Time course of serum neuron-specific enolase levels from infancy to early adulthood in a female patient with beta-propeller protein-associated neurodegeneration.

Beta-propeller protein-associated neurodegeneration (BPAN), a subgroup of neurodegeneration with brain iron accumulation, is typically characterized by non-progressive global developmental delay and seizures in childhood, followed by progressive neurological decline with parkinsonism and dementia in adolescence or early adulthood. It is difficult to clinically identify a patient with BPAN in childhood. Recent studies reported that serum levels of neuron-specific enolase (NSE) were elevated in children with BPAN. We reviewed the time course of serum NSE levels in a 21-year-old female patient genetically diagnosed (a de novo WDR45 variant c.268A > T) with BPAN, which was suspected based on prolonged elevation of serum NSE. There was an overall tendency for serum NSE levels to decrease in a stepwise fashion. The peak serum NSE level was observed during the first 2 years of age and then decreased rapidly in 1 year. High serum NSE levels persisted between 3 and 11 years of age. Subsequently, serum NSE levels decreased and plateaued after 13 years of age. There were tendencies for both blood AST and LDH levels to decrease over time in parallel with serum NSE levels. Serum NSE levels may be a diagnostic biomarker of BPAN in children but becomes of less value in identifying a patient with BPAN after childhood.

Autophagic defects observed in fibroblasts from a patient with ß-propeller protein-associated neurodegeneration.

Beta-propeller protein-associated neurodegeneration (BPAN) is associated with mutations in the autophagy gene WDR45. The aim of this study was to demonstrate autophagic defects in a patient with BPAN. We assayed autophagic markers using western blot analysis and immunocytochemistry and applied transmission electron microscopy (TEM) to visualize the autophagic structures in fibroblasts from a 7-year-old Korean female with WDR45 splice-site mutation (c.977-1G>A; NM_007075.3). The protein and mRNA expression levels of WDR45 gene were decreased in the patient-derived fibroblasts. The amount of increase in LC3-II upon treatment with an autophagy inducer and inhibitor was reduced in mutant cells compared to control cells, suggesting decreased autophagic flux. TEM showed the accumulation of large vacuoles in mutant cells with a decrease of autophagosomes. Our study demonstrated that the WDR45 mutation in this patient impaired autophagy and provided additional insight into ultrastructural changes of autophagic structures.

Beta-propeller protein-associated neurodegeneration presenting Rett-like features: A case report and literature review.

Several patients with beta-propeller protein-associated neurodegeneration (BPAN)/static encephalopathy with neurodegeneration in adulthood have been reported to present Rett syndrome (RTT)-like features. This report presents an individual with BPAN showing clinical features of RTT. Psychomotor delay and epilepsy onset were noted at 1 year, and regression began at 4 years. Screening of the methyl-CpG binding protein 2 (MECP2) did not show variants. At 22 years, basal ganglia iron deposits were found on magnetic resonance imaging (MRI), and the WD-domain repeat 45 gene (WDR45) variant was identified. Review of the literature showed that BPAN with RTT-like features is associated with more epileptic seizures and less deceleration of head growth, breathing irregularities, and cold extremities than classic RTT with MECP2 variants. These clinical presentations may provide clues for differentiating between these two disorders. However, both WDR45 and MECP2 should be screened in patients presenting a clinical picture of RTT without specific MRI findings of BPAN.

How to Build a Complex, Functional Propeller Protein, From Parts.

By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed ß-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo.

Top surface blade residues and the central channel water molecules are conserved in every repeat of the integrin-like ß-propeller structures.

An integrin-like ß-propeller domain contains seven repeats of a four-stranded antiparallel ß-sheet motif (blades). Previously we described a 3D structural motif within each blade of the integrin-type ß-propeller. Here, we show unique structural links that join different blades of the ß-propeller structure, which together with the structural motif for a single blade are repeated in a ß-propeller to provide the functional top face of the barrel, found to be involved in protein-protein interactions and substrate recognition. We compare functional top face diagrams of the integrin-type ß-propeller domain and two non-integrin type ß-propeller domains of virginiamycin B lyase and WD Repeat-Containing Protein 5.

Design of tryptophan-containing mutants of the symmetrical Pizza protein for biophysical studies.

ß-propeller proteins are highly symmetrical, being composed of a repeated motif with four anti-parallel ß-sheets arranged around a central axis. Recently we designed the first completely symmetrical ß-propeller protein, Pizza6, consisting of six identical tandem repeats. Pizza6 is expected to prove a useful building block for bionanotechnology, and also a tool to investigate the folding and evolution of ß-propeller proteins. Folding studies are made difficult by the high stability and the lack of buried Trp residues to act as monitor fluorophores, so we have designed and characterized several Trp-containing Pizza6 derivatives. In total four proteins were designed, of which three could be purified and characterized. Crystal structures confirm these mutant proteins maintain the expected structure, and a clear redshift of Trp fluorescence emission could be observed upon denaturation. Among the derivative proteins, Pizza6-AYW appears to be the most suitable model protein for future folding/unfolding kinetics studies as it has a comparable stability as natural ß-propeller proteins.

Biophysical characterization and structural determination of the potent cytotoxic Psathyrella asperospora lectin.

A lectin with strong cytotoxic effect on human colon cancer HT29 and monkey kidney VERO cells was recently identified from the Australian indigenous mushroom Psathyrella asperospora and named PAL. We herein present its biochemical and structural analysis using a multidisciplinary approach. Glycan arrays revealed binding preference towards N-acetylglucosamine (GlcNAc) and, to a lesser extent, towards sialic acid (Neu5Ac). Submicromolar and millimolar affinity was measured by surface plasmon resonance for GlcNAc and NeuAc, respectively. The structure of PAL was resolved by X-ray crystallography, elucidating both the protein's amino acid sequence as well as the molecular basis rationalizing its binding specificity. Proteins 2017; 85:969-975. © 2016 Wiley Periodicals, Inc.

Novel calcium recognition constructions in proteins: Calcium blade and EF-hand zone.

Metal ions can regulate various cell processes being first, second or third messengers, and some of them, especially transition metal ions, take part in catalysis in many enzymes. As an intracellular ion, Ca2+ is involved in many cellular functions from fertilization and contraction, cell differentiation and proliferation, to apoptosis and cancer. Here, we have identified and described two novel calcium recognition environments in proteins: the calcium blade zone and the EF-hand zone, common to 12 and 8 different protein families, respectively. Each of the two environments contains three distinct structural elements: (a) the well-known characteristic Dx[DN]xDG motif; (b) an adjacent structurally identical segment, which binds metal ion in the same way between the calcium blade zone and the EF-hand zone; and (c) the following structurally variable segment, which distinguishes the calcium blade zone from the EF-hand zone. Both zones have sequence insertions between the last residue of the zone and calcium-binding residues in positions V or VI. The long insertion often connects the active and the calcium-binding sites in proteins. Using the structurally identical segments as an anchor, we were able to construct the classical calmodulin type EF-hand calcium-binding site out of two different calcium-binding motifs from two unrelated proteins.

Elevation of neuron specific enolase and brain iron deposition on susceptibility-weighted imaging as diagnostic clues for beta-propeller protein-associated neurodegeneration in early childhood: Additional case report and review of the literature.

Beta-propeller protein-associated neurodegeneration (BPAN), also known as static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), is a subtype of neurodegeneration with brain iron accumulation (NBIA). BPAN is caused by mutations in an X-linked gene WDR45 that is involved in autophagy. BPAN is characterized by developmental delay or intellectual disability until adolescence or early adulthood, followed by severe dystonia, parkinsonism, and progressive dementia. Brain magnetic resonance imaging (MRI) shows iron deposition in the bilateral globus pallidus (GP) and substantia nigra (SN). Clinical manifestations and laboratory findings in early childhood are limited. We report a 3-year-old girl with BPAN who presented with severe developmental delay and characteristic facial features. In addition to chronic elevation of serum aspartate transaminase, lactate dehydrogenase, creatine kinase, and soluble interleukin-2 receptor, she had persistent elevation of neuron specific enolase (NSE) in serum and cerebrospinal fluid. MRI using susceptibility-weighted imaging (SWI) demonstrated iron accumulation in the GP and SN bilaterally. Targeted next-generation sequencing identified a de novo splice-site mutation, c.831-1G>C in WDR45, which resulted in aberrant splicing evidenced by reverse transcriptase-PCR. Persistent elevation of NSE and iron deposition on SWI may provide clues for diagnosis of BPAN in early childhood.

Structure of the Chlamydia trachomatis immunodominant antigen Pgp3.

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. Left untreated, it can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility. Here we present the structure of the secreted C. trachomatis protein Pgp3, an immunodominant antigen and putative virulence factor. The ∼84-kDa Pgp3 homotrimer, encoded on a cryptic plasmid, consists of globular N- and C-terminal assemblies connected by a triple-helical coiled-coil. The C-terminal domains possess folds similar to members of the TNF family of cytokines. The closest Pgp3 C-terminal domain structural homologs include a lectin from Burkholderia cenocepacia, the C1q component of complement, and a portion of the Bacillus anthracis spore surface protein BclA, all of which play roles in bioadhesion. The N-terminal domain consists of a concatenation of structural motifs typically found in trimeric viral proteins. The central parallel triple-helical coiled-coil contains an unusual alternating pattern of apolar and polar residue pairs that generate a rare right-handed superhelical twist. The unique architecture of Pgp3 provides the basis for understanding its role in chlamydial pathogenesis and serves as the platform for its optimization as a potential vaccine antigen candidate.

Engineering strictosidine synthase: rational design of a small, focused circular permutation library of the ß-propeller fold enzyme.

Strictosidine synthases catalyze the formation of strictosidine, a key intermediate in the biosynthesis of a large variety of monoterpenoid indole alkaloids. Efforts to utilize these biocatalysts for the preparation of strictosidine analogs have however been of limited success due to the high substrate specificity of these enzymes. We have explored the impact of a protein engineering approach called circular permutation on the activity of strictosidine synthase from the Indian medicinal plant Rauvolfia serpentina. To expedite the discovery process, our study departs from the usual process of creating a random protein library, followed by extensive screening. Instead, a small, focused library of circular permutated variants of the six bladed ß-propeller protein was prepared, specifically probing two regions which cover the enzyme active site. The observed activity changes suggest important roles of both regions in protein folding, stability and catalysis.

WDR45-dependent impairment of cell cycle in fibroblasts of patients with beta propeller protein-associated neurodegeneration (BPAN).

De novo mutations in the WDR45 gene have been found in patients affected by Neurodegeneration with Brain Iron Accumulation type 5 (NBIA5 or BPAN), with Non-Transferrin Bound Iron (NTBI) accumulation in the basal ganglia and WDR45-dependent impairment of autophagy. Here we show the downregulation of TFEB and cell cycle impairment in BPAN primary fibroblasts. Noteworthy, TFEB overexpression rescued this impairment, depicting a novel WDR45-dependent cell cycle phenotype.

A Case of Beta-Propeller Protein-Associated Neurodegeneration With a Unique Truncating Variant in the WDR45 Gene and Uncommon Clinical and Radiologic Findings.

Beta-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation, is caused by variants in the WDR45 gene. In this paper, we describe a patient with an atypical presentation of BPAN whose whole exome sequencing revealed a previously unattested truncating variant in the WDR45 gene (c.830+3G>C/p.Leu278Ter), the pathogenicity of which was verified by RNA transcriptomics. A number of uncommon neuroanatomic and clinical findings in our patient are discussed, expanding the phenotype associated with BPAN. This unique case challenges existing genotype-phenotype correlations and highlights the role of X chromosome skewing in shaping the clinical spectrum of BPAN.

Quantitative retrospective natural history modeling of WDR45-related developmental and epileptic encephalopathy - a systematic cross-sectional analysis of 160 published cases.

WDR45-related neurodevelopmental disorder (NDD) is a clinically-heterogenous congenital disorder of macroautophagy/autophagy. The natural history of this ultra-orphan disease remains incompletely understood, leading to delays in diagnosis and lack of quantifiable outcome measures. In this cross-sectional study, we model quantitative natural history data for WDR45-related NDD using a standardized analysis of 160 published cases, representing the largest cohort to date. The primary outcome of this study was survival. Age at disease onset, diagnostic delay and geographic distribution were quantified as secondary endpoints. Our tertiary aim was to explore and quantify the spectrum of WDR45-related phenotypes. Survival estimations showed low mortality until 39 years of age. Median age at onset was 10 months, with a median diagnostic delay of 6.2 years. Geographic distribution appeared worldwide with clusters in North America, East Asia, Western Europe and the Middle East. The clinical spectrum was highly variable with a bi-phasic evolution characterized by early-onset developmental and epileptic encephalopathy during childhood followed by a progressive dystonia-parkinsonism syndrome along with cognitive decline during early adulthood. Female individuals showed milder disease severity. The majority of pathogenic WDR45 variants were predicted to result in a loss of WDR45 expression, without clear genotype-phenotype associations. Our results provide clinical and epidemiological data that may facilitate an earlier diagnosis, enable anticipatory guidance and counseling of affected families and provide the foundation for endpoints for future interventional trials.Abbreviations: BPAN: beta-propeller protein-associated neurodegeneration; CNS: central nervous system; DEE: developmental and epileptic encephalopathy; MRI: magnetic resonance imaging; NBIA: neurodegeneration with brain iron accumulation; NDD: neurodevelopmental disorder; NGS: next-generation sequencing; WDR45/WIPI4: WD repeat domain 45.

Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity.

Functional, biochemical, and preliminary structural properties are reported for three glycoside hydrolases of the recently described glycoside hydrolase (GH) family 159. The genes were cloned from the genomic sequences of different Caldicellulosiruptor strains. This study extends the spectrum of functions of GH159 enzymes. The only activity previously reported for GH159 was hydrolytic activity on ß-galactofuranosides. Activity screening using a set of para-nitrophenyl (pNP) glycosides suggested additional arabinosidase activity on substrates with arabinosyl residues, which has not been previously reported for members of GH159. Even though the thermophilic enzymes investigated-Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A-cleaved pNP-α-l-arabinofuranoside, they were only weakly active on arabinogalactan, and they did not cleave arabinose from arabinan, arabinoxylan, or gum arabic. However, the enzymes were able to hydrolyze the α-1,3-linkage in different arabinoxylan-derived oligosaccharides (AXOS) with arabinosylated xylose at the non-reducing end (A3X, A2,3XX), suggesting their role in the intracellular hydrolysis of oligosaccharides. Crystallization and structural analysis of the apo form of one of the Caldicellulosiruptor enzymes, Ch_Gaf159A, enabled the elucidation of the first 3D structure of a GH159 member. This work revealed a five-bladed ß-propeller structure for GH159 enzymes. The 3D structure and its substrate-binding pocket also provides an explanation at the molecular level for the observed exo-activity of the enzyme. Furthermore, the structural data enabled the prediction of the catalytic amino acids. This was supported by the complete inactivation by mutation of residues D19, D142, and E190 of Ch_Gaf159A.

WDR45, one gene associated with multiple neurodevelopmental disorders.

The WDR45 gene is localized on the X-chromosome and variants in this gene are linked to six different neurodegenerative disorders, i.e., ß-propeller protein associated neurodegeneration, Rett-like syndrome, intellectual disability, and epileptic encephalopathies including developmental and epileptic encephalopathy, early-onset epileptic encephalopathy and West syndrome and potentially also specific malignancies. WDR45/WIPI4 is a WD-repeat ß-propeller protein that belongs to the WIPI (WD repeat domain, phosphoinositide interacting) family. The precise cellular function of WDR45 is still largely unknown, but deletions or conventional variants in WDR45 can lead to macroautophagy/autophagy defects, malfunctioning mitochondria, endoplasmic reticulum stress and unbalanced iron homeostasis, suggesting that this protein functions in one or more pathways regulating directly or indirectly those processes. As a result, the underlying cause of the WDR45-associated disorders remains unknown. In this review, we summarize the current knowledge about the cellular and physiological functions of WDR45 and highlight how genetic variants in its encoding gene may contribute to the pathophysiology of the associated diseases. In particular, we connect clinical manifestations of the disorders with their potential cellular origin of malfunctioning and critically discuss whether it is possible that one of the most prominent shared features, i.e., brain iron accumulation, is the primary cause for those disorders.Abbreviations: ATG/Atg: autophagy related; BPAN: ß-propeller protein associated neurodegeneration; CNS: central nervous system; DEE: developmental and epileptic encephalopathy; EEG: electroencephalograph; ENO2/neuron-specific enolase, enolase 2; EOEE: early-onset epileptic encephalopathy; ER: endoplasmic reticulum; ID: intellectual disability; IDR: intrinsically disordered region; MRI: magnetic resonance imaging; NBIA: neurodegeneration with brain iron accumulation; NCOA4: nuclear receptor coactivator 4; PtdIns3P: phosphatidylinositol-3-phosphate; RLS: Rett-like syndrome; WDR45: WD repeat domain 45; WIPI: WD repeat domain, phosphoinositide interacting.

Emerging Disease-Modifying Therapies in Neurodegeneration With Brain Iron Accumulation (NBIA) Disorders.

Neurodegeneration with Brain Iron Accumulation (NBIA) is a heterogeneous group of progressive neurodegenerative diseases characterized by iron deposition in the globus pallidus and the substantia nigra. As of today, 15 distinct monogenetic disease entities have been identified. The four most common forms are pantothenate kinase-associated neurodegeneration (PKAN), phospholipase A2 group VI (PLA2G6)-associated neurodegeneration (PLAN), beta-propeller protein-associated neurodegeneration (BPAN) and mitochondrial membrane protein-associated neurodegeneration (MPAN). Neurodegeneration with Brain Iron Accumulation disorders present with a wide spectrum of clinical symptoms such as movement disorder signs (dystonia, parkinsonism, chorea), pyramidal involvement (e.g., spasticity), speech disorders, cognitive decline, psychomotor retardation, and ocular abnormalities. Treatment remains largely symptomatic but new drugs are in the pipeline. In this review, we discuss the rationale of new compounds, summarize results from clinical trials, provide an overview of important results in cell lines and animal models and discuss the future development of disease-modifying therapies for NBIA disorders. A general mechanistic approach for treatment of NBIA disorders is with iron chelators which bind and remove iron. Few studies investigated the effect of deferiprone in PKAN, including a recent placebo-controlled double-blind multicenter trial, demonstrating radiological improvement with reduction of iron load in the basal ganglia and a trend to slowing of disease progression. Disease-modifying strategies address the specific metabolic pathways of the affected enzyme. Such tailor-made approaches include provision of an alternative substrate (e.g., fosmetpantotenate or 4'-phosphopantetheine for PKAN) in order to bypass the defective enzyme. A recent randomized controlled trial of fosmetpantotenate, however, did not show any significant benefit of the drug as compared to placebo, leading to early termination of the trials' extension phase. 4'-phosphopantetheine showed promising results in animal models and a clinical study in patients is currently underway. Another approach is the activation of other enzyme isoforms using small molecules (e.g., PZ-2891 in PKAN). There are also compounds which counteract downstream cellular effects. For example, deuterated polyunsaturated fatty acids (D-PUFA) may reduce mitochondrial lipid peroxidation in PLAN. In infantile neuroaxonal dystrophy (a subtype of PLAN), desipramine may be repurposed as it blocks ceramide accumulation. Gene replacement therapy is still in a preclinical stage.

[A case of novel WDR45 mutation with beta-propeller protein-associated neurodegeneration (BPAN) presenting asymmetrical extrapyramidal signs].

Beta-propeller protein-associated neurodegeneration (BPAN) is categorized in Neurodegeneration with brain iron accumulation. The clinical feature of BPAN is global developmental delay in early childhood, followed rapid progression of cognitive disfunction and parkinsonism in adulthood. This case was pointed out intellectual disability at the age of 9, followed left dominant progressive parkinsonism from the age of 31. Brain MRI showed the T1-weighted signal hyperintensity of the substantia nigra with a central band of hypointensity and the T2 star weighted image hypointensity of substantia nigra and globus pallidus presenting dominant at right side. DAT SPECT also showed specific binding ratio decreased dominant in right side. She was diagnosed BPAN based on her genetic test revealing a novel mutation (c.411dupT) in WDR45. No studies reported detailed parkinsonism like laterality in BPAN. This case indicates the left dominant parkinsonism was caused by right dominant iron deposition to substantia nigra and globus pallidus in view of MRI findings and DAT SPECT.

Effect of Polar Amino Acid Residue Substitution by Site-Directed Mutagenesis in the N-terminal Domain of Pseudomonas sp. Phytase on Enzyme Activity.

The N-terminal domain of the Pseudomonas sp. FB15 phytase increases low-temperature activity and catalytic efficiency. In this study, the 3D structure of the N-terminal domain was predicted and substitutions for the amino acid residues of the region assumed to be the active site were made. The activity of mutants, in which alanine (A) was substituted for the original residue, was investigated at various temperatures and pH values. Significant differences in enzymatic activity were observed only in mutant E263A, suggesting that the amino acid residue at position 263 of the N-terminal domain is important in enzyme activity.

A rare cause of epileptic encephalopathy: a beta-propeller protein associated neurodegeneration case with a new mutation and literature review.

In this report, detailed clinical features of a female patient and a new mutation that was not previously identified in the WD repeat-containing protein 45 (WDR45) gene are presented in order to contribute to the information in the literature on the phenotype as well as genotype of Beta-Propeller Protein Associated Neurodegeneration. Whole Exome Sequencing (WES) analysis was done since etiology could not be determined. Our case was admitted to the hospital due to epilepsy, growth retardation and autism. Her family history was unremarkable except consanguineous marriage. She had tonic seizures twice at the age of 7 and 12 months and had continual seizures after 16 months. At the time, electroencephalography and brain MRI were performed twice were determined to be normal. Brain MRI Spectroscopy was also found to be normal at 35 months of age. Metabolic screening tests (acyl carnitine profile, urine organic acids, plasma amino acids, a very long chain fatty acid profile, etc.) were also normal. Genetic screening of the epilepsy panel for epileptic encephalopathies was negative. WES analysis revealed heterozygous previously unreported variant in intron 6 of the WDR45 gene, c.344+5G > A. In conclusion; Beta-Propeller Protein Associated Neurodegeneration should be considered as an option in the diagnosis of female patients with clinical findings of epilepsy, growth retardation and autism, with unspecified etiology.