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This Article Full Text (PDF) All Versions of this Article: 20/3/524 05-5126fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gerard, M. Articles by Engelborghs, Y. Search for Related Content PubMed PubMed Citation Articles by Gerard, M. Articles by Engelborghs, Y.

The aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown by fluorescence correlation spectroscopy

Melanie Gerard^*, Zeger Debyser, Linda Desender, Philipp J. Kahle, Johan Baert, Veerle Baekelandt^||,1 and Yves Engelborghs^*

^* Laboratory of Biomolecular Dynamics, K.U. Leuven, Leuven, Belgium;
Laboratory for Molecular Virology and Gene Therapy, K.U. Leuven, Leuven, Belgium and Interdisciplinary Research Center, K.U. Leuven Campus Kortrijk, Kortrijk, Belgium;
Laboratory of Alzheimer’s and Parkinson’s Disease Research, Department of Biochemistry, Ludwig Maximilians University of Munich, Munich, Germany;
Laboratory for Histology, Interdisciplinary Research Center, K.U. Leuven Campus Kortrijk, Kortrijk, Belgium; and
^|| Laboratory for Neurobiology and Gene Therapy, K.U. Leuven, Leuven, Belgium

¹Correspondence: E-mail: veerle.baekelandt{at}med.kuleuven.be

SPECIFIC AIMS

Alpha-synuclein (-SYN) is a natively unfolded protein of 14 kDa that plays a crucial role in Parkinson’s disease (PD). In disease conditions, -SYN aggregates into amyloid fibrils. In this study we show that this aggregation process is greatly accelerated in vitro, in the presence of FK506 binding proteins (FKBPs). FKBPs are members of the immunophilins, enzymes that bind to immunosuppressant drugs and have a peptidyl-prolyl isomerase (PPIase) activity. This enzymatic activity is believed to help the cis-to-trans conformational change of proline, often the rate-limiting step during the protein folding.

We studied -SYN aggregation with fluorescence correlation spectroscopy (FCS) to investigate the early events in this process. FCS is a sensitive technique that measures the diffusion of fluorescently labeled molecules in vitro or in the cell. The concentration and size of fluorescent molecules can be calculated from the autocorrelation function.

PRINCIPAL FINDINGS

1. Aggregation of purified recombinant -SYN can be measured by FCS
We have demonstrated that the aggregation of recombinant purified -SYN can be followed by FCS. These results were validated by turbidity measurements and thioflavine T fluorescence. The size of -SYN monomers, as calculated with FCS, was comparable to the value determined before with small angle X-ray scattering. We show that we can detect -SYN aggregation intermediates that are 2³ or 3³ times larger than the monomer.

2. E. coli FKBP enhances the aggregation rate of -SYN in vitro.
We have found that the presence of an E. coli FKBP, SlyD, drastically increases the aggregation rate of -SYN. When adding FK506, a well-known inhibitor of PPIase activity, the effect of SlyD on -SYN aggregation decreased in a dose-dependent way.

3. Human FKBP12 enhances the aggregation rate of -SYN
To determine the potential physiological relevance of our findings, we analyzed the effect of human FKBP12 on -SYN aggregation. Similar to the bacterial PPIase, human FKBP12 stimulated -SYN aggregation. FCS measurements were confirmed with turbidity and thioflavin T measurements. Again, the specificity of the pro-aggregatory effect of FKBP12 on -SYN was verified by addition of the inhibitor FK506. This slowed down the aggregation process in a dose-dependent way resulting in complete inhibition of the stimulating effect of FKBP12 (Fig. 1 ).

View larger version (14K): [in this window] [in a new window]   Figure 1. The aggregation of -SYN is influenced by human FKBP12 and its inhibitor FK506. A) Aggregation of 70 µM -SYN was followed over time by FCS in the presence of 30 nM, 120 nM or 1.2 µM FKBP12. B) 70 µM -SYN was incubated with 250 µM spermine as a positive control and 320 nM or 15 µM FKBP12. The absorbance of this mixture at 350 nm was followed in time. C) The aggregation of 70 µM -SYN was followed by turbidity measurement in the presence of 500 nM FKBP12 and 12 or 120 µM FK506. To accelerate the aggregation process, 60 µM spermine was added to each sample.

4. FKBP12 stimulates -SYN fibril formation
Since the -SYN aggregates found in LBs are predominantly straight or twisted fibrils, we investigated the nature of the -SYN aggregates in closer detail. With laser scanning microscopy, large aggregates that precipitated to the bottom of the slide could be visualized. The -SYN aggregates formed upon incubation with FKBP12 predominantly had a fibrillar appearance while some of those displayed a twisted morphology. These findings suggest that aggregates formed in the presence of FKBP12 can be physiologically relevant. Electron microscopy images confirmed that aggregates formed in the presence of FKBP12 have fibrillar morphology.

5. FKBP12 does not coaggregate with -SYN
The accelerated aggregation of -SYN by nanomolar concentrations of SlyD or FKBP12 strongly supports a catalytic role for FKBPs in this process. FCS measurements showed that FKBP12 does not bind to -SYN and -SYN oligomers. Examination of the -SYN aggregates with confocal laser scanning microscopy could not detect FKBP12 in the large -SYN aggregates.

CONCLUSIONS AND SIGNIFICANCE

Our work clearly demonstrates that proteins of the FKBP family accelerate -SYN aggregation in vitro. Two members of this family, E. coli FKBP slyD and the human FKBP12, have been investigated in detail. Since both proteins exerted a similar effect on -SYN aggregation, it is possible that the evolutionary conserved PPIase activity of FKBP is responsible rather than other described functions. The 5 prolines in -SYN are all found in the C terminus, which is believed to have an important regulating effect on the aggregation. Changing the conformation of the prolines may induce aggregation. The hypothesis of a catalytic mechanism is supported by the fact that substoichiometric amounts of FKBP substantially accelerate -SYN aggregation, and this effect is inhibited by FK506. We were unable to detect binding of fluorescent FKBP to -SYN oligomers and fibrils.

We present a novel FCS-based approach to study -SYN aggregation with multiple advantages. However, FCS is better suited to follow oligomerization rather than the formation of large aggregates because of its high sensitivity.

The effects of immunophilin ligands on neurodegenerative diseases have been studied before, although the exact mechanism has not been elucidated. Here we propose that immunophilin ligands can act through the inhibition of the accelerating effect of FKBP proteins on the aggregation of -SYN. Our findings may initiate the development of novel drugs with a higher specificity toward relevant FKBP proteins. We do not yet know which FKBP may be involved in -SYN aggregation in vivo. Two FKBPs are known to be present in the substantia nigra: FKBP12 and FKBP52. It has been shown that the expression of both proteins changes in pathological conditions.

A possible link between FKBPs and PD could be found in the fact that chaperone proteins are induced or redistributed in stress conditions. In the case of FKBP chaperones, this may be not beneficial as it leads to a decrease in the lag time of aggregation of -SYN, which may accelerate the onset of PD. Alternatively, -SYN aggregation and accumulation into Lewy bodies may provide the cell with a defense mechanism against toxicity associated with -SYN derivatives. Indeed, the cytotoxic vs. protective role of -SYN inclusions is still under intense debate. In this case FKBP would instead protect against synucleinopathy and PD. Future experiments are needed to investigate whether the aggregation-inducing role of FKBP can be demonstrated in cell culture and animal models and to identify the involved human FKBP.

View larger version (24K): [in this window] [in a new window]   Figure 2. Step 1) FKBP12 binds to unfolded -SYN (blue, N terminus; red, hydrophobic NAC domain; yellow, C terminus) and changes the conformation of one or several prolines (purple circles). Step 2) Because of the conformational change of the C terminus, the NAC domain becomes unshielded, which induces the interaction with other hydrophobic NAC domains and its folding into ß-sheets. Step 3) The protein continues to fold, which starts the aggregation process. The first steps of the aggregation, such as the formation of the aggregation nucleus and other aggregation intermediates, are reversible. Step 4) Of these aggregation intermediates, short protofibrils are formed. Step 5) These protofibrils rapidly aggregate into mature fibrils.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5126fje;

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This Article Full Text (PDF) All Versions of this Article: 20/3/524 05-5126fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gerard, M. Articles by Engelborghs, Y. Search for Related Content PubMed PubMed Citation Articles by Gerard, M. Articles by Engelborghs, Y.