Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate

Escherichia coli isocitrate lyase was inactivated by diethylpyrocarbonate in a pseudo-first-order process. The enzyme was completely inactivated by modification of a single histidine residue, but slower modification of further residues also occurred. The substrate, isocitrate, and products, glyoxylate and succinate, protected against inactivation by diethylpyrocarbonate but this was not simply due to binding at the active site. Treatment of the inactivated enzyme with hydroxylamine led to only partial recovery of activity. Diethylpyrocarbonate also reacted with sulphydryl groups in isocitrate lyase, as judged by titrations with Nbs2, but this reaction was not responsible for the failure of hydroxylamine to reactivate the enzyme fully. The reactivity of isocitrate lyase to diethylpyrocarbonate declined with pH, following a titration curve for a group of pKa 6.1. Isolation and sequencing of ethoxyformylated peptides showed that the major site of modification by diethylpyrocarbonate was histidine residue 306

Recombinant human nerve growth factor for clinical trials: Protein expression, purification, stability and characterisation of binding to infusion pumps

Nerve growth factor (NGF) has been suggested to be of therapeutic benefit to patients with Alzheimer's disease. One of the early changes in this disease is a loss of cholinergic function within the brain, and NGF is able to rescue cholinergic neurons both in vitro and in vivo. We describe the production of recombinant human β-NGF (rhNGF), using baculovirus infection of insect cells; its purification, formulation and subsequent stability for use in clinical trials. Tests were also carried out to monitor release of protein from infusion pumps and catheters for intracerebroventricular administration (icv). Initial problems with non-specific binding were overcome using a blocking formula. © 2001 Elsevier Science B.V

Specificity in Trk receptor-neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5

AbstractBackground: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease.Results: We have determined the 2.7 Å crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site—unique in each ligand-receptor pair—formed primarily by the ordering of the neurotrophin N terminus.Conclusions: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs

Identification and structure of the nerve growth factor binding site on TrkA

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer’s disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg2) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg2 domain in a new crystal form, refined to 2.0 Å resolution. © 2001 Academic Press

De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Callosum, Axon, Cardiac, Ocular, and Genital Defects

International audienceCadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects)

Loss-of-function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

Epigenetic dysregulation has been associated with many inherited disorders. RBBP5 (HGNC:9888) encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. We identify five unrelated individuals with de novo heterozygous variants in RBBP5. Three nonsense/frameshift and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. Both missense p.(T232I) and p.(E296D) variants affect evolutionarily conserved amino acids located at the interface between RBBP5 and the nucleosome. In Drosophila, overexpression analysis identifies partial loss-of-function mechanisms when the variants are expressed using the fly Rbbp5 or human RBBP5 cDNA. Loss of Rbbp5 leads to a reduction in brain size. The human reference or variant transgenes fail to rescue this loss and expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both missense variants are partial loss-of-function alleles. Haploinsufficiency of RBBP5 observed through de novo null and hypomorphic loss-of-function variants is associated with a syndromic neurodevelopmental disorder. Huang et al. report the first functional validation of candidate pathological variants in RBBP5. We present three truncating p.(K244Nfs*6), p.(W254*), p.(R307*) and two missense p.(T232I), p.(E296D) variants found de novo in affected individuals sharing phenotypes including microcephaly and short stature. RBBP5 is a core member of the COMPASS complex responsible for H3 lysine 4 methylation to activate developmental target genes (COMPASS complex adapted from Namitz et al., 2023). Differentiation of neural stem cells in humans and neuroblasts in Drosophila is conserved allowing for the study of neural development in the fly model organism (neural stem cell/neuroblast differentiation diagram adapted from Kim and Hirth, 2009). We used overexpression and rescue experiments to characterize the missense variants in the fly. Neural progenitor populations were evaluated in the larval brain and tissue specific phenotypes were quantified using adult eye and wing morphology studies. We identify that the truncating and missense variants are loss-of-function alleles. As additional patients are identified, the full phenotypic spectrum of RBBP5-related disorders will be elucidated. Created with Biorender.com. [Display omitted