Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water

Futami, Junichiro; Fujiyama, Haruna; Kinoshita, Rie; Nonomura, Hidenori; Honjo, Tadashi; Tada, Hayato; Matsushita, Hirokazu; Abe, Yoshito; Kakimi, Kazuhiro

doi:10.1371/journal.pone.0113295

Cited by 10 publications

(13 citation statements)

References 37 publications

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“…This discovery has now been extensively confirmed, and become a powerful tool for us and other groups to biophysically characterize Binsoluble^protein (Delak et al 2009;Aguado-Llera et al 2010;Futami et al 2014). Initially, by CD and NMR characterization, we found that Binsoluble^proteins could be divided into three groups according to their conformations in unsalted water (Li et al 2006;Song 2009Song , 2013: group 1, which has no stable secondary and tertiary structures (I of Fig.…”

Section: Aggregation and Self-assembly Into Liquid Droplets And Fibrimentioning

confidence: 86%

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

Song

2017

Biophys Rev

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In his Nobel Lecture, Anfinsen stated Bthe native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment.^As aqueous solutions and membrane systems co-exist in cells, proteins are classified into membrane and non-membrane proteins, but whether one can transform one into the other remains unknown. Intriguingly, many well-folded non-membrane proteins are converted into Binsoluble^and toxic forms by aging-or diseaseassociated factors, but the underlying mechanisms remain elusive. In 2005, we discovered a previously unknown regime of proteins seemingly inconsistent with the classic BSalting-in^dogma: Binsoluble^proteins including the integral membrane fragments could be solubilized in the ion-minimized water. We have thus successfully studied Binsoluble^forms of ALScausing P56S-MSP, L126Z-SOD1, nascent SOD1 and C71G-Profilin1, as well as E. coli S1 fragments. The results revealed that these Binsoluble^forms are either unfolded or co-exist with their unfolded states. Most unexpectedly, these unfolded states acquire a novel capacity of interacting with membranes energetically driven by the formation of helices/loops over amphiphilic/ hydrophobic regions which universally exit in proteins but are normally locked away in their folded native states. Our studies suggest that most, if not all, proteins contain segments which have the dual ability to fold into distinctive structures in aqueous and membrane environments. The abnormal membrane interaction might initiate disease and/or aging processes; and its further coupling with protein aggregation could result in radical proteotoxicity by forming inclusions composed of damaged membranous organelles and protein aggregates. Therefore, environment-transformable sequence-structure relationship may represent a general mechanism for proteotoxicity.

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Section: Aggregation and Self-assembly Into Liquid Droplets And Fibrimentioning

confidence: 86%

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

Song

2017

Biophys Rev

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“…The disulphide-free, flexible conformation of intracellular proteins allows multiple protein interactions in living cells under highly crowded conditions. This structural property is suggested to contribute to the enhanced solubility of intracellular proteins 25 . Conversely, extracellular proteins need to be robust to circulate in the extracellular space.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate

Futami

Miyamoto

Hagimoto

et al. 2017

Sci Rep

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Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

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“…The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. It has been shown that fully denatured mammalian proteins show unusually high solubility in nucleic acid-free pure water [39]. Since the recombinant proteins that are produced as pharmaceuticals are mainly mammalian proteins, the presence of nucleic acids in their preparation is critical for their refolding because the nucleic acids actively participate in the protein aggregation process via direct electrostatic interactions with partially folded or unfolded proteins.…”

Section: Introductionmentioning

confidence: 99%

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

2020

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Background: Inclusion bodies (IBs) are protein aggregates in recombinant bacterial cells containing mainly the target recombinant protein. Although it has been shown that IBs contain functional proteins along with protein aggregates, their direct application as pharmaceuticals is hindered by their heterogeneity and hazardous contaminants with bacterial origin. Therefore, together with the production of soluble species, IBs remain the main source for manufacture of recombinant proteins with medical application. The quality and composition of the IBs affect the refolding yield and further purification of the recombinant protein. The knowledge whether nucleic acids are genuine components or concomitant impurities of the IBs is a prerequisite for the understanding of the IBs formation and for development of optimized protocols for recombinant protein refolding and purification. IBs isolated from Escherichia coli overexpressing human interferon-gamma (hIFNγ), a protein with therapeutic application, were used as a model. Results: IBs were isolated from E. coli LE392 cells transformed with a hIFNγ expressing plasmid under standard conditions and further purified by centrifugation on a sucrose cushion, followed by several steps of sonication and washings with non-denaturing concentrations of urea. The efficiency of the purification was estimated by SDS-PAGE gel electrophoresis and parallel microbiological testing for the presence of residual intact bacteria. Phenol/chloroform extraction showed that the highly purified IBs contain both DNA and RNA. The latter were studied by UV spectroscopy and agarose gel electrophoresis combined with enzymatic treatment and hybridization. DNA was observed as a diffuse fraction mainly in the range of 250 to 1000 bp. RNA isolated by TRIzol ® also demonstrated a substantial molecular heterogeneity. Hybridization with 32 P-labelled oligonucleotides showed that the IBs contain rRNA and are enriched of hIFNγ mRNA. Conclusions: The results presented in this study indicate that the nucleic acids might be intrinsic components rather than co-precipitated impurities in the IBs. We assume that the nucleic acids are active participants in the aggregation of recombinant proteins and formation of the IBs that originate from the transcription and translation machinery of the microbial cell factory. Further studies are needed to ascertain this notion.

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Denatured Mammalian Protein Mixtures Exhibit Unusually High Solubility in Nucleic Acid-Free Pure Water

Cited by 10 publications

References 37 publications

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

Environment-transformable sequence–structure relationship: a general mechanism for proteotoxicity

Evaluation of irreversible protein thermal inactivation caused by breakage of disulphide bonds using methanethiosulphonate

Nucleic acids in inclusion bodies obtained from E. coli cells expressing human interferon-gamma

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