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.     

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.     

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.     

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.     

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.     

Dual loss of ER export and endocytic signals with altered melanosome morphology in the silver mutation of Pmel17

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine-based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.     

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.     

Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of Pmel17 that have been proteolytically processed in endosomes

Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.     

Sialylated core 1 O-glycans influence the sorting of Pmel17/gp100 and determine its capacity to form fibrils

Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form mature, fibrillar, and pigmented organelles. Recently, we reported that the less glycosylated form of Pmel17 (termed iPmel17) is sorted via the plasma membrane in a manner distinct from mature Pmel17 (termed mPmel17), which is sorted directly to melanosomes. To clarify the mechanism(s) underlying the distinct processing and sorting of Pmel17, we generated a highly specific antibody (termed alphaPEP25h) against an epitope within the repeat domain of Pmel17 that is sensitive to changes in O-glycosylation. alphaPEP25h recognizes only iPmel17 and allows analysis of the processing and sorting of iPmel17 when compared with alphaPEP13h, an antibody that recognizes both iPmel17 and mPmel17. Our novel findings using alphaPEP25h demonstrate that iPmel17 differs from mPmel17 not only in its sensitivity to endoglycosidase H, but also in the content of core 1 O-glycans modified with sialic acid. This evidence reveals that iPmel17 is glycosylated differently in the Golgi and that it is sorted through the secretory pathway. Analysis of Pmel17 processing in glycosylation-deficient mutant cells reveals that Pmel17 lacking the correct addition of sialic acid and galactose loses the ability to form fibrils. Furthermore, we show that addition of sialic acid affects the stability and sorting of Pmel17 and reduces pigmentation. Alterations in sialyltransferase activity and substrates differ between normal and transformed melanocytes and may represent a critical change during malignant transformation.     

The repeat domain of the melanosomal matrix protein PMEL17/GP100 is required for the formation of organellar fibers

Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.     

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.     

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.     

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.     

Functional amyloid formation within mammalian tissue

Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.     

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.     

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.     

Amyloid fibril formation of the mouse V(L) domain at acidic pH

The recombinant V(L) domain that represents the variable part of the light chain (type kappa) of mouse monoclonal antibody F11 directed against human spleen ferritin was found to form amyloid fibrils at acidic pH as evidenced by electron microscopy, thioflavin T binding, and apple-green birefringence after Congo red staining. This is the first demonstration of amyloid fibril formation of the mouse V(L) domain. To understand the mechanism of acidic pH-induced amyloid fibril formation, conformational changes of the V(L) domain were studied by one-dimensional NMR, differential scanning calorimetry, analytical ultracentrifugation, hydrophobic dye binding, far-UV circular dichroism, and tryptophan fluorescence. The results indicated accumulation of two intermediate states during acid unfolding, which might be responsible for amyloid fibril formation. The more structured intermediate that exhibited maximal accumulation at pH 3 retained the nativelike secondary structure and a hydrophobic core, but exposed hydrophobic surfaces that bind 8-anilino-1-naphthalenesulfonate. Below pH 2, a more disordered intermediate with dequenched tryptophan fluorescence but still retaining the beta-sheet structure accumulated. The optimal pH of amyloid fibril formation (i.e., pH 4) was close to the optimal pH of the accumulation of the nativelike intermediate, suggesting that the amyloid fibrils might be formed through this intermediate.     

Critical residues in the PMEL/Pmel17 N-terminus direct the hierarchical assembly of melanosomal fibrils

PMEL (also called Pmel17 or gp100) is a melanocyte/melanoma-specific glycoprotein that plays a critical role in melanosome development by forming a fibrillar amyloid matrix in the organelle for melanin deposition. Although ultimately not a component of mature fibrils, the PMEL N-terminal region (NTR) is essential for their formation. By mutational analysis we establish a high-resolution map of this domain in which sequence elements and functionally critical residues are assigned. We show that the NTR functions in cis to drive the aggregation of the downstream polycystic kidney disease (PKD) domain into a melanosomal core matrix. This is essential to promote in trans the stabilization and terminal proteolytic maturation of the repeat (RPT) domain–containing MαC units, precursors of the second fibrillogenic fragment. We conclude that during melanosome biogenesis the NTR controls the hierarchical assembly of melanosomal fibrils.