Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates

Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates

Involvement of Hepatitis C Virus NS5A Hyperphosphorylation Mediated by Casein Kinase I- in Infectious Virus Production

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle

A putative mobile genetic element carrying a novel type IIF restriction-modification system (PluTI)

Genome comparison and genome context analysis were used to find a putative mobile element in the genome of Photorhabdus luminescens, an entomopathogenic bacterium. The element is composed of 16-bp direct repeats in the terminal regions, which are identical to a part of insertion sequences (ISs), a DNA methyltransferase gene homolog, two genes of unknown functions and an open reading frame (ORF) (plu0599) encoding a protein with no detectable sequence similarity to any known protein. The ORF (plu0599) product showed DNA endonuclease activity, when expressed in a cell-free expression system. Subsequently, the protein, named R.PluTI, was expressed in vivo, purified and found to be a novel type IIF restriction enzyme that recognizes 5′-GGCGC/C-3′ (/ indicates position of cleavage). R.PluTI cleaves a two-site supercoiled substrate at both the sites faster than a one-site supercoiled substrate. The modification enzyme homolog encoded by plu0600, named M.PluTI, was expressed in Escherichia coli and shown to protect DNA from R.PluTI cleavage in vitro, and to suppress the lethal effects of R.PluTI expression in vivo. These results suggested that they constitute a restriction–modification system, present on the putative mobile element. Our approach thus allowed detection of a previously uncharacterized family of DNA-interacting proteins

mRNA Display Selection of an Optimized MDM2-Binding Peptide That Potently Inhibits MDM2-p53 Interaction

p53 is a tumor suppressor protein that prevents tumorigenesis through cell cycle arrest or apoptosis of cells in response to cellular stress such as DNA damage. Because the oncoprotein MDM2 interacts with p53 and inhibits its activity, MDM2-p53 interaction has been a major target for the development of anticancer drugs. While previous studies have used phage display to identify peptides (such as DI) that inhibit the MDM2-p53 interaction, these peptides were not sufficiently optimized because the size of the phage-displayed random peptide libraries did not cover all of the possible sequences. In this study, we performed selection of MDM2-binding peptides from large random peptide libraries in two stages using mRNA display. We identified an optimal peptide named MIP that inhibited the MDM2-p53 and MDMX-p53 interactions 29- and 13-fold more effectively than DI, respectively. Expression of MIP fused to the thioredoxin scaffold protein in living cells by adenovirus caused stabilization of p53 through its interaction with MDM2, resulting in activation of the p53 pathway. Furthermore, expression of MIP also inhibited tumor cell proliferation in a p53-dependent manner more potently than DI. These results show that two-stage, mRNA-displayed peptide selection is useful for the rapid identification of potent peptides that target oncoproteins

Use of humanised rat basophilic leukaemia cell line RS-ATL8 for the assessment of allergenicity of Schistosoma mansoni proteins.

BACKGROUND Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites. METHODOLOGY/PRINCIPAL FINDINGS A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line. CONCLUSION/SIGNIFICANCE This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16–27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17–P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60–70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4th ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity

Behavioural observations in gunn rats

The Gunn rat is a hooded mutant of albino rat with various biochemical defects, including a low UDP-glucuronosyl-transferase activity. As a consequence, about half of their offspring are jaundiced from birth, due to high free bilirubin levels, and develop widespread brain damage. The behaviour of both jaundiced and nonjaundiced Gunn rats was studied in four different tests in a shuttle-box and in a stepthrough passive avoidance situation, and compared with that of normal hooded rats. No differences among groups were found in performance of shuttle responses to a tone in a pseudoconditioning paradigm in which tones and shocks were given at random. However, rats from the two Gunn groups made less shuttlings to the tone in two tests that involved an avoidance contingency (each response cancelled one shock). In addition, nonicteric Gunn rats also performed poorly in a classical conditioning test in the shuttle-box (tones and shocks paired on every trial regardless of responses). This last deficiency of non-icteric Gunn rats may be explained by their higher tendency to freeze in situations involving stimulus-stimulus interactions. They also showed a higher latency than that of the two other groups to enter the dark side of the step-through apparatus on their first exposure to it. All animals seemed to learn the passive-avoidance task to the same extent, however, as shown in a retest carried out 48 h later. Both Gunn groups were hypersensitive to the stereotyped-behaviour-inducing action of apomorphine (0.125–1.0 mg/kg, i.p.), but all groups were about equally sensitive to that of d -amphetamine sulfate (0.5–4.0 mg/kg). Since apomorphine is disposed of by glucuronidation, this might be explained by the low UDP-glucuronosyl-transferase activity known to exist in the Gunn animals. The present results show that additional genetic defects have developed by in-breeding in the Gunn population, which are unrelated to brain damage caused by bilirubin, and which can be well characterized from a behavioural standpoint.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46403/1/213_2004_Article_BF00426881.pd