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

Avaliação de linhagens, materiais comerciais e duas populações de milho para tolerância a alumínio.

Neste trabalho realizou-se a avaliacao da tolerancia a Al (4,5 mg.l) de 39 linhagens, 98 materiais comerciais, 167 progenies de uma populacao IAC-Maya e de 466 progenies de uma populacao IAC-Genetica, usando-se a tecnica de solucao nutritiva. Foram usadas na avaliacao as características ICR (indice de crescimento da radicula = comprimento relativo da radicula -CRR (CR+Al.CR-Al)- multiplicado por comprimento relativo da raiz secundaria mais longa - CRRSML (CRSML+Al/CRSML-Al) e CLR (comprimento liquido da radicula = diferenca entre os valores de comprimento da radicula - CR - obtidos no inicio e fim do periodo de crescimento das plantas em presenca de Al). As linhagens e as progenies da populacao IAC-Maya a de milho foram avaliadas através do ICR enquanto que os materiais comerciais e as progenies da populacao IAC-Genetica foram avaliados pelos valores de CLR. Os controles utilizados foram IAC HS1227 (tolerante a Al) e IAC HS7777 (sensivel a Al). O metodo de solucao nutritiva foi eficiente na diferenciacao da tolerancia a Al dentre os materiais testados, evidenciando a ocorrencia de ampla variabilidade genetica para essa caracteristica. As seguintes linhagens e materiais comerciais apresentaram tolerancia a Al (4,5 mg/l): Porto Rico 70. D,2, IP 38-5-3, IP 365-4-I, IA 2992-3-1-2-3, Vic 3-2-3-30-V-6, 490, 519, 532, 536-2 e 820 (linhagens) e AG 82, AG 260, AGROMEM 1022, ASGROW 1255, DINA 03S, DINA 47, IAC Hmd 7974, SS 1243 e UNICAMP 720 (materiais comerciais)

Avaliação de linhagens materiais comerciais e duas populações de milho para tolerância a alumínio.

Avaliacao da tolerancia a Al (4,5mg/l) de 39 linhagens, 98 materiais comerciais, 167 progenies de uma populacao IAC-Maya e de 466 progenies de uma populacao IAC-Taiuba de milho (Zea mays L), usando-se a tecnica de solucao nutritiva. Foram utilizados os seguintes indices: ICR (indice de crescimento da radicula), determinado atraves da multiplicacao dos indices CRR (comprimento relativo da radicula) e CRRSML (comprimento relativo da raiz secundaria mais longa), e CLR (comprimento liquido da radicula) calculado pela diferenca entre os valores de CR (comprimento da radicula) obtidos no inicio e fim do periodo de crescimento das plantas em presenca de aluminio. Os indices CRR e CRRSML foram derivados da relacao obtida entre os valores obtidos na presenca e ausencia de Al. As linhagens e as progenies da populacao IAC-Maya foram avaliadas atraves do ICR, enquanto os demais o foram pelo indice CLR. Os materiais controles foram IAC HS1227 (tolerante a Al) e IAC HS7777 (sensivel a Al). O metodo de solucao nutritiva foi eficiente na diferenciacao da tolerancia a Al dentre os materiais testados, evidenciando a ocorrencia de ampla variabilidade genetica para essa caracteristica. As seguintes linhagens e materiais comerciais apresentaram tolerancia ao Al(4,5 mg/l): Porto Rico 70.D.2, Ip 48-5-3, Ip 365-4-1, IA 2992-3-1-2-3, Vic 3-2-3-30-V6, 490, 519, 532, 535-2 e 820 (linhagens) e AG 82, AG260, AGROMEM 1022, ASGROW 1255, DINA 03 S, DINA 47, IAC Hmd 7974, SS 1243 e UNICAMP 720 (materiais comerciais)

Evaluation of inbred lines, commercial materials, and two maize populations for aluminum tolerance

Avaliação da tolerância a Al (4,5 mg/l) de 39 linhagens, 98 materiais comerciais, 167 progênies de uma população IAC-Maya e de 466 progênies de uma população IAC-Taiuba de milho (Zea mays L), usando-se a técnica de solução nutritiva. Foram utilizados os seguintes índices: ICR (índice de crescimento da radícula), determinado através da multiplicação dos índices CRR  (comprimento relativo da radícula) e CRRSML (comprimento relativo da raiz secundária mais longa), e CLR (comprimento líquido da radícula) calculado pela diferença entre os valores de CR (comprimento da radícula) obtidos no inicio e fim do período de crescimento das plantas em presença de alumínio. Os índices CRR e CRRSML foram 4erivados da relação obtida entre os valores obtidos na presença e ausência de Al. As linhagens e as progênies da população IAC-Maya foram avaliadas através do ICR, enquanto os demais o foram pelo índice CLR. Os materiais controles foram IAC HS1227 (tolerante a Al) e IAC HS7777 (sensível a Al). O método de solução nutritiva foi eficiente na diferenciação da tolerância a Al dentre os materiais testados, evidenciando a ocorrência de ampla variabilidade genética para essa característica. As seguintes linhagens e materiais comerciais apresentaram tolerância ao Al (4,5 mg/l): Porto Rico 70.D.2, Ip 48-5-3, Ip 365-4-1, IA 2992-3-1-2-3, Viç 3-2-3-30-V-6,490,519, 532, 535-2 e 820 (linhagens) e AG 82, AG 260, AGROMEM 1022, ASGROW 1255, DINA 03 5, DINA 47, IAC Hmd 7974,SS 1243 e UNICAMP 720 (materiais comerciais).The evaluation of 39 inbred lines, 98 commercial materials, 167 progenies from, an IAC-Maya population, and 466 progenies from an IAC-Taiuba population of maize (Zea mays L.) for Al tolerance in nutrient solutions was carried out. The following root characteristics were use: CRI (growth radicle index), determined by multiplying RRL (relative radicle length) and RLLSR (relative length of the longest secondary root), and NRL (net radicle length) estimated by the difference between the measurements of RL (radicle length) obtained at the beginning and at the end of the growth period in Al-stressed nutrient solutions. Both indices RRL and RLLSR were determined by dividing the values obtained in solutions with and no added Al. The characteristic GRI was used to evaluate the inbred lines and the IAC-Maya populations, while NRL was used for the commercial materials. The control materials were IAC HS 1227 (Al tolerant) and IAC HS 7777 (Al susceptible). The nutrient solution technique was efficient to differentiate Al tolerance among the maize genotypes tested. A wide genetic variability was found regarding the Al tolerance trait among the maize genotypes tested. The following maize inbred lines and commercial materials were tolerant to Al (4,5 mg/l): Porto Rico 70.D.2, Ip 48-5-3, Ip 365-4-1, IA 2992-3-1-2-3, Viç 3-2-3-30-V-6, 490, 519, 532, 535-2, and 820 (inbred lines) and AG 82, AO 260, AGROMEM 1022, ASGROW 1255, DINA 03S, DINA 47, IAC Hmd 7974, SS 1243, and UNICAMP 720 (commercial materials)

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

Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

Using newer vaccine platforms which have been effective against malaria in rodent models, we tested five immunization regimens against Plasmodium knowlesi in rhesus monkeys. All vaccines included the same four P. knowlesi antigens: the pre-erythrocytic antigens CSP, SSP2, and erythrocytic antigens AMA1, MSP1. We used four vaccine platforms for prime or boost vaccinations: plasmids (DNA), alphavirus replicons (VRP), attenuated adenovirus serotype 5 (Ad), or attenuated poxvirus (Pox). These four platforms combined to produce five different prime/boost vaccine regimens: Pox alone, VRP/Pox, VRP/Ad, Ad/Pox, and DNA/Pox. Five rhesus monkeys were immunized with each regimen, and five Control monkeys received a mock vaccination. The time to complete vaccinations was 420 days. All monkeys were challenged twice with 100 P. knowlesi sporozoites given IV. The first challenge was given 12 days after the last vaccination, and the monkeys receiving the DNA/Pox vaccine were the best protected, with 3/5 monkeys sterilely protected and 1/5 monkeys that self-cured its parasitemia. There was no protection in monkeys that received Pox malaria vaccine alone without previous priming. The second sporozoite challenge was given 4 months after the first. All 4 monkeys that were protected in the first challenge developed malaria in the second challenge. DNA, VRP and Ad5 vaccines all primed monkeys for strong immune responses after the Pox boost. We discuss the high level but short duration of protection in this experiment and the possible benefits of the long interval between prime and boost

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