

The threonine analog beta-hydroxynorvaline is an inhibitor of asparagine-linked glycosylation. In the presence of the analog human fibroblasts synthesized cathepsin D molecules containing two, one, or no oligosaccharides. The nonglycosylated cathepsin D precursor was but a minor species and was degraded within 45 min of its synthesis, presumably in the lumen of the endoplasmic reticulum. The polypeptides with one or two oligosaccharides were normally segregated into lysosomes and their proteolytic maturation was not affected. The stability of mature glycosylated and nonglycosylated cathepsin D polypeptides within the lysosomes, however, was markedly decreased. The recovery of cathepsin D polypeptides was increased in the presence of inhibitors of cysteine and aspartyl-proteinases. These data suggest that the absence of carbohydrate side chains in cathepsin D results in an enhancement of the degradation rate of the precursor in the endoplasmic reticulum, and the replacement of threonine by beta-hydroxynorvaline in an enhanced degradation of the mature cathepsin D in lysosomes.

Studies on convulsants in the isolated frog spinal cord. I. Antagonism of amino acid responses.

1. The isolated frog spinal cord was used to study the effects of picrotoxin, bicuculline, and strychnine on the responses of primary afferents to amino acids. Recording was by sucrose gap technique. 2. A series of neutral amino acids was found to depolarize primary afferents. Optimal activity was obtained by an amino acid whose carboxyl and amino groups were separated by a three-carbon chain length (i.e. GABA). Amino acids with shorter (i.e. beta-alanine, glycine) or longer (i.e. delta-aminovaleric acid, epsilon-aminocaproic acid) distances between the charged groups were less potent. Imidazoleacetic acid was the most potent depolarizing agent tested. 3. Picrotoxin and bicuculline antagonized the primary afferent depolarizations of a number of amino acids tested with equal specificity. Depolarizing responses to standard (10- minus 3 M) concentrations of beta-alanine and taurine were completely blocked by these convulsants, while depolarizations to 10- minus 3 gamma-aminobutyric acid (GABA) were only partially antagonized. Glycine responses were unaffected by these agentsk; Strychnine completely blocked beta-alanine and taurine depolarizations and incompletely antagonized several other neutral amino acids. GABA, glutamate, and glycine depolarizations were not affected. 5. These results suggest that there are at least three distinct populations of neutral amino acid receptors on primary afferent terminals: a GABA-like receptor, a taurine/beta-alanine receptor, and a glycine-like receptor. The strychnine resistance of the glycine responses indictaes that the primary afferent receptors for glycine differ from those on the somata of spinal neurones.