Tài liệu Báo cáo khoa học: Structural features of proinsulin C-peptide oligomeric and amyloid states

Structural features of proinsulin C-peptide oligomeric and

amyloid states

Jesper Lind1,*, Emma Lindahl2,*, Alex Pera´lvarez-Marı´n1,*, Anna Holmlund2

, Hans Jo¨rnvall2 and

Lena Ma¨ler1

1 Department of Biochemistry and Biophysics, Center for Biomembrane Research, The Arrhenius laboratory, Stockholm University, Sweden

2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Keywords

C-peptide; diabetes; oligomer; spectroscopy;

structure

Correspondence

L. Ma¨ler, Department of Biochemistry and

Biophysics, Center for Biomembrane

Research, The Arrhenius laboratory,

Stockholm University, SE-106 91

Stockholm, Sweden

Fax: +46 8 155597

Tel: +46 8 162448

E-mail: [email protected]

Present Address

Department of Molecular and Cell Biology,

Harvard University, Cambridge MA 02138,

USA

*These authors contributed equally to this

work

(Received 27 May 2010, revised 8 July

2010, accepted 13 July 2010)

doi:10.1111/j.1742-4658.2010.07777.x

The formation and structure of proinsulin C-peptide oligomers has been

investigated by PAGE, NMR spectroscopy and dynamic light scattering.

The results obtained show that C-peptide forms oligomers of different

sizes, and that their formation and size distribution is altered by salt and

divalent metal ions, which indicates that the aggregation process is medi￾ated by electrostatic interactions. It is further demonstrated that the size

distribution of the C-peptide oligomers, in agreement with previous studies,

is altered by insulin, which supports a physiologically relevant interaction

between these two peptides. A small fraction of oligomers has previously

been suggested to be in equilibrium with a dominant fraction of soluble

monomers, and this pattern also is observed in the present study. The addi￾tion of modest amounts of sodium dodecyl sulphate at low pH increases

the relative amount of oligomers, and this effect was used to investigate the

details of both oligomer formation and structure by a combination of bio￾physical techniques. The structural properties of the SDS-induced oligo￾mers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR

spectroscopy, demonstrate that soluble aggregates are predominantly in

b-sheet conformation, and that the oligomerization process shows charac￾teristic features of amyloid formation. The formation of large, insoluble,

b-sheet amyloid-like structures will alter the equilibrium between mono￾meric C-peptide and oligomers. This leads to the conclusion that the oligo￾merization of C-peptide may be relevant also at low concentrations.

Structured digital abstract

l MINT-7975828: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind

(MI:0407) by fluorescence technology (MI:0051)

l MINT-7975757: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind

(MI:0407) by nuclear magnetic resonance (MI:0077)

l MINT-7975840: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind

(MI:0407) by circular dichroism (MI:0016)

l MINT-7975708: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind

(MI:0407) by blue native page (MI:0276)

l MINT-7975816: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind

(MI:0407) by dynamic light scattering (MI:0038)

Abbreviations

ATR, attenuated total reflectance; b-C-peptide, biotinylated human C-peptide; CMC, critical micelle concentration; DLS, dynamic light

scattering; ThT, thioflavin T.

FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS 3759