Inhibition of Insulin Amyloid Fibrillation by a Novel Amphipathic Heptapeptide: MECHANISTIC DETAILS STUDIED BY SPECTROSCOPY IN COMBINATION WITH MICROSCOPY.
The aggregation of insulin into amyloid fibers has been a limiting factor in the development of fast acting insulin analogues, creating a demand for excipients that limit aggregation. Despite the potential demand, inhibitors specifically targeting insulin have been few in number. Here we report a non-toxic and serum stable-designed heptapeptide, KR7 (KPWWPRR-NH2), that differs significantly from the primarily hydrophobic sequences that have been previously used to interfere with insulin amyloid fibrillation. Thioflavin T fluorescence assays, circular dichroism spectroscopy, and one-dimensional proton NMR experiments suggest KR7 primarily targets the fiber elongation step with little effect on the early oligomerization steps in the lag time period. From confocal fluorescence and atomic force microscopy experiments, the net result appears to be the arrest of aggregation in an early, non-fibrillar aggregation stage. This mechanism is noticeably different from previous peptide-based inhibitors, which have primarily shifted the lag time with little effect on later stages of aggregation. As insulin is an important model system for understanding protein aggregation, the new peptide may be an important tool for understanding peptide-based inhibition of amyloid formation.
From the Department of Biophysics and.
Bhisma N
BN
Ratha
From the Department of Biophysics and.
Anirban
A
Ghosh
Radiation Biology Branch, National Institutes of Health, Bethesda, Maryland 20814.
Jeffrey R
JR
Brender
Department of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Nilanjan
N
Gayen
From the Department of Biophysics and.
Humaira
H
Ilyas
TIFR Centre for Interdisciplinary Sciences (TCIS), Narsingi, Hyderabad 500075, India, and.
Chilukoti
C
Neeraja
Department of Chemistry, 93/1 APC Road, Bose Institute, Kolkata 700009, India.
Kali P
KP
Das
Department of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII (M), Kolkata 700054, India.
Atin K
AK
Mandal
From the Department of Biophysics and anirbanbhunia@gmail.com bhunia@jcbose.ac.in.
Anirban
A
Bhunia
D000595:Amino Acid Sequence; D000682:Amyloid / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000648:ultrastructure; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000378:metabolism*; D007328:Insulin / Q000737:chemistry / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000737:chemistry / Q000494:pharmacology*; D066329:Protein Aggregates / Q000187:drug effects*
D000682:Amyloid; D007004:Hypoglycemic Agents; D007328:Insulin; D009842:Oligopeptides; D066329:Protein Aggregates
amyloid; atomic force microscopy (AFM); fluorescence anisotropy; inhibitor; insulin
Proc Natl Acad Sci U S A. 2009 Nov 10;106(45):18990-5
19864624
Diabetes Metab. 2006 Jun;32(3):279-84
16799407
Colloids Surf B Biointerfaces. 2012 Apr 1;92:142-50
22178183
Biophys J. 2007 May 15;92(10):3448-58
17325005
Drug Discov Today. 2012 May;17(9-10):505-13
22210119
Philos Trans R Soc Lond B Biol Sci. 2001 Feb 28;356(1406):133-45
11260793
Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9196-201
12093917
J Phys Chem B. 2015 Dec 17;119(50):15395-406
26569375
Structure. 1995 Jun 15;3(6):615-22
8590022
Methods Mol Biol. 2008;494:177-86
18726574
PLoS One. 2015 Jul 14;10(7):e0132309
26172440
PLoS One. 2012;7(6):e36989
22675475
J Phys Chem B. 2014 Mar 27;118(12):3310-6
24611749
Biophys J. 2008 Apr 15;94(8):3241-6
18192359
Biochim Biophys Acta. 2010 Jul;1804(7):1405-12
20399286
J Diabetes Sci Technol. 2012 Mar 01;6(2):265-76
22538135
Biochemistry. 2015 Jan 27;54(3):666-76
25531836
Phys Rev Lett. 2007 Jul 13;99(2):028101
17678264
Curr Protein Pept Sci. 2009 Oct;10(5):509-28
19538143
J Biol Chem. 2016 Jan 22;291(4):2018-2032
26601940
Biochem Biophys Res Commun. 2011 Dec 2;415(4):675-9
22079288
PLoS One. 2013 Aug 29;8(8):e72318
24009675
PLoS One. 2011;6(5):e20244
21625461
PLoS One. 2015 Aug 21;10(8):e0135976
26295714
J Biol Chem. 2014 Dec 26;289(52):35781-94
25378395
Nat Neurosci. 2012 Jan 29;15(3):349-57
22286176
J Phys Chem B. 2014 Feb 6;118(5):1198-206
24428561
Biochemistry. 1990 Nov 20;29(46):10545-55
2271664
Int J Biol Macromol. 2015 Sep;80:610-4
26193678
Biochemistry. 1998 Aug 18;37(33):11516-23
9708987
Am J Med. 2014 May;127(5):450-4
24462809
Biochem Biophys Res Commun. 2014 May 16;447(4):590-5
24747079
J Am Chem Soc. 2001 Jun 27;123(25):6108-17
11414845
Nature. 1989 Apr 13;338(6216):594-6
2648161
J Mol Biol. 2016 Feb 22;428(4):720-725
26410586
J Phys Chem B. 2015 Apr 23;119(16):5145-55
25815792
J Mol Biol. 1998 Nov 27;284(2):477-88
9813131
Chem Commun (Camb). 2013 Apr 25;49(32):3339-41
23505632
Biochemistry. 2003 Oct 7;42(39):11404-16
14516191
J Mol Graph Model. 2004 May;22(5):377-95
15099834
Chemphyschem. 2006 May 12;7(5):1046-9
16596700
Protein Sci. 2006 May;15(5):1133-41
16597825
Biochemistry. 1991 Jun 4;30(22):5505-15
2036420
Biochemistry. 1997 May 13;36(19):5837-45
9153424
Biomacromolecules. 2016 Jun 13;17(6):1998-2009
27116396
Curr Opin Struct Biol. 2007 Feb;17(1):48-57
17251001
Eur Biophys J. 2015 May;44(4):249-61
25794693
J Biol Chem. 2004 Apr 9;279(15):14999-5013
14736893
J Biol Chem. 2004 May 14;279(20):21449-60
14988398
Angew Chem Int Ed Engl. 1999 Jun 14;38(12):1784-1788
29711196
J Phys Chem Lett. 2014 Jun 5;5(11):1864-70
26273866
Prion. 2010 Jan-Mar;4(1):20-5
20305394
J Chem Theory Comput. 2012 Oct 9;8(10):3628-36
26593008
Cell. 2012 Mar 16;148(6):1188-203
22424229
Sci Rep. 2015 Nov 30;5:17599
26616401
Sci Rep. 2014 Jul 10;4:5648
25008537
Biophys J. 2009 Nov 4;97(9):2585-94
19883602
J Mol Biol. 2014 Jan 23;426(2):362-76
24513105
Langmuir. 2015 Apr 14;31(14):4246-54
25803421
J Am Chem Soc. 2003 Feb 19;125(7):1731-7
12580598
Curr Opin Struct Biol. 1998 Apr;8(2):189-94
9631292
Indian J Endocrinol Metab. 2015 Jan-Feb;19(1):174-7
25593849