Functional amyloid--from bacteria to humans

Amyloid--a fibrillar, cross beta-sheet quaternary structure--was first discovered in the context of human disease and tissue damage, and was thought to always be detrimental to the host. Recent studies have identified amyloid fibers in bacteria, fungi, insects, invertebrates and humans that are functional. For example, human Pmel17 has important roles in the biosynthesis of the pigment melanin, and the factor XII protein of the hemostatic system is activated by amyloid. Functional amyloidogenesis in these systems requires tight regulation to avoid toxicity. A greater understanding of the diverse physiological applications of this fold has the potential to provide a fresh perspective for the treatment of amyloid diseases.     

Pathological and functional amyloid formation orchestrated by the secretory pathway

Amyloidogenesis has historically been associated with pathology in a class of neurodegenerative diseases known as amyloid diseases. Recent studies have shown that proteolysis by furin during secretion initiates both variant gelsolin amyloidogenesis, associated with the disease familial amyloidosis of Finnish type, and Pmel17 fiber formation, which is necessary for the functional biogenesis of melanosomes. Proteolysis combined with organelle-dependent environment changes orchestrate amyloidogenesis associated with both pathological processes and a functional pathway.     

Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis

Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.     

Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ～5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel β-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aβ peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.     

Formation of Pmel17 Amyloid Is Regulated by Juxtamembrane Metalloproteinase Cleavage, and the Resulting C-terminal Fragment Is a Substrate for ��-Secretase

The formation of insoluble cross β-sheet amyloid is pathologically associated with disorders such as Alzheimer, Parkinson, and Huntington diseases. One exception is the nonpathological amyloid derived from the protein Pmel17 within melanosomes to generate melanin pigment. Here we show that the formation of insoluble MαC intracellular fragments of Pmel17, which are the direct precursors to Pmel17 amyloid, depends on a novel juxtamembrane cleavage at amino acid position 583 between the furin-like proprotein convertase cleavage site and the transmembrane domain. The resulting Pmel17 C-terminal fragment is then processed by the γ-secretase complex to release a short-lived intracellular domain fragment. Thus, by analogy to the Notch receptor, we designate this cleavage the S2 cleavage site, whereas γ-secretase mediates proteolysis at the intramembrane S3 site. Substitutions or deletions at this S2 cleavage site, the use of the metalloproteinase inhibitor TAPI-2, as well as small interfering RNA-mediated knock-down of the metalloproteinases ADAM10 and 17 reduced the formation of insoluble Pmel17 fragments. These results demonstrate that the release of the Pmel17 ectodomain, which is critical for melanin amyloidogenesis, is initiated by S2 cleavage at a juxtamembrane position.Folding of proteins is a highly regulated process ensuring their correct three-dimensional structure. Under pathological circumstances, a soluble protein can be folded into highly stable cross β-sheet amyloid structures, which are believed to play pathological roles in disorders such as Alzheimer, Parkinson, and Huntington diseases. An exception to this general concept is the physiological amyloid structure of the melanosomal matrix formed by the protein Pmel17. Melanosomes are lysosome-related organelles that contain pigment granules (melanin) in melanocytes and retinal epithelial cells (reviewed in Ref. 1). Melanogenesis is believed to proceed through several sequential maturation steps, classified by melanosomes from stage I to stage IV. Maturation of stage II melanosomes requires the formation of Pmel17 intralumenal fibers (2, 3).Pmel17 (also called gp100, ME20, RPE1, or silver) is a type I transmembrane glycoprotein of up to 668 amino acids in humans (reviewed in Ref. 4). The requirement of Pmel17 for the generation of functional melanin has been shown in a number of different organisms, because, for example, certain point mutations in the Pmel17/silver gene result in hypopigmentation phenotypes (5–7). The most characteristic domain within Pmel17 is a specific lumenal proline/serine/threonine rich repeat domain (see Fig. 1A), that is imperfectly repeated 13 times in the Mα fragment. Importantly, deletion of the rich repeat domain results in a complete loss of fibril formation, pointing to the requirement of Pmel17, and especially the rich repeat domain, in melanin formation (8). Pmel17 exists in different isoforms generated by alternative splicing. Pmel17-i² is the most abundant isoform, whereas the Pmel17-l isoform contains a 7-amino acid insertion close to the transmembrane domain (9, 10).Open in a separate windowFIGURE 1.Effect of the γ-secretase inhibitor DAPT on Pmel17 processing. A, schematic diagram of Pmel17 and epitopes of antibodies. Pmel17 contains five potential N-glycosylation sites indicated by branched structures. The long form of Pmel17, Pmel17-l, is characterized by a seven amino acid insertion (VPGILLT) within the lumenal domain close to the transmembrane domain (TM), which is absent in Pmel17-i. NVS marks a potential N-glycosylation site near this insertion. The epitopes of antibodies αPep13h and HMB45 are indicated. Cleavage by a furin-like PC results in the formation of the Mα and the membrane-bound 26-kDa Mβ fragment, which are connected via disulfide bonds. Release and further processing of the Mα fragment into MαN and MαC fragments results in the formation of fibrils and marks the transition of stage I to stage II melanosomes (dashed line). B, human MNT-1 cells were incubated with increasing amounts of DAPT for 18 h, and then the lysates were separated by SDS-PAGE and analyzed by immunoblotting with αPep13h antibody. DAPT treatment resulted in the accumulation of a C-terminal fragment of Pmel17 (CTF), whereas Pmel17 P1 and Mβ fragment were unchanged. C, probing the Triton-soluble fraction with HMB45 revealed increased amounts of the highly glycosylated P2 form of Pmel17 after DAPT incubation. D, detection of Pmel17 amyloidogenic fragments (MαC) in the SDS-extracted insoluble pellet using antibody HMB45. E, murine B16-FO cells treated with increasing concentrations of DAPT. Immunoblotting using antibodyαPep13h revealed the formation of CTF of similar size as in MNT-1 cells. F, time course analysis of Pmel17, Mβ, and Pmel17-CTF after DAPT treatment. The cell lysates were immunoblotted using αPep13h. Pmel17-CTF was detectable after 10 min of incubation with 1 μm DAPT. G, the size of the Pmel17-CTF was determined using an unstained low molecular range peptide standard. The marker peptides were detected by Ponceau S staining and Pmel17-CTF were detected by immunoblot using αPep13h.Pmel17 traffics through the secretory pathway as a 100-kDa protein (called P1). In the late Golgi compartment it undergoes further glycosylation, resulting in a short lived 120-kDa protein (called P2). P2 is rapidly cleaved within the post-Golgi by a furin-like proprotein convertase (PC) to generate two fragments that remain tethered to each other by disulfide bonds: a C-terminal polypeptide containing the transmembrane domain (Mβ) and a large N-terminal ectodomain (Mα) (2) (Fig. 1A). Consequently, inhibition of this furin-like activity not only prevents the generation of Mα and Mβ fragments but also inhibits the formation of melanosomal striation in HeLa cells (3). These findings suggest that Mα must first be dissociated from the Mβ for melanogenesis to proceed. It is unclear how Mα is released from the membrane. Reduction of disulfide bonds would release Mα from Mβ; alternatively, proteolytic digestion of Mβ should also free Mα from the membrane tether. It has been speculated that, given the presence of lysosomal hydrolases in melanosomes and proteolytic maturation of Pmel17, proteolysis is the more likely mechanism (4). Recently, it was shown that recombinant Mα is able to form amyloid structures in vitro in an unprecedented rapidity, and furthermore, Pmel17 amyloid also accelerated melanin formation (11). These findings demonstrate that mammalian amyloid formed by Pmel17 is functional and physiological.The insoluble pool of Pmel17 in cells consists mostly of truncated Mα C-terminal fragments (MαC) of heterogeneous sizes, indicating that further processing of Mα occurs after its release from the membrane (8, 12). MαC fragments are found in the insoluble fraction of melanocytes as well as in nonmelanotic cells, the latter after overexpression of Pmel17 (8), and are reduced or absent in amelanotic cells (8, 13, 14). Meanwhile, the C-terminal fragment derived from the Mβ fragment and recognized by a C-terminal specific epitope antibody is less stable, indicating rapid turnover (2).The presenilin (PS) family of proteins consists of two homologous integral transmembrane proteins, PS1 and PS2, which are part of the γ-secretase complex. The latter consists of presenilin 1 or 2, nicastrin, APH-1, and PEN-2 (15) and catalyzes the cleavage of the hydrophobic transmembrane domain of a burgeoning list of proteins, also called regulated intramembrane cleavage. Other substrates for the γ-secretase-mediated intramembrane cleavage include Notch, amyloid precursor protein (APP), cadherin (E-cadherin), nectin-1, the low density lipoprotein-related receptor, CD44, ErbB-4, the voltage-gated sodium channel β2-subunit, and the Notch ligands Delta and Jagged. Importantly, in Alzheimer disease, the presenilin-mediated γ-secretase cleavage of APP releases the amyloid β-protein fragment, a peptide believed to play a key role in Alzheimer disease pathogenesis. Interestingly, a recent report described the absence of melanin pigment in presenilin-deficient animals, an observation confirmed by the lack of melanin formation in cells treated with γ-secretase inhibitors (16). The mechanism responsible for this finding is unclear, leading us to ask whether Pmel17 processing is a presenilin-dependent process and, if so, whether this cleavage is involved in melanogenesis.In this study, we show the presence of an endoproteolytic activity that cleaves the extracellular domain of Pmel17-i at a juxtamembrane position between the known PC cleavage site and the transmembrane domain, which we term the S2 cleavage site, by a TAPI-sensitive ADAM (a disintegrin and metalloproteinase protein) protease. This intracellular shedding of Pmel17 after S2 cleavage results in the liberation of the Mα N-terminal ectodomain, the precursor to Pmel17 amyloid, which is able to form insoluble Pmel17 aggregates. The C-terminal transmembrane fragment generated by S2 cleavage is further processed by γ-secretase (S3 cleavage) to release the Pmel17 intracellular domain, which is then rapidly degraded.     

Segmental polymorphism in a functional amyloid

Although amyloid fibrils are generally considered to be causative or contributing agents in amyloid diseases, several amyloid fibrils are also believed to have biological functions. Among these are fibrils formed by Pmel17 within melanosomes, which act as a template for melanin deposition. We use solid-state NMR to show that the molecular structures of fibrils formed by the 130-residue pseudo-repeat domain Pmel17:RPT are polymorphic even within the biologically relevant pH range. Thus, biological function in amyloid fibrils does not necessarily imply a unique molecular structure. Solid-state NMR spectra of three Pmel17:RPT polymorphs show that in all cases, only a subset (∼30%) of the full amino acid sequence contributes to the immobilized fibril core. Although the repetitive nature of the sequence and incomplete spectral resolution prevent the determination of unique chemical shift assignments from two- and three-dimensional solid-state NMR spectra, we use a Monte Carlo assignment algorithm to identify protein segments that are present in or absent from the fibril core. The results show that the identity of the core-forming segments varies from one polymorph to another, a phenomenon known as segmental polymorphism.     

Proprotein convertases process Pmel17 during secretion

Pmel17 is a melanocyte/melanoma-specific protein that traffics to melanosomes where it forms a fibrillar matrix on which melanin gets deposited. Before being cleaved into smaller fibrillogenic fragments the protein undergoes processing by proprotein convertases, a class of serine proteases that typically recognize the canonical motif RX(R/K)R↓. The current model of Pmel17 maturation states that this processing step occurs in melanosomes, but in light of recent reports this issue has become controversial. We therefore addressed this question by thoroughly assessing the processing kinetics of either wild-type Pmel17 or a secreted soluble Pmel17 derivative. Our results demonstrate clearly that processing of Pmel17 occurs during secretion and that it does not require entry of the protein into the endocytic system. Strikingly, processing proceeds even in the presence of the secretion inhibitor monensin, suggesting that Pmel17 is an exceptionally good substrate. In line with this, we find that newly synthesized surface Pmel17 is already quantitatively cleaved. Moreover, we demonstrate that Pmel17 function is independent of the sequence identity of its unconventional proprotein convertase-cleavage motif that lacks arginine in P4 position. The data alter the current view of Pmel17 maturation and suggest that the multistep processing of Pmel17 begins with an early cleavage during secretion that primes the protein for later functional processing.     

A mutation within the transmembrane domain of melanosomal protein Silver (Pmel17) changes lumenal fragment interactions

Melanocytes synthesize and store melanin within tissue-specific organelles, the melanosomes. Melanin deposition takes place along fibrils found within these organelles and fibril formation is known to depend on trafficking of the membrane glycoprotein Silver/Pmel17. However, correctly targeted, full-length Silver/Pmel17 cannot form fibers. Proteolytic processing in endosomal compartments and the generation of a lumenal Mα fragment that is incorporated into amyloid-like structures is also essential. Dominant White (DWhite), a mutant form of Silver/Pmel17 first described in chicken, causes disorganized fibers and severe hypopigmentation due to melanocyte death. Surprisingly, the DWhite mutation is an insertion of three amino acids into the transmembrane domain; the DWhite-Mα fragment is unaffected. To determine the functional importance of the transmembrane domain in organized fibril assembly, we investigated membrane trafficking and multimerization of Silver/Pmel17/DWhite proteins. We demonstrate that the DWhite mutation changes lipid interactions and disulfide bond-mediated associations of lumenal domains. Thus, partitioning into membrane microdomains and effects on conformation explain how the transmembrane region may contribute to the structural integrity of Silver/Pmel17 oligomers or influence toxic, amyloidogenic properties.     

Pmel17 Initiates Premelanosome Morphogenesis within Multivesicular Bodies

Melanosomes are tissue-specific organelles within which melanin is synthesized and stored. The melanocyte-specific glycoprotein Pmel17 is enriched in the lumen of premelanosomes, where it associates with characteristic striations of unknown composition upon which melanin is deposited. However, Pmel17 is synthesized as an integral membrane protein. To clarify its physical linkage to premelanosomes, we analyzed the posttranslational processing of human Pmel17 in pigmented and transfected nonpigmented cells. We show that Pmel17 is cleaved in a post-Golgi compartment into two disulfide-linked subunits: a large lumenal subunit, M alpha, and an integral membrane subunit, M beta. The two subunits remain associated intracellularly, indicating that detectable M alpha remains membrane bound. We have previously shown that Pmel17 accumulates on intralumenal membrane vesicles and striations of premelanosomes in pigmented cells. In transfected nonpigmented cells Pmel17 associates with the intralumenal membrane vesicles of multivesicular bodies; cells overexpressing Pmel17 also display structures resembling premelanosomal striations within these compartments. These results suggest that Pmel17 is sufficient to drive the formation of striations from within multivesicular bodies and is thus directly involved in the biogenesis of premelanosomes.     

Probing fibril dissolution of the repeat domain of a functional amyloid, Pmel17, on the microscopic and residue level

Pmel17 is a human amyloid involved in melanin synthesis. A fragment of Pmel17, the repeat domain (RPT) rich in glutamic acids, forms amyloid only at mildly acidic pH. Unlike pathological amyloids, these fibrils dissolve at neutral pH, supporting a reversible aggregation-disaggregation process. Here, we study RPT dissolution using atomic force microscopy and solution-state nuclear magnetic resonance spectroscopy. Our results reveal asymmetric fibril disassembly proceeding in the absence of intermediates. We suggest that fibril unfolding involves multiple deprotonation events resulting in electrostatic charge repulsion and filament dissolution.     

A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis

Cargo partitioning into intralumenal vesicles (ILVs) of multivesicular endosomes underlies such cellular processes as receptor downregulation, viral budding, and biogenesis of lysosome-related organelles such as melanosomes. We show that the melanosomal protein Pmel17 is sorted into ILVs by a mechanism that is dependent upon lumenal determinants and conserved in non-pigment cells. Pmel17 targeting to ILVs does not require its native cytoplasmic domain or cytoplasmic residues targeted by ubiquitylation and, unlike sorting of ubiquitylated cargo, is insensitive to functional inhibition of Hrs and ESCRT complexes. Chimeric protein and deletion analyses indicate that two N-terminal lumenal subdomains are necessary and sufficient for ILV targeting. Pmel17 fibril formation, which occurs during melanosome maturation in melanocytes, requires a third lumenal subdomain and proteolytic processing that itself requires ILV localization. These results establish an Hrs- and perhaps ESCRT-independent pathway of ILV sorting by lumenal determinants and a requirement for ILV sorting in fibril formation.     

Why Study Functional Amyloids? Lessons from the Repeat Domain of Pmel17

One of the current challenges facing biomedical researchers is the need to develop new approaches in preventing amyloid formation that is associated with disease. While amyloid is generally considered detrimental to the cell, examples of amyloids that maintain a benign nature and serve a specific function exist. Here, we review our work on the repeat domain (RPT) of the functional amyloid Pmel17. Specifically, the RPT domain contributes in generating amyloid fibrils in melanosomes upon which melanin biosynthesis occurs. Amyloid formation of RPT was shown to be pH sensitive, aggregating only under acidic conditions associated with melanosomal pH. Furthermore, preformed fibrils rapidly dissolved at neutral pH to generate benign monomeric species. From a biological perspective, this unique reversible aggregation/disaggregation is a safeguard against an event of releasing RPT fibrils in the cytosol, resulting in rapid fibril unfolding and circumventing cytotoxicity. Understanding how melanosomes preserve a safe environment will address vital questions that remain unanswered with pathological amyloids.     

Lysophospholipid-Containing Membranes Modulate the Fibril Formation of the Repeat Domain of a Human Functional Amyloid,Pmel17

Pmel17 is an important protein for pigmentation in human skin and eyes. Proteolytic fragments from Pmel17 form fibrils upon which melanin is deposited in melanosomes. The repeat domain (RPT) derived from Pmel17 only forms fibrils under acidic melanosomal conditions. Here, we examined the effects of lipids on RPT aggregation to explore whether intramelanosomal vesicles can facilitate fibrillogenesis. Using transmission electron microscopy, circular dichroism, and fluorescence spectroscopy, we monitored fibril formation at the ultrastructural, secondary conformational, and local levels, respectively. Phospholipid vesicles and lysophospholipid (lysolipid) micelles were employed as membrane mimics. The surfactant-like lysolipids are particularly pertinent due to their high content in melanosomal membranes. Interestingly, RPT aggregation kinetics were influenced only by lysolipid-containing phospholipid vesicles. While both vesicles containing either anionic lysophosphatidylglycerol (LPG) or zwitterionic lysophosphatidylcholine (LPC) stimulate aggregation, LPG exerted a greater effect on reducing the apparent nucleation time. A detailed comparison showed distinct behaviors of LPG versus LPC monomers and micelles plausibly originating from their headgroup hydrogen bonding capabilities. Acceleration and retardation of aggregation were observed for LPG monomers and micelles, respectively. Because a specific interaction between LPG and RPT was identified by intrinsic W423 fluorescence and induced α-helical structure, it is inferred that binding of LPG near the C-terminal amyloid core initiates intermolecular association, whereas stabilization of α-helical conformation inhibits β-sheet formation. Contrastingly, LPC promotes RPT aggregation at both submicellar and micellar concentrations via non-specific binding with undetectable secondary structural change. Our findings suggest that protein–lysolipid interactions within melanosomes may regulate amyloid formation in vivo.     

Lysophospholipids induce fibrillation of the repeat domain of Pmel17 through intermediate core-shell structures

Lipids often play an important role in the initial steps of fibrillation. The melanosomal protein Pmel17 forms amyloid in vivo and contains a highly amyloidogenic Repeat domain (RPT), important for melanin biosynthesis. RPT fibrillation is influenced by two lysolipids, the anionic lysophosphatidylglycerol (LPG) and zwitterionic lysophosphatidylcholine (LPC), both present in vivo at elevated concentrations in melanosomes, organelles in which Pmel17 aggregate. Here we investigate the interaction of RPT with both LPG and LPC using small-angle X-ray scattering (SAXS), isothermal titration calorimetry (ITC), electron microscopy, fluorescence and circular dichroism (CD) spectroscopy. Under non-shaking conditions, both lipids promote fibrillation but this is driven by different interactions with RPT. Each RPT binds >40 LPG molecules but only weak interactions are seen with LPC. Above LPG's criticial micelle concentration (cmc), LPG and RPT form connected micelles where RPT binds to the surface as beads on a string with core-shell structures. Binding to LPG only induces α-helical structure well above the cmc, while LPC has no measurable effect on the protein structure. While low (but still super-cmc) concentrations of LPG strongly promote aggregation, at higher LPG concentrations (10 mM), only ~ one RPT binds per micelle, inhibiting amyloid formation. ITC and SAXS reveal some interactions between the zwitterionic lipid LPC and RPT below the cmc but little above the cmc. Nevertheless, LPC only promotes aggregation above the cmc and this process is not inhibited by high LPC concentrations, suggesting that monomers and micelles cooperate to influence amyloid formation.     

Comparing the folding and misfolding energy landscapes of phosphoglycerate kinase

Partitioning of polypeptides between protein folding and amyloid formation is of outstanding pathophysiological importance. Using yeast phosphoglycerate kinase as model, here we identify the features of the energy landscape that decide the fate of the protein: folding or amyloidogenesis. Structure formation was initiated from the acid-unfolded state, and monitored by fluorescence from 10 ms to 20 days. Solvent conditions were gradually shifted between folding and amyloidogenesis, and the properties of the energy landscape governing structure formation were reconstructed. A gradual transition of the energy landscape between folding and amyloid formation was observed. In the early steps of both folding and misfolding, the protein searches through a hierarchically structured energy landscape to form a molten globule in a few seconds. Depending on the conditions, this intermediate either folds to the native state in a few minutes, or forms amyloid fibers in several days. As conditions are changed from folding to misfolding, the barrier separating the molten globule and native states increases, although the barrier to the amyloid does not change. In the meantime, the native state also becomes more unstable and the amyloid more stable. We conclude that the lower region of the energy landscape determines the final protein structure.     

Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres

Amyloid fibres are proteinaceous aggregates associated with several human diseases, including Alzheimer's, Huntington's and Creutzfeldt Jakob's. Disease-associated amyloid formation is the result of proteins that misfold and aggregate into β sheet-rich fibre polymers. Cellular toxicity is readily associated with amyloidogenesis, although the molecular mechanism of toxicity remains unknown. Recently, a new class of 'functional' amyloid fibres was discovered that demonstrates that amyloids can be utilized as a productive part of cellular biology. These functional amyloids will provide unique insights into how amyloid formation can be controlled and made less cytotoxic. Bacteria produce some of the best-characterized functional amyloids, including a surface amyloid fibre called curli. Assembled by enteric bacteria, curli fibres mediate attachment to surfaces and host tissues. Some bacterial amyloids, like harpins and microcinE492, have exploited amyloid toxicity in a directed and functional manner. Here, we review and discuss the functional amyloids assembled by bacteria. Special emphasis will be paid to the biology of functional amyloid synthesis and the connections between bacterial physiology and pathology.     

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.     

Diversity, biogenesis and function of microbial amyloids

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimer's, Parkinson's and Huntington's diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.     

Study of Exosomes Shed New Light on Physiology of Amyloidogenesis

Accumulation of toxic amyloid oligomers, a key feature in the pathogenesis of amyloid-related diseases, results from an imbalance between generation and clearance of amyloidogenic proteins. Cell biology has brought to light the key roles of multivesicular endosomes (MVEs) and their intraluminal vesicles (ILVs), which can be secreted as exosomes, in amyloid generation and clearance. To better understand these roles, we have investigated a relevant physiological model of amyloid formation in pigment cells. These cells have tuned their endosomes to optimize the formation of functional amyloid fibrils from the premelanosome protein (PMEL) and to avoid potential accumulation of toxic species. The functional amyloids derived from PMEL reveal striking analogies with the generation of Aβ peptides. We have recently strengthened these analogies using extracellular vesicles as reporters of the endosomal processes that regulate PMEL melanogenesis. We have shown that in pigmented cells, apolipoprotein E (ApoE) is associated with ILVs and exosomes, and regulates the formation of PMEL amyloid fibrils in endosomes. This process secures the generation of amyloid fibrils by exploiting ILVs as amyloid-nucleating platforms. This physiological model of amyloidogenesis could shed new light on the roles of MVEs and exosomes in conditions with pathological amyloid metabolism, such as Alzheimer’s disease.     

pH-Dependent fibril maturation of a Pmel17 repeat domain isoform revealed by tryptophan fluorescence

The pre-melanosomal protein (Pmel17) aggregates within melanosomes to form functional amyloid fibrils that facilitate melanin polymerization. The repeat domain (RPT) of Pmel17 fibrillates under strict acidic melanosomal pH. Alternative splicing results in a shortened repeat domain (sRPT), which also forms amyloid fibrils. Here, we explored the effects of pH and protein concentration on sRPT aggregation by monitoring the intrinsic fluorescence of the sole tryptophan at position 381 (³⁸¹W). ³⁸¹W emission properties revealed changes of local environment polarity for sRPT fibrils formed at different pH. At pH 4, fibrils formed rapidly with no lag phase. A high ³⁸¹W intensity was observed with a slight blue shift (10 nm). These fibrils underwent further structural rearrangements at intermediate pH (5–6), mirroring that of melanosome maturation, which initiates at pH 4 and increases to near neutral pH. In contrast, typical sigmoidal kinetics were observed at pH 6 with slower rates and ³⁸¹W exhibited quenched emission. Interestingly, biphasic kinetics were observed at pH 5 in a protein concentration-dependent manner. A large ³⁸¹W blue shift (23 nm) was measured, indicating a more hydrophobic environment for fibrils made at pH 5. Consistent with ³⁸¹W fluorescence, Raman spectroscopy revealed molecular level perturbations in sRPT fibrils that were not evident from circular dichroism, transmission electron microscopy, or limited proteolysis analysis. Finally, sRPT fibrils did not form at pH ≥7 and preformed fibrils rapidly disaggregated under these solution conditions. Collectively, this work yields mechanistic insights into pH-dependent sRPT aggregation in the context of melanosome maturation.