There is growing number of evidence supporting the conclusion that the ability to form fibrils is a generic property of the polypeptide chain; i.e., many proteins, perhaps all, are potentially able to form amyloid fibrils under appropriate conditions [Dobson, 1999
; Dobson, 2004b
]. This means that amyloidogenic polypeptides are unrelated in terms of sequence or structure. The experiments with probing of aggregated forms of proteins with antibodies showed that there is a class of structure specific antibodies that recognize protein aggregates (e.g., fibrils), but do not bind to a monomeric form of the same protein (monomers) [O’Nuallain and Wetzel, 2002
]. Importantly, among those structure-specific antibodies are those that distinguish between different aggregated morophologies of the same protein [Glabe, 2004
; Kayed et al., 2003
]. These findings suggest that aggregated forms of protein have different epitopes; in other words, their structural characteristics are different. Importantly, it was also established that some antibodies were able to recognize aggregates formed by different proteins. The striking conclusion based on these experiments was that the same structural morphologies but formed but different proteins are recognized by the same structure-specific antibody. These studies lead to the intriguing conclusion that aggregated protein might have common structural motifs even if they are not structurally close prior to fibrillation, being rich in β-sheet, α-helix, β-helix, or contain both α-helices and β-sheets, be globular proteins with rigid 3D-structure or belong to the realm of natively unfolded (or intrinsically unstructured) proteins [Uversky and Fink, 2004
]. Despite these differences, amyloid fibrils have similar structural features. In fact, based on the results of extensive structural studies on several amyloidogenic proteins a general hypothesis of fibrillogenesis has been formulated: structural transformation of a polypeptide chain into a partially folded conformation represents an important prerequisite for protein fibrillation [Uversky and Fink, 2004
]. Schematically this pathway is shown in .
In protein misfolding diseases, potentially pathogenic misfolded and aggregated forms of a protein can form in different ways:
- The protein may have an intrinsic propensity to assume a pathologic conformation, which becomes evident with aging or at persistently high concentrations;
- The replacement of a single amino acid in the protein, as occurs in hereditary amyloidosis, can increase propensity of protein to misfold and, thus, represents another obvious mechanism of amyloidogeneity;
- Often, proteolytic digestion of the protein precursor might produce an amyloidogenic fragment;
- Interactions (or impaired interactions) with some endogenous factors (e.g., chaperones, intracellular or extracellular matrixes, other proteins) can change conformation of a pathogenic protein and increase its propensity to misfold and aggregate;
- Exposure to internal or external toxins can induce conformational changes in a given protein and facilitate its misfolding and aggregation;
- Malfunction of the antioxidant defense system can lead to the increased production of free radicals, which might induce oxidative modification of a pathogenic protein, or its binding partners;
- Impaired posttranslational modifications (phosphorylation, advanced glycation, deamidation, racemization, etc.) might change protein conformation, facilitate its misfolding and promote aggregation;
- Impaired functioning of proteasome or other proteolytic systems might result in the dramatic increase in the local concentration of the pathogenic protein.
These mechanisms can act independently or in association with one another. In addition to the intrinsic amyloidogenic potential of the pathogenic protein, other factors may act synergistically in amyloid deposition.
What information is lacking for a quantitative description of the system?
Despite the crucial importance of protein misfolding and abnormal interactions, very little is currently known about the molecular and intra-cellular mechanisms underlying these processes. Furthermore, factors that lead to protein misfolding and aggregation in vitro are poorly understood, including the complexities involved in the formation of protein nanoparticles with different morphologies. Although it is well known that incubation of a protein under non-physiological conditions can lead to the formation of fibrillar, pore-like, spherical, or amorphous nano-ensembles with diverse biological consequences, the physiological insults leading to misfolding and the formation of such abnormal complexes are unclear. The mechanisms underlying aggregation in biological systems are even less well understood due to the difficulties in monitoring aggregates in vivo. Clearly, a full understanding of the molecular mechanisms of misfolding and aggregation is essential for the development of rational approaches to prevent protein misfolding that lead to self-assembly in nano-ensembles. Thus, one needs to elaborate novel approaches to determine the molecular basis of protein misfolding and aggregation, and from this fundamental knowledge, to formulate a predictive molecular model that incorporates the cellular nanomachinery and physiological processes involved in protein deposition disorders. In this way, we can establish crucial enabling insights to catalyze the development of new therapeutic advances and novel nanotechnologies for diagnosis.
Misfolded conformations of proteins differ from folded and other aberrant protein conformations by their increased propensity to interact with each other leading to the formation of nano-aggregates. The structure of individual protein molecules within well ordered aggregates can be partially elucidated by traditional structural techniques, including X-ray crystallography, NMR, circular dichroism, fluorescence, and IR spectroscopies (reviewed in [Dobson, 2004a
; Dobson, 2005
]). However, none of these techniques is capable of sensing the misfolded conformation of the protein prior to aggregation. Apparently, the conformation of misfolded protein preceding the aggregation is different from what one can see in aggregates, but to what extent this difference might cause the disease is not clear. The vast majority of current experimental approaches to analyze protein misfolding and aggregation are based on traditional, ensemble techniques that describe the conformational behavior of the entire system and thus do not allow investigators to distinguish between conformational changes in individual protein molecules prior aggregation and changes induced by protein-protein interaction. This leaves open the question on the effect of different factors on folding/misfolding of an individual protein molecule. Conventional structural tools do not allow for the measurement of protein interaction forces or the kinetics of interconversion among different protein conformations in a single protein molecule. The ability to measure these parameters is critical to achieve a quantitative understanding of protein misfolding and aggregation at the nanoscale level. Thus, new experimental tools and approaches are crucial for understanding the protein misfolding phenomenon.
What tools are in place and what additional ones will be required to develop?
Structural methods such as x-ray crystallography, NMR, electron microscopy, and atomic force microscopy (AFM) have provided useful data regarding the secondary structure of proteins in nano-assemblies and the morphologies of self-assembled aggregates. However, we still lack a mechanistic understanding of the processes leading to the misfolding and of the interactions between misfolded conformations leading to protein self-assembly in nano-aggregates. This includes the capability to identify the conformation(s) of a misfolded protein prior to the onset of formation of nano-ensembles. Indeed, there may be several non-canonical protein conformations that exist transiently and that transform from one into the other. To address these questions, we need to utilize new “thinking” and new techniques capable of probing transient conformations of single protein molecules.
The tools currently available to researchers and clinicians essentially provide a population-based picture of a living-dynamical system. Because most studies have involved these ensemble techniques, which average over an enormous number of molecules or freeze the complex system in time, we lack insight into the manifold opportunities for conformational change available to an individual molecule in the complex environment of a cell or tissue. It is evident that the mechanisms of life and disease involve dynamic molecular-scale interactions—most of which occurs under some level of physical stress. An apparently simple process, as examined by existing techniques, may actually have many steps and a variety of important intermediate states—each with their own unique dynamics and response to stress. These intermediate states are likely to be only marginally stable making their transient existence difficult to measure. However, they can be very important to control several pathways. Thus, it is vitally important to establish ultrasensitive methods capable of probing protein conformations and weak intermolecular interactions over a wide range of time scales.
It has been already mentioned that there is rapidly growing evidence that the pathogenesis of protein misfolding diseases is not a result of protein deposition in a form of visible aggregates, which, in fact, represent a final stage of the multi-step molecular association cascade. Rather, the earlier steps in the series of conformational changes and protein-protein interactions are directly tied to pathogenesis. There is now increased understanding of the pathways involved in protein association, and some recent clues have emerged as to the molecular mechanisms of cellular toxicity [Ross and Poirier, 2004
]. Targeting these early events of development of protein misfolding diseases will lead to the development of novel approaches toward rational diagnostics and therapeutics of these disorders.