scholarly journals Inorganic Polyphosphate, Exopolyphosphatase, andPho84-Like Transporters May Be Involved in Copper Resistance inMetallosphaera sedulaDSM 5348T

Archaea ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Matías Rivero ◽  
Constanza Torres-Paris ◽  
Rodrigo Muñoz ◽  
Ricardo Cabrera ◽  
Claudio A. Navarro ◽  
...  
Keyword(s):  
Present Report ◽  
Resistance Mechanism ◽  
Metal Resistance ◽  
Copper Resistance ◽  
Site Directed Mutagenesis ◽  
Pcr Analysis ◽  
Linear Polymers ◽  
Inorganic Polyphosphate ◽  
Metallosphaera Sedula

Polyphosphates (PolyP) are linear polymers of orthophosphate residues that have been proposed to participate in metal resistance in bacteria and archaea. In addition of having a CopA/CopB copper efflux system, the thermoacidophilic archaeonMetallosphaera sedulacontains electron-dense PolyP-like granules and a putative exopolyphosphatase (PPXMsed,Msed_0891) and four presumedpho84-like phosphate transporters (Msed_0846,Msed_0866,Msed_1094, andMsed_1512) encoded in its genome. In the present report, the existence of a possible PolyP-based copper-resistance mechanism inM. sedulaDSM 5348Twas evaluated.M. sedulaDSM 5348Taccumulated high levels of phosphorous in the form of granules, and its growth was affected in the presence of 16 mM copper. PolyP levels were highly reduced after the archaeon was subjected to an 8 mM CuSO4shift. PPXMsedwas purified, and the enzyme was found to hydrolyze PolyPin vitro. Essential residues for catalysis of PPXMsedwere E111 and E113 as shown by a site-directed mutagenesis of the implied residues. Furthermore,M. sedula ppx,pho84-like, andcopTMAgenes were upregulated upon copper exposure, as determined by qRT-PCR analysis. The results obtained support the existence of a PolyP-dependent copper-resistance system that may be of great importance in the adaptation of this thermoacidophilic archaeon to its harsh environment.

scholarly journals Transcriptomes of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula Exposed to Metal “Shock” Reveal Generic and Specific Metal Responses

2016 ◽  
Vol 82 (15) ◽  
pp. 4613-4627 ◽  
Author(s):  
Garrett H. Wheaton ◽  
Arpan Mukherjee ◽  
Robert M. Kelly
Keyword(s):  
Metal Toxicity ◽  
Cellular Response ◽  
Sulfur Metabolism ◽  
Metal Resistance ◽  
Open Reading Frames ◽  
Copper Resistance ◽  
Natural Environments ◽  
Cluster Assembly ◽  
Content Type ◽  
Metallosphaera Sedula

ABSTRACTThe extremely thermoacidophilic archaeonMetallosphaera sedulamobilizes metals by novel membrane-associated oxidase clusters and, consequently, requires metal resistance strategies. This issue was examined by “shocking”M. sedulawith representative metals (Co2+, Cu2+, Ni2+, UO22+, Zn2+) at inhibitory and subinhibitory levels. Collectively, one-quarter of the genome (554 open reading frames [ORFs]) responded to inhibitory levels, and two-thirds (354) of the ORFs were responsive to a single metal. Cu2+(259 ORFs, 106 Cu2+-specific ORFs) and Zn2+(262 ORFs, 131 Zn2+-specific ORFs) triggered the largest responses, followed by UO22+(187 ORFs, 91 UO22+-specific ORFs), Ni2+(93 ORFs, 25 Ni2+-specific ORFs), and Co2+(61 ORFs, 1 Co2+-specific ORF). While one-third of the metal-responsive ORFs are annotated as encoding hypothetical proteins, metal challenge also impacted ORFs responsible for identifiable processes related to the cell cycle, DNA repair, and oxidative stress. Surprisingly, there were only 30 ORFs that responded to at least four metals, and 10 of these responded to all five metals. This core transcriptome indicated induction of Fe-S cluster assembly (Msed_1656-Msed_1657), tungsten/molybdenum transport (Msed_1780-Msed_1781), and decreased central metabolism. Not surprisingly, a metal-translocating P-type ATPase (Msed_0490) associated with a copper resistance system (Cop) was upregulated in response to Cu2+(6-fold) but also in response to UO22+(4-fold) and Zn2+(9-fold). Cu2+challenge uniquely induced assimilatory sulfur metabolism for cysteine biosynthesis, suggesting a role for this amino acid in Cu2+resistance or issues in sulfur metabolism. The results indicate thatM. sedulaemploys a range of physiological and biochemical responses to metal challenge, many of which are specific to a single metal and involve proteins with yet unassigned or definitive functions.IMPORTANCEThe mechanisms by which extremely thermoacidophilic archaea resist and are negatively impacted by metals encountered in their natural environments are important to understand so that technologies such as bioleaching, which leverage microbially based conversion of insoluble metal sulfides to soluble species, can be improved. Transcriptomic analysis of the cellular response to metal challenge provided both global and specific insights into how these novel microorganisms negotiate metal toxicity in natural and technological settings. As genetics tools are further developed and implemented for extreme thermoacidophiles, information about metal toxicity and resistance can be leveraged to create metabolically engineered strains with improved bioleaching characteristics.

Copper Resistance Mechanisms of Biomining Bacteria and Archaea Living under Extremely High Concentrations of Metals

2009 ◽  
Vol 71-73 ◽  
pp. 279-282 ◽  
Author(s):  
A. Orell ◽  
C.A. Navarro ◽  
Carlos A. Jerez
Keyword(s):  
Resistance Mechanisms ◽  
Metal Resistance ◽  
Copper Resistance ◽  
Inorganic Polyphosphate ◽  
Resistance Determinants ◽  
High Metal ◽  
Pi Complex ◽  
Cu Resistance ◽  
High Concentrations ◽  
Cu Tolerance

Extremophiles such as the acidophilic Sulfolobus metallicus (Archaea) and Acidithiobacillus ferrooxidans (Bacteria) can resist Cu (CuSO4) concentrations of 200 mM and 800 mM respectively. These microorganisms are important in biomining processes to extract copper and other metals. A. ferrooxidans grown at low Cu concentrations (5 mM) expressed genes coding for ATPases most likely involved in pumping the metal from the cytoplasm to the periplasm of the bacterium. At 100 mM Cu the previous systems were repressed and there was a great induction in the expression of efflux systems known to use the proton motive force energy to export the metal outside the cell. These Cu-resistance determinants from A. ferrooxidans were found to be functional since when expressed in Escherichia coli they conferred higher Cu tolerance to it. Novel Cu-resistance determinants for A. ferrooxidans were found and characterized. S. metallicus possessed at least 2 CopM metallochaperones and 2 CopA ATPases whose expressions were induced by Cu (5 to 50 mM). Furthermore, we previously reported that both microorganisms accumulate high levels of inorganic polyphosphate (PolyP) and that intracellular Cu concentration stimulates polyP hydrolysis. The resulting Pi would then be transported out of the cell as a metal-Pi complex to detoxify the cells. In addition, our results suggest that at high Cu concentrations polyP could also provide energy for the metal efflux. All the data suggest that both biomining microorganisms use different systems to respond to Cu depending on the extracellular concentrations of the metal and suggest that the presence of different additional systems to respond to Cu may explain the extremely high metal resistance of these extremophiles.

scholarly journals Long-chain polyphosphates impair SARS-CoV-2 infection and replication

2021 ◽  
Vol 14 (690) ◽  
pp. eabe5040
Author(s):  
Veronica Ferrucci ◽  
Dae-Young Kong ◽  
Fatemeh Asadzadeh ◽  
Laura Marrone ◽  
Angelo Boccia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Limited Proteolysis ◽  
High Energy ◽  
Site Directed Mutagenesis ◽  
Linear Polymers ◽  
Amino Acid Residues ◽  
Human Nasal Epithelial Cells ◽  
Antiviral Activities ◽  
Long Chain

Inorganic polyphosphates (polyPs) are linear polymers composed of repeated phosphate (PO43−) units linked together by multiple high-energy phosphoanhydride bonds. In addition to being a source of energy, polyPs have cytoprotective and antiviral activities. Here, we investigated the antiviral activities of long-chain polyPs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In molecular docking analyses, polyPs interacted with several conserved amino acid residues in angiotensin-converting enzyme 2 (ACE2), the host receptor that facilitates virus entry, and in viral RNA-dependent RNA polymerase (RdRp). ELISA and limited proteolysis assays using nano– LC-MS/MS mapped polyP120 binding to ACE2, and site-directed mutagenesis confirmed interactions between ACE2 and SARS-CoV-2 RdRp and identified the specific amino acid residues involved. PolyP120 enhanced the proteasomal degradation of both ACE2 and RdRp, thus impairing replication of the British B.1.1.7 SARS-CoV-2 variant. We thus tested polyPs for functional interactions with the virus in SARS-CoV-2–infected Vero E6 and Caco2 cells and in primary human nasal epithelial cells. Delivery of a nebulized form of polyP120 reduced the amounts of viral positive-sense genomic and subgenomic RNAs, of RNA transcripts encoding proinflammatory cytokines, and of viral structural proteins, thereby presenting SARS-CoV-2 infection in cells in vitro.

scholarly journals Curcumin Regulates ERCC1 Expression and Enhances Oxaliplatin Sensitivity in Resistant Colorectal Cancer Cells through Its Effects on miR-409-3p

2020 ◽  
Vol 2020 ◽  
pp. 1-16
Author(s):  
Wei Han ◽  
Hongli Yin ◽  
Hao Ma ◽  
Yi Wang ◽  
Desong Kong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Drug Resistance ◽  
Cancer Cells ◽  
Resistance Mechanism ◽  
Cancer Cell Line ◽  
Colorectal Cancer Cell Line ◽  
Pcr Analysis ◽  
Colorectal Cancer Cells ◽  
Ercc1 Expression

Background. Oxaliplatin (L-OHP) resistance is a major obstacle to the effective treatment of colorectal cancer. The resistance mechanism(s) of colorectal tumors to L-OHP may be related to the regulation of ERCC1 by cancer-expressed miRNAs, but no in-depth studies on the miRNAs that affect drug resistance have been performed. Curcumin (Cur) can reverse the drug resistance of cancer cells, but its effects on ERCC1 expression and miRNA profiles in colorectal cancer have not been studied. Methods. To study the regulation effect of curcumin on ERCC1 expression and its effects on miRNAs, the L-OHP-resistant colorectal cancer cell line HCT116/L-OHP was established. MTT assays were used to evaluate cell proliferation. Flow cytometry was used to investigate apoptotic induction. Western blot and RT-PCR analysis were used to evaluate the expression of drug-associated ERCC1, Bcl-2, GST-π, MRP, P-gp, and survivin. Results. HCT116//L-OHP cell lines were successfully established. The combination of L-OHP and curcumin could reduce L-OHP resistance in vitro. In addition, combination therapy inhibited the expression of ERCC1, Bcl-2, GST-π, MRP, P-gp, and survivin at the mRNA and protein level. Curcumin was found to inhibit ERCC1 through its ability to modulate miR-409-3p. Conclusion. Curcumin can overcome L-OHP resistance in colorectal cancer cells through its effects on miR-409-3p mediated ERCC1 expression.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

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.

Healing effects of curcumin nanoparticles in deep tissue injury mouse model.

2020 ◽  
Vol 18 ◽  
Author(s):  
Zirui Zhang ◽  
Shangcong Han ◽  
Panpan Liu ◽  
Xu Yang ◽  
Jing Han ◽  
...  
Keyword(s):  
Wound Healing ◽  
Mrna Expression ◽  
Tissue Injury ◽  
Inflammatory Cells ◽  
Pcr Analysis ◽  
Protective Effects ◽  
Deep Tissue ◽  
Deep Tissue Injury

Background: Chronic inflammation and lack of angiogenesis are the important pathological mechanisms in deep tissue injury (DTI). Curcumin is a well-known anti-inflammatory and antioxidant agent. However, curcumin is unstable under acidic and alkaline conditions, and can be rapidly metabolized and excreted in the bile, which shortens its bioactivity and efficacy. Objective: This study aimed to prepare curcumin-loaded poly (lactic-co-glycolic acid) nanoparticles (CPNPs) and to elucidate the protective effects and underlying mechanisms of wound healing in DTI models. Methods: CPNPs were evaluated for particle size, biocompatibility, in vitro drug release and their effect on in vivo wound healing. Results : The results of in vivo wound closure analysis revealed that CPNP treatments significantly improved wound contraction rates (p<0.01) at a faster rate than other three treatment groups. H&E staining revealed that CPNP treatments resulted in complete epithelialization and thick granulation tissue formation, whereas control groups resulted in a lack of compact epithelialization and persistence of inflammatory cells within the wound sites. Quantitative real-time PCR analysis showed that treatment with CPNPs suppressed IL-6 and TNF-α mRNA expression, and up-regulated TGF-β, VEGF-A and IL-10 mRNA expression. Western blot analysis showed up-regulated protein expression of TGF-β, VEGF-A and phosphorylatedSTAT3. Conclusion: Our results showed that CPNPs enhanced wound healing in DTI models, through modulation of the JAK2/STAT3 signalling pathway and subsequent upregulation of pro-healing factors.

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 Single amino acid changes in the mumps virus haemagglutinin–neuraminidase and polymerase proteins are associated with neuroattenuation

2009 ◽  
Vol 90 (7) ◽  
pp. 1741-1747 ◽  
Author(s):  
Tahir H. Malik ◽  
Candie Wolbert ◽  
Laura Nerret ◽  
Christian Sauder ◽  
Steven Rubin
Keyword(s):  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Change ◽  
Mumps Virus ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Supporting Evidence ◽  
L Protein ◽  
Single Amino Acid Change

It has previously been shown that three amino acid changes, one each in the fusion (F; Ala/Thr-91→Thr), haemagglutinin–neuraminidase (HN; Ser-466→Asn) and polymerase (L; Ile-736→Val) proteins, are associated with attenuation of a neurovirulent clinical isolate of mumps virus (88-1961) following serial passage in vitro. Here, using full-length cDNA plasmid clones and site-directed mutagenesis, it was shown that the single amino acid change in the HN protein and to a lesser extent, the change in the L protein, resulted in neuroattenuation, as assessed in rats. The combination of both amino acid changes caused neuroattenuation of the virus to levels previously reported for the clinical isolate following attenuation in vitro. The amino acid change in the F protein, despite having a dramatic effect on protein function in vitro, was previously shown to not be involved in the observed neuroattenuation, highlighting the importance of conducting confirmatory in vivo studies. This report provides additional supporting evidence for the role of the HN protein as a virulence factor and, as far as is known, is the first report to associate an amino acid change in the L protein with mumps virus neuroattenuation.

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