Involvement of calcium ions in amyloid-ß-induced lamin fragmentation.

Amyloid-ß (Aß) peptide, the main pathogenic peptide in Alzheimer's disease, has been shown to induce an increase in cytoplasmic calcium concentration (CCC). In the current study, we explored the cytotoxic signal transduction pathway in 42-amino-acid Aß (Aß42)-treated HeLa cells in relation to the increase in CCC. The increase in CCC was prominent in cells treated twice with oligomeric Aß42. We previously showed that double treatment also promoted Aß-induced lamin fragmentation (AILF), which appears to be mediated by cathepsin L. Apoptotic caspase activation was a downstream event of AILF. The Ca2+ chelator BAPTA-AM suppressed cell death, cathepsin L activation, AILF, and caspase activation in Aß-treated cells. These results indicate that Aß42 induces an increase in CCC, which is an event upstream of the cytotoxic processes. The products of AILF are different from those produced by other cell death-inducing agents, such as staurosporine, which induces caspase-6-mediated lamin fragmentation (CMLF). CMLF was unaffected by BAPTA-AM and was not detected in cells treated with Aß42, indicating that Aß42 peptide induced a specific cytotoxic pathway involving AILF via increased CCC. We confirmed that the same processes (except caspase activation) operated in Aß42-treated neuroblastoma SH-SY5Y cells.

Evidence for a Strong Relationship between the Cytotoxicity and Intracellular Location of ß-Amyloid.

ß-Amyloid (Aß) is a hallmark peptide of Alzheimer's disease (AD). Herein, we explored the mechanism underlying the cytotoxicity of this peptide. Double treatment with oligomeric 42-amino-acid Aß (Aß42) species, which are more cytotoxic than other conformers such as monomers and fibrils, resulted in increased cytotoxicity. Under this treatment condition, an increase in intracellular localization of the peptide was observed, which indicated that the peptide administered extracellularly entered the cells. The cell-permeable peptide TAT-tagged Aß42 (tAß42), which was newly prepared for the study and found to be highly cell-permeable and soluble, induced Aß-specific lamin protein cleavage, caspase-3/7-like DEVDase activation, and high cytotoxicity (5-10-fold higher than that induced by the wild-type oligomeric preparations). Oligomeric species enrichment and double treatment were not necessary for enhancing the cytotoxicity and intracellular location of the fusion peptide. Taiwaniaflavone, an inhibitor of the cytotoxicity of wild-type Aß42 and tAß42, strongly blocked the internalization of the peptides into the cells. These data imply a strong relationship between the cytotoxicity and intracellular location of the Aß peptide. Based on these results, we suggest that agents that can reduce the cell permeability of Aß42 are potential AD therapeutics.

Regulatory role of cathepsin L in induction of nuclear laminopathy in Alzheimer's disease.

Experimental and clinical therapies in the field of Alzheimer's disease (AD) have focused on elimination of extracellular amyloid beta aggregates or prevention of cytoplasmic neuronal fibrillary tangles formation, yet these approaches have been generally ineffective. Interruption of nuclear lamina integrity, or laminopathy, is a newly identified concept in AD pathophysiology. Unraveling the molecular players in the induction of nuclear lamina damage may lead to identification of new therapies. Here, using 3xTg and APP/PS1 mouse models of AD, and in vitro model of amyloid beta42 (Aß42) toxicity in primary neuronal cultures and SH-SY5Y neuroblastoma cells, we have uncovered a key role for cathepsin L in the induction of nuclear lamina damage. The applicability of our findings to AD pathophysiology was validated in brain autopsy samples from patients. We report that upregulation of cathepsin L is an important process in the induction of nuclear lamina damage, shown by lamin B1 cleavage, and is associated with epigenetic modifications in AD pathophysiology. More importantly, pharmacological targeting and genetic knock out of cathepsin L mitigated Aß42 induced lamin B1 degradation and downstream structural and molecular changes. Affirming these findings, overexpression of cathepsin L alone was sufficient to induce lamin B1 cleavage. The proteolytic activity of cathepsin L on lamin B1 was confirmed using mass spectrometry. Our research identifies cathepsin L as a newly identified lamin B1 protease and mediator of laminopathy observed in AD. These results uncover a new aspect in the pathophysiology of AD that can be pharmacologically prevented, raising hope for potential therapeutic interventions.

Amyloid ß cytotoxicity is enhanced or reduced depending on formation of amyloid ß oligomeric forms.

OBJECTIVES: We explored the underlying mechanisms that facilitate reducing and enhancing effects of exogenous proteins on cytotoxicity of amyloid ß (Aß), a main pathogen of Alzheimer's disease, by using an Escherichia coli chaperonin DnaK. RESULTS: DnaK was chosen as a tool, because it, easily available and functionally stable, reduced or enhanced Aß cytotoxicity depending on its concentration. Cytotoxicity was enhanced when the molar ratio of DnaK to Aß42, at 20 µM Aß42, was 0.01-0.5, while reduced cytotoxicity was observed at higher ratios (> 1) at 1 µM Aß42. Significant amounts of oligomeric Aß42 species accumulated concomitantly with enhanced cytotoxicity, whereas the oligomers appeared to form complexes with DnaK in conditions of reduced cytotoxicity. CONCLUSIONS: The difference in cytotoxicity was due to variations in the toxic oligomeric Aß species and DnaK is a useful tool for the study of the Aß ultrastructure formation and toxicity of Aß peptide.

Differential involvement of caspase-6 in amyloid-ß-induced fragmentation of lamin A and B.

Amyloid-ß (Aß), a peptide implicated in Alzheimer's disease, was shown to cause specific fragmentation of lamin proteins, which was mediated by an unidentified protease named nuclear scaffold protease (NSP) independently of caspase-6. Because caspase-6 is responsible for the fragmentation process in many other damage-induced apoptosis, here we further investigated possible involvement of caspase-6 in Aß-induced lamin fragmentation under various conditions. We found that lamin A fragment generated by NSP (named fragment b) disappeared in cells incubated with Aß42 for prolonged periods and this product was preserved by a caspase-6 inhibitor. Furthermore, caspase-6 could remove fragment b in nuclei isolated from Aß42-treated cells (ANU). Lamin B in ANU was fragmented by caspase-6 only after treatment with an alkaline phosphatase. The caspase-mediated fragmentation of lamin B was also achieved with nuclei isolated from cells incubated with Aß42 plus a Cdk5 inhibitor. The results indicate that Aß42 induces NSP-mediated fragmentation of lamin A and the following removal process of fragment b by caspase-6 and an Aß-induced phosphorylation prevents the fragmentation of lamin B by caspase-6. The pathway leading to lamin protein fragmentation in this investigation appears to be specific for Aß and thus the data will provide novel insights into the toxicity of the peptide.

Antimicrobial and anti-inflammatory activities of chemokine CXCL14-derived antimicrobial peptide and its analogs.

CXCL14 is a CXC chemokine family that exhibits antimicrobial activity and contains an amphipathic cationic α-helical region in the C-terminus, a characteristic structure of antimicrobial peptides (AMPs). In this study, we designed three analogs of CXCL1459-75 (named CXCL14-C17) corresponding to the C-terminal α-helix of CXCL14, which displayed potential antimicrobial activity against a wide variety of gram-negative and gram-positive bacteria with minimum inhibitory concentrations of 4-16 µM without mammalian cell toxicity. Furthermore, two CXCL14-C17 analogs (CXCL14-C17-a1 and CXCL14-C17-a3) with improved cell selectivity were engineered by introducing Lys, Arg, or Trp in CXCL14-C17. Additionally, CXCL14-C17 analogs showed much greater synergistic effect (FICI: 0.3125-0.375) with chloramphenicol and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa (MDRPA) than LL-37 did (FICI: 0.75-1.125). CXCL14-C17 analogs were more active against antibiotic-resistant bacteria including methicillin-resistant Staphylococcus aureus (MRSA), MDRPA, and vancomycin-resistant Enterococcus faecium (VREF) than LL-37 and melittin. In particular, CXCL14-C17-a2 and CXCL14-C17-a3 completely inhibited the biofilm formation at sub-MIC and all of the peptides were able to eliminate pre-formed biofilm as well. Membrane depolarization, flow cytometry, sytox green uptake, ONPG hydrolysis and confocal microscopy revealed the possible target of the native peptide (CXCL14-C17) to likely be intracellular, and the amphipathic designed analogs targeted the bacterial membrane. CXCL14-C17 also showed DNA binding characteristic activity similar to buforin-2. Interestingly, CXCL14-C17-a2 and CXCL14-C17-a3 effectively inhibited the production and expression of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, suggesting that these peptides could be promising anti-inflammatory and antimicrobial agents.

Limited activation of the intrinsic apoptotic pathway plays a main role in amyloid-ß-induced apoptosis without eliciting the activation of the extrinsic apoptotic pathway.

Amyloid-ß (Aß), a main pathogenic factor of Alzheimer's disease (AD), induces apoptosis accompanied by caspase activation. However, limited caspase activation and the suppression of the intrinsic apoptotic pathway (IAPW) are frequently observed upon Aß treatment. In this study, we investigated whether these suppressive effects of Aß can be overcome; we also examined the death-related pathways. Single treatments of cells with Aß42 for up to 48 h barely induced caspase activation. In cells treated with Aß42 twice for 2 h followed by 22 h (2+22 h) or for longer durations, the apoptotic protease activating factor-1 (Apaf-1) apoptosome was formed and caspases-3 and -9 were activated to a certain extent, suggesting the activation of the IAPW. However, the Aß42-induced activation of the IAPW differed from that induced by treatment with other agents, such as staurosporine (STS) in that lower amounts of cytochrome c were released from the mitochondria, the majority of procaspase-9 in the Apaf-1 complex was not processed and caspase-3 was activated to a lesser extent in the peptide-treated cells. Thus, it seemed that the IAPW was not fully activated by Aß42. The 30- and 41/43-kDa fragments derived from procaspase-8 were detected, which appear to be produced through the IAPW without death-inducing signaling-complex (DISC) formation, a key feature of the extrinsic apoptotic pathway (EAPW). Bid cleavage was observed only after caspase-3 activity reached its maximal levels, suggesting that the cleavage may contribute in a limited capacity to the amplification process of the IAPW in the Aß-treated cells. Taken together, our data suggest that the IAPW, albeit functional only to a limited extent, plays a major role in Aß42-induced apoptosis without the EAPW.

Oleanane triterpenoids from Akebiae Caulis exhibit inhibitory effects on Aß42 induced fibrillogenesis.

Previous phytochemical investigations of Akebiae Caulis resulted in the isolation of triterpenes, triterpene glycosides, phenylethanoid glycosides and megastigmane glycoside. Amyloid beta (Aß), the main component of the senile plaques detected in Alzheimer's disease, induces cell death. However, only a limited number of studies have addressed the biological and pharmacological effects of Akebiae Caulis. In particular, the inhibitory activity of Akebiae Caulis against Aß42 fibrillogenesis remains unclear. Herein, a new triterpene glycoside, akequintoside F (1), along with nine known compounds pulsatilla saponin A (2), collinsonidin (3), akebonic acid (4), hederagenin (5), 1-(3',4'-dihydroxycinnamoyl) cyclopentane-2,3-diol (6), asperosaponin C (7), leontoside A (8), quinatic acid (9), and quinatoside A (10) were isolated from Akebiae Caulis using repeated column chromatography with silica gel, LiChroprep RP-18, and MCI gel. The chemical structures of compounds 1-10 were illustrated based on 1D and 2D NMR spectroscopy, including 1H-1H COSY, HSQC, HMBC and NOESY spectroscopic analyses. Compound 1 a novel compound and known compounds 6 and 7 were isolated for the first time from this plant. Among these compounds, 1, 3, 4, 5 and 7 displayed significant inhibitory effects on Aß42 induced fibrillogenesis. We present the first report of new compound 1 and the inhibitory effects of components from Akebiae Caulis on Aß42 fibrillogenesis.

ß-Amyloid induces nuclear protease-mediated lamin fragmentation independent of caspase activation.

ß-Amyloid (Aß), a hallmark peptide of Alzheimer's disease, induces both caspase-dependent apoptosis and non-apoptotic cell death. In this study, we examined caspase-independent non-apoptotic cell death preceding caspase activation in Aß42-treated cells. We first determined the optimal treatment conditions for inducing cell death without caspase activation and selected a double-treatment method involving the incubation of cells with Aß42 for 4 and 6 h (4+6 h sample). We observed that levels of lamin A (LA) and lamin B (LB) were reduced in the 4+6 h samples. This reduction was decreased by treatment with suc-AAPF-CMK, an inhibitor of nuclear scaffold (NS) protease, but not by treatment with z-VAD-FMK, a pan-caspase inhibitor. In addition, suc-AAPF-CMK decreased the changes in nuclear morphology observed in cells in the 4+6 h samples, which were different from nuclear fragmentation observed in STS-treated cells. Furthermore, suc-AAPF-CMK inhibited cell death in the 4+6 h samples. LA and LB fragmentation occurred in the isolated nuclei and was also inhibited by suc-AAPF-CMK. Together, these data indicated that the fragmentation of LA and LB in the Aß42-treated cells was induced by an NS protease, whose identity is not clearly determined yet. A correlation between Aß42 toxicity and the lamin fragmentation by NS protease suggests that inhibition of the protease could be an effective method for controlling the pathological process of AD.

The stereochemical effect of SMAP-29 and SMAP-18 on bacterial selectivity, membrane interaction and anti-inflammatory activity.

Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a cathelicidin-related antimicrobial peptide derived from sheep myeloid cells. In order to investigate the effects of L-to-D-amino acid substitution in SMAP-29 on bacterial selectivity, membrane interaction and anti-inflammatory activity, we synthesized its two D-enantiomeric peptides (SMAP-29-E1 and SMAP-29-E2 containing D-Ile and D-allo-Ile, respectively) and two diastereomeric peptides (SMAP-29-D1 and SMAP-29-D2). Additionally, in order to address the effect of L-to-D-amino acid substitution in the N-terminal helical peptide of SMAP-29 (named SMAP-18) on antimicrobial activity, we synthesized its two D-enantiomeric peptides (SMAP-18-E1 and SMAP-18-E2), which are composed of D-amino acids entirely. L-to-D-amino acid substitution in membrane-targeting AMP, SMAP-29 did not affect its antimicrobial activity. However, D-allo-Ile containing-SMAP-29-E2 and SMAP-29-D2 exhibited less hemolytic activity compared to D-Ile containing-SMAP-29-E1 and SMAP-29-D1, respectively. L-to-D-amino acid substitution in intracellular targeting-AMPs, SMAP-18 and buforin-2 improved antimicrobial activity by 2- to eightfold. The improved antimicrobial activity of the D-isomers of SMAP-18 and buforin-2 seems to be due to the stability against proteases inside bacterial cells. Membrane depolarization and dye leakage suggested that the membrane-disruptive mode of SMAP-29-D1 and SMAP-29-D2 is different from that of SMAP-29, SMAP-29-E1, and SMAP-29-E2. L-to-D-amino acid substitution in SMAP-29 improved anti-inflammatory activity in LPS-stimulated RAW 264.7 cells. In summary, we propose here that D-allo-Ile substitution is a more powerful strategy for increasing bacterial selectivity than D-Ile substitution in the design of D-enantiomeric and diastereomeric AMPs. SMAP-29-D1, and SMAP-29-D2 with improved bacterial selectivity and anti-inflammatory activity can serve as promising candidates for the development of anti-inflammatory and antimicrobial agents.

Anamorsin, a novel caspase-3 substrate in neurodegeneration.

Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.

Amyloid ß binds procaspase-9 to inhibit assembly of Apaf-1 apoptosome and intrinsic apoptosis pathway.

Apoptosis is essential in the death process induced by Amyloid-ß (Aß), a major constituent of diffuse plaques found in Alzheimer's disease patients. However, we have found that caspase activation and cell death induced by staurosporine, employed to induce the intrinsic mitochondria-dependent apoptotic pathway, were significantly reduced by 42 amino-acid Aß42, implying that the peptide also has a negative effect on the apoptotic process. The inhibitory effect of Aß42 on the apoptotic pathway is associated with its interaction with procaspase-9 and consequent inhibition of Apaf-1 apoptosome assembly. We detected the inhibitory effect in the early stage (<8h) of apoptosis, but later caspase activation becomes obvious. Thus we inferred that the inhibitory process on apoptosis begins at an early stage, and the later robust activation surpasses it. We propose that the apoptotic manifestation in Aß-treated cells is a combined consequence of those anti- and pro-apoptotic processes.

Bacterial killing mechanism of sheep myeloid antimicrobial peptide-18 (SMAP-18) and its Trp-substituted analog with improved cell selectivity and reduced mammalian cell toxicity.

To develop short antimicrobial peptide with improved cell selectivity and reduced mammalian cell toxicity compared to sheep myeloid antimicrobial peptide-29 (SMAP-29) and elucidate the possible mechanisms responsible for their antimicrobial action, we synthesized a N-terminal 18-residue peptide amide (SMAP-18) from SMAP-29 and its Trp-substituted analog (SMAP-18-W). Due to their reduced hemolytic activity and retained antimicrobial activity, SMAP-18 and SMAP-18-W showed higher cell selectivity than SMAP-29. In addition, SMAP-18 and SMAP-18-W had no cytotoxicity against three different mammalian cells such as RAW 264.7, NIH-3T3 and HeLa cells even at 100 µM. These results suggest that SMAP-18 and SMAP-18-W have potential for future development as novel therapeutic antimicrobial agent. Unlike SMAP-29, SMAP-18 and SMAP-18-W showed relatively weak ability to induce dye leakage from bacterial membrane-mimicking liposomes, N-phenyl-1-napthylamine (NPN) uptake and o-nitrophenyl-ß-galactoside (ONPG) hydrolysis. Similar to SMAP-29, SMAP-18-W led to a significant membrane depolarization (> 80%) against Staphylococcus aureus at 2 × MIC. In contrast, SMAP-18 did not cause any membrane depolarization even at 4 × MIC. In confocal laser scanning microscopy, we observed translocation of SMAP-18 across the membrane in a non-membrane disruptive manner. SMAP-29 and SMAP-18-W were unable to translocate the bacterial membrane. Collectively, we propose here that SMAP-29 and SMAP-18-W kill microorganisms by disrupting/perturbing the lipid bilayer and forming pore/ion channels on bacterial cell membranes, respectively. In contrast, SMAP-18 may kill bacteria via intracellular-targeting mechanism.

Short KR-12 analogs designed from human cathelicidin LL-37 possessing both antimicrobial and antiendotoxic activities without mammalian cell toxicity.

KR-12 (residues 18-29 of LL-37) was known to be the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity. In order to optimize α-helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR-12 analogs. Highest hydrophobic analogs KR-12-a5 and KR-12-a6 displayed greater inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α production and higher LPS-binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR-12 analogs, KR-12-a2, KR-12-a3 and KR-12-a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL-37. KR-12-a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL-37. Also, these KR-12 analogs (KR-12-a2, KR-12-a3, KR-12-a4 and KR-12-a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 µM) against methicillin-resistant Staphylococcus aureus. Taken together, these KR-12 analogs have the potential for future development as a novel class of antimicrobial and anti-inflammatory therapeutic agents.

The inhibitory effects of Escherichia coli maltose binding protein on ß-amyloid aggregation and cytotoxicity.

The aggregation of ß-amyloid (Aß) peptide from its monomeric to its fibrillar form importantly contributes to the development of Alzheimer's disease. Here, we investigated the effects of Escherichia coli maltose binding protein (MBP), which has been previously used as a fusion protein, on Aß42 fibrillization, in order to improve understanding of the self-assembly process and the cytotoxic mechanism of Aß42. MBP, at a sub-stoichiometric ratio with respect to Aß42, was found to have chaperone-like inhibitory effects on ß-sheet fibril formation, due to the accumulation of Aß42 aggregates by sequestration of active Aß42 species as Aß42-MBP complexes. Furthermore, MBP increased the lag time of Aß42 polymerization, decreased the growth rate of fibril extension, and suppressed Aß42 mediated toxicity in human neuroblastoma SH-SY5Y cells. It appears that MBP decreases the active concentration of Aß42 by sequestering it as Aß42-MBP complex, and that this sequestration suppresses ongoing nucleation and retards the growth rate of Aß42 species required for fibril formation. We speculate that inhibition of the growth rate of potent Aß42 species by MBP suppresses Aß42-mediated toxicity in SH-SY5Y cells.

Wild-type, Flemish, and Dutch amyloid-ß exhibit different cytotoxicities depending on Aß40 to Aß42 interaction time and concentration ratio.

Addition of amyloid ß (Aß) peptide Aß40 to Aß42 can delay Aß42 aggregation, but consequent cytotoxicity has been reported to be enhanced or diminished. In the present study, we found that cytotoxicity was enhanced when human neuroblastoma SH-SY5Y cells were incubated in a mixture of wt Aß42 and Aß40wt at a ratio of 1 : 10-20 (0.1 : 1-2 µM) for 24-36 h, whereas the enhancement was detected in cells incubated for longer times (48-60 h) with the less amyloidogenic Flemish Aß40 variant or in cells incubated for as short as 12 h with the more amyloidogenic Dutch variant. Reductions in cytotoxicity by Aß40 were most prominently observed in the Flemish and wt Aß40/Aß42 mixture at ratio 1 : 20 incubated for a short time (~12 h). The most cytotoxic Aß40/Aß42 mixtures were enriched in Aß protofibril-like structures, implying a strong correlation between cytotoxicity and this structure, the formation of which was dependent on amyloidogenic properties and incubation time. The consequences of the interactions were probably because of the different amyloidogenic properties of the Aß40 variants, rather than to those of Aß42, because aggregation rates of Aß40 variants were highly dependent on sequence, whereas those of Aß42 variants were not. These studies highlight a potential role for Aß40 in cytotoxicity and provide novel mechanistic insights into the pathogenesis of each familial Alzheimer's disease-associated Aß40 variant.

Caspase-3-mediated cleavage of PICOT in apoptosis.

Mammalian protein kinase C-interacting cousin of thioredoxin (PICOT) is a multi-domain mono-thiol glutaredoxin that is involved in several signal transduction pathways and is necessary for cell growth and metastasis. Here, we demonstrate that PICOT is a cleavage substrate of the apoptosis-related protein caspase-3. In vitro cleavage assays indicated that PICOT was specifically cleaved by caspase-3. Similarly, endogenous PICOT was cleaved in cell death responses induced by staurosporine and etoposide. These phenomena were blocked in the presence of a pan-caspase inhibitor. Using site-directed mutagenesis, we identified two putative caspase-3 cleavage sequences in PICOT, DRLD(101)/G and EELD(226)/T. Interestingly, overexpression of either PICOT wild type or the D101A/D226A double point mutant accelerated etoposide-induced activation of caspase-3 whereas siRNA-mediated knockdown of PICOT blocked this phenomenon. Our data raise the possibility that the pro-apoptotic role of PICOT is actively regulated via caspase-3-mediated cleavage.

Keampferol-3-O-rhamnoside abrogates amyloid beta toxicity by modulating monomers and remodeling oligomers and fibrils to non-toxic aggregates.

BACKGROUND: Aggregation of soluble, monomeric ß- amyloid (Aß) to oligomeric and then insoluble fibrillar Aß is a key pathogenic feature in development of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aß oligomers, rather than to insoluble fibrils. The use of naturally occurring small molecules for inhibition of Aß aggregation has recently attracted significant interest for development of effective therapeutic strategies against the disease. A natural polyphenolic flavone, Kaempferol-3-O-rhamnoside (K-3-rh), was utilized to investigate its effects on aggregation and cytotoxic effects of Aß42 peptide. Several biochemical techniques were used to determine the conformational changes and cytotoxic effect of the peptide in the presence and absence of K-3-rh. RESULTS: K-3-rh showed a dose-dependent effect against Aß42 mediated cytotoxicity. Anti-amyloidogenic properties of K-3-rh were found to be efficient in inhibiting fibrilogenesis and secondary structural transformation of the peptide. The consequence of these inhibitions was the accumulation of oligomeric structural species. The accumulated aggregates were smaller, soluble, non-ß-sheet and non-toxic aggregates, compared to preformed toxic Aß oligomers. K-3-rh was also found to have the remodeling properties of preformed soluble oligomers and fibrils. Both of these conformers were found to remodel into non-toxic aggregates. The results showed that K-3-rh interacts with different Aß conformers, which affects fibril formation, oligomeric maturation and fibrillar stabilization. CONCLUSION: K-3-rh is an efficient molecule to hinder the self assembly and to abrogate the cytotoxic effects of Aß42 peptide. Hence, K-3-rh and small molecules with similar structure might be considered for therapeutic development against AD.

Prokaryotic selectivity and LPS-neutralizing activity of short antimicrobial peptides designed from the human antimicrobial peptide LL-37.

To develop novel antimicrobial peptides (AMPs) with shorter lengths, improved prokaryotic selectivity and retained lipolysaccharide (LPS)-neutralizing activity compared to human cathelicidin AMP, LL-37, a series of amino acid-substituted analogs based on IG-19 (residues 13-31 of LL-37) were synthesized. Among the IG-19 analogs, the analog a4 showed the highest prokaryotic selectivity, but much lower LPS-neutralizing activity compared to parental LL-37. The analogs, a5, a6, a7 and a8 with higher hydrophobicity displayed LPS-neutralizing activity comparable to that of LL-37, but much lesser prokaryotic selectivity. These results indicate that the proper hydrophobicity of the peptides is crucial to exert the amalgamated property of LPS-neutralizing activity and prokaryotic selectivity. Furthermore, to increase LPS-neutralizing activity of the analog a4 without a remarkable decrease in prokaryotic selectivity, we synthesized Trp-substituted analogs (a4-W1 and a4-W2), in which Phe(5) or Phe(15) of a4 is replaced by Trp. Despite their same prokaryotic selectivity, a4-W2 displayed much higher LPS-neutralizing activity compared to a4-W1. When compared with parental LL-37, a4-W2 showed retained LPS-neutralizing activity and 2.8-fold enhanced prokaryotic selectivity. These results suggest that the effective site for Trp-substitution when designing novel AMPs with higher LPS-neutralizing activity, without a remarkable reduction in prokaryotic selectivity, is the amphipathic interface between the end of the hydrophilic side and the start of the hydrophobic side rather than the central position of the hydrophobic side in their α-helical wheel projection. Taken together, the analog a4-W2 can serve as a promising template for the development of therapeutic agents for the treatment of endotoxic shock and bacterial infection.

Antimicrobial activity, bactericidal mechanism and LPS-neutralizing activity of the cell-penetrating peptide pVEC and its analogs.

pVEC is a cell-penetrating peptide derived from the murine vascular endothelial-cadherin protein. To evaluate the potential of pVEC as antimicrobial peptide (AMP), we synthesized pVEC and its analogs with Trp and Arg/Lys substitution, and their antimicrobial and lipopolysaccharide (LPS)-neutralizing activities were investigated. pVEC and its analogs displayed a potent antimicrobial activity (minimal inhibitory concentration: 4-16 µM) against Gram-positive and Gram-negative bacteria but no or less hemolytic activity (less than 10% hemolysis) even at a concentration of 200 µM. These peptides induced a near-complete membrane depolarization (more than 80%) at 4 µM against Staphylococcus aureus and a significant dye leakage (35-70%) from bacterial membrane-mimicking liposome at a concentration as low as 1 µM. The fluorescence profiles of pVEC and its analogs in dye leakage from liposome and membrane depolarization were similar to those of a frog-derived AMP, magainin 2. These results suggest that pVEC and its analogs kill bacteria by forming a pore or ion channel in the cytoplasmic membrane. pVEC and its analogs significantly inhibited nitric oxide production or tumor necrosis factor-α release in LPS-stimulated mouse macrophage RAW264.7 cells at 10 to 50 µM, in which RAW264.7 were not damaged. Taken together, our results suggest that pVEC and its analogs with potent antimicrobial and LPS-neutralizing activities can serve as AMPs for the treatment of microbial infection and sepsis.