scholarly journals Conversion of a soluble protein into a potent chaperone in vivo

2018 ◽  
Author(s):  
Soon Bin Kwon ◽  
Kisun Ryu ◽  
Ahyun Son ◽  
Hotcherl Jeong ◽  
Keo-Heun Lim ◽  
...  
Keyword(s):  
Protein Folding ◽  
Soluble Protein ◽  
Protein Solubility ◽  
Chaperone Activity ◽  
Surface Charges ◽  
E Coli ◽  
Cellular Factors ◽  
Aggregation Inhibition

AbstractProtein-folding assistance and aggregation inhibition by cellular factors are largely understood in the context of molecular chaperones. As an alternative and complementary model, we previously proposed that, in general, soluble cellular macromolecules including chaperones with large excluded volume and surface charges exhibit the intrinsic chaperone activity to prevent aggregation of their connected polypeptides, irrespective of the connection types, and thus to aid productive protein folding. As a proof of concept, we here demonstrated that a model soluble protein with an inactive protease domain robustly exerted chaperone activity toward various proteins harboring a short protease-recognition tag of 7 residues in Escherichia coli. The chaperone activity of this protein was similar or even superior to that of representative E. coli chaperones in vivo. Furthermore, in vitro refolding experiments confirmed the in vivo results. Our findings revealed that a soluble protein exhibits the intrinsic chaperone activity, which is manifested, upon binding to aggregation-prone proteins. This study gives new insights into the ubiquitous chaperoning role of cellular macromolecules in protein-folding assistance and aggregation inhibition underlying the maintenance of protein solubility and proteostasis in vivo.

scholarly journals The Escherichia coli DjlA and CbpA Proteins Can Substitute for DnaJ in DnaK-Mediated Protein Disaggregation

2004 ◽  
Vol 186 (21) ◽  
pp. 7236-7242 ◽  
Author(s):  
Eyal Gur ◽  
Dvora Biran ◽  
Nelia Shechter ◽  
Pierre Genevaux ◽  
Costa Georgopoulos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Folding ◽  
Protein Targeting ◽  
Chaperone Activity ◽  
E Coli ◽  
Hsp40 Protein

ABSTRACT The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37°C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42°C.

scholarly journals Staphylococcus aureus Trigger Factor is Involved in Biofilm Formation and Cooperates with the Chaperone PpiB

2021 ◽  
Author(s):  
Rebecca A. Keogh ◽  
Rachel L. Zapf ◽  
Andrew Frey ◽  
Emily C. Marino ◽  
Gillian G. Null ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Protein Folding ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Double Mutant ◽  
Chaperone Activity ◽  
Trigger Factor ◽  
Chaperone Proteins

Peptidyl-prolyl cis/trans isomerases (PPIases) are enzymes that assist in protein folding around proline-peptide bonds, and often possess chaperone activity. Staphylococcus aureus encodes three PPIases; PrsA, PpiB and Trigger factor (TF). Previous work by our group demonstrated a role for both PrsA and PpiB in S. aureus, however, TF remains largely unstudied. Here, we identify a role for TF in S. aureus biofilm formation, and demonstrate cooperation between TF and the cytoplasmic PPIase PpiB. Mutation of the tig gene (encoding TF) leads to reduced biofilm development in vitro but no significant attenuation of virulence in a mouse model of infection. To investigate if TF possesses chaperone activity, we analyzed the ability of a tig mutant to survive acid and basic stress. While there was no significant decrease in a tig mutant, a ppiB/tig double mutant exhibited a significant decrease in cell viability after acid and base challenge. We then demonstrate that a ppiB/tig double mutant has exacerbated phenotypes in vitro and in vivo when compared to either single mutant. Finally, in vivo immunoprecipitation of epitope tagged PpiB reveals that PpiB interacts with four times the number of proteins when TF is absent from the cell, suggesting it may be compensating for the loss of TF. Interestingly, the only proteins found to interact with TF are TF itself, FnBPB and the chaperone protein ClpB. Collectively, these results support the first phenotype for S. aureus TF and demonstrate a greater network of cooperation between chaperone proteins in Staphylococcus aureus. IMPORTANCE S. aureus encodes a large number of virulence factors that aid the bacterium in survival and pathogenesis. These virulence factors have a wide variety of functions, however, they must all be properly secreted in order to be functional. Bacterial chaperone proteins often assist in secretion by trafficking proteins to secretion machinery or assisting in proper protein folding. Here, we report that the S. aureus chaperone Trigger factor (TF) contributes to biofilm formation and cooperates with the chaperone PpiB to regulate S. aureus virulence processes. These data highlight the first known role for TF in S. aureus, and suggest that S. aureus chaperone proteins may be involved in a greater regulatory network in the cell.

Protein folding following synthesis in vitro and in vivo: Association of newly synthesized protein with 50S subunit of E. coli ribosome

2008 ◽  
Vol 366 (2) ◽  
pp. 592-597 ◽  
Author(s):  
Arunima Basu ◽  
Dibyendu Samanta ◽  
Arpita Bhattacharya ◽  
Anindita Das ◽  
Debasis Das ◽  
...  
Keyword(s):  
Protein Folding ◽  
E Coli

A Physiologic Regulator of Collagen-Induced Platelet Aggregation: Inhibition by Clq

1981 ◽  
Vol 45 (02) ◽  
pp. 110-115 ◽  
Author(s):  
György Csákó ◽  
Eva A Suba
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Platelet Reactivity ◽  
High Specificity ◽  
Turbidimetric Method ◽  
Specific Manner ◽  
Aggregation Inhibition ◽  
Different Tissues

SummaryPlatelet aggregations were studied by a turbidimetric method in citrated human platelet-rich plasmas (PRP) in vitro. Human Clq inhibited the aggregations caused by collagens derived from different tissues and species. Clq was needed by weight in comparable quantities to collagen for neutralizing the aggregating effect. The dependence of the inhibitory reaction on the preincubation of platelets with Clq and the differences in the occurrence of aggregating substances in supernatants of PRP triggered with collagen in the presence or absence of Clq, confirmed that Clq exerts its effect by preventing fixation of collagen to platelets. In addition, the high specificity of the inhibitory action of Clq for collagen-induced platelet aggregation was demonstrated by results obtained for testing a variety of aggregating agents in combination with Clq and/or collagen.Since normal concentrations of Clq in the blood are in the range of inhibitory doses of Clq for collagen-induced platelet aggregations in vitro and upon activation of complement Clq is known to dissociate from Cl, it is proposed that Clq may participate in a highly specific manner in regulating platelet reactivity to collagen in vivo.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Low endotoxin E. coli strain-derived plasmids reduced rAAV vector-mediated immune responses both in vitro and in vivo.

2021 ◽  
Author(s):  
Qingyun Zheng ◽  
Tianyi Wang ◽  
Xiangying Zhu ◽  
Xiao Tian ◽  
Chen Zhong ◽  
...  
Keyword(s):  
Immune Responses ◽  
Coli Strain ◽  
Raav Vector ◽  
E Coli

scholarly journals Effect of Processing on Fish Protein Antigenicity and Allergenicity

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 969
Author(s):  
Xingyi Jiang ◽  
Qinchun Rao
Keyword(s):  
Fish Species ◽  
Protein Denaturation ◽  
Ex Vivo ◽  
Protein Solubility ◽  
Future Research ◽  
Food Protein ◽  
In Vivo Evaluation ◽  
Fish Allergy

Fish allergy is a life-long food allergy whose prevalence is affected by many demographic factors. Currently, there is no cure for fish allergy, which can only be managed by strict avoidance of fish in the diet. According to the WHO/IUIS Allergen Nomenclature Sub-Committee, 12 fish proteins are recognized as allergens. Different processing (thermal and non-thermal) techniques are applied to fish and fishery products to reduce microorganisms, extend shelf life, and alter organoleptic/nutritional properties. In this concise review, the development of a consistent terminology for studying food protein immunogenicity, antigenicity, and allergenicity is proposed. It also summarizes that food processing may lead to a decrease, no change, or even increase in fish antigenicity and allergenicity due to the change of protein solubility, protein denaturation, and the modification of linear or conformational epitopes. Recent studies investigated the effect of processing on fish antigenicity/allergenicity and were mainly conducted on commonly consumed fish species and major fish allergens using in vitro methods. Future research areas such as novel fish species/allergens and ex vivo/in vivo evaluation methods would convey a comprehensive view of the relationship between processing and fish allergy.

scholarly journals The Interactome of Cancer-Related Lysyl Oxidase and Lysyl Oxidase-Like Proteins

Cancers ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 71
Author(s):  
Sylvain D. Vallet ◽  
Coline Berthollier ◽  
Romain Salza ◽  
Laurent Muller ◽  
Sylvie Ricard-Blum
Keyword(s):  
Protein Folding ◽  
Cancer Progression ◽  
Cellular Response ◽  
Lysyl Oxidase ◽  
Chaperone Activity ◽  
Cross Linking ◽  
Binding Assays ◽  
Vessel Development ◽  
New Interactions

The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

Sign in / Sign up

Export Citation Format

Share Document