Biology subjects

Haskins, N.

Publications and source records attributed to Haskins, N..

4 recordsLinked to original sources

bioRxiv2023

Enhancement of antibody thermostability and affinity by computational design in the absence of antigen

Over the last two decades, therapeutic antibodies have emerged as a rapidly expanding domain within the field biologics. In silico tools that can streamline the process of antibody discovery and optimization are critical to support a pipeline that is growing more numerous and complex every year. In this study, DeepAb, a deep learning model for predicting antibody Fv structure directly from sequence, was used to design 200 potentially stabilized variants of an anti-hen egg lysozyme (HEL) antibody. We sought to determine whether DeepAb can enhance the stability of these antibody variants without relying on or predicting the antibody-antigen interface, and whether this stabilization could increase antibody affinity without impacting their developability profile. The 200 variants were produced through a robust highthroughput method and tested for thermal and colloidal stability (Tonset, Tm, Tagg), affinity (KD) relative to the parental antibody, and for developability parameters (non-specific binding, aggregation propensity, self-association). In the designed clones, 91% and 94% exhibited increased thermal and colloidal stability and affinity, respectively. Of these, 10% showed a significantly increased affinity for HEL (5-to 21-fold increase), with most clones retaining the favorable developability profile of the parental antibody. These data open the possibility of in silico antibody stabilization and affinity maturation without the need to predict the antibody-antigen interface, which is notoriously difficult in the absence of crystal structures.

molecular biology↗

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

AMPK Signaling Regulates Expression of Urea Cycle Enzymes in Response to Changes in Dietary Protein Intake

Abundance of urea cycle enzymes in the liver is regulated by the dietary protein intake. Although urea cycle enzyme levels rise in response to a high protein diet, signaling networks that sense dietary protein intake and trigger changes in expression of urea cycle genes have not been identified. The aim of this study was to identify signaling pathway(s) that respond to changes in protein intake and regulate expression of urea cycle genes in mice and human hepatocytes. Mice were adapted to either control or high (HP) protein diets followed by isolation of liver protein and mRNA and integrated analysis of the proteomic and transcriptome profiles. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways, and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism, which implicated AMPK as a possible regulator. Primary human hepatocytes, treated with AICAR an activator of AMPK, were used to test whether AMPK regulates expression of urea cycle genes. The abundance of CPS1 and OTC mRNA increased in hepatocytes treated with AICAR, which supports a role for AMPK signaling in regulation of the urea cycle. Because AMPK is either a target of drugs used to treat type-2 diabetes, these drugs might increase the expression of urea cycle enzymes in patients with urea cycle disorders, which could be the basis of a new therapeutic approach.\n\nAuthor summaryIntegrated analysis of transcriptional and proteomic profiles of the liver tissue from mice fed different protein content diets revealed that AMPK signaling pathway regulates expression of urea cycle enzymes.

molecular biology↗

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↗