Proline-rich domain of human ALIX contains multiple TSG101-UEV interaction sites and forms phosphorylation-mediated reversible amyloids.
Amino Acid Motifs
Amyloid
/ chemistry
Binding Sites
Calcium-Binding Proteins
/ chemistry
Cell Cycle Proteins
/ chemistry
DNA-Binding Proteins
/ chemistry
Endosomal Sorting Complexes Required for Transport
/ chemistry
Humans
Phosphorylation
Proline
/ genetics
Protein Binding
Protein Domains
Transcription Factors
/ chemistry
NMR
amyloids
intrinsically disordered protein
posttranslational modifications
signal transduction
Journal
Proceedings of the National Academy of Sciences of the United States of America
ISSN: 1091-6490
Titre abrégé: Proc Natl Acad Sci U S A
Pays: United States
ID NLM: 7505876
Informations de publication
Date de publication:
29 09 2020
29 09 2020
Historique:
pubmed:
13
9
2020
medline:
19
12
2020
entrez:
12
9
2020
Statut:
ppublish
Résumé
Proline-rich domains (PRDs) are among the most prevalent signaling modules of eukaryotes but often unexplored by biophysical techniques as their heterologous recombinant expression poses significant difficulties. Using a "divide-and-conquer" approach, we present a detailed investigation of a PRD (166 residues; ∼30% prolines) belonging to a human protein ALIX, a versatile adaptor protein involved in essential cellular processes including ESCRT-mediated membrane remodeling, cell adhesion, and apoptosis. In solution, the N-terminal fragment of ALIX-PRD is dynamically disordered. It contains three tandem sequentially similar proline-rich motifs that compete for a single binding site on its signaling partner, TSG101-UEV, as evidenced by heteronuclear NMR spectroscopy. Global fitting of relaxation dispersion data, measured as a function of TSG101-UEV concentration, allowed precise quantitation of these interactions. In contrast to the soluble N-terminal portion, the C-terminal tyrosine-rich fragment of ALIX-PRD forms amyloid fibrils and viscous gels validated using dye-binding assays with amyloid-specific probes, congo red and thioflavin T (ThT), and visualized by transmission electron microscopy. Remarkably, fibrils dissolve at low temperatures (2 to 6 °C) or upon hyperphosphorylation with Src kinase. Aggregation kinetics monitored by ThT fluorescence shows that charge repulsion dictates phosphorylation-mediated fibril dissolution and that the hydrophobic effect drives fibril formation. These data illuminate the mechanistic interplay between interactions of ALIX-PRD with TSG101-UEV and polymerization of ALIX-PRD and its central role in regulating ALIX function. This study also demonstrates the broad functional repertoires of PRDs and uncovers the impact of posttranslational modifications in the modulation of reversible amyloids.
Identifiants
pubmed: 32917811
pii: 2010635117
doi: 10.1073/pnas.2010635117
pmc: PMC7533887
doi:
Substances chimiques
Amyloid
0
Calcium-Binding Proteins
0
Cell Cycle Proteins
0
DNA-Binding Proteins
0
Endosomal Sorting Complexes Required for Transport
0
PDCD6IP protein, human
0
Transcription Factors
0
Tsg101 protein
0
Proline
9DLQ4CIU6V
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, N.I.H., Intramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
24274-24284Subventions
Organisme : NIAID NIH HHS
ID : P30 AI036214
Pays : United States
Organisme : NIAID NIH HHS
ID : P50 AI150464
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM031749
Pays : United States
Organisme : NIAID NIH HHS
ID : U54 AI150472
Pays : United States
Déclaration de conflit d'intérêts
The authors declare no competing interest.
Références
Nat Rev Mol Cell Biol. 2020 Jan;21(1):25-42
pubmed: 31705132
Proc Natl Acad Sci U S A. 2015 Mar 17;112(11):3374-9
pubmed: 25713345
J Biomol NMR. 2010 Jan;46(1):23-31
pubmed: 19731047
Cell. 2003 Sep 19;114(6):689-99
pubmed: 14505569
Neuron. 2015 May 6;86(3):632-45
pubmed: 25950632
Protein Sci. 1997 Nov;6(11):2359-64
pubmed: 9385638
Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12456-12461
pubmed: 27791180
Angew Chem Int Ed Engl. 1998 Nov 2;37(20):2754-2794
pubmed: 29711117
Nat Rev Mol Cell Biol. 2015 Jan;16(1):18-29
pubmed: 25531225
Biomolecules. 2017 Sep 22;7(4):
pubmed: 28937655
Nature. 2012 Mar 07;483(7389):336-40
pubmed: 22398450
Nat Commun. 2018 Oct 30;9(1):4522
pubmed: 30375386
PLoS One. 2013;8(1):e53785
pubmed: 23372670
Nat Struct Mol Biol. 2008 Jan;15(1):43-9
pubmed: 18066081
Science. 2013 Jan 4;339(6115):85-8
pubmed: 23239624
Annu Rev Phys Chem. 2018 Apr 20;69:273-298
pubmed: 29490200
J Cell Biol. 2016 Feb 29;212(5):499-513
pubmed: 26929449
Protein Sci. 2018 Jan;27(1):146-158
pubmed: 28884933
Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7687-91
pubmed: 18511562
Nano Rev. 2011;2:
pubmed: 22110868
Structure. 2010 Nov 10;18(11):1536-47
pubmed: 21070952
Proc Natl Acad Sci U S A. 2017 Nov 14;114(46):E9855-E9862
pubmed: 29087351
J Mol Biol. 2013 Jun 26;425(12):2100-32
pubmed: 23507311
Traffic. 2011 Oct;12(10):1282-90
pubmed: 21518163
Nat Struct Biol. 2002 Nov;9(11):812-7
pubmed: 12379843
Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8324-7
pubmed: 10890881
Cell. 2003 Sep 19;114(6):701-13
pubmed: 14505570
Oncogene. 2004 Oct 18;23(48):7918-27
pubmed: 15489910
J Biol Chem. 2016 Jul 29;291(31):16307-17
pubmed: 27268056
Biochem J. 1994 Jan 15;297 ( Pt 2):249-60
pubmed: 8297327
J Biol Chem. 2005 Feb 4;280(5):3414-25
pubmed: 15557335
Cell. 2007 Mar 9;128(5):841-52
pubmed: 17350572
Nat Protoc. 2007;2(6):1528-35
pubmed: 17571060
Intrinsically Disord Proteins. 2013 Apr 1;1(1):e24360
pubmed: 28516008
Science. 2000 Mar 24;287(5461):2204-15
pubmed: 10731134
J Mol Biol. 1991 Mar 20;218(2):397-412
pubmed: 2010917
J Am Chem Soc. 2020 Feb 19;142(7):3401-3411
pubmed: 31970984
Sci STKE. 2003 Apr 22;2003(179):RE8
pubmed: 12709533
Sci Rep. 2019 Mar 26;9(1):5137
pubmed: 30914747
Acc Chem Res. 2013 Apr 16;46(4):1039-49
pubmed: 23148796
PeerJ. 2013 May 07;1:e73
pubmed: 23678397
Biochem J. 2010 Oct 1;431(1):93-102
pubmed: 20670214
Sci Rep. 2019 Mar 1;9(1):3265
pubmed: 30824759
J Magn Reson. 2014 Apr;241:3-17
pubmed: 24656076
J Biomol NMR. 2011 Jun;50(2):157-65
pubmed: 21604143
Chem Rev. 2019 Dec 11;119(23):11819-11856
pubmed: 31675223
J Am Chem Soc. 2013 Oct 30;135(43):16133-47
pubmed: 24066695
FEBS J. 2015 Jul;282(14):2612-26
pubmed: 25823410
Biochem Biophys Res Commun. 2004 Apr 30;317(2):401-5
pubmed: 15063772
Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):E878-87
pubmed: 23431150
Methods Enzymol. 1999;309:285-305
pubmed: 10507031
Nat Protoc. 2009;4(10):1513-21
pubmed: 19798084
Biochemistry. 2017 Sep 5;56(35):4646-4655
pubmed: 28776372
Curr Opin Struct Biol. 2005 Oct;15(5):563-70
pubmed: 16140525
J Cell Biol. 2020 Mar 2;219(3):
pubmed: 32049272
Biomolecules. 2017 Sep 22;7(4):
pubmed: 28937634
FASEB J. 2000 Feb;14(2):231-41
pubmed: 10657980
J Biomol NMR. 2011 Feb;49(2):139-49
pubmed: 21234644
Science. 2013 Jan 4;339(6115):82-5
pubmed: 23239623
Nat Rev Mol Cell Biol. 2017 Jan;18(1):5-17
pubmed: 27703243
J Virol. 2011 Sep;85(17):9222-6
pubmed: 21715492
J Biol Chem. 2002 Aug 9;277(32):29108-15
pubmed: 12034747
J Magn Reson. 2003 Jan;160(1):65-73
pubmed: 12565051
Chem Rev. 2014 Jul 9;114(13):6589-631
pubmed: 24773235
Nucleic Acids Res. 2003 Jul 1;31(13):3701-8
pubmed: 12824398
Angew Chem Int Ed Engl. 2018 Mar 1;57(10):2687-2691
pubmed: 29345807
Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10541-6
pubmed: 18641129
Anal Biochem. 1989 Nov 1;182(2):319-26
pubmed: 2610349
Biochem J. 2010 Dec 15;432(3):525-34
pubmed: 20929444
Biochemistry. 2012 Mar 20;51(11):2224-31
pubmed: 22360139