The N terminus of α-synuclein dictates fibril formation.

The generation of α-synuclein (α-syn) truncations from incomplete proteolysis plays a significant role in the pathogenesis of Parkinson's disease. It is well established that C-terminal truncations exhibit accelerated aggregation and serve as potent seeds in fibril propagation. In contrast, mechanistic understanding of N-terminal truncations remains ill defined. Previously, we found that disease-related C-terminal truncations resulted in increased fibrillar twist, accompanied by modest conformational changes in a more compact core, suggesting that the N-terminal region could be dictating fibril structure. Here, we examined three N-terminal truncations, in which deletions of 13-, 35-, and 40-residues in the N terminus modulated both aggregation kinetics and fibril morphologies. Cross-seeding experiments showed that out of the three variants, only ΔN13-α-syn (14‒140) fibrils were capable of accelerating full-length fibril formation, albeit slower than self-seeding. Interestingly, the reversed cross-seeding reactions with full-length seeds efficiently promoted all but ΔN40-α-syn (41-140). This behavior can be explained by the unique fibril structure that is adopted by 41-140 with two asymmetric protofilaments, which was determined by cryogenic electron microscopy. One protofilament resembles the previously characterized bent ß-arch kernel, comprised of residues E46‒K96, whereas in the other protofilament, fewer residues (E61‒D98) are found, adopting an extended ß-hairpin conformation that does not resemble other reported structures. An interfilament interface exists between residues K60‒F94 and Q62‒I88 with an intermolecular salt bridge between K80 and E83. Together, these results demonstrate a vital role for the N-terminal residues in α-syn fibril formation and structure, offering insights into the interplay of α-syn and its truncations.

C-terminal α-synuclein truncations are linked to cysteine cathepsin activity in Parkinson's disease.

A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids-long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCAA53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1-122 and 1-90 from the SNCAA53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.

A characterization of Gaucher iPS-derived astrocytes: Potential implications for Parkinson's disease.

While astrocytes, the most abundant cells found in the brain, have many diverse functions, their role in the lysosomal storage disorder Gaucher disease (GD) has not been explored. GD, resulting from the inherited deficiency of the enzyme glucocerebrosidase and subsequent accumulation of glucosylceramide and its acylated derivative glucosylsphingosine, has both non-neuronopathic (GD1) and neuronopathic forms (GD2 and 3). Furthermore, mutations in GBA1, the gene mutated in GD, are an important risk factor for Parkinson's disease (PD). To elucidate the role of astrocytes in the disease pathogenesis, we generated iAstrocytes from induced pluripotent stem cells made from fibroblasts taken from controls and patients with GD1, with and without PD. We also made iAstrocytes from an infant with GD2, the most severe and progressive form, manifesting in infancy. Gaucher iAstrocytes appropriately showed deficient glucocerebrosidase activity and levels and substrate accumulation. These cells exhibited varying degrees of astrogliosis, Glial Fibrillary Acidic Protein (GFAP) up-regulation and cellular proliferation, depending on the level of residual glucocerebrosidase activity. Glutamte uptake assays demonstrated that the cells were functionally active, although the glutamine transporter EEAT2 was upregulated and EEAT1 downregulated in the GD2 samples. GD2 iAstrocytes were morphologically different, with severe cytoskeletal hypertrophy, overlapping of astrocyte processes, pronounced up-regulation of GFAP and S100ß, and significant astrocyte proliferation, recapitulating the neuropathology observed in patients with GD2. Although astrocytes do not express α-synuclein, when the iAstrocytes were co-cultured with dopaminergic neurons generated from the same iPSC lines, excessive α-synuclein released from neurons was endocytosed by astrocytes, translocating into lysosomes. Levels of aggregated α-synuclein increased significantly when cells were treated with monomeric or fibrillar α-synuclein. GD1-PD and GD2 iAstrocytes also exhibited impaired Cathepsin D activity, leading to further α-synuclein accumulation. Cytokine and chemokine profiling of the iAstrocytes demonstrated an inflammatory response. Thus, in patients with GBA1-associated parkinsonism, astrocytes appear to play a role in α-synuclein accumulation and processing, contributing to neuroinflammation.

Cathepsin K is a potent disaggregase of α-synuclein fibrils.

The intracellular accumulation of α-synuclein (α-syn) amyloid fibrils is a hallmark of Parkinson's disease. Because lysosomes are responsible for degrading aggregated species, enhancing lysosomal function could alleviate the overburden of α-syn. Previously, we showed that cysteine cathepsins (Cts) is the main class of lysosomal proteases that degrade α-syn, and in particular, CtsL was found to be capable of digesting α-syn fibrils. Here, we report that CtsK is a more potent protease for degrading α-syn amyloids. Using peptide mapping by liquid chromatography with mass spectrometry, critical cleavage sites involved in destabilizing fibril structure are identified. CtsK is only able to devour the internal regions after the removal of both N- and C-termini, indicating their protective role of the amyloid core from proteolytic attack. Our results suggest that if overexpressed in lysosomes, CtsK has the potential to ameliorate α-syn pathology.

Structural features of α-synuclein amyloid fibrils revealed by Raman spectroscopy.

Parkinson's disease (PD) is associated with the formation of α-synuclein amyloid fibrils. Elucidating the role of these ß-sheet-rich fibrils in disease progression is crucial; however, collecting detailed structural information on amyloids is inherently difficult because of their insoluble, non-crystalline, and polymorphic nature. Here, we show that Raman spectroscopy is a facile technique for characterizing structural features of α-synuclein fibrils. Combining Raman spectroscopy with aggregation kinetics and transmission electron microscopy, we examined the effects of pH and ionic strength as well as four PD-related mutations (A30P, E46K, G51D, and A53T) on α-synuclein fibrils. Raman spectral differences were observed in the amide-I, amide-III, and fingerprint regions, indicating that secondary structure and tertiary contacts are influenced by pH and to a lesser extent by NaCl. Faster aggregation times appear to facilitate unique fibril structure as determined by the highly reproducible amide-I band widths, linking aggregation propensity and fibril polymorphism. Importantly, Raman spectroscopy revealed molecular-level perturbations of fibril conformation by the PD-related mutations that are not apparent through transmission electron microscopy or limited proteolysis. The amide-III band was found to be particularly sensitive, with G51D exhibiting the most distinctive features, followed by A53T and E46K. Relating to a cellular environment, our data would suggest that fibril polymorphs can be formed in different cellular compartments and potentially result in distinct phenotypes. Our work sets a foundation toward future cellular Raman studies of amyloids.

Cysteine cathepsins are essential in lysosomal degradation of α-synuclein.

A cellular feature of Parkinson's disease is cytosolic accumulation and amyloid formation of α-synuclein (α-syn), implicating a misregulation or impairment of protein degradation pathways involving the proteasome and lysosome. Within lysosomes, cathepsin D (CtsD), an aspartyl protease, is suggested to be the main protease for α-syn clearance; however, the protease alone only generates amyloidogenic C terminal-truncated species (e.g., 1-94, 5-94), implying that other proteases and/or environmental factors are needed to facilitate degradation and to avoid α-syn aggregation in vivo. Using liquid chromatography-mass spectrometry, to our knowledge, we report the first peptide cleavage map of the lysosomal degradation process of α-syn. Studies of purified mouse brain and liver lysosomal extracts and individual human cathepsins demonstrate a direct involvement of cysteine cathepsin B (CtsB) and L (CtsL). Both CtsB and CtsL cleave α-syn within its amyloid region and circumvent fibril formation. For CtsD, only in the presence of anionic phospholipids can this protease cleave throughout the α-syn sequence, suggesting that phospholipids are crucial for its activity. Taken together, an interplay exists between α-syn conformation and cathepsin activity with CtsL as the most efficient under the conditions examined. Notably, we discovered that CtsL efficiently degrades α-syn amyloid fibrils, which by definition are resistant to broad spectrum proteases. This work implicates CtsB and CtsL as essential in α-syn lysosomal degradation, establishing groundwork to explore mechanisms to enhance their cellular activity and levels as a potential strategy for clearance of α-syn.

Taking a Bite Out of Amyloid: Mechanistic Insights into α-Synuclein Degradation by Cathepsin L.

A common hallmark of amyloids is their resistance to an array of proteases, highlighting the difficulty in degrading these disease-related aggregated proteinaceous materials. Here, we report on the potent activity of cathepsin L (CtsL), a lysosomal protease that proteolyzes the Parkinson's disease-related amyloid formed by α-synuclein (α-syn). Using liquid chromatography with mass spectrometry and transmission electron microscopy, an elegant mechanism is revealed on the residue and ultrastructural level, respectively. Specifically, CtsL always truncates α-syn fibrils first at the C-terminus before attacking the internal ß-sheet-rich region between residues 30 and 100. This suggests that only upon removal of the α-syn C-terminus can CtsL gain access to residues within the amyloid core. Interestingly, three of the four mapped sites contain a glycine residue (G36, G41, and G51) that is likely to be involved in a ß-turn in the fibril, whereupon cutting would lead to solvent exposure of internal residues and allow further proteolysis. Via close inspection of the fibril morphology, products resulting from CtsL degradation show imperfections along the fibril axis, with missing protein density as though they have been cannibalized. The ability of CtsL to degrade α-syn amyloid fibrils offers a promising strategy for improving the cellular clearance of aggregated α-syn through the modulation of protease levels and activity.

Reversing the amyloid trend: Mechanism of fibril assembly and dissolution of the repeat domain from a human functional amyloid.

Amyloids are traditionally observed in the context of disease. However, there is growing momentum that these structures can serve a beneficial role where the amyloid carries out a specific function. These so called 'functional amyloids' have all the structural hallmarks of disease-associated amyloids, raising the question as to what differentiates a well-behaved benign amyloid from a lethally destructive one. Here, we review our work on the repeat domain (RPT) from Pmel17, an important functional amyloid involved in melanin biosynthesis. Particularly, we focused our attention on the unique reversible aggregation-disaggregation process of RPT that is controlled strictly by solution pH. This pH dependence of RPT amyloid formation functions as a switch to control fibril assembly and maintains the benign nature that is associated with functional amyloids.

Molecular origin of pH-dependent fibril formation of a functional amyloid.

Fibrils derived from Pmel17 are functional amyloids upon which melanin is deposited. Fibrils of the repeat domain (RPT) of Pmel17 form under strict melanosomal pH (4.5-5.5) and completely dissolve at pH≥6. To determine which Glu residue is responsible for this reversibility, aggregation of single, double, and quadruple Ala and Gln mutants were examined by intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy. Charge neutralization of E404, E422, E425, or E430, which are located in the putative amyloid-forming region, modulated aggregation kinetics. Remarkably, the removal of a single negative charge at E422, one of 16 carboxylic acids, shifted the pH dependence by a full pH unit. Mutation at E404, E425, or E430 had little to no effect. We suggest that protonation at E422 is essential for initiating amyloid formation and that the other Glu residues play an allosteric role in fibril stability.

Suicidal [PSI+] is a lethal yeast prion.

[PSI(+)] is a prion of the essential translation termination factor Sup35p. Although mammalian prion infections are uniformly fatal, commonly studied [PSI(+)] variants do not impair growth, leading to suggestions that [PSI(+)] may protect against stress conditions. We report here that over half of [PSI(+)] variants are sick or lethal. These "killer [PSI(+)]s" are compatible with cell growth only when also expressing minimal Sup35C, lacking the N-terminal prion domain. The severe detriment of killer [PSI(+)] results in rapid selection of nonkiller [PSI(+)] variants or loss of the prion. We also report variants of [URE3], a prion of the nitrogen regulation protein Ure2p, that grow much slower than ure2Δ cells. Our findings give a more realistic picture of the impact of the prion change than does focus on "mild" prion variants.

Emerging insights into the mechanistic link between α-synuclein and glucocerebrosidase in Parkinson's disease.

Mutations in the GBA1 gene, encoding the enzyme glucocerebrosidase, cause the lysosomal storage disorder GD (Gaucher's disease), and are associated with the development of PD (Parkinson's disease) and other Lewy body disorders. Interestingly, GBA1 variants are the most common genetic risk factor associated with PD. Although clinical studies argue a strong case towards a link between GBA1 mutations and the development of PD, mechanistic insights have been lacking. In the present article, we review recent findings that have provided some biochemical evidence to bridge this relationship, focusing on the molecular link between two proteins, α-synuclein and glucocerebrosidase, involved in PD and GD respectively.

Effects of pH on aggregation kinetics of the repeat domain of a functional amyloid, Pmel17.

Pmel17 is a functional amyloidogenic protein whose fibrils act as scaffolds for pigment deposition in human skin and eyes. We have used the repeat domain (RPT, residues 315-444), an essential luminal polypeptide region of Pmel17, as a model system to study conformational changes from soluble unstructured monomers to ß-sheet-containing fibrils. Specifically, we report on the effects of solution pH (4 → 7) mimicking pH conditions of melanosomes, acidic organelles where Pmel17 fibrils are formed. Local, secondary, and fibril structure were monitored via intrinsic Trp fluorescence, circular dichroism spectroscopy, and transmission electron microscopy, respectively. We find that W423 is a highly sensitive probe of amyloid assembly with spectral features reflecting local conformational and fibril morphological changes. A critical pH regime (5 ± 0.5) was identified for fibril formation suggesting the involvement of at least three carboxylic acids in the structural rearrangement necessary for aggregation. Moreover, we demonstrate that RPT fibril morphology can be transformed directly by changing solution pH. Based on these results, we propose that intramelanosomal pH regulates Pmel17 amyloid formation and its subsequent dissolution in vivo.

Structural insights into functional and pathological amyloid.

Amyloid is traditionally viewed as a consequence of protein misfolding and aggregation and is most notorious for its association with debilitating and chronic human diseases. However, a growing list of examples of "functional amyloid" challenges this bad reputation and indicates that many organisms can employ the biophysical properties of amyloid for their benefit. Because of developments in the structural studies of amyloid, a clearer picture is emerging about what defines amyloid structure and the properties that unite functional and pathological amyloids. Here, we review various amyloids and place them within the framework of the latest structural models.

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 ß-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aß peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.

Insights into fluorometabolite biosynthesis in Streptomyces cattleya DSM46488 through genome sequence and knockout mutants.

Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesise fluorine containing natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-l-methionine:fluoride adenosyltransferase (fluorinase, SCATT_41540), 5'-fluoro-5'-deoxyadenosine phosphorylase (SCATT_41550), fluoroacetyl-CoA thioesterase (SCATT_41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT_20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT_p11780) confirm that they are essential for fluorometabolite production. Notably gene disruption of the transaldolase (SCATT_p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesise 4-fluorothreonine, revealing a branchpoint role for the PLP-transaldolase.

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis.

Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solid-state NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine.

Polyketides are among the major classes of bioactive natural products used to treat microbial infections, cancer, and other diseases. Here we describe a pathway to chloroethylmalonyl-CoA as a polyketide synthase building block in the biosynthesis of salinosporamide A, a marine microbial metabolite whose chlorine atom is crucial for potent proteasome inhibition and anticancer activity. S-adenosyl-L-methionine (SAM) is converted to 5'-chloro-5'-deoxyadenosine (5'-ClDA) in a reaction catalyzed by a SAM-dependent chlorinase as previously reported. By using a combination of gene deletions, biochemical analyses, and chemical complementation experiments with putative intermediates, we now provide evidence that 5'-ClDA is converted to chloroethylmalonyl-CoA in a 7-step route via the penultimate intermediate 4-chlorocrotonyl-CoA. Because halogenation often increases the bioactivity of drugs, the availability of a halogenated polyketide building block may be useful in molecular engineering approaches toward polyketide scaffolds.

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.

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.

Characterization of 5-chloro-5-deoxy-D-ribose 1-dehydrogenase in chloroethylmalonyl coenzyme A biosynthesis: substrate and reaction profiling.

SalM is a short-chain dehydrogenase/reductase enzyme from the marine actinomycete Salinispora tropica that is involved in the biosynthesis of chloroethylmalonyl-CoA, a novel halogenated polyketide synthase extender unit of the proteasome inhibitor salinosporamide A. SalM was heterologously overexpressed in Escherichia coli and characterized in vitro for its substrate specificity, kinetics, and reaction profile. A sensitive real-time (13)C NMR assay was developed to visualize the oxidation of 5-chloro-5-deoxy-D-ribose to 5-chloro-5-deoxy-D-ribono-γ-lactone in an NAD(+)-dependent reaction, followed by spontaneous lactone hydrolysis to 5-chloro-5-deoxy-D-ribonate. Although short-chain dehydrogenase/reductase enzymes are widely regarded as metal-independent, a strong divalent metal cation dependence for Mg(2+), Ca(2+), or Mn(2+) was observed with SalM. Oxidative activity was also measured with the alternative substrates D-erythrose and D-ribose, making SalM the first reported stereospecific non-phosphorylative ribose 1-dehydrogenase.