PhD Defence Abolghassemi Fakhree

Functional Insights into membrane bound α-synuclein: surface density, conformation, and localization inside cells

Abolghassemi Fakhree is a PhD student in the Nanobiophysics group. His supervisor is prof.dr.ir. M.M.A.E. Claessens from the faculty of Science and Technology. 

Intrinsically disordered proteins (IDPs) and the intrinsically disordered regions of structured proteins (IDRs) have received an increasing amount of attention during the past two decades. Two major reasons for the increased research efforts in this direction are: 1) IDPs/IDRs are recognized to play a role in the development of diseases, 2) IDPs/IDRs do not have secondary structure which makes it harder to determine their function, and makes their study a challenging task. To complicate matters, not only do IDPs/IDRs not have a fixed structure, they also interact with a range of ligands possibly resulting in multiple functions. Hence IDPs are often “mysterious” proteins.

In order to understand the connection between IDPs/IDRs and diseases, and try to cure IDP related disease it is necessary to “define” the IDP/IDR. The first step to define IDPs/IDRs is to determine what their interaction partners are. Most of IDPs adopt (partial) secondary structure upon binding interaction partners. So, one should determine possible interacting partners of the IDP/IDR and look at the affinity and/or structural association between them, separately or in combination with other interacting partners. By thus characterizing and quantifying the different interactions of a single IDP species, it should be possible to derive the protein/ligand network in which the IDP/IDR serves as a hub. This interaction network represents the physiological role(s)/function(s) of the IDP/IDR of interest. In the present work, we have shown how a number of questions regarding defining the functional form of an IDP, namely alpha-synuclein (αS), can be addressed.

Knowledge of the interaction network becomes important in the design of therapeutic interventions in IDP/IDR related diseased. Since the structure-function dogma is not applicable to IDPs/IDRs, the classic lock-and-key approach to design and target a specific protein with single function as the receptor, will not work and needs adjustment. This adjustment should include specific interaction with one of the many functional forms of the IDP/IDR of interest, without disrupting the required balance in the protein/ligand interaction network of the IDP/IDR.

In the present work, we investigated the biophysical functional features of alpha-synuclein, as a model IDP, in a cellular context. We measured the surface density of αS on cellular vesicles and discussed that based on the determined surface density, αS can act as a membrane remodeling agent. Moreover, based on recent reports on the role of intrinsically disordered domains of protein in the induction of membrane curvature, we suggested a new mechanism of action for membrane remodeling capacity of αS. Furthermore, we linked the determined surface density to the surface density of a suggested interaction partner of αS, synaptobrevin. We thereby made a new possible functional link in the interaction network of αS. As one can see, based on the knowledge from in vitro experiments, determining the cellular copy number of an IDP helped to explain a role, propose a new mechanism of action, and obtain support for one of the suggested interaction partner for the IDP, αS. This was doable because of the techniques that were developed during late 20th century such as expressing fluorescent proteins and single molecule imaging.

Next, we went one step further, and showed that similar to in vitro experiments, αS changes its structure upon binding to cellular vesicles. This is important in two aspects. First, it shows the existence of more than one specific conformationally different form of the IDP inside cells. Since also in the cellular context there is structure, this structure can help us understand the function of the IDP, for example membrane remodeling. Second, our report provided a cellular evidence for the in vitro observations of membrane bound αS with a structure that is distinctly different from the structure in solution. This distinctly different structural sub-population of αS was not resolved in previous reports by other groups using bulk methods. Again, the higher sensitivity of the used single molecule technique made it possible to differentiate populations within cells.

After observing membrane bound structure of αS inside cells, and knowing that αS can modulate membranes, we tested our hypothesis on the second membrane remodeling mechanism of αS, inducing curvature by generating lateral steric pressure. In order to avoid complications by the other possible interaction partners inside cell, such as synaptobrevin and Ca2+, we used an in vitro membrane model. We were able to show that both truncation and elongation of the disordered region of αS, affects the ability of the IDP to remodel membranes into small vesicles. This shows that the disordered domain of the IDP αS plays a role in membrane remodeling. Moreover, we were able to provide a theoretical model for dual mechanism of αS in membrane remodeling. Results of the theoretical model suggest that our hypothesis posed on the membrane remodeling mechanisms of αS is correct.

So what do these observations mean in a cellular context? By careful examination of the literature on the numerous interaction partners of αS, which appears puzzling because it interacts with almost everything inside cells, and following our biophysical findings, we proposed that αS is involved in cellular events that require membrane remodeling, such as endocytosis and exocytosis. So, we went another step further and investigated the colocalization between αS and general parts of endocytosis/exocytosis pathways. Our colocalization findings suggest that αS is mainly associated with the endocytic pathway, from vesicular uptake to recycling and elimination inside cell. And it appears that αS is involved in a specific vesicular uptake/recycling path. Based on our observed colocalization pattern and reported works from literature, this specific path most probably is the uptake of transferrin. The endocytosis path in which αS plays a role generated in this chapter helps to put the puzzle pieces of the function of αS inside cell together. Most of the reported interacting proteins/lipids with αS, and organelles affected by αS, are linked to endocytosis and exocytosis.

In summary, we showed that cellular αS plays a role in membrane remodeling events such as endocytic/exocytic events. This can be argued as part of αS/ligand interaction network and function. Further research is required to bring the whole interaction network and function of αS to the light.