Journal articles: 'LptC protein'

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Sperandeo, Paola, Fion K. Lau, Andrea Carpentieri, Cristina De Castro, Antonio Molinaro, Gianni Dehò, Thomas J. Silhavy, and Alessandra Polissi. "Functional Analysis of the Protein Machinery Required for Transport of Lipopolysaccharide to the Outer Membrane of Escherichia coli." Journal of Bacteriology 190, no. 13 (April 18, 2008): 4460–69. http://dx.doi.org/10.1128/jb.00270-08.

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ABSTRACT Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

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Martorana, Alessandra M., Mattia Benedet, Elisa A. Maccagni, Paola Sperandeo, Riccardo Villa, Gianni Dehò, and Alessandra Polissi. "Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli." Journal of Bacteriology 198, no. 16 (May 31, 2016): 2192–203. http://dx.doi.org/10.1128/jb.00329-16.

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ABSTRACTThe assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made inEscherichia coliof seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level oflptBis a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carriesPa-LptC, the highly divergent LptC orthologue fromPseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carryingPa-LptC which is otherwise defective in interactions with theE. coliLptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery.IMPORTANCEThe asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. InEscherichia colithe LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a transenvelope complex. Here, we show that increased expression of the membrane-associated ABC protein LptB can suppress defects of LptC, which participates in the formation of the periplasmic bridge. This reveals functional interactions between these two components and supports a role of LptB in the assembly of the Lpt machine.

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Lin, Yu-Ling, Li-Yi Chen, Chia-Hung Chen, Yen-Ku Liu, Wei-Tung Hsu, Li-Ping Ho, and Kuang-Wen Liao. "A Soybean Oil-Based Liposome-Polymer Transfection Complex as a Codelivery System for DNA and Subunit Vaccines." Journal of Nanomaterials 2012 (2012): 1–12. http://dx.doi.org/10.1155/2012/427306.

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Inexpensive liposome-polymer transfection complexes (LPTCs) were developed and used as for DNA or protein delivery. The particle sizes of the LPTCs were in the range of 212.2 to 312.1 nm, and the zetapotential was +38.7 mV. LPTCs condensed DNA and protected DNA from DNase I digestion and efficiently delivered LPTC/DNA complexes in Balb/3T3 cells. LPTCs also enhanced the cellular uptake of antigen in mouse macrophage cells and stimulated TNF-αrelease in naïve mice splenocytes, both indicating the potential of LPTCs as adjuvants for vaccines.In vivostudies were performed usingH. pylorirelative heat shock protein 60 as an antigen model. The vaccination of BALB/c mice with LPTC-complexed DNA and protein enhanced the humoral immune response. Therefore, we developed a DNA and protein delivery system using LPTCs that is inexpensive, and we successfully applied it to the development of a DNA and subunit vaccine.

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Dai, Xiaowei, Min Yuan, Yu Lu, Xiaohong Zhu, Chao Liu, Yifan Zheng, Shuyi Si, Lijie Yuan, Jing Zhang, and Yan Li. "Identification of a Small Molecule That Inhibits the Interaction of LPS Transporters LptA and LptC." Antibiotics 11, no. 10 (October 10, 2022): 1385. http://dx.doi.org/10.3390/antibiotics11101385.

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The need for novel antibiotics has become imperative with the increasing prevalence of antibiotic resistance in Gram-negative bacteria in clinics. Acting as a permeability barrier, lipopolysaccharide (LPS) protects Gram-negative bacteria against drugs. LPS is synthesized in cells and transported to the outer membrane (OM) via seven lipopolysaccharide transport (Lpt) proteins (LptA–LptG). Of these seven Lpt proteins, LptC interacts with LptA to transfer LPS from the inner membrane (IM) to the OM, and assembly is aided by LptD/LptE. This interaction among the Lpt proteins is important for the biosynthesis of LPS; therefore, the Lpt proteins, which are significant in the assembly process of LPS, can be a potential target for new antibiotics. In this study, a yeast two-hybrid (Y2H) system was used to screen compounds that could block LPS transport by inhibiting LptA/LptC interaction, which finally disrupts the biosynthesis of the OM. We selected the compound IMB-0042 for this study. Our results suggest that IMB-0042 disrupts LptA/LptC interaction by binding to both LptA and LptC. Escherichia coli cells, when treated with IMB-0042, showed filament morphology, impaired OM integrity, and an accumulation of LPS in the periplasm. IMB-0042 inhibited the growth of Gram-negative bacteria and showed synergistic sensitization to other antibiotics, with low cytotoxicity. Thus, we successfully identified a potential antibacterial agent by using a Y2H system, which blocks the transport of LPS by targeting LptA/LptC interaction in Escherichia coli.

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Hicks, Greg, and Zongchao Jia. "Structural Basis for the Lipopolysaccharide Export Activity of the Bacterial Lipopolysaccharide Transport System." International Journal of Molecular Sciences 19, no. 9 (September 10, 2018): 2680. http://dx.doi.org/10.3390/ijms19092680.

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Gram-negative bacteria have a dense outer membrane (OM) coating of lipopolysaccharides, which is essential to their survival. This coating is assembled by the LPS (lipopolysaccharide) transport (Lpt) system, a coordinated seven-subunit protein complex that spans the cellular envelope. LPS transport is driven by an ATPase-dependent mechanism dubbed the “PEZ” model, whereby a continuous stream of LPS molecules is pushed from subunit to subunit. This review explores recent structural and functional findings that have elucidated the subunit-scale mechanisms of LPS transport, including the novel ABC-like mechanism of the LptB2FG subcomplex and the lateral insertion of LPS into the OM by LptD/E. New questions are also raised about the functional significance of LptA oligomerization and LptC. The tightly regulated interactions between these connected subcomplexes suggest a pathway that can react dynamically to membrane stress and may prove to be a valuable target for new antibiotic therapies for Gram-negative pathogens.

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Xiang, Quanju, Haiyan Wang, Zhongshan Wang, Yizheng Zhang, and Changjiang Dong. "Characterization of lipopolysaccharide transport protein complex." Open Life Sciences 9, no. 2 (February 1, 2014): 131–38. http://dx.doi.org/10.2478/s11535-013-0250-5.

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AbstractLipopolysaccharide (LPS) is an essential component of the outer membranes (OM) of most Gram-negative bacteria, which plays a crucial role in protection of the bacteria from toxic compounds and harsh conditions. The LPS is biosynthesized at the cytoplasmic side of inner membrane (IM), and then transported across the aqueous periplasmic compartment and assembled correctly at the outer membrane. This process is accomplished by seven LPS transport proteins (LptA-G), but the transport mechanism remains poorly understood. Here, we present findings by pull down assays in which the periplasmic component LptA interacts with both the IM complex LptBFGC and the OM complex LptDE in vitro, but not with complex LptBFG. Using purified Lpt proteins, we have successfully reconstituted the seven transport proteins as a complex in vitro. In addition, the LptC may play an essential role in regulating the conformation of LptBFG to secure the lipopolysaccharide from the inner membrane. Our results contribute to the understanding of lipopolysaccharide transport mechanism and will provide a platform to study the detailed mechanism of the LPS transport in vitro.

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Vetterli, Stefan U., Katja Zerbe, Maik Müller, Matthias Urfer, Milon Mondal, Shuang-Yan Wang, Kerstin Moehle, et al. "Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport inEscherichia coli." Science Advances 4, no. 11 (November 2018): eaau2634. http://dx.doi.org/10.1126/sciadv.aau2634.

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With the increasing resistance of many Gram-negative bacteria to existing classes of antibiotics, identifying new paradigms in antimicrobial discovery is an important research priority. Of special interest are the proteins required for the biogenesis of the asymmetric Gram-negative bacterial outer membrane (OM). Seven Lpt proteins (LptA to LptG) associate in most Gram-negative bacteria to form a macromolecular complex spanning the entire envelope, which transports lipopolysaccharide (LPS) molecules from their site of assembly at the inner membrane to the cell surface, powered by adenosine 5′-triphosphate hydrolysis in the cytoplasm. The periplasmic protein LptA comprises the protein bridge across the periplasm, which connects LptB2FGC at the inner membrane to LptD/E anchored in the OM. We show here that the naturally occurring, insect-derived antimicrobial peptide thanatin targets LptA and LptD in the network of periplasmic protein-protein interactions required to assemble the Lpt complex, leading to the inhibition of LPS transport and OM biogenesis inEscherichia coli.

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Schultz, Kathryn M., and Candice S. Klug. "Characterization of and lipopolysaccharide binding to the E. coli LptC protein dimer." Protein Science 27, no. 2 (October 28, 2017): 381–89. http://dx.doi.org/10.1002/pro.3322.

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9

Cina, Nicholas P., and Candice S. Klug. "Characterizing the interactions between the LPS transport protein LptC and the ABC transporter LptB2FG." Biophysical Journal 122, no. 3 (February 2023): 56a. http://dx.doi.org/10.1016/j.bpj.2022.11.511.

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Naclerio, George A., and Herman O. Sintim. "Multiple ways to kill bacteria via inhibiting novel cell wall or membrane targets." Future Medicinal Chemistry 12, no. 13 (July 2020): 1253–79. http://dx.doi.org/10.4155/fmc-2020-0046.

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The rise of antibiotic-resistant infections has been well documented and the need for novel antibiotics cannot be overemphasized. US FDA approved antibiotics target only a small fraction of bacterial cell wall or membrane components, well-validated antimicrobial targets. In this review, we highlight small molecules that inhibit relatively unexplored cell wall and membrane targets. Some of these targets include teichoic acids-related proteins (DltA, LtaS, TarG and TarO), lipid II, Mur family enzymes, components of LPS assembly (MsbA, LptA, LptB and LptD), penicillin-binding protein 2a in methicillin-resistant Staphylococcus aureus, outer membrane protein transport (such as LepB and BamA) and lipoprotein transport components (LspA, LolC, LolD and LolE). Inhibitors of SecA, cell division protein, FtsZ and compounds that kill persister cells via membrane targeting are also covered.

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11

Lee, James, Mingyu Xue, Joseph S. Wzorek, Tao Wu, Marcin Grabowicz, Luisa S. Gronenberg, Holly A. Sutterlin, et al. "Characterization of a stalled complex on the β-barrel assembly machine." Proceedings of the National Academy of Sciences 113, no. 31 (July 20, 2016): 8717–22. http://dx.doi.org/10.1073/pnas.1604100113.

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The assembly of β-barrel proteins into membranes is mediated by an evolutionarily conserved machine. This process is poorly understood because no stable partially folded barrel substrates have been characterized. Here, we slowed the folding of the Escherichia coli β-barrel protein, LptD, with its lipoprotein plug, LptE. We identified a late-stage intermediate in which LptD is folded around LptE, and both components interact with the two essential β-barrel assembly machine (Bam) components, BamA and BamD. We propose a model in which BamA and BamD act in concert to catalyze folding, with the final step in the process involving closure of the ends of the barrel with release from the Bam components. Because BamD and LptE are both soluble proteins, the simplest model consistent with these findings is that barrel folding by the Bam complex begins in the periplasm at the membrane interface.

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Tran, An X., Changjiang Dong, and Chris Whitfield. "Structure and Functional Analysis of LptC, a Conserved Membrane Protein Involved in the Lipopolysaccharide Export Pathway inEscherichia coli." Journal of Biological Chemistry 285, no. 43 (August 18, 2010): 33529–39. http://dx.doi.org/10.1074/jbc.m110.144709.

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Tran, An X., Changjiang Dong, and Chris Whitfield. "Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway inEscherichia coli." Journal of Biological Chemistry 292, no. 45 (November 10, 2017): 18731. http://dx.doi.org/10.1074/jbc.aac117.000510.

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Marchesini, María Inés, Ansgar Poetsch, Leticia Soledad Guidolín, and Diego J. Comerci. "Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion." Microorganisms 10, no. 1 (January 6, 2022): 114. http://dx.doi.org/10.3390/microorganisms10010114.

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Rhomboids are intramembrane serine proteases highly conserved in the three domains of life. Their key roles in eukaryotes are well understood but their contribution to bacterial physiology is still poorly characterized. Here we demonstrate that Brucella abortus, the etiological agent of the zoonosis called brucellosis, encodes an active rhomboid protease capable of cleaving model heterologous substrates like Drosophila melanogaster Gurken and Providencia stuartii TatA. To address the impact of rhomboid deletion on B. abortus physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. About 50% of the B. abortus predicted proteome was identified by quantitative proteomics under two experimental conditions and 108 differentially represented proteins were detected. Membrane associated proteins that showed variations in concentration in the mutant were considered as potential rhomboid targets. This class included nitric oxide reductase subunit C NorC (Q2YJT6) and periplasmic protein LptC involved in LPS transport to the outer membrane (Q2YP16). Differences in secretory proteins were also addressed. Differentially represented proteins included a putative lytic murein transglycosylase (Q2YIT4), nitrous-oxide reductase NosZ (Q2YJW2) and high oxygen affinity Cbb3-type cytochrome c oxidase subunit (Q2YM85). Deletion of rhomboid had no obvious effect in B. abortus virulence. However, rhomboid overexpression had a negative impact on growth under static conditions, suggesting an effect on denitrification enzymes and/or high oxygen affinity cytochrome c oxidase required for growth in low oxygen tension conditions.

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Sperandeo, Paola, Rachele Cescutti, Riccardo Villa, Cristiano Di Benedetto, Daniela Candia, Gianni Dehò, and Alessandra Polissi. "Characterization of lptA and lptB, Two Essential Genes Implicated in Lipopolysaccharide Transport to the Outer Membrane of Escherichia coli." Journal of Bacteriology 189, no. 1 (October 20, 2006): 244–53. http://dx.doi.org/10.1128/jb.01126-06.

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ABSTRACT The outer membrane (OM) of gram-negative bacteria is an asymmetric lipid bilayer that protects the cell from toxic molecules. Lipopolysaccharide (LPS) is an essential component of the OM in most gram-negative bacteria, and its structure and biosynthesis are well known. Nevertheless, the mechanisms of transport and assembly of this molecule in the OM are poorly understood. To date, the only proteins implicated in LPS transport are MsbA, responsible for LPS flipping across the inner membrane, and the Imp/RlpB complex, involved in LPS targeting to the OM. Here, we present evidence that two Escherichia coli essential genes, yhbN and yhbG, now renamed lptA and lptB, respectively, participate in LPS biogenesis. We show that mutants depleted of LptA and/or LptB not only produce an anomalous LPS form, but also are defective in LPS transport to the OM and accumulate de novo-synthesized LPS in a novel membrane fraction of intermediate density between the inner membrane (IM) and the OM. In addition, we show that LptA is located in the periplasm and that expression of the lptA-lptB operon is controlled by the extracytoplasmic σ factor RpoE. Based on these data, we propose that LptA and LptB are implicated in the transport of LPS from the IM to the OM of E. coli.

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Botos, Istvan, Nicholas Noinaj, and Susan K. Buchanan. "Insertion of proteins and lipopolysaccharide into the bacterial outer membrane." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1726 (June 19, 2017): 20160224. http://dx.doi.org/10.1098/rstb.2016.0224.

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The bacterial outer membrane contains phospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. Both proteins and LPS must be frequently inserted into the outer membrane to preserve its integrity. The protein complex that inserts LPS into the outer membrane is called LptDE, and consists of an integral membrane protein, LptD, with a separate globular lipoprotein, LptE, inserted in the barrel lumen. The protein complex that inserts newly synthesized outer-membrane proteins (OMPs) into the outer membrane is called the BAM complex, and consists of an integral membrane protein, BamA, plus four lipoproteins, BamB, C, D and E. Recent structural and functional analyses illustrate how these two complexes insert their substrates into the outer membrane by distorting the membrane component (BamA or LptD) to directly access the lipid bilayer. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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Benedet, Mattia, Federica A. Falchi, Simone Puccio, Cristiano Di Benedetto, Clelia Peano, Alessandra Polissi, and Gianni Dehò. "The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter." PLOS ONE 11, no. 8 (August 16, 2016): e0161354. http://dx.doi.org/10.1371/journal.pone.0161354.

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Wenzel, Cory Q., Frank St. Michael, Jacek Stupak, Jianjun Li, Andrew D. Cox, and James C. Richards. "Functional Characterization of Lpt3 and Lpt6, the Inner-Core Lipooligosaccharide Phosphoethanolamine Transferases from Neisseria meningitidis." Journal of Bacteriology 192, no. 1 (October 23, 2009): 208–16. http://dx.doi.org/10.1128/jb.00558-09.

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ABSTRACT The lipooligosaccharide (LOS) of Neisseria meningitidis contains heptose (Hep) residues that are modified with phosphoethanolamine (PEtn) at the 3 (3-PEtn) and/or 6 (6-PEtn) position. The lpt3 (NMB2010) and lpt6 (NMA0408) genes of N. meningitidis, which are proposed to encode the required HepII 3- and 6-PEtn transferases, respectively, were cloned and overexpressed as C-terminally polyhistidine-tagged fusion proteins in Escherichia coli and found to localize to the inner membrane, based on sucrose density gradient centrifugation. Lpt3-His6 and Lpt6-His6 were purified from Triton X-100-solubilized membranes by nickel chelation chromatography, and dot blot analysis of enzymatic reactions with 3-PEtn- and 6-PEtn-specific monoclonal antibodies demonstrated conclusively that Lpt3 and Lpt6 are phosphatidylethanolamine-dependent LOS HepII 3- and 6-PEtn transferases, respectively, and that both enzymes are capable of transferring PEtn to both fully acylated LOS and de-O-acylated (de-O-Ac) LOS. Further enzymatic studies using capillary electrophoresis-mass spectrometry (MS) demonstrated that both Lpt3 and Lpt6 are capable of transferring PEtn to de-O-Ac LOS molecules already containing PEtn at the 6 and 3 positions of HepII, respectively, demonstrating that there is no obligate order of PEtn addition in the generation of 3,6-di-PEtn LOS moieties in vitro.

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Schultz, Kathryn M., and Candice S. Klug. "High-Pressure EPR Spectroscopy Studies of the E. coli Lipopolysaccharide Transport Proteins LptA and LptC." Applied Magnetic Resonance 48, no. 11-12 (September 21, 2017): 1341–53. http://dx.doi.org/10.1007/s00723-017-0948-z.

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Hsueh, Yi-Ching, Eva-M. Brouwer, Julian Marzi, Oliver Mirus, and Enrico Schleiff. "Functional properties of LptA and LptD in Anabaena sp. PCC 7120." Biological Chemistry 396, no. 9-10 (September 1, 2015): 1151–62. http://dx.doi.org/10.1515/hsz-2014-0322.

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Abstract Lipopolysaccharides (LPS) are central components of the outer membrane and consist of Lipid A, the core polysaccharide, and the O-antigen. The synthesis of LPS is initiated at the cytosolic face of the cytoplasmic membrane. The subsequent transport to and across the outer membrane involves multiple lipopolysaccharide transport (Lpt) proteins. Among those proteins, the periplasmic-localized LptA and the outer membrane-embedded LptD participate in the last steps of transfer and insertion of LPS into the outer membrane. While the process is described for proteobacterial model systems, not much is known about the machinery in cyanobacteria. We demonstrate that anaLptD (alr1278) of Anabaena sp. PCC 7120 is important for cell wall function and its pore domain shows a Lipid A sensitive cation-selective gating behavior. The N-terminal domain of anaLptD recognizes anaLptA (alr4067), but not ecLptA. Furthermore, anaLptA specifically interacts with the Lipid A from Anabaena sp. PCC 7120 only, while anaLptD binds to Lipid A isolated from Escherichia coli as well. Based on the comparative analysis of proteins from E. coli and Anabaena sp. we discuss the properties of the cyanobacterial Lpt system.

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Svanberg Frisinger, Frida, Bimal Jana, Stefano Donadio, and Luca Guardabassi. "In silico Prediction and Prioritization of Novel Selective Antimicrobial Drug Targets in Escherichia coli." Antibiotics 10, no. 6 (May 25, 2021): 632. http://dx.doi.org/10.3390/antibiotics10060632.

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Novel antimicrobials interfering with pathogen-specific targets can minimize the risk of perturbations of the gut microbiota (dysbiosis) during therapy. We employed an in silico approach to identify essential proteins in Escherichia coli that are either absent or have low sequence identity in seven beneficial taxa of the gut microbiota: Faecalibacterium, Prevotella, Ruminococcus, Bacteroides, Lactobacillus, Lachnospiraceae and Bifidobacterium. We identified 36 essential proteins that are present in hyper-virulent E. coli ST131 and have low similarity (bitscore < 50 or identity < 30% and alignment length < 25%) to proteins in mammalian hosts and beneficial taxa. Of these, 35 are also present in Klebsiella pneumoniae. None of the proteins are targets of clinically used antibiotics, and 3D structure is available for 23 of them. Four proteins (LptD, LptE, LolB and BamD) are easily accessible as drug targets due to their location in the outer membrane, especially LptD, which contains extracellular domains. Our results indicate that it may be possible to selectively interfere with essential biological processes in Enterobacteriaceae that are absent or mediated by unrelated proteins in beneficial taxa residing in the gut. The identified targets can be used to discover antimicrobial drugs effective against these opportunistic pathogens with a decreased risk of causing dysbiosis.

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Tusi, Solaleh Khoramian, Lien Nguyen, Kiruphagaran Thangaraju, Jian Li, John D. Cleary, Tao Zu, and Laura P. W. Ranum. "The alternative initiation factor eIF2A plays key role in RAN translation of myotonic dystrophy type 2 CCUG•CAGG repeats." Human Molecular Genetics 30, no. 11 (April 15, 2021): 1020–29. http://dx.doi.org/10.1093/hmg/ddab098.

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Abstract Repeat-associated non-ATG (RAN) proteins have been reported in 11 microsatellite expansion disorders but the factors that allow RAN translation to occur and the effects of different repeat motifs and alternative AUG-like initiation codons are unclear. We studied the mechanisms of RAN translation across myotonic dystrophy type 2 (DM2) expansion transcripts with (CCUG) or without (CAGG) efficient alternative AUG-like codons. To better understand how DM2 LPAC and QAGR RAN proteins are expressed, we generated a series of CRISPR/Cas9-edited HEK293T cell lines. We show that LPAC and QAGR RAN protein levels are reduced in protein kinase R (PKR)−/− and PKR-like endoplasmic reticulum kinase (PERK)−/− cells, with more substantial reductions of CAGG-encoded QAGR in PKR−/− cells. Experiments using mutant eIF2α-S51A HEK293T cells show that p-eIF2α is required for QAGR production. In contrast, LPAC levels were only partially reduced in these cells, suggesting that both non-AUG and close-cognate initiation occur across CCUG RNAs. Overexpression of the alternative initiation factor eIF2A increases LPAC and QAGR protein levels but, notably, has a much larger effect on QAGR expressed from CAGG-expansion RNAs that lack efficient close-cognate codons. The effects of eIF2A on increasing LPAC are consistent with previous reports that eIF2A affects CUG-initiation translation. The observation that eIF2A also increases QAGR proteins is novel because CAGG expansion transcripts do not contain CUG or similarly efficient close-cognate AUG-like codons. For QAGR but not LPAC, the eIF2A-dependent increases are not seen when p-eIF2α is blocked. These data highlight the differential regulation of DM2 RAN proteins and eIF2A as a potential therapeutic target for DM2 and other RAN diseases.

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Batistela, Emanuele, Mayara Peron Pereira, Juliany Torres Siqueira, Silvia Paula-Gomes, Neusa Maria Zanon, Eduardo Brandt Oliveira, Luiz Carlos Carvalho Navegantes, et al. "Decreased rate of protein synthesis, caspase-3 activity, and ubiquitin–proteasome proteolysis in soleus muscles from growing rats fed a low-protein, high-carbohydrate diet." Canadian Journal of Physiology and Pharmacology 92, no. 6 (June 2014): 445–54. http://dx.doi.org/10.1139/cjpp-2013-0290.

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The aim of this study was to investigate the changes in the rates of both protein synthesis and breakdown, and the activation of intracellular effectors that control these processes in soleus muscles from growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The mass and the protein content, as well as the rate of protein synthesis, were decreased in the soleus from LPHC-fed rats. The availability of amino acids was diminished, since the levels of various essential amino acids were decreased in the plasma of LPHC-fed rats. Overall rate of proteolysis was also decreased, explained by reductions in the mRNA levels of atrogin-1 and MuRF-1, ubiquitin conjugates, proteasome activity, and in the activity of caspase-3. Soleus muscles from LPHC-fed rats showed increased insulin sensitivity, with increased levels of insulin receptor and phosphorylation levels of AKT, which probably explains the inhibition of both the caspase-3 activity and the ubiquitin–proteasome system. The fall of muscle proteolysis seems to represent an adaptive response that contributes to spare proteins in a condition of diminished availability of dietary amino acids. Furthermore, the decreased rate of protein synthesis may be the driving factor to the lower muscle mass gain in growing rats fed the LPHC diet.

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Damron, F. Heath, Jennifer Napper, M. Allison Teter, and Hongwei D. Yu. "Lipotoxin F of Pseudomonas aeruginosa is an AlgU-dependent and alginate-independent outer membrane protein involved in resistance to oxidative stress and adhesion to A549 human lung epithelia." Microbiology 155, no. 4 (April 1, 2009): 1028–38. http://dx.doi.org/10.1099/mic.0.025833-0.

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Chronic lung infection with P. aeruginosa and excessive neutrophil-associated inflammation are major causes of morbidity and mortality in patients with cystic fibrosis (CF). Overproduction of an exopolysaccharide known as alginate leads to the formation of mucoid biofilms that are resistant to antibiotics and host defences. Alginate overproduction or mucoidy is controlled by a stress-related ECF sigma factor AlgU/T. Mutation in the anti-sigma factor MucA is a known mechanism for conversion to mucoidy. Recently, we showed that inactivation of a kinase (KinB) in nonmucoid strain PAO1 results in overproduction of alginate. Here, we report the initial characterization of lipotoxin F (LptF, PA3692), an OmpA-like outer membrane protein that exhibited increased expression in the mucoid PAO1kinB mutant. The lipotoxin family of proteins has been previously shown to induce inflammation in lung epithelia, which may play a role in CF disease progression. Expression of LptF was observed to be AlgU-dependent and upregulated in CF isolates. Deletion of lptF from the kinB mutant had no effect on alginate production. Deletion of lptF from PAO1 caused a differential susceptibility to oxidants that can be generated by phagocytes. The lptF and algU mutants were more sensitive to hypochlorite than PAO1. However, the lptF mutant displayed increased resistance to hydrogen peroxide. LptF also contributed to adhesion to A549 human lung epithelial cells. Our data suggest that LptF is an outer membrane protein that may be important for P. aeruginosa survival in harsh environments, including lung colonization in CF.

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Wahl, Devin, David Raubenheimer, Rafael de Cabo, David Sinclair, Stephen Simpson, Samantha Solon-Biet, and David Le Couteur. "Comparing the Effects of Low-Protein and High-Carbohydrate Diets and Caloric Restriction on Brain Aging in Mice." Innovation in Aging 4, Supplement_1 (December 1, 2020): 846. http://dx.doi.org/10.1093/geroni/igaa057.3103.

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Abstract The Geometric Framework for Nutrition (GFN) has revealed that ad-libitum low-protein, high-carbohydrate (LPHC) diets improve cardiometabolic health and extend lifespan in rodents, but it is not known whether these diets are also beneficial for brain health. Here, we utilized previous results from GFN studies and compared hippocampus biology and memory in mice subjected to 20% calorie restriction (CR) or provided ad-libitum access to several LPHC diets. RNA expression in the hippocampus of 15-month-old mice were similar between mice fed CR and LPHC diets. Nutrient-sensing proteins, including SIRT1, MTOR, and PGC1-alpha, were also influenced by diet; however, the effects varied by sex. CR and LPHC diets were associated with increased dendritic spines in dentate gyrus neurons. Mice fed CR and LPHC diets had modest improvements in the Barnes maze spatial recognition memory paradigm and novel object recognition test. LPHC diets recapitulate some of the benefits of CR on brain aging. Part of a symposium sponsored by the Nutrition Interest Group.

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Chang, Kai-Fu, Jinghua Tsai Chang, Xiao-Fan Huang, Yu-Ling Lin, Kuang-Wen Liao, Chien-Wei Huang, and Nu-Man Tsai. "Antitumor Effects of N-Butylidenephthalide Encapsulated in Lipopolyplexs in Colorectal Cancer Cells." Molecules 25, no. 10 (May 21, 2020): 2394. http://dx.doi.org/10.3390/molecules25102394.

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Colorectal cancer (CRC) is the third most common type of cancer and the second most common cause of cancer-related death in the world. N-Butylidenephthalide (BP), a natural compound, inhibits several cancers, such as hepatoma, brain tumor and colon cancer. However, due to the unstable structure, the activity of BP is quickly lost after dissolution in an aqueous solution. A polycationic liposomal polyethylenimine and polyethylene glycol complex (LPPC), a new drug carrier, encapsulates both hydrophobic and hydrophilic compounds, maintains the activity of the compound, and increases uptake of cancer cells. The purpose of this study is to investigate the antitumor effects and protection of BP encapsulated in LPPC in CRC cells. The LPPC encapsulation protected BP activity, increased the cytotoxicity of BP and enhanced cell uptake through clathrin-mediated endocytosis. Moreover, the BP/LPPC-regulated the expression of the p21 protein and cell cycle-related proteins (CDK4, Cyclin B1 and Cyclin D1), resulting in an increase in the population of cells in the G0/G1 and subG1 phases. BP/LPPC induced cell apoptosis by activating the extrinsic (Fas, Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways. Additionally, BP/LPPC combined with 5-FU synergistically inhibited the growth of HT-29 cells. In conclusion, LPPC enhanced the antitumor activity and cellular uptake of BP, and the BP/LPPC complex induced cell cycle arrest and apoptosis, thereby causing death. These findings suggest the putative use of BP/LPPC as an adjuvant cytotoxic agent for colorectal cancer.

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O'Connor, Ellen T., Andrzej Piekarowicz, Karen V. Swanson, J. McLeod Griffiss, and Daniel C. Stein. "Biochemical Analysis of Lpt3, a Protein Responsible forPhosphoethanolamine Addition to Lipooligosaccharide ofPathogenic Neisseria." Journal of Bacteriology 188, no. 3 (February 1, 2006): 1039–48. http://dx.doi.org/10.1128/jb.188.3.1039-1048.2006.

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ABSTRACT The inner core of neisserial lipooligosaccharide (LOS) contains heptose residues that can be decorated by phosphoethanolamine (PEA). PEA modification of heptose II (HepII) can occur at the 3, 6, or 7 position(s). We used a genomic DNA sequence of lpt3, derived from Neisseria meningitidis MC58, to search the genomic sequence of N. gonorrhoeae FA1090 and identified a homolog of lpt3 in N. gonorrhoeae. A PCR amplicon containing lpt3 was amplified from F62ΔLgtA, cloned, mutagenized, and inserted into the chromosome of N. gonorrhoeae strain F62ΔLgtA, producing strain F62ΔLgtAlpt3::Tn5. LOS isolated from this strain lost the ability to bind monoclonal antibody (MAb) 2-1-L8. Complementation of this mutation by genetic removal of the transposon insertion restored MAb 2-1-L8 binding. Mass spectrometry analysis of LOS isolated from the F62ΔLgtA indicated that this strain contained two PEA modifications on its LOS. F62ΔLgtAlpt3::Tn5 lacked a PEA modification on its LOS, a finding consistent with the hypothesis that lpt3 encodes a protein mediating PEA addition onto gonococcal LOS. The DNA encoding lpt3 was cloned into an expression vector and Lpt3 was purified. Purified Lpt3 was able to mediate the addition of PEA to LOS isolated from F62ΔLgtAlpt3::Tn5.

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Franco, Juliana Gastão, Egberto Gaspar de Moura, Josely Correa Koury, Paula Affonso Trotta, Aline Cordeiro, Luana Lopes Souza, Norma Aparecida dos Santos Almeida, et al. "Resveratrol reduces lipid peroxidation and increases sirtuin 1 expression in adult animals programed by neonatal protein restriction." Journal of Endocrinology 207, no. 3 (September 24, 2010): 319–28. http://dx.doi.org/10.1677/joe-10-0124.

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Resveratrol (Res) has been associated with protective effects against oxidative stress. This study evaluated the effect of Res over lipid peroxidation, antioxidant defense, hepatic sirtuin 1 (SIRT1), which up-regulates antioxidant enzymes, and copper/zinc superoxide dismutase (Cu/Zn SOD) in adult offspring whose mothers were protein restricted during lactation. Lactating Wistar rats were divided into control (C) group, which were fed a normal diet (23% protein), and low-protein and high-carbohydrate (LPHC) group, which were fed a diet containing 8% protein. After weaning (21 days), C and LPHC offspring were fed a normal diet until they were 180 days old. At the 160th day, animals were separated into four groups as follows: control, control+Res, LPHC, and LPHC+Res. Resveratrol was given for 20 days (30 mg/kg per day by gavage). LPHC animals showed a higher total antioxidant capacity (TAC) without change in lipid peroxidation and SIRT1 expression. The treatment with Res increased TAC only in the control group without effect on lipid peroxidation and SIRT1. LPHC animals treated with Res had lower lipid peroxidation and higher protein and mRNA expression of SIRT1 without any further increase in TAC. No significant difference in liver Cu/Zn SOD expression was observed among the groups. In conclusion, maternal protein restriction during lactation programs the offspring for a higher antioxidant capacity, and these animals seem to respond to Res treatment with a lower lipid peroxidation and higher hepatic SIRT1 expression that we did not observe in the Res-treated controls. It is probable that the protective effect can be attributed to Res activating SIRT1, only in the LPHC-programed group.

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Pankov, Genady, Alice Dawson, and William N. Hunter. "The structure of lipopolysaccharide transport protein B (LptB) from Burkholderia pseudomallei." Acta Crystallographica Section F Structural Biology Communications 75, no. 4 (March 13, 2019): 227–32. http://dx.doi.org/10.1107/s2053230x19001778.

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The thick outer membrane (OM) of Gram-negative bacteria performs an important protective role against hostile environments, supports cell integrity, and contributes to surface adhesion and in some cases also to virulence. A major component of the OM is lipopolysaccharide (LPS), a complex glycolipid attached to a core containing fatty-acyl chains. The assembly and transport of lipid A, the membrane anchor for LPS, to the OM begins when a heteromeric LptB2FG protein complex extracts lipid A from the outer leaflet of the inner membrane. This process requires energy, and upon hydrolysis of ATP one component of the heteromeric assembly, LptB, triggers a conformational change in LptFG in support of lipid A transport. A structure of LptB from the intracellular pathogen Burkholderia pseudomallei is reported here. LptB forms a dimer that displays a relatively fixed structure irrespective of whether it is in complex with LptFG or in isolation. Highly conserved sequence and structural features are discussed that allow LptB to fuel the transport of lipid A.

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Malke, Horst. "Cytoplasmic Membrane Lipoprotein LppC ofStreptococcus equisimilis Functions as an Acid Phosphatase." Applied and Environmental Microbiology 64, no. 7 (July 1, 1998): 2439–42. http://dx.doi.org/10.1128/aem.64.7.2439-2442.1998.

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ABSTRACT The function of the streptococcal cytoplasmic membrane lipoprotein, LppC, was identified with isogenic Streptococcus equisimilis H46A and Escherichia coli JM109 strain pairs differing in whether they contained [H46A and JM109(pLPP2)] or lacked (H46A lppC::pLPP10 and JM109) the functional lppC gene for comparative phosphatase determinations under acidic conditions. lppC-directed acid phosphatase activity was demonstrated zymographically and by specific enzymatic activity assays, with whole cells or cell membrane preparations as enzyme sources. LppC acid phosphatase showed optimum activity at pH 5, and the enzyme activity was unaffected by Triton X-100, l-(+)-tartaric acid, or EDTA. Database searches revealed significant structural homology of LppC to the Streptococcus pyogenes LppA,Flavobacterium meningosepticum OplA, Helicobacter pylori HP1285, and Haemophilus influenzae Hel [e (P4)] proteins. These results suggest a possible function for these proteins and establish a novel function of streptococcal cell membrane lipoproteins.

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Navarro-Meza, Mónica, Mauricio Díaz-Muñoz, Perla Belén García-Solano, Raquel Cobián-Cervantes, Éricka A. de los Ríos-Arellano, Felipe Santoyo Telles, and Mariela Camacho-Barrón. "Effects of Low Protein-High Carbohydrate Diet during Early and Late Pregnancy on Respiratory Quotient and Visceral Adiposity." Oxidative Medicine and Cellular Longevity 2022 (April 7, 2022): 1–11. http://dx.doi.org/10.1155/2022/3878581.

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Background. Low Protein-High Carbohydrate (LPHC) diet during pregnancy is considered a nutritional and health problem related to the development of maternal metabolic alterations, such as fatty liver and obesity in the perinatal and postnatal period. It is known that increase in visceral adiposity tissue (VAT) modulates maternal metabolic rate, with the respiratory quotient (RQ) being a parameter related to that variable; however, it is unknown whether LPHC intake during pregnancy affects the VAT and the RQ. In this study, we examine if consumption of LPHC during pregnancy modifies the VAT and RQ in early and late periods of pregnancy. Methods. This is a longitudinal and cross-sectional study with Wistar rats during gestation (G) (3, 8, 15, and 20) and nonpregnant rats. Rats were fed with a control diet with 63/18% carbohydrate/protein and an experimental diet with 79/6% carbohydrate/protein. We studied water and food consumption and metabolic parameters such as RQ and energy expenditure (EE), calculated by indirect calorimetry. In the cross-sectional study, we determined visceral fat, as well as the concentration of free fatty acids, insulin, glucose, and lipid profile in serum. Results. Nonpregnant rats with LPHC intake decreased significantly in VAT (86%) and the RQ (18%); in pregnant rats in early (8G) and late pregnancy (15G) in LPHC diet, both parameters (VAT and RQ) (25%-92%) increased during light time. When comparing time points during pregnancy in the control and LPHC groups, the RQ increased in 15G during daytime compared to 8G during the night period (17 and 5%, respectively). In late pregnancy, LPHC intake and triacylglyceride levels increased and cholesterol and glucose decreased (45 and 26%, respectively), in comparison to nonpregnant rats. Conclusions. LPHC intake in nonpregnant rats decreases the RQ and VAT. Interestingly, the opposite occurs in early pregnancy: the RQ and VAT increased, and this correlates with free fatty acid (FFA) levels. The increase in RQ and VAT during light time in early pregnancy increased mobilization of carbohydrate and protein metabolism. These results suggest that LPHC intake during pregnancy increases the glucose metabolism as a compensatory mechanism for energy needs in the fetus and the mother in early pregnancy.

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Daimon, Yasushi, Shin-ichiro Narita, Ryoji Miyazaki, Yohei Hizukuri, Hiroyuki Mori, Yoshiki Tanaka, Tomoya Tsukazaki, and Yoshinori Akiyama. "Reversible autoinhibitory regulation ofEscherichia colimetallopeptidase BepA for selective β-barrel protein degradation." Proceedings of the National Academy of Sciences 117, no. 45 (October 22, 2020): 27989–96. http://dx.doi.org/10.1073/pnas.2010301117.

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Escherichia coliperiplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer-membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD. The underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features: In particular, the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246–mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

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Buzelle, Samyra L., Maísa P. Santos, Amanda M. Baviera, Carbene F. Lopes, Maria A. R. Garófalo, Luiz C. C. Navegantes, Isis C. Kettelhut, Valéria E. Chaves, and Nair H. Kawashita. "A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats." Canadian Journal of Physiology and Pharmacology 88, no. 12 (December 2010): 1157–65. http://dx.doi.org/10.1139/y10-096.

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The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity — i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P — was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.

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Pereira, Mayara P., Samyra L. Buzelle, Emanuele Batistela, Diego L. Doneda, Suélem A. de França, Maísa P. dos Santos, Cláudia M. B. Andrade, et al. "High glucose uptake in growing rats adapted to a low-protein, high-carbohydrate diet determines low fasting glycemia even with high hepatic gluconeogenesis." Canadian Journal of Physiology and Pharmacology 92, no. 6 (June 2014): 460–66. http://dx.doi.org/10.1139/cjpp-2013-0346.

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The our objective was to investigate the adaptations induced by a low-protein, high-carbohydrate (LPHC) diet in growing rats, which by comparison with the rats fed a control (C) diet at displayed lower fasting glycemia and similar fasting insulinemia, despite impairment in insulin signaling in adipose tissues. In the insulin tolerance test the LPHC rats showed higher rates of glucose disappearance (30%) and higher tolerance to overload of glucose than C rats. The glucose uptake by the soleus muscle, evaluated in vivo by administration of 2-deoxy-[14C]glucose, increased by 81%. The phosphoenolpyruvate carboxykinase content and the incorporation of [1-14C]pyruvate into glucose was also higher in the slices of liver from the LPHC rats than in those from C rats. The LPHC rats showed increases in l-lactate as well as in other gluconeogenic precursors in the blood. These rats also had a higher hepatic production of glucose, evaluated by in situ perfusion. The data obtained indicate that the main substrates for gluconeogenesis in the LPHC rats are l-lactate and glycerol. Thus, we concluded that the fasting glycemia in the LPHC animals was maintained mainly by increases in the hepatic gluconeogenesis from glycerol and l-lactate, compensating, at least in part, for the higher glucose uptake by the tissues.

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Saidi, Oussama, Emmanuelle Rochette, Giovanna Del Sordo, Paul Peyrel, Jérôme Salles, Eric Doré, Etienne Merlin, Stéphane Walrand, and Pascale Duché. "Isocaloric Diets with Different Protein-Carbohydrate Ratios: The Effect on Sleep, Melatonin Secretion and Subsequent Nutritional Response in Healthy Young Men." Nutrients 14, no. 24 (December 13, 2022): 5299. http://dx.doi.org/10.3390/nu14245299.

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This study aimed to determine the short-term effect of two isocaloric diets differing in the ratio of protein–carbohydrate on melatonin levels, sleep, and subsequent dietary intake and physical activity in healthy young men. Twenty-four healthy men took part in a crossover design including two sessions of three days on isocaloric diets whether high-protein, low-carbohydrate (HPLC) or low-protein, high-carbohydrate (LPHC) followed by 24-h free living assessments. Sleep was measured by ambulatory polysomnography pre-post-intervention. Melatonin levels were assessed on the third night of each session on eight-point salivary sampling. Physical activity was monitored by accelerometry. On day 4, participants reported their 24-h ad-libitum dietary intake. LPHC resulted in better sleep quality and increased secretion of melatonin compared to HPLC. A significant difference was noted in sleep efficiency (p < 0.05) between the two sessions. This was mainly explained by a difference in sleep onset latency (p < 0.01) which was decreased during LPHC (PRE: 15.8 ± 7.8 min, POST: 11.4 ± 4.5 min, p < 0.001). Differences were also noted in sleep staging including time spent on REM (p < 0.05) and N1 (p < 0.05). More importantly, REM latency (PRE: 97.2 ± 19.9 min, POST 112.0 ± 20.7 min, p < 0.001) and cortical arousals (PRE: 7.2 ± 3.9 event/h, POST 8.5 ± 3.3 event/h) increased in response to HPLC diet but not LPHC. On day 4, 24-h ad-libitum energy intake was higher following HPLC compared to LPHC (+64 kcal, p < 0.05) and explained by increased snacking behavior (p < 0.01) especially from carbohydrates (p < 0.05). Increased carbohydrates intake was associated with increased cortical arousals.

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Schultz, Kathryn M., Tanner J. Lundquist, and Candice S. Klug. "Lipopolysaccharide binding to the periplasmic protein LptA." Protein Science 26, no. 8 (April 30, 2017): 1517–23. http://dx.doi.org/10.1002/pro.3177.

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Chang, Kai-Fu, Xiao-Fan Huang, Yu-Ling Lin, Kuang-Wen Liao, Ming-Chang Hsieh, Jinghua Tsai Chang, and Nu-Man Tsai. "Positively Charged Nanoparticle Delivery of n-Butylidenephthalide Enhances Antitumor Effect in Hepatocellular Carcinoma." BioMed Research International 2021 (March 19, 2021): 1–14. http://dx.doi.org/10.1155/2021/8817875.

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Hepatocellular carcinoma (HCC) is the second and sixth leading cause of cancer death in men and woman in 185 countries statistics, respectively. n-Butylidenephthalide (BP) has shown anti-HCC activity, but it also has an unstable structure that decreases its potential antitumor activity. The aim of this study was to investigate the cell uptake, activity protection, and antitumor mechanism of BP encapsulated in the novel liposome LPPC in HCC cells. BP/LPPC exhibited higher cell uptake and cytotoxicity than BP alone, and combined with clinical drug etoposide (VP-16), BP/LPPC showed a synergistic effect against HCC cells. Additionally, BP/LPPC increased cell cycle regulators (p53, p-p53, and p21) and decreased cell cycle-related proteins (Rb, p-Rb, CDK4, and cyclin D1), leading to cell cycle arrest at the G0/G1 phase in HCC cells. BP/LPPC induced cell apoptosis through activation of both the extrinsic (Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways and activated the caspase cascade to trigger HCC cell death. In conclusion, the LPPC complex improved the antitumor activity of BP in terms of cytotoxicity, cell cycle regulation and cell apoptosis, and BP/LPPC synergistically inhibited cell growth during combination treatment with VP-16 in HCC cells. Therefore, BP/LPPC is potentially a good candidate for clinical drug development or for use as an adjuvant for clinical drugs as a combination therapy for hepatocellular carcinoma.

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CHEN, Sharon C. A., Lesley C. WRIGHT, John C. GOLDING, and Tania C. SORRELL. "Purification and characterization of secretory phospholipase B, lysophospholipase and lysophospholipase/transacylase from a virulent strain of the pathogenic fungus Cryptococcus neoformans." Biochemical Journal 347, no. 2 (April 10, 2000): 431–39. http://dx.doi.org/10.1042/bj3470431.

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Infection caused by the fungus Cryptococcus neoformans is potentially fatal. A highly active extracellular phospholipase, demonstrating phospholipase B (PLB), lysophospholipase (LPL) and lysophospholipase/transacylase (LPTA) activities, was purified to homogeneity from C. neoformans using (NH4)2SO4 fractionation, and hydrophobic-interaction, anion-exchange and gel-filtration chromatography. All three enzyme activities co-purified as a single protein with an apparent molecular mass of 70-90 kDa by SDS/PAGE and 160-180 kDa by gel filtration. The ratio of the three activities remained constant after each purification step. The amino acid composition, as well as the sequences of the N-terminus and of five internal peptide fragments were novel. The protein was an acidic glycoprotein containing N-linked carbohydrate moieties, with pI values of 5.5 and 3.5. The apparent Vmax values for PLB and LPL activities were 12.3 and 870 μmol/min per mg of protein respectively; the corresponding Km values were approx. 185.3 and 92.2 μM. The enzyme was active only at acidic pH (pH optimum of 4.0 for PLB and 4.0-5.0 for LPL and LPTA). Enzyme activity did not require added cations, but was inhibited by Fe3+. LPL and LPTA activities were decreased by 0.1% (v/v) Triton X-100 to 50% of the control value. Palmitoylcarnitine (0.5 mM) inhibited PLB (97% inhibition) and LPL and LPTA activities (35% inhibition) competitively. All phospholipids except phosphatidic acid were degraded by PLB, but dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylcholine were the preferred substrates. This is the first complete description of the purification and properties of a phospholipase, which may be involved in virulence, from a pathogenic fungus.

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Simamora, Cico Jhon Karunia, and Sukmawati Sukmawati. "Identifikasi dan Karakterisasi Aktivitas Ekstrak Kasar Enzim Lipase Isolat Bakteri Lipolitik Lptk 19 Asal Tempe Biji Karet." Median : Jurnal Ilmu Ilmu Eksakta 12, no. 1 (April 13, 2020): 28. http://dx.doi.org/10.33506/md.v12i1.834.

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Lipase adalah enzim yang larut dalam air yang memiliki kemampuan untuk menghidrolisis triasilgliserol untuk melepaskan asam lemak bebas dan gliserol. Penelitian ini bertujuan untuk mengidentifikasi dan mengkarakterisasi aktivitas ekstrak kasar enzim lipase. Pelaksanaan penelitian meliputi peremajaan isolat, pengujian kualitatif produksi lipase yang ditunjukkan dengan adanya zona bening di sekitar koloni pada medium LAOR, dan dilanjutkan dengan pengujian kuantitatif aktivitas enzim lipase, uji aktivitas spesifik enzim, dan identifikasi biokimia isolat bakteri LpTk 19. Penelitian ini menunjukkan hasil uji kualitatif bakteri LpTK 19 pada media LAOR yang diinkubasi selama 48 jam mencapai 90,59%. Sementara nilai aktivitas enzim tertinggi didapatkan pada jam ke-60 sebesar 487,5 U/mL, kadar protein tertinggi pada jam ke-48 sebesar 12,4 mg/mL, dan aktivitas spesifik tertinggi berada pada jam ke-60 sebesar 325,0 U/mg. Berdasarkan uji biokimia diketahui isolate bakteri LpTk 19 termasuk dalam golongan bakteri Gram positif, yang termasuk family Bacillaceae, dengan jenis Bacillus sp.

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Zhang, Qi, Cuiping Zhang, Xiaoyu Li, Yanan Yu, Kun Liang, Xinzhi Shan, Kun Zhao, Qinghui Niu, and Zibin Tian. "WISP1 Is Increased in Intestinal Mucosa and Contributes to Inflammatory Cascades in Inflammatory Bowel Disease." Disease Markers 2016 (2016): 1–11. http://dx.doi.org/10.1155/2016/3547096.

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Inflammatory bowel disease (IBD) is mainly characterized by intestinal tissue damage, which is caused by excessive autoimmune responses poorly controlled by corresponding regulatory mechanisms. WISP1, which belongs to the CCN protein family, is a secreted matricellular protein regulating several inflammatory pathways, such as Wnt/β-catenin pathway, and has been reported in several diseases including cancer. Here we examined the expression, regulatory mechanisms, and functions of WISP1 in IBD. WISP1 mRNA and protein expression was upregulated in colonic biopsies and lamina propria mononuclear cells (LPMC) of IBD patients compared with those of healthy controls. Tumor necrosis factor- (TNF-)αinduced WISP1 expression in LPMC from healthy controls. Consistently, WISP1 mRNA expression was downregulated in colonic biopsies from IBD patients who had achieved clinical remission with infliximab (IFX). Furthermore, WISP1 expression was also found to be increased in colons from 2,4,6-trinitrobenzenesulfonic acid- (TNBS-) induced mice compared with those from control mice. Further studies confirmed that administration of rWISP1 could aggravate TNBS-induced colitis in vivo. Therefore, we concluded that WISP1 is increased in IBD and contributes to the proinflammatory cascades in the gut.

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Schmidt, Robert, Russ B. Altman, and Mark Gerstein. "LPFC: An internet library of protein family core structures." Protein Science 6, no. 1 (January 1997): 246–48. http://dx.doi.org/10.1002/pro.5560060127.

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Juibari, Aref Doozandeh, Sina Ramezani, and Mohammad Hosein Rezadoust. "Bioinformatics analysis of various signal peptides for periplasmic expression of parathyroid hormone in E.coli." Journal of Medicine and Life 12, no. 2 (April 2019): 184–91. http://dx.doi.org/10.25122/jml-2018-0049.

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Hypoparathyroidism is a rare endocrine disease which is characterized by the deficiency of serum calcium levels. RhPTH is prescribed as a therapy for the management of refractory hypoparathyroidism. The aim of this study is to investigate 32 signal peptides of gram-negative bacterial origin and evaluate their potential for efficient secretion of recombinant human PTH (1–84)In E.coli to obtain higher expression of recombinant PTH in bacterial systems by using this fusion partner. SignalP and ProtParam servers were employed to predict the presence and location of signal peptide cleavage sites in protein sequence and computation of various physical and chemical parameters of protein respectively. Also, SOLpro server was applied for prediction of the protein solubility. Then ProtComp and SecretomeP online servers were employed to determine protein location. The evaluations showed that theoretically two signal peptides Lipopolysaccharide export system protein LptA (lptA) and Periplasmic pH-dependent serine endoprotease DegQ (degQ) are the most appropriate signal peptides examined. Due to the lack of post-translational modification in PTH, its periplasmic expression has preferences. Based on the results of this study, using bioinformatics and reliable servers signal peptides with appropriate secretory potential can be obtained which lead to the highest expression level.

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Consoli, Elisa, Jean-François Collet, and Tanneke den Blaauwen. "The Escherichia coli Outer Membrane β-Barrel Assembly Machinery (BAM) Anchors the Peptidoglycan Layer by Spanning It with All Subunits." International Journal of Molecular Sciences 22, no. 4 (February 12, 2021): 1853. http://dx.doi.org/10.3390/ijms22041853.

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Gram-negative bacteria possess a three-layered envelope composed of an inner membrane, surrounded by a peptidoglycan (PG) layer, enclosed by an outer membrane. The envelope ensures protection against diverse hostile milieus and offers an effective barrier against antibiotics. The layers are connected to each other through many protein interactions. Bacteria evolved sophisticated machineries that maintain the integrity and the functionality of each layer. The β-barrel assembly machinery (BAM), for example, is responsible for the insertion of the outer membrane integral proteins including the lipopolysaccharide transport machinery protein LptD. Labelling bacterial cells with BAM-specific fluorescent antibodies revealed the spatial arrangement between the machinery and the PG layer. The antibody detection of each BAM subunit required the enzymatic digestion of the PG layer. Enhancing the spacing between the outer membrane and PG does not abolish this prerequisite. This suggests that BAM locally sets the distance between OM and the PG layer. Our results shed new light on the local organization of the envelope.

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Vrielink, Alice, Anandhi Anandan, Susannah Piek, Isabel Moares, and Charlene Kahler. "Structure of an endotoxin modifying enzyme and virulence factor in Neisseria." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1047. http://dx.doi.org/10.1107/s2053273314089529.

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Multiple drug resistance (MDR) in Gram-negative bacteria represents one of the most intractable problems facing modern medicine. Not only is antibiotic resistance incrementally increasing during clinical treatment of infections, but also the evolution and acquisition of new mechanisms of antibiotic resistance lead to the sudden loss of the capacity to treat infections. The most recent superbug, MDR-Neisseria gonorrhoeae, causes the untreatable sexually transmitted infection gonorrhoeae. Chronic gonococcal infections have a high morbidity rate and, due to the explosion in cases worldwide, the community burden is enormous. N. gonorrhoeae colonizes the mucosal surfaces of the human body and has a number of virulence mechanisms that prevent clearance by the human immune system. The most important of these mechanisms is decoration of the lipooligosaccharide lipid A headgroups with phosphoethanolamine (PEA) by the enzyme, lipid A PEA transferase (LptA). Inactivation of the LptA results in the complete loss of PEA groups from lipid A, loss of bacterial colonisation of epithelial cells (Takahashi et al., 2008), increased sensitivity to cationic antimicrobial peptides (Tzeng et al., 2005) and reduced resistance to human complement mediated killing (Lewis et al., 2013). LptA knockouts of N. gonorrhoeae also result in the complete loss of virulence in models of human and mouse infections. Based on these findings we have undertaken a structure-guided approach to develop inhibitors of LptA that will assist in controlling infection and transmission by this important human pathogen. LptA is a membrane protein that interacts with two different lipid substrates. We have determined the crystal structure of the enzyme to 2.75Å resolution. The structure provides insights into the mechanism of substrate binding and catalysis and suggests that significant conformational changes occur through its catalytic cycle.

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Goode, Triona, Joe O'Connell, Wen-Zhe Ho, Gerald C. O'Sullivan, J. Kevin Collins, Steven D. Douglas, and Fergus Shanahan. "Differential Expression of Neurokinin-1 Receptor by Human Mucosal and Peripheral Lymphoid Cells." Clinical Diagnostic Laboratory Immunology 7, no. 3 (May 1, 2000): 371–76. http://dx.doi.org/10.1128/cdli.7.3.371-376.2000.

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ABSTRACT Substance P (SP) has been implicated in peripheral and mucosal neuroimmunoregulation. However, confusion remains regarding immunocyte expression of the receptor for SP, neurokinin-1 receptor (NK-1R), and whether there is differential NK-1R expression in the mucosal versus the peripheral immune system. In the same assay systems, we examined the expression of NK-1R in human lamina propria mononuclear cells (LPMC), peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), monocytes, and monocyte-derived macrophages (MDM). Using standard reverse transcription (RT)-PCR, mRNA expression of both the long and the short isoforms of the NK-1R was evident in LPMC but not in PBMC, PBL, monocytes, or MDM. However, by using nested RT-PCR NK-1R mRNA expression was detected in PBMC, PBL, monocytes, and MDM. This level of expression was found to represent one NK-1R mRNA transcript in >1,000 cells. In contrast, by using competitive RT-PCR we demonstrate that LPMC express a more biologically significant level of eight NK-1R mRNA transcripts per cell. Flow cytometric detection of NK-1R expression at the protein level was evident in LPMC but not in PBMC. These findings illustrate the extreme sensitivity of nested RT-PCR and the advantages of competitive RT-PCR in comparative studies of receptor expression in different cell populations. This study suggests that, under normal conditions, readily detectable expression of NK-1R in human mononuclear cells occurs at the mucosal level rather than in the peripheral circulation.

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Pham, T. D. "LPC Cepstral Distortion Measure for Protein Sequence Comparison." IEEE Transactions on Nanobioscience 5, no. 2 (June 2006): 83–88. http://dx.doi.org/10.1109/tnb.2006.875029.

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WRIGHT, Lesley C., Jackie PAYNE, Rosemary T. SANTANGELO, Mukoma F. SIMPANYA, Sharon C. A. CHEN, Fred WIDMER, and Tania C. SORRELL. "Cryptococcal phospholipases: a novel lysophospholipase discovered in the pathogenic fungus Cryptococcus gattii." Biochemical Journal 384, no. 2 (November 23, 2004): 377–84. http://dx.doi.org/10.1042/bj20041079.

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The pathogenic fungus Cryptococcus neoformans produces an extracellular PLB1 (phospholipase B1), shown previously to be a virulence factor. A novel phospholipase (LPL1) with only LPL (lysophospholipase) and LPTA (transacylase) activities has now been characterized in C. gattii, and found to be a 66-kDa glycoprotein (by SDS/PAGE), with a native molecular mass of 670 kDa. The pI was 6.3, and it was active at high temperatures (to 70 °C), as well as at both acidic and neutral pH values. It was stimulated by calcium and palmitoyl carnitine at pH 7.0, but not at pH 5.0, and palmitoyl lysophosphatidylcholine was the preferred substrate. Sequencing indicated that LPL1 is a novel cryptococcal lysophospholipase, and not the gene product of CnLYSO1 or PLB1. A protein with only LPL and LPTA activities was subsequently isolated from two strains of C. neoformans var. grubii. A PLB1 enzyme was isolated from both C. gattii and a highly virulent strain of C. neoformans var. grubii (H99). In both cases, all three enzyme activities (PLB, LPL and LPTA) were present in one 95–120 kDa glycoprotein (by SDS/PAGE) with pI 3.9–4.3. Characterization of PLB1 from C. gattii showed that it differed from that of C. neoformans in its larger native mass (275 kDa), high PLB activity relative to LPL and LPTA, and preference for saturated lipid substrates. Differences in the properties between the secreted phospholipases of the two cryptococcal species could contribute to phenotypic differences that determine their respective environmental niches and different clinical manifestations.

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Wieczorek, Alicja, Anna Sendobra, Akshey Maniyeri, Magdalena Sugalska, Gracjana Klein, and Satish Raina. "A New Factor LapD Is Required for the Regulation of LpxC Amounts and Lipopolysaccharide Trafficking." International Journal of Molecular Sciences 23, no. 17 (August 26, 2022): 9706. http://dx.doi.org/10.3390/ijms23179706.

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Lipopolysaccharide (LPS) constitutes the major component of the outer membrane and is essential for bacteria, such as Escherichia coli. Recent work has revealed the essential roles of LapB and LapC proteins in regulating LPS amounts; although, if any additional partners are involved is unknown. Examination of proteins co-purifying with LapB identified LapD as a new partner. The purification of LapD reveals that it forms a complex with several proteins involved in LPS and phospholipid biosynthesis, including FtsH-LapA/B and Fab enzymes. Loss of LapD causes a reduction in LpxC amounts and vancomycin sensitivity, which can be restored by mutations that stabilize LpxC (mutations in lapB, ftsH and lpxC genes), revealing that LapD acts upstream of LapB-FtsH in regulating LpxC amounts. Interestingly, LapD absence results in the substantial retention of LPS in the inner membranes and synthetic lethality when either the lauroyl or the myristoyl acyl transferase is absent, which can be overcome by single-amino acid suppressor mutations in LPS flippase MsbA, suggesting LPS translocation defects in ΔlapD bacteria. Several genes whose products are involved in cell envelope homeostasis, including clsA, waaC, tig and micA, become essential in LapD’s absence. Furthermore, the overproduction of acyl carrier protein AcpP or transcriptional factors DksA, SrrA can overcome certain defects of the LapD-lacking strain.

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Soltan, Mohamed A., Nada Elbassiouny, Helmy Gamal, Eslam B. Elkaeed, Refaat A. Eid, Muhammad Alaa Eldeen, and Ahmed A. Al-Karmalawy. "In Silico Prediction of a Multitope Vaccine against Moraxella catarrhalis: Reverse Vaccinology and Immunoinformatics." Vaccines 9, no. 6 (June 18, 2021): 669. http://dx.doi.org/10.3390/vaccines9060669.

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Moraxella catarrhalis (M. catarrhalis) is a Gram-negative bacterium that can cause serious respiratory tract infections and middle ear infections in children and adults. M. catarrhalis has demonstrated an increasing rate of antibiotic resistance in the last few years, thus development of an effective vaccine is a major health priority. We report here a novel designed multitope vaccine based on the mapped epitopes of the vaccine candidates filtered out of the whole proteome of M. catarrhalis. After analysis of 1615 proteins using a reverse vaccinology approach, only two proteins (outer membrane protein assembly factor BamA and LPS assembly protein LptD) were nominated as potential vaccine candidates. These proteins were found to be essential, outer membrane, virulent and non-human homologs with appropriate molecular weight and high antigenicity score. For each protein, cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL) and B cell lymphocyte (BCL) epitopes were predicted and confirmed to be highly antigenic and cover conserved regions of the proteins. The mapped epitopes constituted the base of the designed multitope vaccine where suitable linkers were added to conjugate them. Additionally, beta defensin adjuvant and pan-HLA DR-binding epitope (PADRE) peptide were also incorporated into the construct to improve the stimulated immune response. The constructed multitope vaccine was analyzed for its physicochemical, structural and immunological characteristics and it was found to be antigenic, soluble, stable, non-allergenic and have a high affinity to its target receptor. Although the in silico analysis of the current study revealed that the designed multitope vaccine has the ability to trigger a specific immune response against M. catarrhalis, additional translational research is required to confirm the effectiveness of the designed vaccine.

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Ganendren, Ranjini, Fred Widmer, Vatsala Singhal, Christabel Wilson, Tania Sorrell, and Lesley Wright. "In Vitro Antifungal Activities of Inhibitors of Phospholipases from the Fungal Pathogen Cryptococcus neoformans." Antimicrobial Agents and Chemotherapy 48, no. 5 (May 2004): 1561–69. http://dx.doi.org/10.1128/aac.48.5.1561-1569.2004.

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ABSTRACT Secreted phospholipase B is a proven virulence factor for the pathogenic fungus Cryptococcus neoformans and exhibits three phospholipase activities in the one protein. These are phospholipase B (PLB), lysophospholipase (LPL), and lysophospholipase transacylase (LPTA). Our aim was to investigate the feasibility of using this enzyme as a target for antifungal therapy. We determined in C. neoformans var. grubii strain H99 that 82% of PLB activity was secreted but that 64% of LPL activity and 70% of LPTA activity were cell associated. Cell-associated activities (cytosolic and membrane) were further characterized, since it is likely that any fungicidal effect would depend on inhibition of these enzymes. Four commercially available compounds with structural similarities to phospholipid substrates were tested as inhibitors. These were alexidine dihydrochloride (compound A), dioctadecyldimethylammonium bromide (compound O), 1,12 bis-(tributylphosphonium)dodecane dibromide (compound P), and decamethonium dibromide (compound D). The best phospholipase inhibitors (compounds A and P) were also the most potent antifungal agents by the standard broth microdilution test. Compound A was highly selective for secreted and cell-associated PLB activities and showed no inhibition of mammalian phospholipase A 2 at 0.25 μM. Compound O, which was specific for secretory and cytosolic LPL and LPTA and membrane-associated PLB, was not antifungal. We conclude that inhibitors of cryptococcal phospholipases can be selective for fungal enzymes and intrinsically antifungal. They also provide tools for assessing the relative importance of the various enzyme activities in virulence. Our results enable further rational structure-function studies to validate the use of phospholipases as antifungal targets.

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Journal articles on the topic 'LptC protein'

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