Artykuły w czasopismach: „Fic Proteins”

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Li, Qinghong, Yue Sun i Sven C. D. van IJzendoorn. "A Link between Intrahepatic Cholestasis and Genetic Variations in Intracellular Trafficking Regulators". Biology 10, nr 2 (4.02.2021): 119. http://dx.doi.org/10.3390/biology10020119.

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Intrahepatic cholestasis is characterized by the accumulation of compounds in the serum that are normally secreted by hepatocytes into the bile. Genes associated with familial intrahepatic cholestasis (FIC) include ATP8B1 (FIC1), ABCB11 (FIC2), ABCB4 (FIC3), TJP2 (FIC4), NR1H4 (FIC5) and MYO5B (FIC6). With advanced genome sequencing methodologies, additional mutated genes are rapidly identified in patients presenting with idiopathic FIC. Notably, several of these genes, VPS33B, VIPAS39, SCYL1, and AP1S1, together with MYO5B, are functionally associated with recycling endosomes and/or the Golgi apparatus. These are components of a complex process that controls the sorting and trafficking of proteins, including those involved in bile secretion. These gene variants therefore suggest that defects in intracellular trafficking take a prominent place in FIC. Here we review these FIC-associated trafficking genes and their variants, their contribution to biliary transporter and canalicular protein trafficking, and, when perturbed, to cholestatic liver disease. Published variants for each of these genes have been summarized in table format, providing a convenient reference for those who work in the intrahepatic cholestasis field.

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2

Molloy, Sheilagh. "Controlling Fic proteins". Nature Reviews Microbiology 10, nr 3 (13.02.2012): 160. http://dx.doi.org/10.1038/nrmicro2757.

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Welner, Ditte, Emil Dedic, Hans van Leeuwen, Ed Kuijper, Rene Jorgensen, Jorgensen i Rene Jorgensen. "Structural characterisation of a Fic protein from Clostridium difficile". Acta Crystallographica Section A Foundations and Advances 70, a1 (5.08.2014): C814. http://dx.doi.org/10.1107/s2053273314091852.

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Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of gram-negative bacteria have recently been demonstrated to catalyse the transfer of an AMP moiety from ATP to small host GTPases (1,2). This post-translational modification has received considerable interest and a role for adenylylation in pathology and physiology is emerging. We have structurally characterised a newly identified Fic protein of the pathogenic gram-positive bacterium Clostridium difficile. A constitutively active inhibitory helix mutant of C. difficile Fic was purified, crystallised and data collected to 1.7 Å resolution. The structure confirms C. difficult Fic protein as an ATP binding protein and reveal a binding site similar to other confirmed virulent Fic proteins. Surprisingly, this gram-positive Fic protein does not seem to target GTPases in humans and currently target identification is being chased. The current status of the project will be presented.

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4

Roy, Craig R., i Jacqueline Cherfils. "Structure and function of Fic proteins". Nature Reviews Microbiology 13, nr 10 (24.08.2015): 631–40. http://dx.doi.org/10.1038/nrmicro3520.

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Roy, C. R., i S. Mukherjee. "Bacterial FIC Proteins AMP Up Infection". Science Signaling 2, nr 62 (10.03.2009): pe14. http://dx.doi.org/10.1126/scisignal.262pe14.

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Stanger, Frédéric V., Björn M. Burmann, Alexander Harms, Hugo Aragão, Adam Mazur, Timothy Sharpe, Christoph Dehio, Sebastian Hiller i Tilman Schirmer. "Intrinsic regulation of FIC-domain AMP-transferases by oligomerization and automodification". Proceedings of the National Academy of Sciences 113, nr 5 (19.01.2016): E529—E537. http://dx.doi.org/10.1073/pnas.1516930113.

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Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (αinh) that partly obstructs the active site. For the single-domain class III Fic proteins, the αinh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins.

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Heinrich, J. N., R. P. Ryseck, H. Macdonald-Bravo i R. Bravo. "The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine". Molecular and Cellular Biology 13, nr 4 (kwiecień 1993): 2020–30. http://dx.doi.org/10.1128/mcb.13.4.2020-2030.1993.

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We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.

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8

Heinrich, J. N., R. P. Ryseck, H. Macdonald-Bravo i R. Bravo. "The product of a novel growth factor-activated gene, fic, is a biologically active "C-C"-type cytokine." Molecular and Cellular Biology 13, nr 4 (kwiecień 1993): 2020–30. http://dx.doi.org/10.1128/mcb.13.4.2020.

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We have characterized a new member of the superfamily of proinflammatory peptides encoded by a growth factor-inducible gene, fic, previously isolated by differential screening of a cDNA library of mRNA from serum-stimulated NIH 3T3 cells. Immunoprecipitation analyses showed that the protein was rapidly induced following serum stimulation and secreted unglycosylated into the medium. The fic protein, FIC, shows highest sequence homology (57%) to human and rabbit monocyte chemoattractant protein 1 (MCP-1), an established monocyte activator. To determine the biological activity of FIC and to compare it with that of mouse MCP-1 (muMCP-1), both proteins were expressed in the baculovirus system. FIC and muMCP-1 were purified to near homogeneity by a two-step chromatography protocol. Both proteins elicited changes in intracellular calcium concentration in human monocytes. The effect was dependent on external Ca2+ and was inhibited by pretreatment of cells with pertussis toxin. FIC did not desensitize human monocytes to the three related cytokines muMCP-1, human MCP-1 (huMCP-1), and huMCP-2. However, pretreatment with muMCP-1 or huMCP-1, but not with huMCP-2, desensitized human monocytes to FIC. Specific binding of [125I]FIC was found in human monocytes, mouse monocytic cultured cells, and human endothelial cells but not in lymphocytes, neutrophils, or primary mouse fibroblasts. Scatchard analysis of the binding of [125I]FIC to human monocytes showed the presence of two classes of receptors, with apparent KdS of 1.2 and 7.7 nM and receptor numbers per cell of 2,400 and 6,300, respectively. FIC, muMCP-1, and huMCP-1 competed to the same extent for the binding of [125I]FIC to human monocytes, contrary to huMCP-2, which competed very ineffectively, if at all.

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9

Zekarias, Bereket, Seema Mattoo, Carolyn Worby, Jason Lehmann, Ricardo F. Rosenbusch i Lynette B. Corbeil. "Histophilus somni IbpA DR2/Fic in Virulence and Immunoprotection at the Natural Host Alveolar Epithelial Barrier". Infection and Immunity 78, nr 5 (22.02.2010): 1850–58. http://dx.doi.org/10.1128/iai.01277-09.

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ABSTRACT Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. To relate cellular mechanisms to pathogenesis and immune protection, we studied the cytotoxicity of the Histophilus somni immunoglobulin-binding protein A (IbpA) direct repeat 2 Fic domain (DR2/Fic) for natural host target cells. Live virulent IbpA-producing H. somni strain 2336, a cell-free culture supernatant (CCS) of this strain, or recombinant DR2/Fic (rDR2/Fic) caused dramatic retraction and rounding of bovine alveolar type 2 (BAT2) epithelial cells. IbpA-deficient H. somni strain 129Pt and a Fic motif His298Ala mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the presence of [α-32P]ATP, which resulted in adenylylation of Rho GTPases and cytoskeletal disruption. Since IbpA is not secreted by type III or type IV secretion systems, we determined whether DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. Although H. somni did not invade BAT2 cells, DR2/Fic was internalized by cells treated with H. somni, CCS, or the rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration assays showed that large numbers of strain 2336 bacteria migrated between retracted BAT2 cells, but IbpA-deficient strain 129Pt did not cross a monolayer unless the monolayer was pretreated with strain 2336 CCS or rDR2/Fic protein. Antibody to rDR2/Fic or passively protective convalescent-phase serum blocked IbpA-mediated cytotoxicity and inhibited H. somni transmigration across BAT2 monolayers, confirming the role of DR2/Fic in pathogenesis and corresponding to the results for in vivo protection in previous animal studies.

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Schirmer, Tilman, Tjaart A. P. de Beer, Stefanie Tamegger, Alexander Harms, Nikolaus Dietz, David M. Dranow, Thomas E. Edwards, Peter J. Myler, Isabelle Phan i Christoph Dehio. "Evolutionary Diversification of Host-Targeted Bartonella Effectors Proteins Derived from a Conserved FicTA Toxin-Antitoxin Module". Microorganisms 9, nr 8 (31.07.2021): 1645. http://dx.doi.org/10.3390/microorganisms9081645.

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Proteins containing a FIC domain catalyze AMPylation and other post-translational modifications (PTMs). In bacteria, they are typically part of FicTA toxin-antitoxin modules that control conserved biochemical processes such as topoisomerase activity, but they have also repeatedly diversified into host-targeted virulence factors. Among these, Bartonella effector proteins (Beps) comprise a particularly diverse ensemble of FIC domains that subvert various host cellular functions. However, no comprehensive comparative analysis has been performed to infer molecular mechanisms underlying the biochemical and functional diversification of FIC domains in the vast Bep family. Here, we used X-ray crystallography, structural modelling, and phylogenetic analyses to unravel the expansion and diversification of Bep repertoires that evolved in parallel in three Bartonella lineages from a single ancestral FicTA toxin-antitoxin module. Our analysis is based on 99 non-redundant Bep sequences and nine crystal structures. Inferred from the conservation of the FIC signature motif that comprises the catalytic histidine and residues involved in substrate binding, about half of them represent AMP transferases. A quarter of Beps show a glutamate in a strategic position in the putative substrate binding pocket that would interfere with triphosphate-nucleotide binding but may allow binding of an AMPylated target for deAMPylation or another substrate to catalyze a distinct PTM. The β-hairpin flap that registers the modifiable target segment to the active site exhibits remarkable structural variability. The corresponding sequences form few well-defined groups that may recognize distinct target proteins. The binding of Beps to promiscuous FicA antitoxins is well conserved, indicating a role of the antitoxin to inhibit enzymatic activity or to serve as a chaperone for the FIC domain before translocation of the Bep into host cells. Taken together, our analysis indicates a remarkable functional plasticity of Beps that is mostly brought about by structural changes in the substrate pocket and the target dock. These findings may guide future structure–function analyses of the highly versatile FIC domains.

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11

Okuyama, Ryo. "Chronological Analysis of First-in-Class Drugs Approved from 2011 to 2022: Their Technological Trend and Origin". Pharmaceutics 15, nr 7 (22.06.2023): 1794. http://dx.doi.org/10.3390/pharmaceutics15071794.

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The discovery and development of first-in-class (FIC) drugs are becoming increasingly important due to increasing reimbursement pressure and personalized medication. To investigate the technological trends and origin of FIC drugs, the FIC drugs approved in the U.S. from January 2011 to December 2022 were analyzed. The analysis shows that previous major target families, viz. enzymes, G-protein coupled receptors, transporters, and transcription factors, are no longer considered major in recent years. Instead, the shares of secreted proteins/peptides and mRNAs have continuously increased from 2011–2014 to 2019–2022, suggesting that the target family of FIC drugs has shifted to molecules previously considered challenging as drug targets. Small molecules were predominant in 2011–2014, followed by a large increase in antibody medicines in 2015–2018 and further diversification of antibody medicine modalities in 2019–2022. Nucleic acid medicine has also continuously increased its share, suggesting that diversifying modalities supports the creation of FIC drugs toward challenging target molecules. Over half of FIC drugs were created by small and medium enterprises (SMEs), especially young companies established in the 1990s and 2000s. All SMEs that produced more than one FIC drug approved in 2019–2022 have the strong technological capability in a specific modality. Investment in modality technologies and facilitating mechanisms to translate academic modality technologies to start-ups might be important for enhancing FIC drug development.

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12

Armendariz-Borunda, J., C. P. Simkevich, N. Roy, R. Raghow, A. H. Kang i J. M. Seyer. "Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression". Biochemical Journal 304, nr 3 (15.12.1994): 817–24. http://dx.doi.org/10.1042/bj3040817.

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Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.

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13

Harms, Alexander, Frédéric V. Stanger i Christoph Dehio. "Biological Diversity and Molecular Plasticity of FIC Domain Proteins". Annual Review of Microbiology 70, nr 1 (8.09.2016): 341–60. http://dx.doi.org/10.1146/annurev-micro-102215-095245.

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Welner, Ditte, Emil Dedic, Hans C. van Leeuwen, Ed Kuijper, Morten Jannik Bjerrum, Ole Østergaard i René Jørgensen. "Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein fromClostridium difficile". Acta Crystallographica Section F Structural Biology Communications 70, nr 6 (25.05.2014): 827–31. http://dx.doi.org/10.1107/s2053230x1400987x.

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Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacteriumClostridium difficile. A constitutively active inhibitory helix mutant ofC. difficileFic was overexpressed inEscherichia coli, purified and crystallized by the vapour-diffusion technique. Preliminary X-ray crystallographic analysis shows that the crystals diffract to at least 1.68 Å resolution at a synchrotron X-ray source. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 80.8,c= 144.7 Å, α = β = γ = 90°. Two molecules per asymmetric unit corresponds to a Matthews coefficient of 2.37 Å3 Da−1and a solvent content of 48%.

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15

Engel, Philipp, Arnaud Goepfert, Frédéric V. Stanger, Alexander Harms, Alexander Schmidt, Tilman Schirmer i Christoph Dehio. "Adenylylation control by intra- or intermolecular active-site obstruction in Fic proteins". Nature 482, nr 7383 (22.01.2012): 107–10. http://dx.doi.org/10.1038/nature10729.

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Kokaska, Ashlynn. "Assessing the Role of Fic (Filamentation Induced by cAMP) Proteins in E. coli". Journal of Purdue Undergraduate Research 5, nr 1 (13.08.2015): 90–91. http://dx.doi.org/10.5703/1288284315667.

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Truttmann, Matthias C., David Pincus i Hidde L. Ploegh. "Chaperone AMPylation modulates aggregation and toxicity of neurodegenerative disease-associated polypeptides". Proceedings of the National Academy of Sciences 115, nr 22 (14.05.2018): E5008—E5017. http://dx.doi.org/10.1073/pnas.1801989115.

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Proteostasis is critical to maintain organismal viability, a process counteracted by aging-dependent protein aggregation. Chaperones of the heat shock protein (HSP) family help control proteostasis by reducing the burden of unfolded proteins. They also oversee the formation of protein aggregates. Here, we explore how AMPylation, a posttranslational protein modification that has emerged as a powerful modulator of HSP70 activity, influences the dynamics of protein aggregation. We find that adjustments of cellular AMPylation levels in Caenorhabditis elegans directly affect aggregation properties and associated toxicity of amyloid-β (Aβ), of a polyglutamine (polyQ)-extended polypeptide, and of α-synuclein (α-syn). Expression of a constitutively active C. elegans AMPylase FIC-1(E274G) under its own promoter expedites aggregation of Aβ and α-syn, and drastically reduces their toxicity. A deficiency in AMPylation decreases the cellular tolerance for aggregation-prone polyQ proteins and alters their aggregation behavior. Overexpression of FIC-1(E274G) interferes with cell survival and larval development, underscoring the need for tight control of AMPylase activity in vivo. We thus define a link between HSP70 AMPylation and the dynamics of protein aggregation in neurodegenerative disease models. Our results are consistent with a cytoprotective, rather than a cytotoxic, role for such protein aggregates.

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Ushijima, H., C. M. Tsiapalis, T. Daum, H. C. Schröder, E. Matthes, J. W. Engels, M. Mag, J. Muth i W. E. G. Müller. "Synergistic Anti-Human Immunodeficiency Viral (HIV-1) Effect of the Immunomodulator Ampligen (Mismatched Double-Stranded RNA) with Inhibitors of Reverse Transcriptase and HIV-1 Regulatory Proteins". Antiviral Chemistry and Chemotherapy 4, nr 6 (grudzień 1993): 315–21. http://dx.doi.org/10.1177/095632029300400602.

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The potent antiviral effect of double stranded RNA, such as the mismatched poly(l)·poly(C12U) [Ampligen], 2′,3′-dideoxy-3′-fluorothymidine (FddThd) and antisense oligodeoxynucleotides (ODN) has been established in in vitro systems using cells infected with the human immunodeficiency virus type 1 (HIV-1). We report here that the immunomodulator poly(l)·poly(C12U) interacts synergistically with (1) the reverse transcriptase inhibitor FddThd (FIC value: 0.43), (2) the modified (5′- and 3′-end capped thioates) antisense ODN-4 directed against the splice acceptor site of the HIV-1/ tat gene (FIC value: 0.66) and (3) also with pyronin Y, a compound which prevents binding of HIV-1 Rev protein to the HIV-1 RRE element. These data suggest that combinations of poly(l)·poly(C12U), a stimulator of the natural antiviral protection system of the cells, with compounds targeting HIV1-specific processes should be considered as candidate treatments of AIDS patients.

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Hatala, Patrícia, Andrea Lajos, Máté Mackei, Csilla Sebők, Patrik Tráj, Júlia Vörösházi, Zsuzsanna Neogrády i Gábor Mátis. "Feline Uroepithelial Cell Culture as a Novel Model of Idiopathic Cystitis: Investigations on the Effects of Norepinephrine on Inflammatory Response, Oxidative Stress, and Barrier Function". Veterinary Sciences 10, nr 2 (8.02.2023): 132. http://dx.doi.org/10.3390/vetsci10020132.

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Feline idiopathic cystitis (FIC) is one of the most common urinary tract disorders in domestic cats. As stress is suggested to play a key role in the pathogenesis of FIC, the effects of norepinephrine (NE) as a stress mediator were investigated on a novel feline primary uroepithelial cell culture, serving as an in vitro model of the disease. The uroepithelial cells gained from the mucosa of the bladder of a euthanized cat were cultured for 6 days and were acutely exposed to NE (10, 100, and 1000 µM) for 1 h. NE increased the metabolic activity of the cultured cells and elevated the extracellular concentrations of the pro-inflammatory mediators interleukin-6 (IL-6) and stromal cell derived factor 1 (SDF-1), confirming that NE can trigger an inflammatory response in the uroepithelium. Cellular protein carbonyl levels were increased by NE exposure, while malondialdehyde and glucose regulated protein 78 concentrations remained unchanged, indicating that NE may provoke the oxidative damage of proteins without inducing lipid peroxidation or endoplasmic reticulum stress. Further, it can be strongly suggested that an acute NE challenge might diminish the barrier function of uroepithelial cells, as reflected by the decreased glycosaminoglycan concentration, claudin-4 protein expression, and reduced TER values of the NE-treated cell cultures. Based on these results, short-term NE exposure mimicking acute stress can provoke an inflammatory response and decrease the barrier integrity of cultured feline uroepithelial cells. Hence, it is highly expected that stress-associated NE release may play an important mediatory role in the pathogenesis of FIC.

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Liu, Meili, Zhe Huai, Hongwei Tan i Guangju Chen. "Investigation of the Detailed AMPylated Reaction Mechanism for the Huntingtin Yeast-Interacting Protein E Enzyme HYPE". International Journal of Molecular Sciences 22, nr 13 (29.06.2021): 6999. http://dx.doi.org/10.3390/ijms22136999.

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AMPylation is a prevalent posttranslational modification that involves the addition of adenosine monophosphate (AMP) to proteins. Exactly how Huntingtin-associated yeast-interacting protein E (HYPE), as the first human protein, is involved in the transformation of the AMP moiety to its substrate target protein (the endoplasmic reticulum chaperone binding to immunoglobulin protein (BiP)) is still an open question. Additionally, a conserved glutamine plays a vital key role in the AMPylation reaction in most filamentation processes induced by the cAMP (Fic) protein. In the present work, the detailed catalytic AMPylation mechanisms in HYPE were determined based on the density functional theory (DFT) method. Molecular dynamics (MD) simulations were further used to investigate the exact role of the inhibitory glutamate. The metal center, Mg2+, in HYPE has been examined in various coordination configurations, including 4-coordrinated, 5-coordinated and 6-coordinated. DFT calculations revealed that the transformation of the AMP moiety of HYPE with BiP followed a sequential pathway. The model with a 4-coordinated metal center had a barrier of 14.7 kcal/mol, which was consistent with the experimental value and lower than the 38.7 kcal/mol barrier of the model with a 6-coordinated metal center and the 31.1 kcal/mol barrier of the model with a 5-coordinated metal center. Furthermore, DFT results indicated that Thr518 residue oxygen directly attacks the phosphorus, while the His363 residue acts as H-bond acceptor. At the same time, an MD study indicated that Glu234 played an inhibitory role in the α-inhibition helix by regulating the hydrogen bond interaction between Arg374 and the Pγ of the ATP molecule. The revealed sequential pathway and the inhibitory role of Glu234 in HYPE were inspirational for understanding the catalytic and inhibitory mechanisms of Fic-mediated AMP transfer, paving the way for further studies on the physiological role of Fic enzymes.

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Vorotyntseva, M. V. "The effect of glutenins on grain quality as one of the complex polygenic traits in the genus Triticum (a review)". Proceedings on applied botany, genetics and breeding 182, nr 1 (3.04.2021): 168–85. http://dx.doi.org/10.30901/2227-8834-2021-1-168-185.

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Evaluation of plant breeding material, based on protein markers, gives an opportunity to perform rapid and reliable selection and control the transfer of desired traits from parents to their progeny. A search for new and stable protein markers is needed to identify genotypes with high grain quality. Such storage proteins in wheat as glutenins have been studied profoundly enough. Full characterization of individual protein fractions and components can be found in many scienti fic publications, while studying genetic patterns of protein accumulation in the grain of different wheat cultivars and using high-molecular-weight (HMW) and lowmolecular-weight (LMW) glutenin subunits (GS) for genotype identi fication remain high in the research agenda. This is a comprehensive review of scienti fic publications about the structure and molecular organization of glutenins and a comparative analysis of 22 research papers about the degree of their effect on grain quality indicators: SDS-sedimentation volume (ml), grain/ flour protein content (%; 14% m.b.; 12,5% m.b.), mixing time (min), mixing tolerance (min; mm), bread loaf volume (cm3; ml), dough strength (10 -4 J), and P/L ratio. As a result of reviewing, the best alleles (subunits) of glutenin were identi fied, namely: Glu-A1а, Glu-B1(h, f, b), Glu-D1d, Glu-A3d, and Glu-B3d.

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Keay, Susan K., Lori A. Birder i Toby C. Chai. "Evidence for Bladder Urothelial Pathophysiology in Functional Bladder Disorders". BioMed Research International 2014 (2014): 1–15. http://dx.doi.org/10.1155/2014/865463.

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Understanding of the role of urothelium in regulating bladder function is continuing to evolve. While the urothelium is thought to function primarily as a barrier for preventing injurious substances and microorganisms from gaining access to bladder stroma and upper urinary tract, studies indicate it may also function in cell signaling events relating to voiding function. This review highlights urothelial abnormalities in bladder pain syndrome/interstitial cystitis (BPS/IC), feline interstitial cystitis (FIC), and nonneurogenic idiopathic overactive bladder (OAB). These bladder conditions are typified by lower urinary tract symptoms including urinary frequency, urgency, urgency incontinence, nocturia, and bladder discomfort or pain. Urothelial tissues and cells from affected clinical subjects and asymptomatic controls have been compared for expression of proteins and mRNA. Animal models have also been used to probe urothelial responses to injuries of the urothelium, urethra, or central nervous system, and transgenic techniques are being used to test specific urothelial abnormalities on bladder function. BPS/IC, FIC, and OAB appear to share some common pathophysiology including increased purinergic, TRPV1, and muscarinic signaling, increased urothelial permeability, and aberrant urothelial differentiation. One challenge is to determine which of several abnormally regulated signaling pathways is most important for mediating bladder dysfunction in these syndromes, with a goal of treating these conditions by targeting specific pathophysiology.

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Marlaire, Simon, i Christoph Dehio. "Bartonella effector protein C mediates actin stress fiber formation via recruitment of GEF-H1 to the plasma membrane". PLOS Pathogens 17, nr 1 (28.01.2021): e1008548. http://dx.doi.org/10.1371/journal.ppat.1008548.

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Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called ‘invasome’. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal ‘Bep intracellular delivery’ (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal ‘filamentation induced by cAMP’ (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane.

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Wronkowska, Małgorzata, Dorota Szawara-Nowak, Mariusz Konrad Piskuła i Henryk Zieliński. "Bioaccessibility of Maillard Reaction Products from Biscuits Formulated from Buckwheat Flours Fermented by Selected Lactic Acid Bacteria". Microorganisms 11, nr 4 (29.03.2023): 883. http://dx.doi.org/10.3390/microorganisms11040883.

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The in vitro bioaccessibility of the soluble protein and Maillard reaction products (MRPs) such as furosine (an early indicator of the MR), free FIC (fluorescent intermediate compounds), and FAST index (fluorescence of advanced MRPs and tryptophan), and the level of melanoidins defined by the browning index were analyzed in biscuits formulated from raw and roasted common buckwheat flours fermented by select lactic acid bacteria (LAB). The content of soluble proteins in fermented buckwheat flour and biscuits before and after digestion in vitro was significantly dependent on the LAB applied and the type of flour used and was the highest in the digested biscuits, indicating increased bioaccessibility. Generally, in all analyzed biscuits a lower furosine content was observed as compared to control samples, and its high bioaccessibility was noted after digestion. The free FIC in biscuits was strain-dependent, resulting in low bioaccessibility with the exception of biscuits obtained from both types of flours fermented by Streptococcus thermophilus MK-10. Compared to control biscuits obtained from raw buckwheat flour, the almost twice-increased FAST index was found for samples fermented by L. plantarum IB or Streptococcus thermophilus MK-10. After digestion, at least a fivefold higher value of the browning index was noted in control and tested biscuits, indicating the high bioaccessibility of melanoidins. This study indicates that fermentation of buckwheat flours by selected lactic acid bacteria seems to be a good way to obtain a product with high bioaccessibility of MRPs. However, further research on their functional properties is needed.

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Virolainen, Minttu S., Cecilie L. Søltoft, Per A. Pedersen i Lars Ellgaard. "Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD". International Journal of Molecular Sciences 23, nr 5 (23.02.2022): 2458. http://dx.doi.org/10.3390/ijms23052458.

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The human Fic domain-containing protein (FICD) is a type II endoplasmic reticulum (ER) membrane protein that is important for the maintenance of ER proteostasis. Structural and in vitro biochemical characterisation of FICD AMPylase and deAMPylase activity have been restricted to the soluble ER-luminal domain produced in Escherichia coli. Information about potentially important features, such as structural motifs, modulator binding sites or other regulatory elements, is therefore missing for the approximately 100 N-terminal residues including the transmembrane region of FICD. Expressing and purifying the required quantity and quality of membrane proteins is demanding because of the low yields and poor stability often observed. Here, we produce full-length FICD by combining a Saccharomyces cerevisiae-based platform with green fluorescent protein (GFP) tagging to optimise the conditions for expression, solubilisation and purification. We subsequently employ these conditions to purify milligram quantities of His-tagged FICD per litre of culture, and show that the purified, detergent-solubilised membrane protein is an active deAMPylating enzyme. Our work provides a straightforward methodology for producing not only full-length FICD, but also other membrane proteins in S. cerevisiae for structural and biochemical characterisation.

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Ernst, Stefan, Felix Ecker, Marietta S. Kaspers, Philipp Ochtrop, Christian Hedberg, Michael Groll i Aymelt Itzen. "Legionella effector AnkX displaces the switch II region for Rab1b phosphocholination". Science Advances 6, nr 20 (maj 2020): eaaz8041. http://dx.doi.org/10.1126/sciadv.aaz8041.

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The causative agent of Legionnaires disease, Legionella pneumophila, translocates the phosphocholine transferase AnkX during infection and thereby posttranslationally modifies the small guanosine triphosphatase (GTPase) Rab1 with a phosphocholine moiety at S76 using cytidine diphosphate (CDP)–choline as a cosubstrate. The molecular basis for Rab1 binding and enzymatic modification have remained elusive because of lack of structural information of the low-affinity complex with AnkX. We combined thiol-reactive CDP-choline derivatives with recombinantly introduced cysteines in the AnkX active site to covalently capture the heterocomplex. The resulting crystal structure revealed that AnkX induces displacement of important regulatory elements of Rab1 by placing a β sheet into a conserved hydrophobic pocket, thereby permitting phosphocholine transfer to the active and inactive states of the GTPase. Together, the combination of chemical biology and structural analysis reveals the enzymatic mechanism of AnkX and the family of filamentation induced by cyclic adenosine monophosphate (FIC) proteins.

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Dietz, Nikolaus, Markus Huber, Isabel Sorg, Arnaud Goepfert, Alexander Harms, Tilman Schirmer i Christoph Dehio. "Structural basis for selective AMPylation of Rac-subfamily GTPases by Bartonella effector protein 1 (Bep1)". Proceedings of the National Academy of Sciences 118, nr 12 (15.03.2021): e2023245118. http://dx.doi.org/10.1073/pnas.2023245118.

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Small GTPases of the Ras-homology (Rho) family are conserved molecular switches that control fundamental cellular activities in eukaryotic cells. As such, they are targeted by numerous bacterial toxins and effector proteins, which have been intensively investigated regarding their biochemical activities and discrete target spectra; however, the molecular mechanism of target selectivity has remained largely elusive. Here we report a bacterial effector protein that selectively targets members of the Rac subfamily in the Rho family of small GTPases but none in the closely related Cdc42 or RhoA subfamilies. This exquisite target selectivity of the FIC domain AMP-transferase Bep1 from Bartonella rochalimae is based on electrostatic interactions with a subfamily-specific pair of residues in the nucleotide-binding G4 motif and the Rho insert helix. Residue substitutions at the identified positions in Cdc42 enable modification by Bep1, while corresponding Cdc42-like substitutions in Rac1 greatly diminish modification. Our study establishes a structural understanding of target selectivity toward Rac-subfamily GTPases and provides a highly selective tool for their functional analysis.

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Truttmann, Matthias C., Victor E. Cruz, Xuanzong Guo, Christoph Engert, Thomas U. Schwartz i Hidde L. Ploegh. "The Caenorhabditis elegans Protein FIC-1 Is an AMPylase That Covalently Modifies Heat-Shock 70 Family Proteins, Translation Elongation Factors and Histones". PLOS Genetics 12, nr 5 (3.05.2016): e1006023. http://dx.doi.org/10.1371/journal.pgen.1006023.

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Iseppi, Ramona, Carla Condò i Patrizia Messi. "Synergistic Inhibition of Methicillin-Resistant Staphylococcus aureus (MRSA) by Melaleuca alternifolia Chell (Tea Tree) and Eucalyptus globulus Labill. Essential Oils in Association with Oxacillin". Antibiotics 12, nr 5 (3.05.2023): 846. http://dx.doi.org/10.3390/antibiotics12050846.

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The presence of antibiotic-resistant bacteria has become a major therapeutic priority. This trend indicates the need for alternative agents to antibiotics, such as natural compounds of plant origin. By assessing membrane permeability, we investigated the antimicrobial activity of Melaleuca alternifolia and Eucalyptus globulus essential oils (EOs) against three strains of methicillin-resistant Staphylococcus aureus (MRSA). Using the checkerboard method, the efficacy of single EOs, in association with each other or in combination with oxacillin, was quantified by calculating the fractional inhibitory concentrations (FIC Index). All EOs showed a reduction in bacterial load, an alteration of membrane permeability which leads to an increase in its function, resulting in the release of nucleic acids and proteins. The treatment with EO–oxacillin combinations and associated EO–EO resulted in a synergistic effect in most of the tests performed. EO–EO association showed a high activity in the alteration of the membrane, increasing the permeability to about 80% in all the MRSA strains treated. In conclusion, the combination of EOs and antibiotics represents a valid therapeutic support against MRSA bacteria, allowing for a decrease in the antibiotic concentration needed for therapeutic use.

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Li, Jinyang, Sheng Tong, Farrukh Raza Amin, Habiba Khalid, Kai Chen, Xiaoguang Zhao, Jinling Cai i Demao Li. "Enhancing the Activity of a Self-Inducible Promoter in Escherichia coli through Saturation Mutation and High-Throughput Screening". Fermentation 9, nr 5 (13.05.2023): 468. http://dx.doi.org/10.3390/fermentation9050468.

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The use of self-inducible promoters is a promising strategy to address metabolic imbalances caused by overexpression. However, the low activity of natural self-inducible promoters hinders their widespread application. To overcome this limitation, we selected the fic promoter as a model promoter to create an enhanced self-inducible promoter library using saturation mutations and high-throughput screening. Sequence analysis revealed that these promoters share certain characteristics, including semi-conservation in the −35 hexamer, highly conserved cytosine in the −17 motif (compared to −13 for other promoters), and moderate A+T content between positions −33 and −18 in the spacer region. Additionally, the discriminator region of these promotors features high A+T content in the first five bases. We identified PficI-17, PficII-33, and PficIII-14 promoters as the optional promoters in the −35 hexamer, spacer region, and discriminator mutation libraries, respectively. These promotors were used as representatives to measure the specific fluorescence and OD600 nm dynamics in different media and to confirm their effect on the expression of different proteins, including egfp (enhanced green fluorescence protein) and rfp (red fluorescence protein). Overall, our findings provide valuable guidance for modifying promoters and developing a promoter library suitable for regulating target genes.

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31

Kuok, Chiu-Fai, Sai-On Hoi, Chi-Fai Hoi, Chi-Hong Chan, Io-Hong Fong, Cheong-Kei Ngok, Li-Rong Meng i Pedro Fong. "Synergistic antibacterial effects of herbal extracts and antibiotics on methicillin-resistant Staphylococcus aureus: A computational and experimental study". Experimental Biology and Medicine 242, nr 7 (24.01.2017): 731–43. http://dx.doi.org/10.1177/1535370216689828.

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Antibiotic resistance has become a serious global concern, and the discovery of antimicrobial herbal constituents may provide valuable solutions to overcome the problem. In this study, the effects of therapies combining antibiotics and four medicinal herbs on methicillin-resistant Staphylococcus aureus (MRSA) were investigated. Specifically, the synergistic effects of Magnolia officinalis, Verbena officinalis, Momordica charantia, and Daphne genkwa in combination with oxacillin or gentamicin against methicillin-resistant (ATCC43300) and methicillin-susceptible (ATCC25923) S. aureus were examined. In vitro susceptibility and synergistic testing were performed to measure the minimum inhibitory concentration and fractional inhibitory concentration (FIC) index of the antibiotics and medicinal herbs against MRSA and methicillin-susceptible S. aureus. To identify the active constituents in producing these synergistic effects, in silico molecular docking was used to investigate the binding affinities of 139 constituents of the four herbs to the two common MRSA inhibitory targets, penicillin binding proteins 2a (PBP2a) and 4 (PBP4). The physicochemical and absorption, distribution, metabolism, and excretion properties and drug safety profiles of these compounds were also analyzed. D. genkwa extract potentiated the antibacterial effects of oxacillin against MRSA, as indicated by an FIC index value of 0.375. M. officinalis and V. officinalis produced partial synergistic effects when combined with oxacillin, whereas M. charantia was found to have no beneficial effects in inhibiting MRSA. Overall, tiliroside, pinoresinol, magnatriol B, and momorcharaside B were predicted to be PBP2a or PBP4 inhibitors with good drug-like properties. This study identifies compounds that deserve further investigation with the aim of developing therapeutic agents to modulate the effect of antibiotics on MRSA. Impact statement Antibiotic resistant is a well-known threat to global health and methicillin-resistant Staphylococcus aureus is one of the most significant ones. These resistant bacteria kill thousands of people every year and therefore a new effective antimicrobial treatment is necessary. This study identified the herbs and their associated bioactive ingredients that can potential the effects of current antibiotics. These herbs have long history of human usage in China and have well-defined monograph in the Chinese Pharmacopeia. These indicate their relatively high clinical safety and may have a quicker drug development process than that of a new novel antibiotic. Based on the results of this study, the authors will perform further in vitro and animal studies, aiming to accumulate significant data for the application of clinical trial.

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Toudji, Gerard A., Emmanuel Thiombiano, Simplice D. Karou, Kokou Anani, Yao Adjrah, Holaly E. Gbekley, Martin Kiendrebeogo, Yaovi Ameyapoh i Jacques Simpore. "ANTIBACTERIAL AND ANTI-INFLAMMATORY ACTIVITIES OF CRUDE EXTRACTS OF THREE TOGOLESE MEDICINAL PLANTS AGAINST ESBL KLEBSIELLA PNEUMONIAE STRAINS". African Journal of Traditional, Complementary and Alternative medicines 15, nr 1 (29.12.2017): 42. http://dx.doi.org/10.21010/ajtcam.vi15.1.5.

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Background: Pithecellobium dulce (Roxb.) Benth., Securidaca longepedunculata Fresn and Cryptolepis sanguinolenta (Lindl.) Schlt are three plants widely used in the Togolese traditional medicine to treat microbial infections. Some studies reported their antibacterial activity alone but until know there no data concerning their possible interaction with conventional antibiotics. The main objective of this study was to investigate the antibacterial activity of the association of the crude extracts of the three plants with some conventional antibiotics. We further evaluate the antioxidant and the anti-inflammatory activities of the extracts on rat’s model. Materials and methods:The antimicrobial activity was evaluated by the broth microdilution assay and the Fractional inhibitory concentrations (FIC) determined by the checkerboard method. The anti-inflammatory activity was evaluated using the Carrageenan- induced rat paw edema model. The antioxidant activities and the phenol contents were determined by spectrophotometry. Results: The MICs of hydroethanolic extract of plants ranged from 3.125 to 100 mg/mL on Klebsiella pneumoniae strains. Synergistic action was observed only with the combination of Imipenem/P. dulce, imipenem/C. sanguinolenta, amikacin/P. dulce and amikacin/C. sanguinolenta against the ESBL negative Klebsiella pneumoniae strain. Of the 21 associations, 15 were antagonistic on the ESBL-producing strains. The indifference effect was observed with the combination of the extract of Securidaca longepedunculata and the following antibiotics imipenem, amikacin, tetracyclin, ciprofloxacin, Cefotaxim; and Sulfametoxazol+Trimethoprim. The in vitro anti-inflammatory with Lipoxygenase inhibition activity was best with C. sanguinolenta extract while the in vivo paw edema model revealed that S. longepedunculata was the highest reducer of paw edema. In addition white blood cells count and biochemical parameters such as total proteins and immunoglobulins were significantly affected by the administration of plant extracts. Conclusion: This study revealed that the three plants although they may inhibit the bacterial growth by themselves, but there is also a possible synergistic action with the commercial antibiotics. Further investigations are needed to identify the active compounds and their mechanism of action.

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Devi, Lalitha, Himesh Makala, Lavanya Pothana, Khemlal Nirmalkar i Sandeep Goel. "Comparative efficacies of six different media for cryopreservation of immature buffalo (Bubalus bubalis) calf testis". Reproduction, Fertility and Development 28, nr 7 (2016): 872. http://dx.doi.org/10.1071/rd14171.

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Buffalo calves have a high mortality rate (~80%) in commercial dairies and testis cryopreservation can provide a feasible option for the preservation of germplasm from immature males that die before attaining sexual maturity. The aim of the present study was to evaluate combinations of 10 or 20% dimethylsulfoxide (DMSO) with 0, 20 or 80% fetal bovine serum (FBS) for cryopreservation of immature buffalo testicular tissues, subjected to uncontrolled slow freezing. Tissues cryopreserved in 20% DMSO with 20% FBS (D20S20) showed total, tubular and interstitial cell viability, number of early apoptotic and DNA-damaged cells, surviving germ and proliferating cells and expression of testicular cell-specific proteins (POU class 5 homeobox (POU5F1), vimentin (VIM) and actin α2 (ACTA2)) similar to that of fresh cultured control (FCC; P > 0.05). Expression of cytochrome P450, family 11, subfamily A (CYP11A1) protein and testosterone assay showed that only tissues cryopreserved in D20S20 had Leydig cells and secretory functions identical to that of FCC (P > 0.05). High expression of superoxide dismutase2 (SOD2), cold-inducible RNA-binding protein (CIRBP) and RNA-binding motif protein3 (RBM3) proteins in cryopreserved tissues indicated involvement of cell signalling pathways regulating cellular protective mechanisms. Similarity in expression of pro-apoptosis proteins transcription factor tumour protein P53 (TP53) and BCL2-associated X protein (BAX) in D20S20 cryopreserved tissues to that of FCC (P > 0.05) suggested lower apoptosis and DNA damage as key reasons for superior cryopreservation.

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Choi, Kwang-Pil, Nathan Kendrick i Lacy Daniels. "Demonstration that fbiC Is Required by Mycobacterium bovis BCG for Coenzyme F420 and FO Biosynthesis". Journal of Bacteriology 184, nr 9 (1.05.2002): 2420–28. http://dx.doi.org/10.1128/jb.184.9.2420-2428.2002.

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ABSTRACT Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F420 were identified. Four independent mutants that could not make F420 or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC− mutant with fbiC restored the F420 phenotype. These data demonstrate that fbiC is essential for F420 production and that FbiC participates in a portion of the F420 biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F420. Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (∼95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.

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Bond, Lisa M., David A. Tumbarello, John Kendrick-Jones i Folma Buss. "Small-molecule inhibitors of myosin proteins". Future Medicinal Chemistry 5, nr 1 (styczeń 2013): 41–52. http://dx.doi.org/10.4155/fmc.12.185.

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Hoatlin, Maureen E., Tracy A. Christianson, Winnie W. Keeble, Adam T. Hammond, Yu Zhi, Michael C. Heinrich, Paula A. Tower i Grover C. Bagby Jr. "The Fanconi Anemia Group C Gene Product Is Located in Both the Nucleus and Cytoplasm of Human Cells". Blood 91, nr 4 (15.02.1998): 1418–25. http://dx.doi.org/10.1182/blood.v91.4.1418.

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Abstract The Fanconi anemia (FA) complementation group C (FAC) protein gene encodes a cytoplasmic protein with a predicted Mrof 63,000. The protein's function is unknown, but it has been hypothesized that it either mediates resistance to DNA cross-linking agents or facilitates repair after exposure to such factors. The protein also plays a permissive role in the growth of colony-forming unit–granulocyte/macrophage (CFU-GM), burst-forming unit–erythroid (BFU-E), and CFU-erythroid (CFU-E). Attributing a specific function to this protein requires an understanding of its intracellular location. Recognizing that prior study has established the functional importance of its cytoplasmic location, we tested the hypothesis that FAC protein can also be found in the nucleus. Purified recombinant Escherichia coli–derived FAC antigens were used to create antisera able to specifically identify an Mr = 58,000 protein in lysates from human Epstein-Barr virus (EBV)-transformed cell lines by immunoblot analysis. Subcellular fractionation of the cell lysates followed by immunoblot analysis revealed that the majority of the FAC protein was cytoplasmic, as reported previously; however, approximately 10% of FAC protein was reproducibly detected in nuclear fractions. These results were reproducible by two different fractionation methods, and included markers to control for contamination of nuclear fractions by cytoplasmic proteins. Moreover, confocal image analysis of human 293 cells engineered to express FAC clearly demonstrated that FAC protein is located in both cytoplasmic and nuclear compartments, consistent with data obtained from fractionation of the FA cell lines. Finally, complementation of the FAC defect using retroviral-mediated gene transfer resulted in a substantial increase in nuclear FAC protein. Therefore, while cytoplasmic localization of this protein appears to be functionally important, it may also exert some essential nuclear function.

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Banner, Miriam A., John G. Cunniffe, Robin L. Macintosh, Timothy J. Foster, Holger Rohde, Dietrich Mack, Emmy Hoyes, Jeremy Derrick, Mathew Upton i Pauline S. Handley. "Localized Tufts of Fibrils on Staphylococcus epidermidis NCTC 11047 Are Comprised of the Accumulation-Associated Protein". Journal of Bacteriology 189, nr 7 (2.02.2007): 2793–804. http://dx.doi.org/10.1128/jb.00952-06.

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ABSTRACT Staphylococcus epidermidis is both a human skin commensal and an opportunistic pathogen, causing infections linked to implanted medical devices. This paper describes localized tufts of fibrillar appendages on a subpopulation (25%) of wild-type (WT) S. epidermidis NCTC 11047 cells. The fibrils (122.2 ± 10.8 nm long) are usually in a lateral position on the cells. Fibrillar (Fib+) and nonfibrillar (Fib−) subpopulations were separated (enriched) by 34 sequential partitions of WT cells between a buffer phase and a hexadecane phase. Following enrichment, hydrophobic cells from the hexadecane phase comprised 70% Fib+ cells and the less hydrophobic cells from the buffer phase entirely comprised Fib− cells. The Fib+ and Fib− subpopulations did not revert on subculture (34 times) on solid medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell surface proteins from WT, Fib+, and Fib− cells revealed two high-molecular-mass proteins (280 kDa and 230 kDa) on the WT and Fib+ cells that were absent from the Fib− cells. Amino acid sequencing revealed that fragments of both the 280- and 230-kDa proteins had 100% identity to the accumulation-associated protein (Aap). Aap is known to cause biofilm formation if it is truncated by loss of the terminal A domain. Immunogold staining with anti-Aap antibodies labeled tuft fibrils of the WT and Fib+ cells but not the cell surface of Fib− cells. The tufts were labeled with N-terminally directed antibodies (anti-A domain), showing that the fibrillar Aap was not truncated on the cell surface. Thus, the presence of full-length Aap correlated with the low biofilm-forming abilities of both WT and Fib+ S. epidermidis NCTC 11047 populations. Reverse transcription-PCR showed that aap was transcribed in both Fib+ and Fib− cells. We therefore propose that full-length Aap is expressed on cells of S. epidermidis NCTC 11047 as tufts of short fibrils and that fibril expression is regulated at a posttranscriptional level.

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Kukić, Predrag, i Jens Erik Nielsen. "Electrostatics in proteins and protein–ligand complexes". Future Medicinal Chemistry 2, nr 4 (kwiecień 2010): 647–66. http://dx.doi.org/10.4155/fmc.10.6.

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Wirmer-Bartoschek, Julia, i Stefan Bartoschek. "NMR in drug discovery on membrane proteins". Future Medicinal Chemistry 4, nr 7 (maj 2012): 869–75. http://dx.doi.org/10.4155/fmc.12.46.

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Ndubaku, Chudi, Frederick Cohen, Eugene Varfolomeev i Domagoj Vucic. "Targeting inhibitor of apoptosis proteins for therapeutic intervention". Future Medicinal Chemistry 1, nr 8 (listopad 2009): 1509–25. http://dx.doi.org/10.4155/fmc.09.116.

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Nahleh, Z., A. Tfayli, A. Najm, A. El Sayed i Z. Nahle. "Heat shock proteins in cancer: targeting the ‘chaperones’". Future Medicinal Chemistry 4, nr 7 (maj 2012): 927–35. http://dx.doi.org/10.4155/fmc.12.50.

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Carnegie, Graeme K., i Brian T. Burmeister. "A-Kinase Anchoring Proteins That Regulate Cardiac Remodeling". Journal of Cardiovascular Pharmacology 58, nr 5 (listopad 2011): 451–58. http://dx.doi.org/10.1097/fjc.0b013e31821c0220.

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Andersen, Ib. "Determination of specific proteins by the FIA principle". Journal of Automatic Chemistry 12, nr 2 (1990): 53–59. http://dx.doi.org/10.1155/s1463924690000074.

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The following analytes have been investigated: urine albumin (u-albumin), plasma-transferrin (p-transferrin), p-haptoglobin, p-IgG, p-IgA, p-IgM, and p-orosomucoid. An unmodified commercial analytical system FIA Star (Tecator) with a two-channel injector (40 μl) was used. The prediluted plasma samples and antibodies are allowed to react for 33 s before the change in turbidity is measured as a Peak maximum at 405 nm. The optimal concentrations of calibrators and antibodies have been determined to secure antibody excess. Response time (i.e. delay between aspiration of a sample and presentation of the result in absorption units) is 75 s. Automatic print-out of the absorbance profile and movement of the sample rack further accounted for 21 s per sample, so the throughput is reduced to 75 determinations per 2 h. Results are available within an hour, compared to two-12 days with the present methods (electroimmunoassays). Parallel analyses with established methods/analysers show excellent agreement for u-albumin, p-transferrin and p-haptoglobin. For p-IgG, p-IgA and p-IgM the reaction time of 33 s is insufficient because their relative molecular masses (i.e. the size of the molecules) are so high, 150.000-971.000. Five minutes is a more adequate reaction time, which makes a serial analyser such as FIA Star unsuitable for larger workloads of samples of immunoglobulins. The plasma concentration of Orosomucoid is low, resulting in high sample blanks. It is therefore recommended that the reaction is followed kinetically if a serial analyser is used.

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Hoshino, Taizo, Jianxiang Wang, Marcel P. Devetten, Nobuhisa Iwata, Sachiko Kajigaya, Robert J. Wise, Johnson M. Liu i Hagop Youssoufian. "Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression". Blood 91, nr 11 (1.06.1998): 4379–86. http://dx.doi.org/10.1182/blood.v91.11.4379.

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Abstract The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

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45

Hoshino, Taizo, Jianxiang Wang, Marcel P. Devetten, Nobuhisa Iwata, Sachiko Kajigaya, Robert J. Wise, Johnson M. Liu i Hagop Youssoufian. "Molecular Chaperone GRP94 Binds to the Fanconi Anemia Group C Protein and Regulates Its Intracellular Expression". Blood 91, nr 11 (1.06.1998): 4379–86. http://dx.doi.org/10.1182/blood.v91.11.4379.411k14_4379_4386.

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Streszczenie:

The FAC protein encoded by the gene defective in Fanconi anemia (FA) complementation group C binds to at least three ubiquitous cytoplasmic proteins in vitro. We used here the complete coding sequence ofFAC in a yeast two-hybrid screen to identify interacting proteins. The molecular chaperone GRP94 was isolated twice from a B-lymphocyte cDNA library. Binding was confirmed by coimmunoprecipitation of FAC and GRP94 from cytosolic, but not nuclear, lysates of transfected COS-1 cells, as well as from mouse liver cytoplasmic extracts. Deletion mutants of FAC showed that residues 103-308 were required for interaction with GRP94, and a natural splicing mutation within the IVS-4 of FAC that removes residues 111-148 failed to bind GRP94. Ribozyme-mediated inactivation of GRP94 in the rat NRK cell line led to significantly reduced levels of immunoreactive FAC and concomitant hypersensitivity to mitomycin C, similar to the cellular phenotype of FA. Our results demonstrate that GRP94 interacts with FAC both in vitro and in vivo and regulates its intracellular level in a cell culture model. In addition, the pathogenicity of the IVS-4 splicing mutation in the FAC gene may be mediated in part by its inability to bind to GRP94.

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46

Wirstlein, Przemysław Krzysztof, Piotr Jasiński, Marcin Rajewski, Tomasz Goździewicz i Jana Skrzypczak. "Complement inhibitory proteins expression in placentas of thrombophilic women". Folia Histochemica et Cytobiologica 50, nr 3 (8.10.2012): 460–67. http://dx.doi.org/10.5603/fhc.2012.0064.

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Gojis, Ondrej, Martina Kubecova, Jozef Rosina, Jana Vranova, Martin Celko, Denisa Frajerova, Jan Zmrhal i in. "Expression of selected proteins in breast cancer brain metastases". Folia Histochemica et Cytobiologica 51, nr 3 (7.11.2013): 213–18. http://dx.doi.org/10.5603/fhc.2013.0030.

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Borzym-Kluczyk, Małgorzata, Iwona Radziejewska i Barbara Darewicz. "Glycosylation of proteins in healthy and pathological human renal tissues". Folia Histochemica et Cytobiologica 50, nr 4 (23.12.2012): 599–604. http://dx.doi.org/10.5603/fhc.2012.0084.

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KUBO, Hiroaki, Yuesheng HUANG, Toshio KINOSHITA i Hiroyuki NAKAZAWA. "Sensitive determination of proteins by fia with coulometric detection." Bunseki kagaku 38, nr 9 (1989): 454–57. http://dx.doi.org/10.2116/bunsekikagaku.38.9_454.

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Courtney, MA, MH Stoler, VJ Marder i PJ Haidaris. "Developmental expression of mRNAs encoding platelet proteins in rat megakaryocytes". Blood 77, nr 3 (1.02.1991): 560–68. http://dx.doi.org/10.1182/blood.v77.3.560.560.

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Abstract The committed bone marrow megakaryocyte (MK) progenitor undergoes a series of highly regulated stages of development resulting in a large multi-nucleated platelet-producing cell. We studied the developmental expression of the mRNA for two alpha granule proteins, fibronectin (FN) and fibrinogen gamma chain (gamma-FIB), and a cytoskeletal protein, actin, in MKs from marrow of Sprague-Dawley rats. By the method of in situ RNA:RNA hybridization, we showed that mRNAs for the alpha granule proteins were expressed most abundantly in a population of 15-microns diameter promegakaryocytes and in cells as small as 10 microns whose identity as immature MKs was inferred by positive staining for platelet- and MK-specific markers. gamma-FIB and FN mRNAs were present in reduced abundance in a small proportion of intermediate MKs; however, little or no expression was seen in mature platelet-producing MKs. In contrast, high levels of actin mRNA were expressed predominantly in mature, multi- nucleated MKs, and less abundantly in the immature forms. These results suggest that FN and gamma-FIB are transcribed early in MK development to permit translation and packaging of the protein into alpha granules, after which transcription ceases. On the other hand, transcription of actin occurs continuously throughout development, with highest levels in mature platelet-producing MKs, in which actin is needed for shape changes and intracellular movement of organelles. Our data suggest that in situ RNA:RNA hybridization for platelet-specific markers will provide additional criteria by which to establish MK lineage in immature marrow progenitors.

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