Heon Seok

Clostridium difficile toxin A decreases acetylation of tubulin leading to microtubule depolymerization through activation of histone deacetylase 6 and this mediates acute inflammation

Clostridium difficile toxin A is known to cause actin disaggregation through the enzymatic inactivation of intracellular Rho proteins. Based on the rapid and severe cell rounding of toxin A-exposed cells, we speculated that toxin A may be involved in post-translational modification of tubulin, leading to microtubule instability. In the current study, we observed that toxin A strongly reduced α-tubulin acetylation in human colonocytes and mouse intestine. Fractionation analysis demonstrated that toxin A-induced α-tubulin deacetylation yielded monomeric tubulin, indicating the presence of microtubule depolymerization. Inhibition of the glucosyltransferase activity against Rho proteins of toxin A by UDP-2′,3′-dialdehyde significantly abrogated toxin A-induced α-tubulin deacetylation. In colonocytes treated with trichostatin A (TSA), an inhibitor of the HDAC6 tubulin deacetylase, toxin A-induced α-tubulin deacetylation and loss of tight junction were completely blocked. Administration of TSA also attenuated proinflammatory cytokine production, mucosal damage, and epithelial cell apoptosis in mouse intestine exposed to toxin A. These results suggest that toxin A causes microtubule depolymerization by activation of HDAC6-mediated tubulin deacetylation. Indeed, blockage of HDAC6 by TSA markedly attenuates α-tubulin deacetylation, proinflammatory cytokine production, and mucosal damage in a toxin A-induced mouse enteritis model. Tubulin deacetylation is an important component of the intestinal inflammatory cascade following toxin A-mediated Rho inactivation in vitro and in vivo.

The insect peptide, coprisin, prevents Clostridium difficile-mediated acute inflammation and mucosal damage through selective antimicrobial activity

ABSTRACT Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.

PDZ Domain-containing 1 (PDZK1) Protein Regulates Phospholipase C-β3 (PLC-β3)-specific Activation of Somatostatin by Forming a Ternary Complex with PLC-β3 and Somatostatin Receptors

Background: The four PLC-β subtypes (β1–β4) have different roles in GPCR-mediated signaling despite having similar structures and regulatory modes. Results: PDZK1 mediates the physical coupling of PLC-β3 to SSTRs using different PDZ domains. Conclusion: PLC-β3 is specifically involved in SSTR-mediated signaling via its interaction with PDZK1. Significance: The subtype-specific role of PLC-β is mediated by differential interactions with PDZ proteins and GPCRs. Phospholipase C-β (PLC-β) is a key molecule in G protein-coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, we identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, we next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca2+ mobilization, which further attenuated subsequent ERK1/2 phosphorylation. Taken together, our results strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST.

The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 μM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

Downregulation of erythropoietin receptor by overexpression of phospholipase C-gamma 1 is critical for decrease on focal adhesion in transformed cells

BackgroundPhospholipase C-γl (PLC-γl) is known to play a critical role in cell adhesion and migration and is highly expressed in metastatic tumors. In the current study, we found that cells transformed by PLC overexpression (PLC-γl cells) exhibited a marked decrease in expression of the Epo receptor (EpoR). Here, we assessed the role of EpoR-dependent signaling pathways in PLC-γl-dependent regulation of cell adhesion and migration.MethodsExpression and phosphorylation of EpoR and its functional role in PLC-γl cells were evaluated by immunoblot analysis or cell adhesion assay. The mechanism for PLC-γ1-induced EpoR downregulation was analyzed by blockage of proteosomal degradation with MG132. EpoR expression was also confirmed in colorectal cancer tissues in which PLC-γl was highly expressed.ResultsEpoR was present on rat fibroblasts, where it functionally active and capable of increasing cell adhesion and migratory activity. However, PLC-γl cells significantly decreased the Epo-dependent effects via ubiquitination-proteosomal degradation of EpoR. A marked decrease of EpoR expression was confirmed in colorectal cancer tissues that showed high-level of PLC-γl expression.ConclusionThe Epo/EpoR complex plays a critical role in the adhesion and migration of rat fibroblasts, and its functional inactivation is associated with PLC-γl-dependent reduction of cell-matrix adhesion and this also affects cell migration.

Neurotropic and neuroprotective activities of the earthworm peptide Lumbricusin

We recently isolated a polypeptide from the earthworm Lumbricus terrestris that is structurally similar to defensin, a well-known antibacterial peptide. An 11-mer antibacterial peptide (NH2-RNRRWCIDQQA), designated Lumbricusin, was synthesized based on the amino acid sequence of the isolated polypeptide. Since we previously reported that CopA3, a dung beetle peptide, enhanced neuronal cell proliferation, we here examined whether Lumbricusin exerted neurotropic and/or neuroprotective effects. Lumbricusin treatment induced a time-dependent increase (∼51%) in the proliferation of human neuroblastoma SH-SY5Y cells. Lumbricusin also significantly inhibited the apoptosis and decreased viability induced by treatment with 6-hydroxy dopamine, a Parkinsons disease-mimicking agent. Immunoblot analyses revealed that Lumbricusin treatment increased ubiquitination of p27(Kip1) protein, a negative regulator of cell-cycle progression, in SH-SY5Y cells, and markedly promoted its degradation. Notably, adenoviral-mediated over-expression of p27(Kip1) significantly blocked the antiapoptotic effect of Lumbricusin in 6-hydroxy dopamine-treated SH-SY5Y cells. These results suggest that promotion of p27(Kip1) degradation may be the main mechanism underlying the neuroprotective and neurotropic effects of Lumbricusin.

Insect peptide CopA3-induced protein degradation of p27Kip1 stimulates proliferation and protects neuronal cells from apoptosis

We recently demonstrated that the antibacterial peptide, CopA3 (a D-type disulfide dimer peptide, LLCIALRKK), inhibits LPS-induced macrophage activation and also has anticancer activity in leukemia cells. Here, we examined whether CopA3 could affect neuronal cell proliferation. We found that CopA3 time-dependently increased cell proliferation by up to 31 ± 2% in human neuroblastoma SH-SY5Y cells, and up to 29 ± 2% in neural stem cells isolated from neonatal mouse brains. In both cell types, CopA3 also significantly inhibited the apoptosis and viability losses caused by 6-hydroxy dopamine (a Parkinson disease-mimicking agent) and okadaic acid (an Alzheimers disease-mimicking agent). Immunoblotting revealed that the p27Kip1 protein (a negative regulator of cell cycle progression) was markedly degraded in CopA3-treated SH-SY5Y cells. Conversely, an adenovirus expressing p27Kip1 significantly inhibited the antiapoptotic effects of CopA3 against 6-hydroxy dopamine- and okadaic acid-induced apoptosis, and decreased the neurotropic effects of CopA3. These results collectively suggest that CopA3-mediated protein degradation of p27Kip1 may be the main mechanism through which CopA3 exerts neuroprotective and neurotropic effects.

The insect peptide CopA3 inhibits lipopolysaccharide-induced macrophage activation.

We recently demonstrated that the insect peptide CopA3 (LLCIALRKK), a disulfide‐linked dimeric peptide, exerts antimicrobial and anti‐inflammatory activities in a mouse colitis model. Here, we examined whether CopA3 inhibited activation of macrophages by LPS. Exposure of an unseparated mouse peritoneal cell population or isolated peritoneal macrophages to LPS markedly increased secretion of IL‐6 and TNF‐α; these effects were significantly inhibited by CopA3 treatment. The inhibitory effect of CopA3 was also evident in murine macrophage cell line, RAW 264.7. Western blotting revealed that LPS‐induced activation of STAT1 and STAT5 in macrophages was significantly inhibited by CopA3. Inhibition of JAK (STAT1/STAT5 kinase) with AG490 markedly reduced the production of IL‐6 and TNF‐α in macrophages. Collectively, these observations suggest that CopA3 inhibits macrophage activation by inhibiting activating phosphorylations of the transcription factors, STAT1 and STAT5, and blocking subsequent production of IL‐6 and TNF‐α and indicate that CopA3 may be useful as an immune‐modulating agent. Copyright

The Insect Peptide CopA3 Increases Colonic Epithelial Cell Proliferation and Mucosal Barrier Function to Prevent Inflammatory Responses in the Gut

The epithelial cells of the gut form a physical barrier against the luminal contents. The collapse of this barrier causes inflammation, and its therapeutic restoration can protect the gut against inflammation. EGF enhances mucosal barrier function and increases colonocyte proliferation, thereby ameliorating inflammatory responses in the gut. Based on our previous finding that the insect peptide CopA3 promotes neuronal growth, we herein tested whether CopA3 could increase the cell proliferation of colonocytes, enhance mucosal barrier function, and ameliorate gut inflammation. Our results revealed that CopA3 significantly increased epithelial cell proliferation in mouse colonic crypts and also enhanced colonic epithelial barrier function. Moreover, CopA3 treatment ameliorated Clostridium difficile toxin As-induced inflammation responses in the mouse small intestine (acute enteritis) and completely blocked inflammatory responses and subsequent lethality in the dextran sulfate sodium-induced mouse model of chronic colitis. The marked CopA3-induced increase of colonocyte proliferation was found to require rapid protein degradation of p21Cip1/Waf1, and an in vitro ubiquitination assay revealed that CopA3 directly facilitated ubiquitin ligase activity against p21Cip1/Waf1. Taken together, our findings indicate that the insect peptide CopA3 prevents gut inflammation by increasing epithelial cell proliferation and mucosal barrier function.