scholarly journals PHR1 and PHR2 ofCandida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

1999 ◽  
Vol 181 (22) ◽  
pp. 7070-7079 ◽  
Author(s):  
William A. Fonzi
Keyword(s):  
Temporal Analysis ◽  
Surface Proteins ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Fivefold Increase ◽  
Conditional Expression

ABSTRACT PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and β-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between β-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in β-1,3-glucan-associated β-1,6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between β-1,6- and β-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process β-1,3-glucans and make available acceptor sites for the attachment of β-1,6-glucans.

scholarly journals Molecular Characterization, Expression and Functional Analysis of Chicken STING

2018 ◽  
Vol 19 (12) ◽  
pp. 3706 ◽  
Author(s):  
Jin-Shan Ran ◽  
Jie Jin ◽  
Xian-Xian Zhang ◽  
Ye Wang ◽  
Peng Ren ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Molecular Characterization ◽  
Poly I:C ◽  
Interferon Response ◽  
Type I ◽  
Sequence Alignments ◽  
Multiple Sequence ◽  
Pathogen Invasion

Innate immunity is an essential line of defense against pathogen invasion which is gained at birth, and the mechanism involved is mainly to identify pathogen-associated molecular patterns through pattern recognition receptors. STING (stimulator of interferon genes) is a signal junction molecule that hosts the perception of viral nucleic acids and produces type I interferon response, which plays a crucial role in innate immunity. However, relatively few studies have investigated the molecular characterization, tissue distribution, and potential function of STING in chickens. In this study, we cloned the full-length cDNA of chicken STING that is composed of 1341 bp. Sequence analyses revealed that STING contains a 1140-bp open-reading frame that probably encodes a 379-amino acid protein. Multiple sequence alignments showed that the similarity of the chicken STING gene to other birds is higher than that of mammals. Real-time polymerase chain reaction (PCR) assays revealed that STING is highly expressed in the spleen, thymus and bursa of fabricious in chickens. Furthermore, we observed that STING expression was significantly upregulated both in vitro and in vivo following infection with Newcastle disease virus (NDV). STING expression was also significantly upregulated in chicken embryo fibroblasts upon stimulation with poly(I:C) or poly(dA:dT). Taken together, these findings suggest that STING plays an important role in antiviral signaling pathways in chickens.

scholarly journals Leucines 193 and 194 at the N-Terminal Domain of the XylS Protein, the Positive Transcriptional Regulator of the TOL meta-Cleavage Pathway, Are Involved in Dimerization

2003 ◽  
Vol 185 (10) ◽  
pp. 3036-3041 ◽  
Author(s):  
Raquel Ruíz ◽  
Silvia Marqués ◽  
Juan L. Ramos
Keyword(s):  
Carbon Metabolism ◽  
Transcriptional Regulator ◽  
Transcriptional Regulators ◽  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Signal Recognition ◽  
Sequence Alignments ◽  
Conserved Domain ◽  
Lexa Protein

ABSTRACT Members of the AraC/XylS family of transcriptional regulators are usually organized in two domains: a conserved domain made up of 100 amino acids and frequently located at the C-terminal end, involved in DNA binding; and an N-terminal nonconserved domain involved in signal recognition, as is the case for regulators involved in the control of carbon metabolism (R. Tobes and J. L. Ramos, Nucleic Acids Res. 30:318-321, 2002). The XylS protein, which is extremely insoluble, controls expression of the meta-cleavage pathway for alkylbenzoate metabolism. We fused the N-terminal end of XylS to the maltose-binding protein (MBP) in vitro and found in glutaraldehyde cross-linking assays that the protein dimerized. Experiments with a chimeric N-terminal XylS linked to a ′LexA protein showed that the dimer was stabilized in the presence of alkylbenzoates. Sequence alignments with AraC and UreR allowed us to identify three residues, Leu193, Leu194, and Ile205, as potentially being involved in dimerization. Site-directed mutagenesis of XylS in which each of the above residues was replaced with Ala revealed that Leu193 and Leu194 were critical for activity and that a chimera in which LexA was linked to the N terminus of XylSLeu193Ala or XylSLeu194Ala was not functional. Dimerization of the chimeras MBP-N-XylSLeu193Ala and MBP-N-XylSLeu194Ala was not observed in cross-linking assays with glutaraldehyde.

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals Breakdown of Filamentous Myofibrils by the UPS–Step by Step

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 110
Author(s):  
Dina Aweida ◽  
Shenhav Cohen
Keyword(s):  
Protein Quality ◽  
Protein Structures ◽  
Ubiquitin Proteasome System ◽  
Surface Proteins ◽  
Catalytic Core ◽  
Complex Protein ◽  
Ubiquitin Proteasome ◽  
Aaa Atpases

Protein degradation maintains cellular integrity by regulating virtually all biological processes, whereas impaired proteolysis perturbs protein quality control, and often leads to human disease. Two major proteolytic systems are responsible for protein breakdown in all cells: autophagy, which facilitates the loss of organelles, protein aggregates, and cell surface proteins; and the ubiquitin-proteasome system (UPS), which promotes degradation of mainly soluble proteins. Recent findings indicate that more complex protein structures, such as filamentous assemblies, which are not accessible to the catalytic core of the proteasome in vitro, can be efficiently degraded by this proteolytic machinery in systemic catabolic states in vivo. Mechanisms that loosen the filamentous structure seem to be activated first, hence increasing the accessibility of protein constituents to the UPS. In this review, we will discuss the mechanisms underlying the disassembly and loss of the intricate insoluble filamentous myofibrils, which are responsible for muscle contraction, and whose degradation by the UPS causes weakness and disability in aging and disease. Several lines of evidence indicate that myofibril breakdown occurs in a strictly ordered and controlled manner, and the function of AAA-ATPases is crucial for their disassembly and loss.

scholarly journals Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

2021 ◽  
Vol 22 (13) ◽  
pp. 7099
Author(s):  
Pradeep Kumar Kopparapu ◽  
Meghshree Deshmukh ◽  
Zhicheng Hu ◽  
Majd Mohammad ◽  
Marco Maugeri ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Responses ◽  
Surface Proteins ◽  
Host Cells ◽  
Immune Stimulation ◽  
Domains Of Life ◽  
Major Surface ◽  
Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

scholarly journals Additional evidence for a proform to tropoelastin from chick aorta

1979 ◽  
Vol 177 (2) ◽  
pp. 559-567 ◽  
Author(s):  
C S Heng-Khoo ◽  
R B Rucker ◽  
K W Buckingham
Keyword(s):  
Basic Protein ◽  
Cross Linking ◽  
Protein Subunit ◽  
Deficient Diet ◽  
Culture In Vitro ◽  
One Step

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10–20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.

scholarly journals Antigenicity of the infected-erythrocyte and merozoite surfaces in Falciparum malaria.

1979 ◽  
Vol 150 (5) ◽  
pp. 1241-1254 ◽  
Author(s):  
S G Langreth ◽  
R T Reese
Keyword(s):  
Plasmodium Falciparum ◽  
Falciparum Malaria ◽  
Infected Erythrocyte ◽  
Falciparum Infection ◽  
Human Erythrocytes ◽  
Cross Linking ◽  
Common Antigens

The antigenicity of altered structures induced by Plasmodium falciparum in the membranes of infected Aotus monkey and human erythrocytes was examined. Antisera were obtained from monkeys made immune to malaria. Bound antibodies were shown to be localized on the knob protrusions of infected erythrocytes of both human and monkey origin and from both in vitro and in vivo infections. Therefore, P. falciparum infection has produced similar antigenic changes in the erythrocyte surfaces of both man and monkey. Uninfected erythrocytes and all knobless-infected erythrocytes bound no antibody from immune sera. Strains of P. falciparum from widely different geographic areas that were cultured in vitro in human erythrocytes induced structures (knobs) which have common antigenicity. Merozoites were agglutinated by cross-linking of their cell coats when incubated with immune sera. The binding of ferritin-labeled antibody was heavy on the coats of both homologous and heterologous strains of the parasite, indicating that the merozoite surfaces of these strains share common antigens.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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