LIPAD (LRRK2/Luebeck International Parkinson's Disease) Study Protocol: Deep Phenotyping of an International Genetic Cohort.

Background: Pathogenic variants in the Leucine-rich repeat kinase 2 (LRRK2) gene are the most common known monogenic cause of Parkinson's disease (PD). LRRK2-linked PD is clinically indistinguishable from idiopathic PD and inherited in an autosomal dominant fashion with reduced penetrance and variable expressivity that differ across ethnicities and geographic regions. Objective: To systematically assess clinical signs and symptoms including non-motor features, comorbidities, medication and environmental factors in PD patients, unaffected LRRK2 pathogenic variant carriers, and controls. A further focus is to enable the investigation of modifiers of penetrance and expressivity of LRRK2 pathogenic variants using genetic and environmental data. Methods: Eligible participants are invited for a personal or online examination which comprises completion of a detailed eCRF and collection of blood samples (to obtain DNA, RNA, serum/plasma, immune cells), urine as well as household dust. We plan to enroll 1,000 participants internationally: 300 with LRRK2-linked PD, 200 with LRRK2 pathogenic variants but without PD, 100 PD patients with pathogenic variants in the GBA or PRKN genes, 200 patients with idiopathic PD, and 200 healthy persons without pathogenic variants. Results: The eCRF consists of an investigator-rated (1 h) and a self-rated (1.5 h) part. The first part includes the Movement Disorder Society Unified Parkinson's Disease Rating, Hoehn &Yahr, and Schwab & England Scales, the Brief Smell Identification Test, and Montreal Cognitive Assessment. The self-rating part consists of a PD risk factor, food frequency, autonomic dysfunction, and quality of life questionnaires, the Pittsburgh Sleep Quality Inventory, and the Epworth Sleepiness as well as the Hospital Anxiety and Depression Scales. The first 15 centers have been initiated and the first 150 participants enrolled (as of March 25th, 2021). Conclusions: LIPAD is a large-scale international scientific effort focusing on deep phenotyping of LRRK2-linked PD and healthy pathogenic variant carriers, including the comparison with additional relatively frequent genetic forms of PD, with a future perspective to identify genetic and environmental modifiers of penetrance and expressivity Clinical Trial Registration:ClinicalTrials.gov, NCT04214509.

The Rostock International Parkinson's Disease (ROPAD) Study: Protocol and Initial Findings.

BACKGROUND: Genetic stratification of Parkinson's disease (PD) patients facilitates gene-tailored research studies and clinical trials. The objective of this study was to describe the design of and the initial data from the Rostock International Parkinson's Disease (ROPAD) study, an epidemiological observational study aiming to genetically characterize ~10,000 participants. METHODS: Recruitment criteria included (1) clinical diagnosis of PD, (2) relative of participant with a reportable LRRK2 variant, or (3) North African Berber or Ashkenazi Jew. DNA analysis involved up to 3 successive steps: (1) variant (LRRK2) and gene (GBA) screening, (2) panel sequencing of 68 PD-linked genes, and (3) genome sequencing. RESULTS: Initial data based on the first 1360 participants indicated that the ROPAD enrollment strategy revealed a genetic diagnostic yield of ~14% among a PD cohort from tertiary referral centers. CONCLUSIONS: The ROPAD screening protocol is feasible for high-throughput genetic characterization of PD participants and subsequent prioritization for gene-focused research efforts and clinical trials. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Inverse control of Rab proteins by Yersinia ADP-ribosyltransferase and glycosyltransferase related to clostridial glucosylating toxins.

We identified a glucosyltransferase (YGT) and an ADP-ribosyltransferase (YART) in Yersinia mollaretii, highly related to glucosylating toxins from Clostridium difficile, the cause of antibiotics-associated enterocolitis. Both Yersinia toxins consist of an amino-terminal enzyme domain, an autoprotease domain activated by inositol hexakisphosphate, and a carboxyl-terminal translocation domain. YGT N-acetylglucosaminylates Rab5 and Rab31 at Thr52 and Thr36, respectively, thereby inactivating the Rab proteins. YART ADP-ribosylates Rab5 and Rab31 at Gln79 and Gln64, respectively. This activates Rab proteins by inhibiting GTP hydrolysis. We determined the crystal structure of the glycosyltransferase domain of YGT (YGTG) in the presence and absence of UDP at 1.9- and 3.4-Å resolution, respectively. Thereby, we identified a previously unknown potassium ion-binding site, which explains potassium ion-dependent enhanced glycosyltransferase activity in clostridial and related toxins. Our findings exhibit a novel type of inverse regulation of Rab proteins by toxins and provide new insights into the structure-function relationship of glycosyltransferase toxins.

A cysteine protease-like domain enhances the cytotoxic effects of the Photorhabdus asymbiotica toxin PaTox.

The nematode mutualistic bacterium Photorhabdus asymbiotica produces a large virulence-associated multifunctional protein toxin named PaTox. A glycosyltransferase domain and a deamidase domain of this large toxin function as effectors that specifically target host Rho GTPases and heterotrimeric G proteins, respectively. Modification of these intracellular regulators results in toxicity toward insects and mammalian cells. In this study, we identified a cysteine protease-like domain spanning PaTox residues 1844-2114 (PaToxP), upstream of these two effector domains and characterized by three conserved amino acid residues (Cys-1865, His-1955, and Asp-1975). We determined the crystal structure of the PaToxP C1865A variant by native single-wavelength anomalous diffraction of sulfur atoms (sulfur-SAD). At 2.0 Å resolution, this structure revealed a catalytic site typical for papain-like cysteine proteases, comprising a catalytic triad, oxyanion hole, and typical secondary structural elements. The PaToxP structure had highest similarity to that of the AvrPphB protease from Pseudomonas syringae classified as a C58-protease. Furthermore, we observed that PaToxP shares structural homology also with non-C58-cysteine proteases, deubiquitinases, and deamidases. Upon delivery into insect larvae, PaToxP alone without full-length PaTox had no toxic effects. Yet, PaToxP expression in mammalian cells was toxic and enhanced the apoptotic phenotype induced by PaTox in HeLa cells. We propose that PaToxP is a C58-like cysteine protease module that is essential for full PaTox activity.

Structural evidence of intramolecular propeptide inhibition of the aspzincin metalloendopeptidase AsaP1.

The Gram-negative bacterium Aeromonas salmonicida is a fish pathogen for various fish species worldwide. Aeromonas salmonicida subsp. achromogenes produces the extracellular, toxic zinc endopeptidase AsaP1. Crystal structure analyses at 2.0 Å resolution of two proteolytically inactive AsaP1 variants show the polypeptide folding of the protease domain and the propeptide domain. These first crystal structure analyses of a precursor of a deuterolysin-like aspzincin protease provide insights into propeptide function, and specific substrate binding. A lysine side chain of the propeptide binds in the hydrophobic S1'-pocket interacting with three carboxylate side chains. An AsaP1 variant with a lysine to alanine exchange identifies the chaperone function of the propeptide.

A bacterial toxin catalyzing tyrosine glycosylation of Rho and deamidation of Gq and Gi proteins.

Entomopathogenic Photorhabdus asymbiotica is an emerging pathogen in humans. Here, we identified a P. asymbiotica protein toxin (PaTox), which contains a glycosyltransferase and a deamidase domain. PaTox mono-O-glycosylates Y32 (or Y34) of eukaryotic Rho GTPases by using UDP-N-acetylglucosamine (UDP-GlcNAc). Tyrosine glycosylation inhibits Rho activation and prevents interaction with downstream effectors, resulting in actin disassembly, inhibition of phagocytosis and toxicity toward insects and mammalian cells. The crystal structure of the PaTox glycosyltransferase domain in complex with UDP-GlcNAc determined at 1.8-Å resolution represents a canonical GT-A fold and is the smallest glycosyltransferase toxin known. (1)H-NMR analysis identifies PaTox as a retaining glycosyltransferase. The glutamine-deamidase domain of PaTox blocks GTP hydrolysis of heterotrimeric Gαq/11 and Gαi proteins, thereby activating RhoA. Thus, PaTox hijacks host GTPase signaling in a bidirectional manner by deamidation-induced activation and glycosylation-induced inactivation of GTPases.

Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing.

AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37 kDa pre-pro-protein and processed to an active enzyme of 19kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1(rec) in E. coli results in a protease of 22 kDa that is not secreted. AsaP1(rec) induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1(wt). The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease. Four different AsaP1 mutants (AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A), and AsaP1(Y309F)) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1(E294A), AsaP1(E294Q), and AsaP1(Y309A)) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37 kDa. Caseinolytic active AsaP1(rec) and a mutant with reduced activity, AsaP1(Y309F), were processed to a size of 22 kDa. Furthermore, AsaP1(rec) is able to process the inactive mutants to the mature size of 22 kDa, allowing the conclusion that AsaP1 is autocatalytically processed. All four mutants AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A) and AsaP1(Y309F) are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives.

The crystal structure of an esterase from the hyperthermophilic microorganism Pyrobaculum calidifontis VA1 explains its enantioselectivity.

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E > 100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E = 2-4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/ß-hydrolase fold as core domain with a cap structure at the C-terminal end of the ß-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/ß-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83-87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.

Cloning, functional expression, biochemical characterization, and structural analysis of a haloalkane dehalogenase from Plesiocystis pacifica SIR-1.

A haloalkane dehalogenase (DppA) from Plesiocystis pacifica SIR-1 was identified by sequence comparison in the NCBI database, cloned, functionally expressed in Escherichia coli, purified, and biochemically characterized. The three-dimensional (3D) structure was determined by X-ray crystallography and has been refined at 1.95 Å resolution to an R-factor of 21.93%. The enzyme is composed of an α/ß-hydrolase fold and a cap domain and the overall fold is similar to other known haloalkane dehalogenases. Active site residues were identified as Asp123, His278, and Asp249 and Trp124 and Trp163 as halide-stabilizing residues. DppA, like DhlA from Xanthobacter autotrophicus GJ10, is a member of the haloalkane dehalogenase subfamily HLD-I. As a consequence, these enzymes have in common the relative position of their catalytic residues within the structure and also show some similarities in the substrate specificity. The enzyme shows high preference for 1-bromobutane and does not accept chlorinated alkanes, halo acids, or halo alcohols. It is a monomeric protein with a molecular mass of 32.6 kDa and exhibits maximum activity between 33 and 37°C with a pH optimum between pH 8 and 9. The K(m) and k(cat) values for 1-bromobutane were 24.0 mM and 8.08 s(-1). Furthermore, from the 3D-structure of DppA, it was found that the enzyme possesses a large and open active site pocket. Docking experiments were performed to explain the experimentally determined substrate preferences.

Influence of temperature during crystallization setup on precipitate formation and crystal shape of a metalloendopeptidase.

It is well known that protein crystallization is affected by several different parameters such as the composition of the reservoir solution, the protein concentration, the pH and the temperature. An effect of different temperatures during setup of crystallization experiments was observed for a metalloendopeptidase (AsaP1(E294A)). Spontaneous protein precipitation was reduced and the crystal shape could be improved by decreasing the temperature during crystallization setup.

Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I.

DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2(1)2(1)2. The DppA crystal diffracted X-rays to 1.9 A resolution using an in-house X-ray generator.

Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres.

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn(2+) ion as the active-site centre whereas others are only active with Fe(2+) (or Co(2+), Ni(2+) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.

Crystallization and preliminary X-ray diffraction studies of AsaP1_E294A and AsaP1_E294Q, two inactive mutants of the toxic zinc metallopeptidase AsaP1 from Aeromonas salmonicida subsp. achromogenes.

Two mutants of the toxic extracellular zinc endopeptidase AsaP1 (AsaP1_E294Q and AsaP1_E294A) of Aeromonas salmonicida subsp. achromogenes were expressed in Escherichia coli and crystallized by the vapour-diffusion method. Crystals were obtained using several precipitants and different protein concentrations. Protein crystals were found in a monoclinic (C2) as well as an orthorhombic (P2(1)2(1)2(1)) space group. The crystals belonging to the monoclinic space group C2 had unit-cell parameters a = 103.4, b = 70.9, c = 54.9 A, beta = 109.3 degrees for AsaP1_E294A, and a = 98.5, b = 74.5, c = 54.7 A, beta = 112.4 degrees for AsaP1_E294Q. The unit-cell parameters of the orthorhombic crystal obtained for AsaP1_E294A were a = 57.9, b = 60.2, c = 183.6 A. The crystals of the two different mutants diffracted X-rays beyond 2.0 A resolution.