Viral particles of human severe acute respiratory syndrome coronavirus (SARS CoV) consist of three virion structural proteins, including spike protein, membrane protein, and envelope protein. In this report, virus-like particles were assembled in insect cells by the co-infection with recombinant baculoviruses, which separately express one of these three virion proteins. We found that the membrane and envelope proteins are sufficient for the efficient formation of virus-like particles and could be visualized by electron microscopy. Sucrose gradient purification followed by Western blot analysis and immunogold labeling showed that the spike protein could be incorporated into the virus like particle also. The construction of engineered virus-like particles bearing resemblance to the authentic one is an important step towards the development of an effective vaccine against infection of SARS CoV.

Antimicrobial peptides (AMPs) act either through membrane lysis or by attacking intracellular targets. Intracellular targeting AMPs are a resource for antimicrobial agent development. Several AMPs have been identified as intracellular targeting peptides; however, the intracellular targets of many of these peptides remain unknown. In the present study, we used an Escherichia coli proteome microarray to systematically identify the protein targets of three intracellular targeting AMPs: bactenecin 7 (Bac7), a hybrid of pleurocidin and dermaseptin (P-Der), and proline-arginine-rich peptide (PR-39). In addition, we also included the data of lactoferricin B (LfcinB) from our previous study for a more comprehensive analysis. We analyzed the unique protein hits of each AMP in the Kyoto Encyclopedia of Genes and Genomes. The results indicated that Bac7 targets purine metabolism and histidine kinase, LfcinB attacks the transcription-related activities and several cellular carbohydrate biosynthetic processes, P-Der affects several catabolic processes of small molecules, and PR-39 preferentially recognizes proteins involved in RNA-and folate-metabolism-related cellular processes. Moreover, both Bac7 and LfcinB target purine metabolism, whereas LfcinB and PR-39 target lipopolysaccharide biosynthesis. This suggested that LfcinB and Bac7 as well as LfcinB and PR-39 have a synergistic effect on antimicrobial activity, which was validated through antimicrobial assays. Furthermore, common hits of all four AMPs indicated that all of them target arginine decarboxylase, which is a crucial enzyme for Escherichia coli survival in extremely acidic environments. Thus, these AMPs may display greater inhibition to bacterial growth in extremely acidic environments. We have also confirmed this Natural antimicrobial peptides (AMPs)1 are an evolutionarily conserved defense system of organisms against invading microorganisms. AMPs are effective against a wide range of microorganisms including bacteria, fungi, parasites, and some viruses (1). In general, AMPs are cationic peptides with fewer than 50 amino acid residues. To date, two main mechanisms of action have been identified for AMPs acting as antimicrobial agents (2): (1) membrane integrity disruption and (2) intracellular activity inhibition. Although the basic properties of all AMPs are similar, each AMP has a unique structure and microbial intracellular activity inhibition mechanism. For example, indolicidin inhibits DNA synthesis (3), whereas buforin II binds to DNA and RNA (4), mersacidin (a lantibiotic) binds to Lipid II and blocks peptidoglycan metabolism (5), tachyplesin I binds to the minor groove of DNA duplexes (6), microcin B17 inhibits DNA gyrase (thus influencing DNA replication) (7,8), microcin J25 recognizes the secondary channel of RNA polymerase (inhibiting transcription) (9), and pyrrhocoricin inhibits the biological function of the heat shock protein, DnaK (10). However, because no systematic study has been reported, the intracellular targets of numerous AMPs, such as b...

Antimicrobial peptides have been considered well-deserving candidates to fight the battle against microorganisms due to their broad-spectrum antimicrobial activities. Several studies have suggested that membrane disruption is the basic mechanism of AMPs that leads to killing or inhibiting microorganisms. Also, AMPs have been reported to interact with macromolecules inside the microbial cells such as nucleic acids (DNA/RNA), protein synthesis, essential enzymes, membrane septum formation and cell wall synthesis. Proteins are associated with many intracellular mechanisms of cells, thus protein targets may be specifically involved in mechanisms of action of AMPs. AMPs like pyrrhocoricin, drosocin, apidecin and Bac 7 are documented to have protein targets, DnaK and GroEL. Moreover, the intracellular targeting AMPs are reported to influence more than one protein targets inside the cell, suggesting for the multiple modes of actions. This complex mechanism of intracellular targeting AMPs makes them more difficult for the development of resistance. Herein, we have summarized the current status of AMPs in terms of their mode of actions, entry to cytoplasm and inhibition of macromolecules. To reveal the mechanism of action, we have focused on AMPs with intracellular protein targets. We have also included the use of high-throughput proteome microarray to determine the unidentified AMP protein targets in this review.

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF) 1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 M of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P 2 -specific binding proteins (PI(3,5)P 2 -BPs). We not only recovered many proteins that possessed known PI(3,5)P 2 -binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P 2 -BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P 2 -BPs were similar to those associated with PI(3,5)P 2 , including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P 2 -BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P 2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P 2 -binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies. Cell viability and physiological functions are maintained through a complex biomolecular interaction network. One of the key components in the regulatory system includes lipidprotein interactions that mediate various cell responses and metabolisms. Increasing evidence shows that such interactions have profound influences on cell polarization, the cell cycle, and other cellular processes. To date, in vitro characterizations of lipid interactions with other biomolecules are often conducted using artificial membrane models, such as liposomal nanovesicles, to mimic biological membranes. Liposomal nanovesicles, termed liposomes, are spherical vesicles that are surrounded by phospholipid bilayers in which the lipid of interest can be incorporated. An important benefit of liposomes is the ease in which a large number of fluorescent molecules can be encapsulated so that the liposome binding signals can be greatly enhanced for detection (1-4). Therefore, liposomes have become a practical and popular tool for