scholarly journals Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by HslUV proteolysis

2018 ◽  
Author(s):  
Bruno Fauvet ◽  
Andrija Finka ◽  
Marie-Pierre Castanié-Cornet ◽  
Anne-Marie Cirinesi ◽  
Pierre Genevaux ◽  
...  
Keyword(s):  
Molecular Chaperones ◽  
Quantitative Proteomics ◽  
Protein Solubility ◽  
Label Free ◽  
Wild Type ◽  
Respiratory Enzymes ◽  
Genetic Studies ◽  
E Coli ◽  
Physiological Processes ◽  
Knockout Mutants

AbstractWhereas in eukaryotic cells, the Hsp90s are profusely-studied molecular chaperones controlling protein homeostasis together with Hsp70s, in bacteria, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild typeE. coli, and from knockout mutantsΔdnaKdnaJ(ΔKJ),ΔhtpG(ΔG) andΔdnaKdnaJΔhtpG(ΔKJG) and compared their growth rates under heat-stress also withΔdnaKdnaJΔhslV. Whereas, expectedly, mutant ΔG showed no proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that ΔKJG showed higher levels of metabolic and respiratory enzymes and both ΔKJG andΔdnaKdnaJΔhslVgrew better at 37oC than ΔKJ. The results indicate that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates, preferably by the HslUV protease.Significance statement:The molecular chaperones Hsp70 and Hsp90 are among the most abundant and well-conserved proteins in all realms of life, forming together the core of the cellular proteostasis network. In eukaryotes, Hsp90 functions in collaboration with Hsp70; we studied this collaboration inE. coli, combining genetic studies with label-free quantitative proteomics in which both protein abundance and protein solubility were quantified. Bacteria lacking Hsp70 (DnaK) and its co-chaperone DnaJ (ΔdnaKdnaJ) grew slower and contained significantly less key metabolic and respiratory enzymes. Unexpectedly, an additional deletion of the Hsp90(htpG)gene partially restored the WT phenotype. Deletion of the HslV protease in the ΔdnaKdnaJ background also improved growth, suggesting that bacterial Hsp90 mediates the degradation of Hsp70 substrates, preferentially through HslV.At 37oCΔdnaKdnaJ E. colimutants grow slower than wild type cells. Quantitative proteomics shows that compared to wild type cells,ΔdnaKdnaJcells grown at 30oC contain significantly less key metabolic and respiratory enzymes. Unexpectedly, deletion of theHtpGgene in the ΔdnaKdnaJbackground ameliorates growth at 37oC and partially restores the cellular levels of some metabolic and respiratory enzymes.

scholarly journals Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70–Hsp40 Substrates

2021 ◽  
Vol 8 ◽  
Author(s):  
Bruno Fauvet ◽  
Andrija Finka ◽  
Marie-Pierre Castanié-Cornet ◽  
Anne-Marie Cirinesi ◽  
Pierre Genevaux ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Wild Type ◽  
Triple Mutant ◽  
Respiratory Enzymes ◽  
Physiological Processes ◽  
Knockout Mutants ◽  
Shock Proteins ◽  
Control Protein

In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70–Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.

scholarly journals The effect of lipocalin-2 (LCN2) on apoptosis: a proteomics analysis study in an LCN2 deficient mouse model

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Dongming Wu ◽  
Xiaopeng Wang ◽  
Ye Han ◽  
Yayun Wang
Keyword(s):  
Knockout Mice ◽  
Quantitative Proteomics ◽  
Energy Homeostasis ◽  
Metabolic Diseases ◽  
Differentially Expressed Proteins ◽  
Label Free ◽  
Lipocalin 2 ◽  
Wild Type ◽  
Proteomics Analysis ◽  
Associated Proteins

Abstract Background Recent studies have shown that lipocalin-2 (LCN2) has multiple functions involved in various biological and pathological processes including energy homeostasis, cancer, inflammation, and apoptosis. We aimed to investigate the effect of LCN2 on apoptosis that influences the pathogenetic process of metabolic diseases and cancer. Methods We performed a proteomics analysis of livers taken from LCN2-knockout mice and wild type mice by using label-free LC-MS/MS quantitative proteomics. Results Proteomic analysis revealed that there were 132 significantly differentially expressed proteins (49 upregulated and 83 downregulated) among 2140 proteins in the liver of LCN2-knockout mice compared with wild type mice. Of these, seven apoptosis-associated proteins were significantly upregulated and seven apoptosis-associated proteins downregulated. Conclusion Proteomics demonstrated that there were seven upregulated and seven downregulated apoptosis-associated proteins in liver of LCN2-knockout mice. It is important to clarify the effect of LCN2 on apoptosis that might contribute to the pathogenesis of insulin resistance, cancer, and various nervous system diseases.

Mechanism of pyridoxine 5'-phosphate accumulation in PLPBP protein-deficiency

2022 ◽  
Author(s):  
Tomokazu Ito ◽  
Honoka Ogawa ◽  
Hisashi Hemmi ◽  
Diana M. Downs ◽  
Tohru Yoshimura
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Vitamin B ◽  
Wild Type ◽  
Salvage Pathway ◽  
Intracellular Accumulation ◽  
Pyridoxal Kinase ◽  
Genetic Studies ◽  
E Coli ◽  
Metabolic Systems

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B 6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B 6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. The accumulated PNP could affect diverse metabolic systems through inhibition of some PLP-dependent enzymes. In this study, we investigated the as yet unclear mechanism of intracellular accumulation of PNP by the loss of PLPBP protein encoded by yggS in E. coli . Genetic studies using several PLPBP-deficient strains of E. coli lacking known enzyme(s) in the de novo or salvage pathway of vitamin B 6 , which includes pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies with the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa . We show that disruption of PLPBP lead to perturb PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. Importance A PLP-binding protein PLPBP from the conserved COG0325 family has recently been recognized as a key player in vitamin B 6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B 6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of the PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of the PNP accumulation was poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and its disruption may lead to the accumulation of PNP in PLPBP-deficiency.

scholarly journals GlpD and PlsB Participate in Persister Cell Formation in Escherichia coli

2006 ◽  
Vol 188 (14) ◽  
pp. 5136-5144 ◽  
Author(s):  
Amy L. Spoering ◽  
Marin Vulić ◽  
Kim Lewis
Keyword(s):  
Escherichia Coli ◽  
Cell Formation ◽  
Wild Type ◽  
Expression Library ◽  
Genetic Studies ◽  
Bacterial Populations ◽  
E Coli ◽  
Overexpression Strain ◽  
Multidrug Tolerance ◽  
Glycerol 3 Phosphate Dehydrogenase

ABSTRACT Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.

Increased ultraviolet radiation sensitivity ofEscherichia coligrown at low temperature

2014 ◽  
Vol 60 (5) ◽  
pp. 327-331 ◽  
Author(s):  
Suhas Mangoli ◽  
Devashish Rath ◽  
Manish Goswami ◽  
Narendra Jawali
Keyword(s):  
Dna Repair ◽  
Low Temperature ◽  
Ultraviolet Radiation ◽  
Excision Repair ◽  
Radiation Sensitivity ◽  
Genetic Studies ◽  
E Coli ◽  
Knockout Mutants ◽  
Highly Sensitive ◽  
Nucleotide Excision

The repair of DNA damage caused by ultraviolet radiation (UVR) is well understood in both lower and higher organisms. Genetic studies carried out at optimum temperature for growth, 37 °C in Escherichia coli, have revealed the major pathways of DNA repair. We show that E. coli cells grown at 20 °C are more sensitive to UVR than cells grown at 37 °C. The analysis of knockout mutants demonstrates that cells impaired in recombinational DNA repair pathways show increased UV sensitivity at 20 °C. Cells with mutations in the nucleotide excision repair (NER) pathway genes are highly sensitive to UVR when grown at 37 °C and retain that sensitivity when grown at 20 °C, whereas wild-type cells are not sensitive when grown at 37 °C but become more sensitive to UVR when grown at low temperatures. Our results taken along with reports from the literature suggest that the UVR sensitivity of E. coli cells at low temperature could be due to impaired NER function.

scholarly journals Quantitative proteomics reveals the antifungal effect of canthin-6-one isolated from Ailanthus altissima against Fusarium oxysporum f. sp. cucumerinum in vitro

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250712
Author(s):  
Yongchun Li ◽  
Meirong Zhao ◽  
Zhi Zhang
Keyword(s):  
Antifungal Activity ◽  
Fusarium Oxysporum ◽  
Quantitative Proteomics ◽  
Ailanthus Altissima ◽  
Label Free ◽  
Physiological Processes ◽  
Parallel Reaction Monitoring ◽  
Nitrogen Nutrients ◽  
New Perspective

Canthin-6-one, one of the main alkaloid compounds extracted from Ailanthus altissima, has recently attracted increasing interest for its antifungal activity. To evaluate the potential of canthin-6-one in controlling plant fungal diseases, we investigated the antifungal activity of canthin-6-one isolated from A. altissima against Fusarium oxysporum f. sp. cucumerinum (Foc) in vitro. The mycelial growth rate and micro-broth dilution were used to test antifungal activity. Furthermore, label-free quantitative proteomics and parallel reaction monitoring (PRM) techniques were applied to analyze the antifungal mechanism. It was found that canthin-6-one significantly inhibited the growth of Foc, and had higher inhibitory action than chlorothalonil at the same concentration. Proteomic analysis showed that the expression of 203 proteins altered significantly after canthin-6-one treatment. These differentially expressed proteins were mainly involved in amino acid biosynthesis and nitrogen metabolism pathways. These results suggest that canthin-6-one significantly interferes with the metabolism of amino acids. Therefore, it affects nitrogen nutrients and disturbs the normal physiological processes of fungi, and ultimately leads to the death of pathogens. This study provides a natural plant antifungal agent and a new perspective for the study of antifungal mechanisms.

Development of Algorithms for Mass Spectrometry-based Label-free Quantitative Proteomics*

2011 ◽  
Vol 38 (6) ◽  
pp. 506-518 ◽  
Author(s):  
Wei ZHANG ◽  
Ji-Yang ZHANG ◽  
Hui LIU ◽  
Han-Chang SUN ◽  
Chang-Ming XU ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Label Free

The Effect of Mianserin on Lifespan of Caenorhabditis elegans is Abolished by Glucose

2021 ◽  
Vol 13 ◽  
Author(s):  
Abdullah Almotayri ◽  
Jency Thomas ◽  
Mihiri Munasinghe ◽  
Markandeya Jois
Keyword(s):  
Caenorhabditis Elegans ◽  
Scaffolding Protein ◽  
Lifespan Extension ◽  
Wild Type ◽  
E Coli ◽  
C Elegans ◽  
Risk Of Death ◽  
Increased Risk ◽  
Antidepressant Use ◽  
Extension Effect

Background: The antidepressant mianserin has been shown to extend the lifespan of Caenorhabditis elegans (C. elegans), a well-established model organism used in aging research. The extension of lifespan in C. elegans was shown to be dependent on increased expression of the scaffolding protein (ANK3/unc-44). In contrast, antidepressant use in humans is associated with an increased risk of death. The C. elegans in the laboratory are fed Escherichia coli (E. coli), a diet high in protein and low in carbohydrate, whereas a typical human diet is high in carbohydrates. We hypothesized that dietary carbohydrates might mitigate the lifespan-extension effect of mianserin. Objective: To investigate the effect of glucose added to the diet of C. elegans on the lifespan-extension effect of mianserin. Methods: Wild-type Bristol N2 and ANK3/unc-44 inactivating mutants were cultured on agar plates containing nematode growth medium and fed E. coli. Treatment groups included (C) control, (M50) 50 μM mianserin, (G) 73 mM glucose, and (M50G) 50 μM mianserin and 73 mM glucose. Lifespan was determined by monitoring the worms until they died. Statistical analysis was performed using the Kaplan-Meier version of the log-rank test. Results: Mianserin treatment resulted in a 12% increase in lifespan (P<0.05) of wild-type Bristol N2 worms but reduced lifespan by 6% in ANK3/unc-44 mutants, consistent with previous research. The addition of glucose to the diet reduced the lifespan of both strains of worms and abolished the lifespan-extension by mianserin. Conclusion: The addition of glucose to the diet of C. elegans abolishes the lifespan-extension effects of mianserin.

scholarly journals The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit

2021 ◽  
Author(s):  
D Israel ◽  
S Khan ◽  
C R Warren ◽  
J J Zwiazek ◽  
T M Robson
Keyword(s):  
Vapour Pressure Deficit ◽  
Air Humidity ◽  
Wild Type ◽  
Knockout Mutant ◽  
Evaporative Demand ◽  
Co2 Transport ◽  
Whole Plant ◽  
Knockout Mutants ◽  
Leaf Level ◽  
Low Humidity

Abstract The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm) or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of lacking PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than the wild type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes of humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

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