Host expression system modulates recombinant Hsp70 activity through post-translational modifications.
Escherichia coli
Pichia pastoris
DnaK
Heat-shock protein
post-translational modifications
Journal
The FEBS journal
ISSN: 1742-4658
Titre abrégé: FEBS J
Pays: England
ID NLM: 101229646
Informations de publication
Date de publication:
06 Mar 2020
06 Mar 2020
Historique:
received:
16
07
2019
revised:
22
01
2020
accepted:
03
03
2020
entrez:
8
3
2020
pubmed:
8
3
2020
medline:
8
3
2020
Statut:
aheadofprint
Résumé
The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.
Identifiants
pubmed: 32144867
doi: 10.1111/febs.15279
pmc: PMC7483562
mid: NIHMS1596528
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NCI NIH HHS
ID : R01 CA176326
Pays : United States
Organisme : NCI NIH HHS
ID : R15 CA208773
Pays : United States
Informations de copyright
This article is protected by copyright. All rights reserved.
Références
Sci Rep. 2019 Nov 7;9(1):16260
pubmed: 31700027
J Biol Chem. 2008 Sep 26;283(39):26477-83
pubmed: 18658155
Proc Natl Acad Sci U S A. 2011 Jan 11;108(2):733-8
pubmed: 21187371
Annu Rev Genet. 1988;22:631-77
pubmed: 2853609
Molecules. 2018 Nov 01;23(11):
pubmed: 30388847
BMC Genomics. 2017 Apr 17;18(1):301
pubmed: 28412930
Annu Rev Biochem. 2013;82:323-55
pubmed: 23746257
Eur J Immunol. 2006 Sep;36(9):2304-14
pubmed: 16909434
Cell Stress Chaperones. 2016 Jul;21(4):609-16
pubmed: 27075190
Trends Biochem Sci. 2017 May;42(5):355-368
pubmed: 28314505
Biochem Biophys Res Commun. 2009 Sep 18;387(2):229-33
pubmed: 19555663
J Biol Chem. 2003 Jan 3;278(1):174-9
pubmed: 12403778
Trends Biochem Sci. 2017 Dec;42(12):932-935
pubmed: 29102083
Immunology. 2007 Aug;121(4):462-72
pubmed: 17346283
Nat Rev Immunol. 2002 Aug;2(8):569-79
pubmed: 12154376
J Biol Chem. 2005 Jun 17;280(24):23349-55
pubmed: 15826944
Anal Chem. 2002 Oct 15;74(20):5383-92
pubmed: 12403597
Biochem Biophys Res Commun. 2004 Nov 12;324(2):511-7
pubmed: 15474457
Cytokine. 2016 Sep;85:123-9
pubmed: 27337694
Cell Mol Life Sci. 2005 Mar;62(6):670-84
pubmed: 15770419
Front Immunol. 2016 Aug 09;7:303
pubmed: 27555846
Philos Trans R Soc Lond B Biol Sci. 2018 Jun 19;373(1749):
pubmed: 29735737
Sci Rep. 2011 Sep 13;1:
pubmed: 22034591
J Biol Chem. 2005 Apr 8;280(14):13593-9
pubmed: 15695803
Curr Opin Microbiol. 2015 Apr;24:47-52
pubmed: 25625314
Biochemistry. 1999 Mar 30;38(13):4165-76
pubmed: 10194333
Methods. 2007 Nov;43(3):229-37
pubmed: 17920520
Nat Rev Mol Cell Biol. 2019 Nov;20(11):665-680
pubmed: 31253954
Cell. 1980 Jul;20(3):679-89
pubmed: 6774811
Cell Rep. 2018 Nov 27;25(9):2605-2616.e7
pubmed: 30485823
Front Immunol. 2012 May 04;3:95
pubmed: 22566973
J Cell Sci. 2001 Apr;114(Pt 7):1273-82
pubmed: 11256994
Cell Rep. 2015 May 5;11(5):759-69
pubmed: 25921532
Anal Chem. 2003 Sep 1;75(17):4646-58
pubmed: 14632076
J Immunol. 2006 Dec 1;177(11):7849-57
pubmed: 17114456
Life Sci. 2019 Mar 15;221:187-195
pubmed: 30716336
Nat Med. 2000 Apr;6(4):435-42
pubmed: 10742151
J Cell Physiol. 1989 Feb;138(2):257-66
pubmed: 2918030
Exp Mol Med. 2019 Sep 26;51(9):1-14
pubmed: 31558706
Appl Microbiol Biotechnol. 2011 Feb;89(4):939-48
pubmed: 21125266
Immunity. 2002 Sep;17(3):353-62
pubmed: 12354387
Nat Commun. 2018 Aug 28;9(1):3482
pubmed: 30154416
Biochim Biophys Acta. 2013 May;1829(5):443-54
pubmed: 23459247
Trends Biochem Sci. 2018 Apr;43(4):285-300
pubmed: 29501325
Proteomics. 2008 Oct;8(19):4083-99
pubmed: 18780348
Annu Rev Biochem. 1986;55:1151-91
pubmed: 2427013
Biol Chem. 2003 Feb;384(2):267-79
pubmed: 12675520
Front Immunol. 2012 Apr 09;3:75
pubmed: 22566956