Journal of Molecular Biology
ReviewWhy Study Functional Amyloids? Lessons from the Repeat Domain of Pmel17
Introduction
Amyloids are traditionally associated in the context of disease; however, the emerging concept of “functional amyloids” has redefined this historic interpretation of amyloid [1]. Many examples of so called “functional amyloids” have been discovered that serve a beneficial role where the amyloid carries out a specific function. Examples have been identified in bacteria [2], fungi [3], plants [4] and humans [5], [6], [7], offering molecular and cellular insights of using amyloids to carry out a function. Some of these functions are associated with bacterial physiology [8], hormone storage [6] and human reproduction [9]. With new functional amyloids constantly being discovered, a better understanding of what differentiates them from its pathological counterpart is emerging.
One thought is that oligomers are the true pathogenic agents that are longer lived during pathological amyloid formation [10], [11]. For example, soluble oligomers of Aβ correlate better with Alzheimer's disease severity than the insoluble fibrillar deposits that are present in amyloid plaques, suggesting that oligomeric forms are the toxic species [12]. In the case of functional amyloids, strict control would circumvent the buildup of toxic oligomers. This has been demonstrated with the rapid aggregation kinetics of the Orb2 protein [13], thereby bypassing oligomeric intermediate buildup. Tight kinetic control from the functional bacterial amyloid “Curli” shows cell regulation at several steps by other proteins which facilitate localization and nucleate rapid polymerization of the protein CsgA [8]. Thus, Escherichia coli has evolved ways to safely assemble amyloid and transport it to the extracellular matrix to carry out its specific function.
Sequestration of amyloids within membrane compartments is another possible mechanism to alleviate the detrimental effect caused by forming amyloid. For example, peptide hormones [6] and acrosomal matrix proteins [14] are stored in endocrine granules and acrosomes, respectively. These organelles provide an acidic environment optimal for the assembly of amyloid fibrils and prevent interaction with unwanted cellular components.
In this review, we highlight our work on the repeat (RPT) domain of Pmel17 that forms amyloid fibrils only at the biologically relevant pH of melanosomes [15], [16]. Through a controlled series of proteolytic events of Pmel17, the RPT domain is released and self-assembles at acidic pH to form a fibrillar scaffold upon which melanin is deposited. This pH-sensitive aggregation process was also shown to be reversible, with fibril disassembly occurring at near neutral pH. The ability to control fibril formation has strong biological implications. Unlike pathological amyloids, RPT fibrils rapidly dissolving at neutral pH would ensure that in the event of escaping from their melanosomal environment, they would disassemble and maintain a soluble benign form. This sensitive pH-dependent mechanism sequesters fibril formation only in the melanosome where it aids in melanin synthesis.
Section snippets
Pmel17 and Its Structural Role in Melanin Synthesis
Pmel17 is a transmembrane protein that is proteolytically processed to generate intralumenal fibrils in melanosomes, acidic organelles that synthesize and store melanin [17]. The direct involvement of Pmel17 in melanin synthesis was shown using a Pmel17 knockout mutation in mice, where a 40%–50% reduction in melanin content in hair was demonstrated [18]. This decline in melanin content suggests the fibrils might be responsible for enhancing melanin synthesis. To generate melanin, a multitude of
Defining the Fibril-Forming Region of Pmel17
Melanosomes were first shown to contain amyloid fibrils using the amyloidogenic dyes thioflavin S and Congo Red [5]. To associate Pmel17 to these structures, a Triton X-100-insoluble melanosome fraction showed colocalization between thioflavin S-stained particles and Pmel17 immunofluorescence. Direct involvement of Pmel17 in amyloid formation was demonstrated using a recombinant Mα (residues 25–467) fragment expressed in E. coli which resulted in rapid aggregation upon dilution out of
Mechanistic Insights into pH-dependent RPT Amyloid Formation
During melanosome maturation, the pH environment changes from a starting pH ~ 4.0 in stages I and II to near neutral pH (~ pH 6.0) in melanized stage IV melanosomes. Knowing RPT aggregates into amyloid at pH 5 prompted a more detailed study of aggregation as a function of pH. Using an array of techniques that include ThT (an amyloidogenic dye) and intrinsic Trp (W423) fluorescence (Fig. 1d), light scattering and TEM were used to track amyloid formation [37]. At pH 4.0–4.5, aggregation occurred
Melanosomal Membrane Lipids Modulate RPT Fibril Formation
It has been suggested that Pmel17 processing and liberation of the amyloid-forming region (RPT domain) occurs in conjunction with ILVs [40], [41] (Fig. 3a). This association is most evident during stage I melanosome development where prefibrillar aggregates formed from the proteolytic processing of Pmel17 appear to emanate from ILVs [19]. Recently, apolipoprotein E was shown to be associated with ILVs and regulate the formation of Pmel17 amyloid fibrils in endosomes [42]. As the melanosome
Probing Disassembly of RPT Fibrils as a Function of pH
The strict pH dependence of RPT fibril formation was determined to be reversible, with preformed fibrils at pH 5 rapidly disassembling when exposed to neutral pH [34], [37], [38], [45]. This unique characteristic is in total contrast to pathological amyloids that generally resist the harshest of treatments. However, in the case of functional amyloids, this observation has been shown from many peptide hormones [6], which have a reversible aggregation/disaggregation process. In the acidic
RPT Amyloid Enhances Melanin Synthesis at Acidic pH
To recapitulate the involvement of RPT in melanin synthesis, an in vitro assay was performed using RPT fibrils, tyrosinase and l-3,4-dihydroxyphenylalanine [34], [39]. Under acidic pH, tyrosinase activity is known to be significantly diminished, with optimal activity at pH 6.8 [46]. However, in the presence of RPT fibrils at pH 5, a 6-fold increase in melanin content was observed when compared to a reaction containing only soluble RPT [34]. This enhancement offers support for the direct
RPT Fibrils Have an In-register Parallel β-Sheet Structure
Using solid-state NMR spectroscopy, preformed RPT fibrils formed at pH 5 were shown by selective 13C-labeled amino acids to adopt the typical in-register parallel β-sheet architecture observed for pathological amyloids [39]. By labeling with 13C-labeled amino acid (Ala, Val or Met) in the carbonyl position, the closest intra/intermolecular 13C distance between β-stands in a β-sheet was approximately 0.5 nm, suggestive of in-register parallel structure. ssNMR measurements of three independently
Pmel17 Orthologs Contain Amyloid Forming RPT Domains
Pmel17 is conserved across many species, with the RPT domain being the most varied in sequence identity [25]. This may suggest that the diversity in the number and sequence of repeats in RPT makes it an improbable candidate for the amyloid core for an evolutionarily conserved functional amyloid. To observe if fibrils are formed from these regions, the repeat sequences from mouse (mRPT), zebrafish (zRPT) and a known splice variant of the human Pmel17 (sRPT) sequence where 42 amino acids is
Controversy Surrounding a Structural Role for RPT
While our work highlights RPT as highly amyloidogenic under melanosomal pH conditions, there is controversy surrounding whether RPT forms amyloid in vivo. Here, we discuss reasons supporting our in vitro work and addressing prior concerns. First, the statement that the RPT domain is heavily O-glycosylated and for that reason cannot be considered to participate in amyloid formation in vivo is misinterpreted. Yes, the RPT domain is O-glycosylated, but the only known glycosylation sites are in the
Conclusion and Future Direction
In this review, we focus on our work on the RPT domain of Pmel17 that forms amyloid fibrils only at the physiological pH (pH 4.0–5.5) of early-stage melanosomes. Our work provides an elegant mechanism that shows a reversible aggregation/disaggregation process that is governed by pH. The strict pH dependence was largely controlled by the protonation of residue E422. Strikingly, removal of a single negative charge at E422 out of 16 carboxylic acids shifted the pH dependence by a full pH unit. The
Acknowledgments
This work is supported by the Intramural Research Program at the National Institutes of Health, National Heart, Lung, and Blood Institute.
Declarations of interest: None.
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