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.

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.

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).

Structural Insights into α-Synuclein Fibril Polymorphism: Effects of Parkinson's Disease-Related C-Terminal Truncations.

Lewy bodies, hallmarks of Parkinson's disease, contain C-terminally truncated (ΔC) α-synuclein (α-syn). Here, we report fibril structures of three N-terminally acetylated (Ac) α-syn constructs, Ac1-140, Ac1-122, and Ac1-103, solved by cryoelectron microscopy. Both ΔC-α-syn variants exhibited faster aggregation kinetics, and Ac1-103 fibrils efficiently seeded the full-length protein, highlighting their importance in pathogenesis.

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.

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.

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 β-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.

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.

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.

Segmental Deuteration of α-Synuclein for Neutron Reflectometry on Tethered Bilayers.

Neutron reflectometry (NR) is uniquely suited for studying protein interaction with phospholipid bilayers along the bilayer normal on an angstrom scale. However, NR on its own cannot discern specific membrane-bound regions due to a lack of scattering contrast within a protein. Here we report the successful coupling of native chemical ligation (NCL) and NR to study α-synuclein (α-syn), a membrane-binding neuronal protein central in Parkinson's disease.

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.

Sporadic distribution of prion-forming ability of Sup35p from yeasts and fungi.

Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p's from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms.

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.

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.

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.

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.

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.

The yin and yang of amyloid: insights from α-synuclein and repeat domain of Pmel17.

Amyloid has been traditionally viewed in the context of disease. However, the emerging concept of 'functional amyloid' has taken a new direction into how we view amyloid. Recent studies have identified amyloid fibrils ranging from bacteria to humans that have a beneficial role, instead of being associated with a misfolded state that has been implicated in diseases such as Alzheimer's, Parkinson's and prion diseases.

Bacterial self-resistance to the natural proteasome inhibitor salinosporamide A.

Proteasome inhibitors have recently emerged as a therapeutic strategy in cancer chemotherapy, but susceptibility to drug resistance limits their efficacy. The marine actinobacterium Salinispora tropica produces salinosporamide A (NPI-0052, marizomib), a potent proteasome inhibitor and promising clinical agent in the treatment of multiple myeloma. Actinobacteria also possess 20S proteasome machinery, raising the question of self-resistance.

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.

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.

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 β-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.

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.

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.