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1.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(5):519-528
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. 相似文献
2.
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. 相似文献
3.
pH-Dependent fibril maturation of a Pmel17 repeat domain isoform revealed by tryptophan fluorescence
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(10):961-969
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. 相似文献
4.
Ralf M. Leonhardt Nathalie Vigneron Jia Shee Hee Morven Graham Peter Cresswell 《Molecular biology of the cell》2013,24(7):964-981
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. 相似文献
5.
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. 相似文献
6.
McGlinchey RP Shewmaker F Hu KN McPhie P Tycko R Wickner RB 《The Journal of biological chemistry》2011,286(10):8385-8393
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. 相似文献
7.
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. 相似文献
8.
Hoashi T Muller J Vieira WD Rouzaud F Kikuchi K Tamaki K Hearing VJ 《The Journal of biological chemistry》2006,281(30):21198-21208
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. 相似文献
9.
Jonathan S. Wall Angela Williams Craig Wooliver Emily B. Martin Xiaolin Cheng R. Eric Heidel Stephen J. Kennel 《Biochemistry and Biophysics Reports》2016
BackgroundPolybasic helical peptides, such as peptide p5, bind human amyloid extracts and synthetic amyloid fibrils. When radiolabeled, peptide p5 has been shown to specifically bind amyloid in vivo thereby allowing imaging of the disease. Structural requirements for heparin and amyloid binding have been studied using analogues of p5 that modify helicity and chirality.MethodsPeptide-ligand interactions were studied using CD spectroscopy and solution-phase binding assays with radiolabeled p5 analogues. The interaction of a subset of peptides was further studied by using molecular dynamics simulations.ResultsDisruption of the peptide helical structure reduced peptide binding to heparin and human amyloid extracts. The all-D enantiomer and the β-sheet-structured peptide bound all substrates as well as, or better than, p5. The interaction of helical and β-sheet structured peptides with Aβ fibrils was modeled and shown to involve both ionic and non-ionic interactions.ConclusionsThe α-helical secondary structure of peptide p5 is important for heparin and amyloid binding; however, helicity is not an absolute requirement as evidenced by the superior reactivity of a β-sheet peptide. The differential binding of the peptides with heparin and amyloid fibrils suggests that these molecular interactions are different. The all-D enantiomer of p5 and the β-sheet peptide are candidates for amyloid targeting reagents in vivo.General SignificanceEfficient binding of polybasic peptides with amyloid is dependent on the linearity of charge spacing in the context of an α-helical secondary structure. Peptides with an α-helix or β-sheet propensity and with similar alignment of basic residues is optimal. 相似文献
10.
《生物化学与生物物理学报:生物膜》2018,1860(9):1840-1847
α-Synuclein (α-Syn) is a presynaptic protein that is accumulated in its amyloid form in the brains of Parkinson's patients. Although its biological function remains unclear, α-syn has been suggested to bind to synaptic vesicles and facilitate neurotransmitter release. Recently, studies have found that α-syn induces membrane tubulation, highlighting a potential mechanism for α-syn to stabilize highly curved membrane structures which could have both functional and dysfunctional consequences. To understand how membrane remodeling by α-syn affects amyloid formation, we have studied the α-syn aggregation process in the presence of phosphatidylglycerol (PG) micellar tubules, which were the first reported example of membrane tubulation by α-syn. Aggregation kinetics, β-sheet content, and macroscopic protein-lipid structures were observed by Thioflavin T fluorescence, circular dichroism spectroscopy and transmission electron microscopy, respectively. Collectively, the presence of PG micellar tubules formed at a stochiometric (L/P = 1) ratio was found to stimulate α-syn fibril formation. Moreover, transmission electron microscopy and solid-state nuclear magnetic resonance spectroscopy revealed the co-assembly of PG and α-syn into fibril structures. However, isolated micellar tubules do not form fibrils by themselves, suggesting an important role of free α-syn monomers during amyloid formation. In contrast, fibrils did not form in the presence of excess PG lipids (≥L/P = 50), where most of the α-syn molecules are in a membrane-bound α-helical form. Our results provide new mechanistic insights into how membrane tubules modulate α-syn amyloid formation and support a pivotal role of protein–lipid interaction in the dysfunction of α-syn. 相似文献
11.
Theos AC Truschel ST Tenza D Hurbain I Harper DC Berson JF Thomas PC Raposo G Marks MS 《Developmental cell》2006,10(3):343-354
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. 相似文献
12.
Premelanosome amyloid-like fibrils are composed of only golgi-processed forms of Pmel17 that have been proteolytically processed in endosomes 总被引:1,自引:0,他引:1
Harper DC Theos AC Herman KE Tenza D Raposo G Marks MS 《The Journal of biological chemistry》2008,283(4):2307-2322
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. 相似文献
13.
Melanin, the major determinant of skin colour, is a tyrosine‐based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post‐tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin–protein interaction is not nonspecific. 相似文献
14.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2019,1867(5):502-507
The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-β structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma. 相似文献
15.
Sharma S Wagh S Govindarajan R 《Pigment cell research / sponsored by the European Society for Pigment Cell Research and the International Pigment Cell Society》2002,15(2):127-133
Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific. 相似文献
16.
Karunakar Kar Irene Arduini Kenneth W. Drombosky Patrick C.A. van der Wel Ronald Wetzel 《Journal of molecular biology》2014
Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins. 相似文献
17.
《Journal of molecular biology》2023,435(17):168194
The protein tau is involved in several neurogenerative diseases such as Alzheimer’s Disease, where tau content and fibrillation have been linked to disease progression. Tau colocalizes with phospholipids and glycosaminoglycans in vivo. We investigated if and how tau fibrillation can be induced by two lysophospholipids, namely the zwitterionic 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC) and the anionic 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPG) as well as the glycosaminoglycan heparin. We used a range of biophysical techniques including small-angle X-ray scattering, Thioflavin T fluorescence, and SDS-PAGE, collecting data at various time points to obtain structural information on each phase of the fibrillation. We find that LPC does not induce fibrillation or interact with tau. Low concentrations of LPG induce fibrillation by formation of small hydrophobic clusters with monomeric tau. At higher LPG concentrations, a core–shell complex is formed where tau wraps around LPG micelles with regions extending away from the micelles. For heparin, loosely associated oligomers are formed rapidly with around ten tau molecules. Fibrils formed with either LPG or heparin show similar final cross-section dimensions. Furthermore, SDS-resistant oligomers are observed for both LPG and heparin. Our study demonstrates that tau fibrillation can be induced by two different biologically relevant cofactors leading to structurally different initial states but similar cross-sectional dimensions for the fibrils. Structural information about initial states prior to fibril formation is important both to gain a better understanding of the onset of fibrillation in vivo, and for the development of targeted drugs that can reduce or abolish tau fibrillation. 相似文献
18.
Markus P. Kummer Hiroko Maruyama Claudia Huelsmann Sandra Baches Sascha Weggen Edward H. Koo 《The Journal of biological chemistry》2009,284(4):2296-2306
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. 相似文献
19.
Khurana R Coleman C Ionescu-Zanetti C Carter SA Krishna V Grover RK Roy R Singh S 《Journal of structural biology》2005,151(3):229-238
Thioflavin T is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. In aqueous solutions, thioflavin T was found to exist as micelles at concentrations commonly used to monitor fibrils by fluorescence assay ( approximately 10-20 microM). Specific conductivity changes were measured at varying concentration of thioflavin T and the critical micellar concentration was calculated to be 4.0+/-0.5 microM. Interestingly, changes in the fluorescence excitation and emission of thioflavin T were also dependent on the micelle formation. The thioflavin T micelles of 3 nm diameter were directly visualized using atomic force microscopy, and bound thioflavin T micelles were observed along the fibril length for representative fibrils. Increasing concentration of thioflavin T above the critical micellar concentration shows increased numbers of micelles bound along the length of the amyloid fibrils. Thioflavin T micelles were disrupted at low pH as observed by atomic force microscopy and fluorescence enhancement upon binding of thioflavin T to amyloid fibrils also reduced by several-fold upon decreasing the pH to below 3. This suggests that positive charge on the thioflavin T molecule has a role in its micelle formation that then bind the amyloid fibrils. Our data suggests that the micelles of thioflavin T bind amyloid fibrils leading to enhancement of fluorescence emission. 相似文献
20.
Valencia JC Rouzaud F Julien S Chen KG Passeron T Yamaguchi Y Abu-Asab M Tsokos M Costin GE Yamaguchi H Jenkins LM Nagashima K Appella E Hearing VJ 《The Journal of biological chemistry》2007,282(15):11266-11280
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. 相似文献