Biology subjects

Morizono, H.

Publications and source records attributed to Morizono, H..

6 recordsLinked to original sources

bioRxiv2025

Functional Profiling of 2,193 ASS1 Missense Variants: Insights into Variant Pathogenicity and Epistatic Interactions in Citrullinemia Type I

Sequence variants in the urea cycle gene argininosuccinate synthase (ASS1) cause Citrullinemia type 1 (CTLN1), a rare autosomal recessive disease. Mechanistically, reduction in argininosuccinate synthetase (ASS) enzyme activity impairs the urea cycle, leading to an accumulation of citrulline and neurotoxic ammonia. Disease severity varies according to the degree of enzyme impairment, ranging from severe neonatal forms (classic citrullinemia) to milder, late-onset forms that may manifest in childhood or adulthood. We established a high-throughput yeast functional assay of human ASS and individually measured the impact of 2193 amino acid substitutions, representing 90% of all single nucleotide variant (SNV)-accessible substitutions. When benchmarked against existing clinical variant annotation, our assay distinguishes known benign variants from strong loss of function pathogenic variants, enabling identification of a functional score threshold below which variants show clinically relevant impairment of ASS activity. Using the ACMG OddsPath framework, our assay meets PS3_supporting criteria for pathogenicity classification and achieves full PS3-level strength when variants observed as homozygotes in other primates are used as benign proxies for calibration. These results provide direct functional evidence to inform reclassification of ASS1 missense variants, supporting their clinical interpretation and diagnostic utility. Mapping functional scores onto the protein structure, we confirmed that residues involved in catalysis are highly sensitive to substitution. In addition, we identified residues from adjacent subunits of the ASS homotetramer that form compound active sites. Assaying these positions revealed a capacity for intragenic complementation consistent with a variant sequestration model: a form of positive epistasis in which deleterious variants from different subunits are sequestered into only a subset of active sites, restoring function in the remaining variant-free sites. The discovery of intragenic complementation in ASS reveals a novel mode of functional interaction with clinical implications for interpreting variant combinations in heterozygous individuals.

genetics↗

bioRxiv2024

Dysregulated airway epithelial antiviral immunity in Down Syndrome impairs type III IFN response and amplifies airway inflammation during RSV infection.

Trisomy 21 (TS21), also known as Down syndrome (DS), increases pediatric mortality risk from respiratory syncytial virus (RSV) by nine-fold, yet its underlying immunological basis remains unclear. Here, we investigated RSV-induced immunological responses in TS21 airway epithelial cells (AECs), the primary site of respiratory virus entry and host defense. TS21 AECs exhibit hyperactive interferon (IFN) signaling and reduced RSV infectivity, but they also show impaired type-III IFN responses during viral infection. Furthermore, TS21 AECs demonstrate heightened production of proinflammatory mediators CXCL5 and CXCL10 both before and after RSV exposure. Infants with DS suffering from severe viral bronchiolitis demonstrate dysregulated airway immune responses in vivo, characterized by diminished type-III IFN levels and increased CXCL5/CXCL10 secretion. Our results indicate that RSV severity in DS is not due to impaired viral control but to dysregulated airway proinflammatory responses, offering new therapeutic opportunities to mitigate the severity of RSV infection in children with DS.

immunology↗

bioRxiv2022

The functional impact of 1,570 SNP-accessible missense variants in human OTC

Deleterious mutations in the X-linked gene encoding ornithine transcarbamylase (OTC) cause the most common urea cycle disorder, OTC deficiency. This rare, but highly actionable disease can present with severe neonatal onset in males or with later onset in either sex. Neonatal onset patients appear normal at birth but rapidly develop hyperammonemia, which can progress to cerebral edema, coma and death, outcomes ameliorated by rapid diagnosis and treatment. Existing biochemical assays have limitations, including the sensitivity of the citrulline assays used in newborn screening panels. With prior knowledge of variant pathogenicity, DNA sequence-based diagnostics would provide an alternative screening method. Here, we develop a high throughput functional assay for human OTC and measure the impact of 1,570 variants, 84% of all SNP-accessible missense mutations. Our assay scores agree well with existing clinical significance calls, distinguishing known benign from pathogenic variants and variants with neonatal onset from late-onset disease presentation. Further, use of an intronless expression construct allows us to measure the impact of amino acid changes at splice sites independent of their effect on splicing, thereby separating the contribution of splicing and protein coding changes to aid the analysis of molecular mechanisms underlying pathogenicity. Finally, we assess the utility of our functional data on OTC variant curation by using the current ACMG/AMP guidelines to reclassify variants. Inclusion of our data as PS3/BS3 substantially improves variant interpretation. Thus, our dataset is of high clinical utility and illustrates the power of functional assays to inform interpretation of existing and novel genetic variation.

genetics↗

bioRxiv2020

Mitochondrial Enzymes of the Urea Cycle Cluster at the Inner Mitochondrial Membrane

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other perhaps in a multiprotein complex. This view is supported by mutations in surface residues of the urea cycle proteins that impair urea genesis in the patients but do not affect protein stability or catalytic activity. Further, we find one third of the NAGS, CPS1 and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM), and co-immunoprecipitate. Our in silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a region we call variable segment present only in the mammalian NAGS protein. We experimentally confirmed that NAGS variable segment mediates the interaction of NAGS with CPS1. Use of Gated-Stimulation Emission Depletion (gSTED) super resolution microscopy showed that in situ, NAGS, CPS1 and OTC are organized into clusters. These results are consistent with mitochondrial urea cycle proteins forming a cluster instead of functioning either independently or in a rigid multienzyme complex.

biochemistry↗

bioRxiv2018

Baseline human gut microbiota profile in healthy people and standard reporting template

A comprehensive knowledge of the types and ratios of microbes that inhabit the healthy human gut is necessary before any kind of pre-clinical or clinical study can be performed that attempts to alter the microbiome to treat a condition or improve therapy outcome. To address this need we present an innovative scalable comprehensive analysis workflow, a healthy human reference microbiome list and abundance profile (GutFeelingKB), and a novel Fecal Biome Population Report (FecalBiome) with clinical applicability. GutFeelingKB provides a list of 157 organisms (8 phyla, 18 classes, 23 orders, 38 families, 59 genera and 109 species) that forms the baseline biome and therefore can be used as healthy controls for studies related to dysbiosis. The incorporation of microbiome science into routine clinical practice necessitates a standard report for comparison of an individuals microbiome to the growing knowledgebase of \"normal\" microbiome data. The FecalBiome and the underlying technology of GutFeelingKB address this need. The knowledgebase can be useful to regulatory agencies for the assessment of fecal transplant and other microbiome products, as it contains a list of organisms from healthy individuals. In addition to the list of organisms and abundances the study also generated a list of contigs of metagenomics dark matter. In this study, metagenomic dark matter represents sequences that cannot be mapped to any known sequence but can be assembled into contigs of 10,000 nucleotides or higher. These sequences can be used to create primers to study potential novel organisms. All data is freely available from https://hive.biochemistry.gwu.edu/gfkb and NCBIs Short Read Archive.

microbiology↗

bioRxiv2018

Impaired ureagenesis due to arginine-insensitive N-acetylglutamate synthase

The urea cycle protects the central nervous system from ammonia toxicity by converting ammonia to non-toxic urea. N-acetylglutamate synthase (NAGS) is an enzyme that catalyzes the formation of N-acetylglutamate (NAG), an allosteric activator of carbamylphosphate synthetase 1 (CPS1), the rate limiting enzyme of the urea cycle. Enzymatic activity of mammalian NAGS doubles in the presence of L-arginine but the physiological significance of NAGS activation by L-arginine is unknown. Previously, we have described the creation of a NAGS knockout (Nags-/-) mouse, which develops hyperammonemia without N-carbamylglutamate and L-citrulline supplementation (NCG+Cit). In order to investigate the effect of L-arginine on ureagenesis in vivo, we used adeno associated virus (AAV) mediated gene transfer to deliver either wild-type or E354A mutant mouse NAGS (mNAGS), which is not activated by L-arginine, to Nags-/- mice. The ability of the E354A mNAGS mutant protein to rescue Nags-/- mice was determined by measuring their activity on the voluntary wheel following NCG+Cit withdrawal. The Nags-/- mice that received E354A mNAGS remained apparently healthy and active but had elevated plasma ammonia concentration despite similar expression levels of the E354A mNAGS and control wild-type NAGS proteins. The corresponding mutation in human NAGS (NP 694551.1:p.E360D) that abolishes binding and activation by L-arginine was also identified in a patient with hyperammonemia due to NAGS deficiency. Taken together, our results suggest that L-arginine binding to the NAGS enzyme is essential for normal ureagenesis.

molecular biology↗