UBR-1 enzyme network regulates glutamate homeostasis to affect organismal behavior and developmental viability

Abstract

Johanson-Blizzard Syndrome (JBS) is an autosomal recessive spectrum disorder associated with the UBR-1 ubiquitin ligase that features developmental delay including motor abnormalities. Here, we demonstrate that C. elegans UBR-1 regulates high-intensity locomotor behavior and developmental viability via both ubiquitin ligase and scaffolding mechanisms. Super-resolution imaging with CRISPR-engineered UBR-1 and genetic results demonstrated that UBR-1 is expressed and functions in the nervous system including in pre-motor interneurons. To decipher mechanisms of UBR-1 function, we deployed CRISPR-based proteomics using C. elegans which identified a cadre of glutamate metabolic enzymes physically associated with UBR-1 including GLN-3, GOT-2.2, GFAT-1 and GDH-1. Similar to UBR-1, all four glutamate enzymes are genetically linked to human developmental and neurological deficits. Proteomics, multi-gene interaction studies, and pharmacological findings indicated that UBR-1, GLN-3 and GOT-2.2 form a signaling axis that regulates glutamate homeostasis. Developmentally, UBR-1 is expressed in embryos and functions with GLN-3 to regulate viability. Overall, our results suggest UBR-1 is an enzyme hub in a GOT-2.2/UBR-1/GLN-3 axis that maintains glutamate homeostasis required for efficient locomotion and organismal viability. Given the prominent role of glutamate within and outside the nervous system, the UBR-1 glutamate homeostatic network we have identified could contribute to JBS etiology.

Fig 1.. UBR-1 regulates high-intensity locomotor behavior in C. elegans

A. Schematic of UBR-1 and annotated protein domains showing deletion allele (tm5996) and CRISPR engineered RING inactivation alleles, LD1 (bgg116) and LD2 (bgg212). B. Still frame images show impaired locomotion in ubr-1 mutants swimming including altered tail movement (arrowheads). C. Schematic of MWT tracking for high-intensity locomotor swimming behavior. D. 30 sec MWT animal traces show individual ubr-1 mutant with impaired locomotion. E. Quantitation of locomotor defects in ubr-1 mutants and UBR-1 transgenic rescue. Shown are MWT plots of average locomotor speed (left) and expanded quantitation at 90 minutes (right) for indicated genotypes. F. AlphaFold prediction of UBR-1 structure. Inset shows RING domain (teal) and CRISR-engineered residues that are altered in ubr-1 LD1 (dark blue) and LD2 (green) mutants. Also shown are predicted coordination sites for Zn²⁺ ions (red circles), UBR box 1 (purple) and box 2 (orange). G. Quantitation indicates locomotor speed is reduced in ubr-1 LD1 and LD2 mutants. For E and G, MWT plots (solid lines, left) represent average speed of all recorded animals (4 animals/well, 4–5 wells per genotype per experiment, and 3–4 independent experiments) and shaded regions are SEM. Bars represent average of all wells for indicated time point, dots represent single wells tracked (4 animals/well), and error bars are SEM. Significance for plots (genotype annotations, left) tested with pairwise two-way ANOVA, and significance for bars with dots (right) tested using one-way ANOVA with Bonferroni’s post-hoc correction for multiple comparisons. **** p<0.0001. *** p<0.001

Fig 2:. UBR-1 is expressed broadly in head neurons including pre-motor interneurons where it functions to regulate locomotor behavior.

A. Schematic of GFP::UBR-1 generated by CRISPR engineering. B. Diagram of C. elegans head where cell bodies of sensory, interneurons and pre-motor interneurons as well as axonal nerve ring are located. C. Super-resolution images of adult C. elegans showing CRISPR engineered GFP::UBR-1 is expressed and localized to neuronal cell bodies (arrows) and nerve ring (dashed oval). D. Diagram shows pre-motor interneurons in head and tail of C. elegans with axons entering ventral nerve cord (VNC). Interneurons occur on both sides of head but only one side is depicted. E and F. Super-resolution images of GFP::UBR-1 (green) in cell bodies (arrows) and axons (arrowhead) of pre-motor interneurons in head (E) and tail (F). Pre-motor interneurons visualized using Pglr-1::RFP (magenta). G. Quantitation of locomotor speed indicates UBR-1 expressed in pre-motor interneurons partially rescues locomotor defects in ubr-1 mutants. For G, significance for data shown in plots (genotype annotations, left) tested with pairwise two-way ANOVA, and significance for bars with dots (right) tested using one-way ANOVA and Bonferroni’s post-hoc correction. ** p<0.01 Scale bar is 10 μm.

Fig 3:. Large-scale CRISPR-based proteomics with UBR-1 identifies glutamate enzyme mini-network.

A. Schematics depict GFP::UBR-1 and GFP::SL2::UBR-1 CRISPR engineered constructs used for AP-proteomics. B. Summary of CRISPR-based AP-proteomics workflow for C. elegans. C. Example of silver stain (left) and immunoblot (right) of anti-GFP purified samples used for proteomics. D. Example of single proteomics experiment with proteins identified for GFP::UBR-1 sample (test sample, y-axis) and GFP::SL2::UBR-1 sample (negative control, x-axis). Only protein hits with 1.5-fold or greater enrichment in GFP::UBR-1 sample (dotted line) were plotted. Highlighted proteins are UBR-1 affinity target (gray) and glutamate metabolic enzymes: GDH-1, GOT-2.2, GLN-3, AND GFAT-1 (green). E. Diagram of STRING computational network analysis for GOT-2.2, GDH-1, GLN-3, and GFAT-1. Pink lines highlight protein-protein interactions identified in this study between UBR-1 and glutamate enzyme mini-network. F. Summary of mass spectrometry results and statistics for 10 independent UBR-1 proteomics experiments from C. elegans. G. Quantitative analysis of glutamate enzyme enrichment in GFP::UBR-1 proteomics compared to GFP::SL2::UBR-1 (SL2) negative controls. For F and G, significance tested using Mann-Whitney and adjusted for multiple comparisons with 5% false-discovery rate (FDR). ** p<0.01, * p<0.05

Fig 4:. GOT-2.2/UBR-1/GLN-3 axis regulates locomotor activity.

A) Schematic showing opposing effects of GOT-2.2 and GLN-3 on glutamate metabolism. B and C. Protein diagrams of GOT-2.2 and GLN-3 isoforms with CRISPR engineered three-frame STOP cassettes. D. Quantitation of locomotor swimming speed showing sustained locomotion in got-2.2(bgg204) mutants and early fatigue in gln-3(bgg206) mutants in MWT plots (left). Expanded quantitation is shown for got-2.2 at 150 mins (middle) and gln-3 at 95 mins (right). E. Quantitation shows ubr-1; got-2.2 double mutants have similar decrease in speed as ubr-1 mutants, and significant decrease compared to got-2.2 single mutants. F. Quantitation shows ubr-1; gln-3 double mutants have similar decrease in speed to ubr-1 single mutants. For D-F, MWT plots (solid lines, left) represent average speed of all recorded animals (4 animals/well, 5 wells per genotype per experiment, and 3–4 independent experiments) and shaded regions are SEM. Bars (right) represent average of all wells for indicated time point, dots represent single wells tracked (4 animals/well), and error bars are SEM. Significance for plots (genotype annotations, left) tested with pairwise two-way ANOVA, and significance for bars with dots (right) tested using one-way ANOVA with Bonferroni’s correction for multiple comparisons. **** p<0.0001. *** p<0.001, ** p<0.01

Fig 5:. GOT-2.2/UBR-1/GLN-3 axis regulates glutamate homeostasis to affect cholinergic motor neurons and locomotor behavior.

A. Simplified C. elegans sensorimotor circuit showing how aldicarb acetylcholine esterase (AChE) inhibitor alters ACh motor neuron function. Right panels depict how altering glutamate homeostasis influences ACh motor neuron function and aldicarb sensitivity. B. Quantitative MWT results show ubr-1 mutants are hypersensitive to aldicarb treatment (arrow). Shown are normalized MWT plots of average locomotor speed (left) and expanded quantitation at 45 mins (right). C. Quantitative results show got-2.2 mutants are aldicarb resistant, and both gln-3 and ubr-1 mutants display aldicarb hypersensitivity. D. Quantitation shows aldicarb resistance is suppressed in ubr-1; got-2.2 double mutants compared to got-2.2 single mutants. E. Quantitation shows aldicarb hypersensitivity is enhanced in ubr-1; gln-3 double mutants compared to gln-3 single mutants. F. Quantitative LC-MS results show increased whole-animal glutamate levels in gln-3 mutants. For B-E, MWT plots (solid lines, left) represent average speed of all recorded animals (4 animals/well, 5 wells per genotype per experiment, and 3–4 independent experiments) and shaded regions are SEM. MWT plots are normalized to average speed over 10 min baseline prior to aldicarb treatment. Bars (right) represent average of all wells for indicated time point, dots represent single wells tracked (4 animals/well), and error bars are SEM. Significance for plots (genotype annotations, left) tested with pairwise two-way ANOVA, and significance for bars with dots (right) tested using one-way ANOVA and Bonferroni’s post-hoc correction for multiple comparisons. For F, lines are mean, dots are single LC-MS result, and error bars are SEM. Significance determined using Student’s t test with Bonferonni correction **** p<0.0001. *** p<0.001, ** p<0.01, * p<0.05

Fig 6:. UBR-1 is expressed in gonad and functions with GLN-3 to regulate developmental viability.

A. Diagram of C. elegans adult gonad with germ cells, oocytes and embryos. B and C. Super-resolution images showing CRISPR engineered GFP::UBR-1 is expressed in germ cells and oocytes (B) as well as germ cells and embryos (C). D. Representative plates showing offspring laid by a single adult animal after 96 hrs for indicated genotypes. E. Quantitation shows reduced total viable offspring for ubr-1 and gln-3 mutants compared to wt. Viability is not further reduced in ubr-1(tm5996); gln-3 double mutants compared to gln-3 single mutants. Dots represent total viable offspring laid over 96 hrs from a single adult. Significance was tested with one-way ANOVA and Bonferroni’s post-hoc correction for multiple comparisons. **** p<0.0001, ns = not significant Scale bar is 10 μm

Fig 7:. GOT-2.2/UBR-1/GLN-3 axis regulates glutamate homeostasis to affect high-intensity locomotor behavior and developmental viability.

Summary shows that UBR-1 functions as an enzyme hub in the GOT-2.2/UBR-1/GLN-3 axis that is required to maintain glutamate homeostasis. UBR-1 axis regulation of glutamate homeostasis affects ACh motor neuron function and cellular metabolism leading to outcomes on locomotor behavior, sensitivity to aldicarb perturbation of ACh motor neuron function, and developmental viability.