Direct observation of the interconversion of normal and toxic forms of α-synuclein.

Here, we use single-molecule techniques to study the aggregation of α-synuclein, the protein whose misfolding and deposition is associated with Parkinson's disease. We identify a conformational change from the initially formed oligomers to stable, more compact proteinase-K-resistant oligomers as the key step that leads ultimately to fibril formation. The oligomers formed as a result of the structural conversion generate much higher levels of oxidative stress in rat primary neurons than do the oligomers formed initially, showing that they are more damaging to cells. The structural conversion is remarkably slow, indicating a high kinetic barrier for the conversion and suggesting that there is a significant period of time for the cellular protective machinery to operate and potentially for therapeutic intervention, prior to the onset of cellular damage. In the absence of added soluble protein, the assembly process is reversed and fibrils disaggregate to form stable oligomers, hence acting as a source of cytotoxic species.

Discriminating α-synuclein strains in Parkinson's disease and multiple system atrophy.

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.

Computer-assisted semi-rational design enhanced the enzymatic activity and protein stability of Proteinase K in calcium-free conditions.

Wild-type Proteinase K binds to two Ca2+ ions, which play an important role in regulating enzymaticactivity and maintaining protein stability. Therefore, a predetermined concentration of Ca2+ must be added during the use of Proteinase K, which increases its commercial cost. Herein, we addressed this challenge using a computational strategy to engineer a Proteinase K mutant that does not require Ca2+ and exhibits high enzymatic activity and protein stability. In the absence of Ca2+, the best mutant, MT24 (S17W-S176N-D260F), displayed an activity approximately 9.2-fold higher than that of wild-type Proteinase K. It also exhibited excellent protein stability, retaining 56.2 % of its enzymatic activity after storage at 4 °C for 5 days. The residual enzymatic activity was 65-fold higher than that of the wild-type Proteinase K under the same storage conditions. Structural analysis and molecular dynamics simulations suggest that the introduction of new hydrogen bond and π-π stacking at the Ca2+ binding sites due to the mutation may be the reasons for the increased enzymatic activity and stability of MT24.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Improvement of RNA In Situ Hybridisation for Grapevine Fruits and Ovules.

The European grapevine (Vitis vinifera L.) is one of the world's most widely cultivated and economically important fruit crops. Seedless fruits are particularly desired for table grapes, with seedlessness resulting from stenospermocarpy being an important goal for cultivar improvement. The establishment of an RNA in situ hybridisation (ISH) system for grape berries and ovules is, therefore, important for understanding the molecular mechanisms of ovule abortion in stenospermocarpic seedless cultivars. We improved RNA in situ hybridisation procedures for developing berries and ovules by targeting two transcription factor genes, VvHB63 and VvTAU, using two seeded varieties, 'Red Globe' and 'Pinot Noir', and two seedless cultivars, 'Flame Seedless' and 'Thompson Seedless'. Optimisation focused on the time of proteinase K treatment, probe length, probe concentration, hybridisation temperature and post-hybridisation washing conditions. The objectives were to maximise hybridisation signals and minimise background interference, while still preserving tissue integrity. For the target genes and samples tested, the best results were obtained with a pre-hybridisation proteinase K treatment of 30 min, probe length of 150 bp and concentration of 100 ng/mL, hybridisation temperature of 50 °C, three washes with 0.2× saline sodium citrate (SSC) solution and blocking with 1% blocking reagent for 45 min during the subsequent hybridisation. The improved ISH system was used to study the spatiotemporal expression patterns of genes related to ovule development at a microscopic level.

Characterization of Treponema denticola Major Surface Protein (Msp) by Deletion Analysis and Advanced Molecular Modeling.

Treponema denticola, a keystone pathogen in periodontitis, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp forms an oligomeric outer membrane complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular processes in host cells. T. denticola msp is an ortholog of the Treponema pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. We recently identified the primary Msp surface-exposed epitope and proposed a model of the Msp protein as a ß-barrel protein similar to Gram-negative bacterial porins. Here, we report fine-scale Msp mutagenesis demonstrating that both the N and C termini as well as the centrally located Msp surface epitope are required for native Msp oligomer expression. Removal of as few as three C-terminal amino acids abrogated Msp detection on the T. denticola cell surface, and deletion of four residues resulted in complete loss of detectable Msp. Substitution of a FLAG tag for either residues 6 to 13 of mature Msp or an 8-residue portion of the central Msp surface epitope resulted in expression of full-length Msp but absence of the oligomer, suggesting roles for both domains in oligomer formation. Consistent with previously reported Msp N-glycosylation, proteinase K treatment of intact cells released a 25 kDa polypeptide containing the Msp surface epitope into culture supernatants. Molecular modeling of Msp using novel metagenome-derived multiple sequence alignment (MSA) algorithms supports the hypothesis that Msp is a large-diameter, trimeric outer membrane porin-like protein whose potential transport substrate remains to be identified. IMPORTANCE The Treponema denticola gene encoding its major surface protein (Msp) is an ortholog of the T. pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. Using a combined strategy of fine-scale mutagenesis and advanced predictive molecular modeling, we characterized the Msp protein and present a high-confidence model of its structure as an oligomer embedded in the outer membrane. This work adds to knowledge of Msp-like proteins in oral treponemes and may contribute to understanding the evolutionary and potential functional relationships between T. denticola Msp and the orthologous T. pallidum Tpr proteins.

Investigation of the Thermal Stability of Proteinase K for the Melt Processing of Poly(l-lactide).

The enzymatic degradation of aliphatic polyesters offers unique opportunities for various use cases in materials science. Although evidently desirable, the implementation of enzymes in technical applications of polyesters is generally challenging due to the thermal lability of enzymes. To prospectively overcome this intrinsic limitation, we here explored the thermal stability of proteinase K at conditions applicable for polymer melt processing, given that this hydrolytic enzyme is well established for its ability to degrade poly(l-lactide) (PLLA). Using assorted spectroscopic methods and enzymatic assays, we investigated the effects of high temperatures on the structure and specific activity of proteinase K. Whereas in solution, irreversible unfolding occurred at temperatures above 75-80 °C, in the dry, bulk state, proteinase K withstood prolonged incubation at elevated temperatures. Unexpectedly little activity loss occurred during incubation at up to 130 °C, and intermediate levels of catalytic activity were preserved at up to 150 °C. The resistance of bulk proteinase K to thermal treatment was slightly enhanced by absorption into polyacrylamide (PAM) particles. Under these conditions, after 5 min at a temperature of 200 °C, which is required for the melt processing of PLLA, proteinase K was not completely denatured but retained around 2% enzymatic activity. Our findings reveal that the thermal processing of proteinase K in the dry state is principally feasible, but equally, they also identify needs and prospects for improvement. The experimental pipeline we establish for proteinase K analysis stands to benefit efforts directed to this end. More broadly, our work sheds light on enzymatically degradable polymers and the thermal processing of enzymes, which are of increasing economical and societal relevance.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

Disease Inhibiting Effect of Strain Bacillus subtilis EG21 and Its Metabolites Against Potato Pathogens Phytophthora infestans and Rhizoctonia solani.

Potato production worldwide is plagued by several disease-causing pathogens that result in crop and economic losses estimated to billions of dollars each year. To this day, synthetic chemical applications remain the most widespread control strategy despite their negative effects on human and environmental health. Therefore, obtainment of superior biocontrol agents or their naturally produced metabolites to replace fungicides or to be integrated into practical pest management strategies has become one of the main targets in modern agriculture. Our main focus in the present study was to elucidate the antagonistic potential of a new strain identified as Bacillus subtilis EG21 against potato pathogens Phytophthora infestans and Rhizoctonia solani using several in vitro screening assays. Microscopic examination of the interaction between EG21 and R. solani showed extended damage in fungal mycelium, while EG21 metabolites displayed strong anti-oomycete and zoosporecidal effect on P. infestans. Mass spectrometry (MS) analysis revealed that EG21 produced antifungal and anti-oomycete cyclic lipopeptides surfactins (C12 to C19). Further characterization of EG21 confirmed its ability to produce siderophores and the extracellular lytic enzymes cellulase, pectinase and chitinase. The antifungal activity of EG21 cell-free culture filtrate (CF) was found to be stable at high-temperature/pressure treatment and extreme pH values and was not affected by proteinase K treatment. Disease-inhibiting effect of EG21 CF against P. infestans and R. solani infection was confirmed using potato leaves and tubers, respectively. Biotechnological applications of using microbial agents and their bioproducts for crop protection hold great promise to develop into effective, environment-friendly and sustainable biocontrol strategies. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Biochemical and Neuropathological Findings in a Creutzfeldt-Jakob Disease Patient with the Rare Val180Ile-129Val Haplotype in the Prion Protein Gene.

Genetic Creutzfeldt-Jakob disease (gCJD) associated with the V180I mutation in the prion protein (PrP) gene (PRNP) in phase with residue 129M is the most frequent cause of gCJD in East Asia, whereas it is quite uncommon in Caucasians. We report on a gCJD patient with the rare V180I-129V haplotype, showing an unusually long duration of the disease and a characteristic pathological PrP (PrPSc) glycotype. Family members carrying the mutation were fully asymptomatic, as commonly observed with this mutation. Neuropathological examination showed a lesion pattern corresponding to that commonly reported in Japanese V180I cases with vacuolization and gliosis of the cerebral cortexes, olfactory areas, hippocampus and amygdala. PrP was deposited with a punctate, synaptic-like pattern in the cerebral cortex, amygdala and olfactory tract. Western blot analyses of proteinase-K-resistant PrP showed the characteristic two-banding pattern of V180I gCJD, composed of mono- and un-glycosylated isoforms. In line with reports on other V180I cases in the literature, Real-Time Quaking Induced Conversion (RT-QuIC) analyses did not demonstrate the presence of seeding activity in the cerebrospinal fluid and olfactory mucosa, suggesting that this haplotype also may result in a reduced seeding efficiency of the pathological PrP. Further studies are required to understand the origin, penetrance, disease phenotype and transmissibility of 180I-129V haplotype in Caucasians.

Structural Perturbations of Exon-Skipping Edits within the Dystrophin D20:24 Region.

Exon skipping is a disease-modifying therapy in which oligonucleotide analogues mask specific exons, eliminating them from the mature mRNA, and also the cognate protein. That is one possible therapeutic aim, but it can also be used to restore the reading frame for diseases caused by frameshift mutations, which is the case for Duchenne muscular dystrophy (DMD). DMD most commonly arises as a result of large exonic deletions that create a frameshift and abolish protein expression. Loss of dystrophin protein leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is restoration of protein expression by reading frame correction. However, the therapeutically expressed protein is missing both the region of the underlying genetic defect and the therapeutically skipped exon. How removing some region from the middle of a protein affects its structure and function is unclear. Many different underlying deletions are known, and exon skipping can be applied in many ways, in some cases in different ways to the same defect. These vary in how severely perturbative they are, with possible clinical consequences. In this study, we examine a systematic, comprehensive panel of exon edits in a region of dystrophin and identify for the first time exon edits that are minimally perturbed and appear to keep the structural stability similar to that of wild-type protein. We also identify factors that appear to be correlated with how perturbative an edit is.

An automated, high-throughput methodology optimized for quantitative cell-free mitochondrial and nuclear DNA isolation from plasma.

Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of ∼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.

The use of a two-step removal protocol and optimized culture conditions improve development and quality of zona free mouse embryos.

The zona pellucida (ZP) plays an important role in both the fertilization and embryonic development. For the successful handling of early stage blastomeres for differentiation analysis, the production of identical twins or quadruplets, nuclear transfer or gene introduction requires the removal of the ZP (ZPR). Although single use of either acidic Tyrode's solution or pronase are commonly used for ZPR, long-term exposure to these agents can result in the inhibition of development with the collapse of the three-dimensional blastomere structure. Here, we demonstrate the benefits of using a two-step combined ZPR method, which relies upon a customized well-of-well (cWOW) system with smaller well size, on developmental competence and the quality of the zona free (ZF) mouse embryos. We first isolated 2-cell embryos using acid Tyrode's solution and then cultured these embryos using either commercially available or cWOW, which had a smaller microwell size. The rate of blastocyst was significantly increased by use of cWOW when compared to other culture systems. Then we evaluated the use of a two-step ZPR protocol, relying on acid Tyrode's solution and proteinase K, and subsequent culture in the cWOW system. Although acid Tyrode's solution treatment alone reduced ZPR time, blastomere morphology became wrinkled, significant decrease in blastocyst rate associated with increased number of apoptotic cells and increased expression of apoptosis-related genes were observed. Using proteinase K alone increased ZPR time and significantly decreased the blastocyst rate, but did not induce an increase in apoptotic cell number or apoptosis-related gene expression. In contrast, two-step method significantly reduced ZPR time and improved blastocyst rate by increasing the total number of cells in these wells an reducing the number of apoptotic cells in these experiments. These results suggest that the two-step ZPR protocol is beneficial for reducing the toxic effects of zona removal on ZF embryo development and quality when combined with a suitable culture system.

Speciation of Transition-Metal-Substituted Keggin-Type Silicotungstates Affected by the Co-crystallization Conditions with Proteinase K.

We report on the synthesis of the tetrasubstituted sandwich-type Keggin silicotungstates as the pure Na salts Na14[(A-α-SiW10O37)2{Co4(OH)2(H2O)2}]·37H2O (Na{SiW10Co2}2) and Na14[(A-α-SiW10O37)2{Ni4(OH)2(H2O)2}]·77.5H2O (Na{SiW10Ni2}2), which were prepared by applying a new synthesis protocol and characterized thoroughly in the solid state by single-crystal and powder X-ray diffraction, IR spectroscopy, thermogravimetric analysis, and elemental analysis. Proteinase K was applied as a model protein and the polyoxotungstate (POT)-protein interactions of Na{SiW10Co2}2 and Na{SiW10Ni2}2 were studied side by side with the literature-known K5Na3[A-α-SiW9O34(OH)3{Co4(OAc)3}]·28.5H2O ({SiW9Co4}) featuring the same number of transition metals. Testing the solution behavior of applied POTs under the crystallization conditions (sodium acetate buffer, pH 5.5) by time-dependent UV/vis spectroscopy and electrospray ionization mass spectrometry speciation studies revealed an initial dissociation of the sandwich POTs to the disubstituted Keggin anions HxNa5-x[SiW10Co2O38]3- and HxNa5-x[SiW10Ni2O38]3- ({SiW10M2}, M = CoII and NiII) followed by partial rearrangement to the monosubstituted compounds (α-{SiW11Co} and α-{SiW11Ni}) after 1 week of aging. The protein crystal structure analysis revealed monosubstituted α-Keggin POTs in two conserved binding positions for all three investigated compounds, with one of these positions featuring a covalent attachment of the POT anion to an aspartate carboxylate. Despite the presence of both mono- and disubstituted anions in a crystallization mixture, proteinase K selectively binds to monosubstituted anions because of their preferred charge density for POT-protein interaction.

α-Synuclein BAC transgenic mice exhibit RBD-like behaviour and hyposmia: a prodromal Parkinson's disease model.

Parkinson's disease is one of the most common movement disorders and is characterized by dopaminergic cell loss and the accumulation of pathological α-synuclein, but its precise pathogenetic mechanisms remain elusive. To develop disease-modifying therapies for Parkinson's disease, an animal model that recapitulates the pathology and symptoms of the disease, especially in the prodromal stage, is indispensable. As subjects with α-synuclein gene (SNCA) multiplication as well as point mutations develop familial Parkinson's disease and a genome-wide association study in Parkinson's disease has identified SNCA as a risk gene for Parkinson's disease, the increased expression of α-synuclein is closely associated with the aetiology of Parkinson's disease. In this study we generated bacterial artificial chromosome transgenic mice harbouring SNCA and its gene expression regulatory regions in order to maintain the native expression pattern of α-synuclein. Furthermore, to enhance the pathological properties of α-synuclein, we inserted into SNCA an A53T mutation, two single-nucleotide polymorphisms identified in a genome-wide association study in Parkinson's disease and a Rep1 polymorphism, all of which are causal of familial Parkinson's disease or increase the risk of sporadic Parkinson's disease. These A53T SNCA bacterial artificial chromosome transgenic mice showed an expression pattern of human α-synuclein very similar to that of endogenous mouse α-synuclein. They expressed truncated, oligomeric and proteinase K-resistant phosphorylated forms of α-synuclein in the regions that are specifically affected in Parkinson's disease and/or dementia with Lewy bodies, including the olfactory bulb, cerebral cortex, striatum and substantia nigra. Surprisingly, these mice exhibited rapid eye movement (REM) sleep without atonia, which is a key feature of REM sleep behaviour disorder, at as early as 5 months of age. Consistent with this observation, the REM sleep-regulating neuronal populations in the lower brainstem, including the sublaterodorsal tegmental nucleus, nuclei in the ventromedial medullary reticular formation and the pedunculopontine nuclei, expressed phosphorylated α-synuclein. In addition, they also showed hyposmia at 9 months of age, which is consistent with the significant accumulation of phosphorylated α-synuclein in the olfactory bulb. The dopaminergic neurons in the substantia nigra pars compacta degenerated, and their number was decreased in an age-dependent manner by up to 17.1% at 18 months of age compared to wild-type, although the mice did not show any related locomotor dysfunction. In conclusion, we created a novel mouse model of prodromal Parkinson's disease that showed RBD-like behaviour and hyposmia without motor symptoms.

Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study.

Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

Proteinase K is an activator for the male-dependent spermiogenesis pathway in Caenorhabditis elegans: Its application to pharmacological dissection of spermiogenesis.

Caenorhabditis elegans spermiogenesis involves spermatid activation into spermatozoa. Activation occurs through either SPE-8 class-dependent or class-independent pathways. Pronase (Pron) activates the SPE-8 class-dependent pathway, whereas no in vitro tools are available to stimulate the SPE-8 class-independent pathway. Thus, whether there is a functional relationship between these two pathways is currently unclear. In this study, we found that proteinase K (ProK) can activate the SPE-8 class-independent pathway. In vitro spermiogenesis assays using Pron and ProK suggested that SPE-8 class proteins act in the hermaphrodite- and male-dependent spermiogenesis pathways and that some spermatid proteins presumably working downstream of spermiogenesis pathways, including MAP kinases, are preferentially involved in the SPE-8 class-dependent pathway. We screened a library of chemicals, and a compound that we named DDI-1 inhibited both Pron- and ProK-induced spermiogenesis. To our surprise, several DDI-1 analogues that are structurally similar to DDI-1 blocked Pron, but not ProK, induced spermiogenesis. Although the mechanism by which DDI-1 blocks spermiogenesis is yet unknown, we have begun to address this issue by selecting two DDI-1-resistant mutants. Collectively, our data support a model in which C. elegans male and hermaphrodite spermiogenesis each has its own distinct, parallel pathway.

Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species.

Although misfolding of normal prion protein (PrPC) into abnormal conformers (PrPSc) is critical for prion disease pathogenesis our current understanding of the underlying molecular pathophysiology is rudimentary. Exploiting an electrophysiology paradigm, herein we report that at least modestly proteinase K (PK)-resistant PrPSc (PrPres) species are acutely synaptotoxic. Brief exposure to ex vivo PrPSc from two mouse-adapted prion strains (M1000 and MU02) prepared as crude brain homogenates (cM1000 and cMU02) and cell lysates from chronically M1000-infected RK13 cells (MoRK13-Inf) caused significant impairment of hippocampal CA1 region long-term potentiation (LTP), with the LTP disruption approximating that reported during the evolution of murine prion disease. Proof of PrPSc (especially PrPres) species as the synaptotoxic agent was demonstrated by: significant rescue of LTP following selective immuno-depletion of total PrP from cM1000 (dM1000); modestly PK-treated cM1000 (PK+M1000) retaining full synaptotoxicity; and restoration of the LTP impairment when employing reconstituted, PK-eluted, immuno-precipitated M1000 preparations (PK+IP-M1000). Additional detailed electrophysiological analyses exemplified by impairment of post-tetanic potentiation (PTP) suggest possible heightened pre-synaptic vulnerability to the acute synaptotoxicity. This dysfunction correlated with cumulative insufficiency of replenishment of the readily releasable pool (RRP) of vesicles during repeated high-frequency stimulation utilised for induction of LTP. Broadly comparable results with LTP and PTP impairment were obtained utilizing hippocampal slices from PrPC knockout (PrPo/o) mice, with cM1000 serial dilution assessments revealing similar sensitivity of PrPo/o and wild type (WT) slices. Size fractionation chromatography demonstrated that synaptotoxic PrP correlated with PK-resistant species >100kDa, consistent with multimeric PrPSc, with levels of these species >6 ng/ml appearing sufficient to induce synaptic dysfunction. Biochemical analyses of hippocampal slices manifesting acute synaptotoxicity demonstrated reduced levels of multiple key synaptic proteins, albeit with noteworthy differences in PrPo/o slices, while such changes were absent in hippocampi demonstrating rescued LTP through treatment with dM1000. Our findings offer important new mechanistic insights into the synaptic impairment underlying prion disease, enhancing prospects for development of targeted effective therapies.

High-resolution crystal structures of a "half sandwich"-type Ru(II) coordination compound bound to hen egg-white lysozyme and proteinase K.

The high-resolution X-ray crystal structures of the adducts formed between the "half sandwich"-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes.