| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2004 as doi:10.1096/fj.03-1072fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;
* Departamento de Ultraestrutura e Biologia Celular, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil;
Department of Medical Biochemistry and Biophysics, Ume
University, Ume, Sweden; and
Department of Chemistry, University of Cambridge, Cambridge, UK
2Correspondence: Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21944-590, Brazil. E-mail: ferreira{at}bioqmed.ufrj.br
SPECIFIC AIMS
We have used hydrostatic pressure as a tool to populate partially unfolded intermediate states of human lysozyme and its disease-associated variants. We show that combined use of high pressure and temperature leads to the formation of amyloidogenic states of WT human lysozyme and its disease-associated variants (Ile56Thr or Asp67His) at physiological pH. Lysozyme remains soluble during application of pressure but amyloid fibrils, protofibrils, or globular aggregates are readily formed upon decompression.
PRINCIPAL FINDINGS
1. Lysozyme aggregation induced by compression/decompression
The effects of pressure and high temperature on wild-type (WT) and variant forms of lysozyme were investigated using right-angle light scattering (LS). Samples were compressed to 3.5 kbar at 23°C, then temperature was increased to 57°C over 
1 h. The heat/pressure treatment caused little or no changes in the LS of WT or mutant lysozymes (Fig. 1
A–C, left panels), indicating that aggregates were not formed in significant amounts by any of the proteins. After reaching 57°C, samples were equilibrated for additional periods (ranging from 30 min to 3 h; data not shown), with no changes in LS (Fig. 1A-C
, right panels). The pressure was then quickly released (within 1 min) and LS was measured after a pressure of 1 atm was re-established. Marked increases in LS were observed for WT and mutant lysozyme samples over periods of minutes to hours (Fig. 1A-C
, right panels), indicating protein aggregation. The increase in LS was not reversed by cooling the samples back to 23°C (Fig. 1A-C
, right panels). Moreover, further increases in LS were observed for the I56T and D67H lysozyme variants, suggesting that aggregation continued even at lower temperatures. SDS-PAGE analysis of WT and variant lysozyme samples after the pressure cycle revealed no proteolysis or degradation induced by the pressure/heat treatment (not shown).
|
2. Lysozyme amyloid fibrils and/or protofibrils are formed upon decompression
Figure 2
A shows that WT lysozyme exhibited a strong thioflavin T fluorescence peak 12 h after decompression, whereas a control nonpressurized sample kept under otherwise identical conditions (including the temperature cycle) exhibited virtually no fluorescence. This enhanced fluorescence suggests that aggregates of WT lysozyme generated in the pressurized samples possess tinctorial properties characteristic of amyloid structure. Decompressed D67H lysozyme exhibited lower thioflavin T emission (63% relative to the thioflavin T emission of WT lysozyme) whereas decompressed I56T lysozyme exhibited only 13% of the thioflavin T fluorescence relative to WT protein (Fig. 2A
).
|
Direct evidence that the compression/decompression cycle induced the formation of amyloid aggregates was obtained by transmission electron microscopy (EM). Figure 2B-D
shows EM analysis of samples of WT, I56T, or D67H lysozyme that had been subjected to the pressure treatment described above and negatively stained 12 h after decompression. Protein aggregates were observed in all compressed/decompressed samples, albeit with different morphologies. For WT lysozyme, isolated fibrils and bundles of fibrils, all with the morphology characteristic of amyloid structures, were found in the samples examined by EM (Fig. 2B
). For D67H lysozyme, a large number of amyloid fibrils of various lengths, as well as smaller curvilinear structures resembling protofibrils, were seen in all EM samples (Fig. 2C
). For I56T lysozyme, however, samples stained 12 h after decompression did not exhibit distinct amyloid fibrils, but abundant clusters of roughly globular aggregates (Fig. 2D
). Control samples kept under exactly the same experimental conditions (including the temperature cycle) but that did not undergo pressurization were also prepared. Careful examination of these samples by LS, thioflavin T fluorescence and EM failed to reveal fibrillar or other types of aggregates (not shown).
3. Pressure effects on intrinsic and ANS fluorescence
The effects of pressure and temperature on the intrinsic fluorescence and ANS binding of WT and variant lysozymes were investigated. In their native states, all three proteins exhibited intrinsic fluorescence emission maxima at 330 nm. Compression to 3.5 kbar at 23°C led to small (2–4 nm) red shifts in fluorescence emission of the three proteins. Heating to 57°C had little or no additional effect on the emission of WT or variant lysozymes.
ANS is weakly fluorescent in aqueous buffer but exhibits a marked increase in emission upon binding to hydrophobic domains in proteins. It has been shown that the partially unfolded amyloidogenic state of lysozyme formed at the midpoint of its thermal denaturation transition binds ANS. Fluorescence of ANS in the presence of WT and variant lysozymes was unaffected by compression to 3.5 kbar at 57°C. However, ANS fluorescence increased significantly in decompressed lysozyme samples.
CONCLUSIONS
Amyloid diseases are of great clinical importance, and amyloid oligomers, protofibrils, and/or fibrils have been implicated as primary causes of cell death and tissue degeneration in several such diseases. Understanding the formation of amyloidogenic conformations of proteins and the mechanisms of aggregation may lead to novel therapeutic approaches to prevent or reverse the formation of amyloid structures in disease states. Developing an approach to promote the formation of the aggregation-prone conformation of a protein may provide insight into the process of amyloidogenesis. Here, we have shown that such an approach may be achieved through the combined use of high pressure and temperature in studies of human lysozyme and its disease-associated variants.
WT lysozyme and its variants remain soluble at high pressure, and aggregation takes place only upon decompression (Fig. 3
). Conceivably, pressure perturbs hydrophobic interactions that are important for the stability of lysozyme aggregates and prevents any amyloidogenic intermediates formed in solution from undergoing self-association.
|
Pressure-induced unfolding of lysozyme has been reported using only extreme conditions of pH and temperature, suggesting that application of pressure under our experimental conditions causes only subtle conformational changes in lysozyme. Nevertheless, the changes appear to be significant enough that decompression leads to aggregation and formation of amyloid fibrils, protofibrils, or globular aggregates (Fig. 3)
. It may well be that under our conditions involving elevated temperature, a partially unfolded intermediate state of lysozyme is formed under pressure and stimulates nucleation of the aggregation. Upon decompression, such nuclei could then lead to further aggregation, resulting in the formation of aggregates. In the absence of pressure treatment, addition of preaggregated lysozyme fibrils (seeds) to solutions of lysozyme at neutral pH does not lead to formation of amyloid fibrils (L. A. Morozova-Roche, unpublished observations). Prolonged incubation of lysozyme at acidic pH leads to partial proteolysis, which creates another complication in interpreting the mechanism of amyloid fibril formation from lysozyme. These observations underline the importance of pressure-induced conformational changes in promoting acquisition of the amyloidogenic state of lysozyme at physiological pH.
Disease-associated lysozyme variants I56T and D67H exhibit an initial tendency to form globular aggregates or protofibrils, respectively, upon decompression (Fig. 2)
whereas WT lysozyme, which is not associated with disease, readily forms mature amyloid fibrils. These differences may be related to differences in structural stability associated with the point mutations in the lysozyme variants, making them more prone to forming early oligomeric toxic species.
In conclusion, we show that pressure provides a convenient means to produce amyloid fibrils, protofibrils, and globular aggregates from WT lysozyme and its disease-associated variants. These results suggest that this method may be applicable to studies of the aggregation of other amyloidogenic proteins, as there appear to be common features in the aggregation behavior of different proteins. Targeting amyloid aggregation may bring about new therapeutic strategies for important human diseases. Therefore, the use of pressure to generate amyloidogenic states of proteins may represent an important contribution to the development of a detailed understanding of the initial stages of aggregation, and hence assist in drug design approaches that target inhibitors of amyloidogenesis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1072fje
1 These authors contributed equally to this work. 
This article has been cited by other articles:
|
|
 |
|
  E. Herczenik and M. F. B. G. Gebbink Molecular and cellular aspects of protein misfolding and disease FASEB J, July 1, 2008; 22(7): 2115 - 2133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Calamai, F. Chiti, and C. M. Dobson Amyloid Fibril Formation Can Proceed from Different Conformations of a Partially Unfolded Protein Biophys. J., December 1, 2005; 89(6): 4201 - 4210. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
