Clin Infect Dis . 2012 May 9; Buyck JM, Plésiat P, Traore H, Vanderbist F, Tulkens PM, Van Bambeke F BackgroundMacrolides show high MICs against Pseudomonas aeruginosa (PA) when tested in recommended media (Cation-Adjusted Muller-Hinton Broth [CA-MHB]). Nevertheless, azithromycin is successfully used in Cystic Fibrosis patients, supposedly due to “non-antibiotic effects”.MethodsCA-MHB and RPMI-1640 (used for growing eukaryotic cells) were compared for azithromycin MICs (+/- Phe-Arg-β-naphthylamide [PAβN], an efflux inhibitor), [(14)C]-clarithromycin accumulation, azithromycin-induced protein synthesis inhibition, oprM (encoding the outer membrane protein coupled with MexAB and MexXY efflux systems) expression, outer membrane permeability (OMP; tested with 1-N-phenylnaphthylamine and nitrocefin), and synergy (checkerboard assay) between azithromycin and outer-membrane disrupting agents (OMDA).
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PLoS Pathog. 2012 Apr; 8(4): e1002639 A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses. View original post here:
PLoS Pathog. 2012 Apr; 8(4): e1002639 A small open reading frame (ORF), pipo, overlaps with the P3 coding region of the potyviral polyprotein ORF. Previous evidence suggested a requirement for pipo for efficient viral cell-to-cell movement. Here, we provide immunoblotting evidence that the protein PIPO is expressed as a trans-frame protein consisting of the amino-terminal half of P3 fused to PIPO (P3N-PIPO). P3N-PIPO of Turnip mosaic virus (TuMV) fused to GFP facilitates its own cell-to-cell movement. Using a yeast two-hybrid screen, co-immunoprecipitation assays, and bimolecular fluorescence complementation (BiFC) assays, we found that P3N-PIPO interacts with host protein PCaP1, a cation-binding protein that attaches to the plasma membrane via myristoylation. BiFC revealed that it is the PIPO domain of P3N-PIPO that binds PCaP1 and that myristoylation of PCaP1 is unnecessary for interaction with P3N-PIPO. In PCaP1 knockout mutants (pcap1) of Arabidopsis, accumulation of TuMV harboring a GFP gene (TuMV-GFP) was drastically reduced relative to the virus level in wild-type plants, only small localized spots of GFP were visible, and the plants showed few symptoms. In contrast, TuMV-GFP infection in wild-type Arabidopsis yielded large green fluorescent patches, and caused severe stunting. However, viral RNA accumulated to high level in protoplasts from pcap1 plants indicating that PCaP1 is not required for TuMV RNA synthesis. In contrast to TuMV, the tobamovirus Oilseed rape mosaic virus did not require PCaP1 to infect Arabidopsis plants. We conclude that potyviral P3N-PIPO interacts specifically with the host plasma membrane protein PCaP1 to participate in cell-to-cell movement. We speculate that PCaP1 links a complex of viral proteins and genomic RNA to the plasma membrane by binding P3N-PIPO, enabling localization to the plasmodesmata and cell-to-cell movement. The PCaP1 knockout may contribute to a new strategy for recessive resistance to potyviruses.
Protein Expr Purif. 2012 Apr 5; Prostaglandin H synthase-1 and -2 (PGHS-1 and PGHS-2, EC 1.14.99.1) are membrane associated glycoproteins that catalyze the first two steps in prostaglandin synthesis. As the enzymes play an important regulatory role in several physiological and pathophysiological processes, recombinant PGHS isoforms are widely used in biomedical research. In the present study, we expressed human PGHS-2 (hPGHS-2) with and without a six histidine sequence tag (His(6) tag) near the amino- or carboxy-terminus of the protein in the Pichia pastoris (P. pastoris) expression system using native or yeast signal sequences. The recombinant His(6) tagged hPGHS-2 was purified using Ni-affinity and anion exchange chromatography, whereas the purification of the C-terminally His(6) tagged hPGHS-2 was more efficient. K(m), k(cat) and IC(50) values were determined to characterize the protein. The data obtained indicate that both the N- and C-terminally His(6) tagged hPGHS-2 are functional and the catalytic properties of the recombinant protein and the enzyme produced in other expression systems are comparable. As the yeast culture is easy to handle, the P. pastoris system could serve as an alternative to the most commonly used baculovirus-insect cell expression system for the production of the recombinant PGHS-2. The rest is here: In combination with robust cell free synthesis systems such as those developed by Volker Dõtsch and colleagues (see Klammt et al. for a review, and the Membrane Protein Expression and Purification Core page of this website for detailed protocols), the … Recent successes are indeed encouraging and the advantages provided by combining the two approaches represent an untapped resource in the analysis of the structure and dynamics of membrane proteins. Read this article:
Mol Ecol. 2012 Apr 10; The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO(2) on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO(2) on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO(2) -driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane-associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO(2) -driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO(3) deposition. The skeletal organic matrix effects included elevated expression of adult-type galaxins and some secreted acidic proteins, but down-regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral-specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification. Follow this link:
Mol Ecol. 2012 Apr 10; The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO(2) on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO(2) on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO(2) -driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane-associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO(2) -driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO(3) deposition. The skeletal organic matrix effects included elevated expression of adult-type galaxins and some secreted acidic proteins, but down-regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral-specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification. See the original post: Proc Natl Acad Sci U S A . 2012 Apr 9; Whelan RS, Konstantinidis K, Wei AC, Chen Y, Reyna DE, Jha S, Yang Y, Calvert JW, Lindsten T, Thompson CB, Crow MT, Gavathiotis E, Dorn GW, O’Rourke B, Kitsis RN The defining event in apoptosis is mitochondrial outer membrane permeabilization (MOMP), allowing apoptogen release. In contrast, the triggering event in primary necrosis is early opening of the inner membrane mitochondrial permeability transition pore (mPTP), precipitating mitochondrial dysfunction and cessation of ATP synthesis Link:
Microb Cell Fact. 2012 Apr 4; 11(1): 42 ABSTRACT: BACKGROUND: Caffeic acid (3,4-dihydroxycinnamic acid) is a natural phenolic compound derived from the plant phenylpropanoid pathway. Caffeic acid and its phenethyl ester (CAPE) have attracted increasing attention for their various pharmaceutical properties and health-promoting effects. Nowadays, large-scale production of drugs or drug precursors via microbial approaches provides a promising alternative to chemical synthesis and extraction from plant sources. RESULTS: We first identified that an Escherichia coli native hydroxylase complex previously characterized as the 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) was able to convert p-coumaric acid to caffeic acid efficiently. This critical enzymatic step catalyzed in plants by a membrane-associated cytochrome P450 enzyme, p-coumarate 3-hydroxylase (C3H), is difficult to be functionally expressed in prokaryotic systems. Moreover, the performances of two tyrosine ammonia lyases (TALs) from Rhodobacter species were compared after overexpression in E. coli. The results indicated that the TAL from R. capsulatus (Rc) possesses higher activity towards both tyrosine and L-dopa. Based on these findings, we further designed a dual pathway leading from tyrosine to caffeic acid consisting of the enzymes 4HPA3H and RcTAL. This heterologous pathway extended E. coli native tyrosine biosynthesis machinery and was able to produce caffeic acid (12.1 mg/L) in minimal salt medium. Further improvement in production was accomplished by boosting tyrosine biosynthesis in E. coli, which involved the alleviation of tyrosine-induced feedback inhibition and carbon flux redirection. Finally, the titer of caffeic acid reached 50.2 mg/L in shake flasks after 48-hour cultivation. CONCLUSION: We have successfully established a novel pathway and constructed an E. coli strain for the production of caffeic acid. This work forms a basis for further improvement in production, as well as opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from caffeic acid. In addition, we have identified that TAL is the rate-limiting enzyme in this pathway. Thus, exploration for more active TALs via bio-prospecting and protein engineering approaches is necessary for further improvement of caffeic acid production. Here is the original post:
PLoS One. 2012; 7(3): e34037 Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which is induced in cells undergoing a collapse of Δψ. Consistent with the notion that LrgA modulates murein hydrolase activity, COL grown in the presence of amicoumacin A showed reduced autolysis, which was primarily caused by lower hydrolase activity. To gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by a serial passage method was carried out. Single point mutations generating codon substitutions were uncovered in ksgA (encoding RNA dimethyltransferase), fusA (elongation factor G), dnaG (primase), lacD (tagatose 1,6-bisphosphate aldolase), and SACOL0611 (a putative glycosyl transferase). The codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance reside in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability. Read more here: Microb Drug Resist . 2012 Mar 20; Dziarski R, Kashyap DR, Gupta D Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and function in antibacterial immunity. Go here to read the rest: Microb Drug Resist . 2012 Mar 20; Dziarski R, Kashyap DR, Gupta D Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and function in antibacterial immunity. We have revealed a novel mechanism of bacterial killing by innate immune system, in which mammalian PGRPs bind to bacterial cell wall or outer membrane and exploit bacterial stress defense response to kill bacteria See original here:
J Biol Chem. 2012 Mar 16; Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification, and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be S194 in the STAR-related sterol-binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to S194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR allowing this protein to induce maximal mitochondrial steroid formation. Originally posted here: Administrative Core · Information/Dissemination Core · Membrane Protein Expression/Purification · Chemical Synthesis · Synthetic Antigen Binder (SAB) Generation and Crystallography · Computational Modeling · Phage … Read this article: Structural Platform for Autolytic Activity of an Intact NS2B-NS3 Protease Complex from Dengue Virus.
Biochemistry. 2012 Mar 8; Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing of the single-chain polyprotein precursor into ten functional proteins. This vital process relies on the two-component virus-encoded protease complex, of which the non-structural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated and studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane with fos-choline-14 detergent, and purified to apparent homogeneity. The detergent-solubilized protease complex retained autolytic activity, and intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further established a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage, and provide evidence for the importance of the two distal β-hairpins in activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form. See the original post here: |
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