Natural SEL1L variants modify ERAD, proteasome function, and survival in a Drosophila model of NGLY1 deficiency

Natural SEL1L variants modify ERAD, proteasome function, and survival in a Drosophila model of NGLY1 deficiency | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Natural SEL1L variants modify ERAD, proteasome function, and survival in a Drosophila model of NGLY1 deficiency Travis K. Tu’ifua , Clement Y. Chow doi: https://doi.org/10.1101/2025.03.06.641902 Travis K. Tu’ifua 1 Department of Human Genetics, University of Utah School of Medicine , Salt Lake City, Utah, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Clement Y. Chow 1 Department of Human Genetics, University of Utah School of Medicine , Salt Lake City, Utah, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: cchow{at}genetics.utah.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease caused by autosomal recessive loss-of-function mutations in the NGLY1 gene. NGLY1 removes N-linked glycans from glycoproteins in the cytoplasm and is thought to help clear misfolded proteins from the endoplasmic reticulum (ER) through the ER associated degradation (ERAD) pathway. Despite this, the physiological significance of NGLY1 in ERAD is not understood. The best characterized substrate of NGLY1 is NRF1, a transcription factor that upregulates proteasome expression and the proteasome bounce-back response. We previously performed a genetic modifier screen using a Drosophila model of NGLY1 deficiency and identified potential modifiers that alter the lethality of the model. We identified two protein-coding variants in Hrd3 / SEL1L : S780P and Δ806-809 . Both variants are localized to the SEL1L cytoplasmic tail, an uncharacterized domain. SEL1L is a component of the ERAD complex that retrotranslocates misfolded proteins from the ER to the cytoplasm for degradation. We used CRISPR to generate fly lines carrying these SEL1L variants in a common genetic background and tested them with our model of NGLY1 deficiency. Validating our previous screen, the SEL1L P780 and SEL1L Δ806-809 variants increase the survival of the NGLY1 deficiency model, compared to the SEL1L S780 variant. To determine how these SEL1L variants were modifying lethality in NGLY1 deficiency, we interrogated the ERAD and NRF1 signaling pathways. We found that the SEL1L P780 and SEL1L Δ806-809 variants improve ERAD function in an NGLY1- dependent manner, further implicating NGLY1 in general ERAD function. We also found that these variants protect against changes in larval size and survival caused by proteasome inhibition in heterozygous NGLY1 null flies. These results provide new insights into the role of SEL1L in the disease pathogenesis of NGLY1 deficiency. SEL1L is a strong candidate modifier gene in patients, where variability in presentation is common. AUTHOR’S SUMMARY NGLY1 deficiency is a debilitating rare genetic disorder. There are currently no treatment options for NGLY1 deficiency and NGLY1 biology remains poorly understood. We previously performed a genetic modifier screen in a Drosophila model of NGLY1 deficiency and identified a number of candidate modifier genes that impacted the survival of our model. Modifier genes can help reveal NGLY1 biology and NGLY1 deficiency disease pathogenesis. In this study, we follow-up on two natural protein-coding variants of Hrd3 (the fly version of the human gene, SEL1L ) that increased the survival of our NGLY1 deficiency model. SEL1L is a critical component of an important quality control pathway called the endoplasmic reticulum associated degradation (ERAD) pathway. We discovered that these SEL1L variants enhance ERAD and modify NGLY1 deficiency sensitivity to proteasome inhibition. This study confirms SEL1L as an important modifier gene of NGLY1 deficiency. Further study of the ERAD and proteasome degradation pathways may reveal additional candidate modifier genes of NGLY1 deficiency and potential targets for therapeutic development. INTRODUCTION N-glycanase 1 (NGLY1) deficiency is an ultra-rare disease and the first identified congenital disorder of deglycosylation (CDDG). The disease is caused by autosomal recessive loss-of-function mutations in the NGLY1 gene ( 1 , 2 ). NGLY1 is a cytosolic deglycosylating enzyme that removes N-linked glycans from proteins. NGLY1 is thought to be a component of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway, an important cellular quality control mechanism which removes misfolded proteins from the ER to the cytosol for degradation ( 3 , 4 ). However, the loss of NGLY1 shows little effect on ERAD and does not prevent the degradation of misfolded proteins ( 5 – 7 ). Therefore, despite its known function as a deglycosylating enzyme, the physiological significance of NGLY1 and disease pathogenesis remains poorly understood. NGLY1 deficiency is marked by extensive phenotypic heterogeneity, even among patients with identical NGLY1 mutations ( 2 , 8 ), suggesting the presence of genetic modifiers. In a previous genetic screen, we identified 61 potential modifier genes that were associated with changes in survival in our Drosophila model of NGLY1 deficiency ( 9 ). From this screen, our top hit was Ncc69 ( Drosophila ortholog for human NKCC1/2 ), which encodes for a conserved ion transporter and we showed that it is both a substrate of NGLY1 and a modifier of NGLY1 deficiency ( 9 ). Another interesting candidate modifier gene we identified in the screen was Hrd3 (hereon referred to by the human ortholog SEL1L ). Through a genome-wide association study (GWAS), we identified a natural missense variant in SEL1L that was associated with increased survival in the Drosophila NGLY1 deficiency model ( 9 ). This SEL1L variant is a substitution of serine 780 ( SEL1L S780 ) for a proline ( SEL1L P780 ). Additionally, we identified a private protein-coding deletion in the strain showing near complete rescue of NGLY1 deficiency lethality. This second variant is a deletion of amino acids 806 to 809 ( SEL1L Δ806-809 ). Both variants are 26 amino acids apart and are located in the cytoplasmic tail of SEL1L. SEL1L is a single-pass ER membrane protein and a critical, well-established component of ERAD. ERAD functions alongside other quality control mechanisms such as the unfolded protein response (UPR) and autophagy to maintain ER homeostasis and prevent ER stress ( 10 – 12 ). The SEL1L-Hrd1 ERAD complex is the most conserved branch of ERAD from yeast to humans and translocates misfolded proteins from the ER to the cytosol for proteasomal degradation ( 13 – 15 ). The luminal domain of SEL1L assists in the recognition of ERAD substrates in the ER lumen and the transmembrane domain helps move proteins through the ER membrane to the cytosol ( 16 ). The cytoplasmic tail of SEL1L is a highly disordered region across species and its function is unknown. In previous studies, both SEL1L and NGLY1 were identified as genetic modifiers of NRF1, a transcription factor responsible for the proteasome bounce-back response ( 17 , 18 ). NRF1 is co-translated and glycosylated in the ER before ERAD machinery translocates NRF1 to the cytosol ( 19 ). NGLY1 deglycosylation and DDI1/2 protease cleavage activate NRF1. Deglycosylation of N-linked glycans by NGLY1 results in the deamidation of asparagine to aspartate residues. This amino acid editing is necessary and sufficient for NRF1 activation, localization, and function ( 17 , 18 ). The protease DDI1/2 cleaves NRF1 to release the activated protein from its ER tether ( 17 , 18 , 20 ). Under healthy, homeostatic conditions, activated NRF1 is constitutively degraded by the proteasome. Under conditions of proteasomal stress, however, NRF1 is not degraded, accumulates in the cytosol, and is transported to the nucleus where it acts as a transcription factor and upregulates genes that increase proteasome function, including proteasome subunit genes ( 17 , 18 , 21 ). This activation of NRF1 is known as the proteasome bounce-back response. NGLY1 and ERAD machinery are necessary for NRF1 activation and the loss of either prevents the proteasome bounce-back response ( 17 , 18 ). In this study, we characterized the functional consequences of the two new SEL1L variants identified in our NGLY1 deficiency genetic screen. We placed the SEL1L variants on an isogenic background to test the effects each variant has on both SEL1L and NGLY1. The SEL1L variants increase survival in our NGLY1 deficiency model, validating the observations from the modifier screen. The SEL1L variants also enhance ERAD in an NGLY1-dependent manner and provide a protective fitness advantage during proteasome inhibition. Our results suggest that SEL1L is a modifier of NGLY1 and that interactions between SEL1L , NGLY1 , and NRF1 underly these observed changes in fitness. These genetic interactions are potential targets for NGLY1 deficiency treatment. RESULTS SEL1L P780 and SEL1L Δ806-809 variants increase survival of NGLY1 deficiency model In a previous study, we crossed our NGLY1 deficiency Drosophila model, which uses the GAL4/UAS system to ubiquitously express RNAi against NGLY1 , with nearly 200 strains of the Drosophila Genetic Reference Panel (DGRP) ( 9 , 22 ). On a standard laboratory background, the NGLY1 deficiency model has ∼30% survival to adulthood ( 7 , 9 ). In the DGRP strains, the NGLY1 deficiency model showed survival ranging from 0 to 100%, indicating that lethality is highly modifiable by genetic background. We performed a GWAS to identify candidate modifier genes associated with increased survival. One of the top associated variants was the S780P missense variant in SEL1L . The SEL1L P780 minor allele was associated with increased survival of the NGLY1 deficiency model, compared to the common SEL1L S780 allele. We also discovered that the DGRP strain with nearly 100% survival in the screen (DGRP strain 379) harbored a private SEL1L variant, resulting in the deletion of amino acids 806-809 ( SEL1L Δ806-809 ). This strain also carries the more common S780 allele. SEL1L Δ806-809 was not formally identified through the GWAS because it is a private variant in a single DGRP strain. Because these two variants were both in the cytoplasmic tail of SEL1L, a functionally uncharacterized region of the protein, we sought to understand how these variants were affecting survival of the NGLY1 deficiency model. We used CRISPR to place each of the SEL1L variants ( Fig 1A ) onto the same isogenetic background, creating three strains that are homozygous for SEL1L S780 , SEL1L P780 , or SEL1L Δ806-809 . This allowed us to test for phenotypic differences specific to each SEL1L variant. Download figure Open in new tab Fig 1. SEL1L variants increase NGLY1 deficiency survival. (A) Amino acid sequences for the three SEL1L variant alleles. Red boxes highlight differences between alleles. (B) SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes significantly increase the proportion surviving (∼70%) with NGLY1 knockdown when compared to SEL1L (S/S, +/+) (45%, p<0.0001). Chi-squared test. (C) Lifespan of NGLY1 KD flies shows no significant difference in survival between SEL1L genotypes. Cox proportional hazard regression analysis In our original screen, we crossed a strain carrying both a GAL4 and NGLY1 RNAi transgene with strains of the DGRP. Lethality was scored based on survival of the F1 flies, which had half of their genomes coming from the NGLY1 RNAi strain and half from the different DGRP strains. Because the NGLY1 RNAi strain is homozygous for the common SEL1L S780 allele, any new SEL1L variant introduced by the DGRP strain in the F1 generation was heterozygous with the SEL1L S780 allele. Therefore, the relevant SEL1L genotypes from our screen are: SEL1L (S/S, +/+) , SEL1L (S/P, +/+) , or SEL1L (S/S, +/DEL) . We focus our analyses on these three SEL1L genotypes throughout this study. Based on the original DGRP screen, we expected that the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes would increase survival of the NGLY1 model, compared to the SEL1L (S/S, +/+) genotype. To validate the results of the screen, we crossed the same NGLY1 RNAi strain used in the modifier screen with each of our new SEL1L variant CRISPR strains to generate flies that have the exact SEL1L genotypes from the screen. The proportion surviving was determined in the same manner as the original screen, by dividing the number of NGLY1 knockdown flies by the largest balancer class in its cross ( 9 ). There was significantly increased survival of NGLY1 knockdown flies with the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes compared to the SEL1L (S/S, +/+) genotype ( Fig 1B and S1 Data). The SEL1L (S/S, +/+) genotype had a 45% proportion surviving compared to ∼70% in the SEL1L (S/P, +/+) (p<0.001) and SEL1L (S/S, +/DEL) (p<0.001) genotypes. This result nicely replicates our previous genetic screen that showed increased survival in the DGRP lines with these particular SEL1L variants and suggests that the SEL1L P780 and SEL1L Δ806-809 alleles are protective against NGLY1 deficiency. We also evaluated the lifespan of these surviving NGLY1 knockdown flies, but found no significant differences in survival between the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) and the SEL1L (S/S, +/+) genotype ( Fig 1C and S1 Data), suggesting that the interaction occurs during development. SEL1L variants impact sensitivity to proteasome inhibition in NGLY1 +/- flies We next interrogated pathways that involve both SEL1L and NGLY1 to understand how these SEL1L variants are protecting against NGLY1 deficiency. Both NGLY1 and SEL1L were previously identified as modifier genes of NRF1 , which encodes for an important transcription factor that upregulates proteasome genes in response to proteasomal stress ( 17 , 18 ). The importance of NGLY1 in NRF1 function is well established. NGLY1 mutants have reduced proteasome function and are exquisitely sensitive to proteasome stress because NRF1 is not processed ( 17 , 18 , 23 , 24 ). Previous studies have shown that heterozygous NGLY1 null larvae, which are otherwise normal, are sensitive to proteasome inhibition, leading to larval size defects ( 23 , 24 ). Although SEL1L was identified as a genetic modifier of NRF1, its role in NRF1 signaling has not been determined. We hypothesized that these SEL1L variants would affect NRF1 signaling and modify phenotypes in an NGLY1 deficiency model. We tested whether the SEL1L variants affect proteasome sensitivity in NGLY deficient Drosophila using the proteasome inhibitor bortezomib (BTZ). Homozygous NGLY1 null Drosophila are embryonic lethal, but heterozygous NGLY1 null flies are phenotypically normal when unchallenged. We used heterozygous NGLY1 null flies as a model of NGLY1 deficiency because of their known increased sensitivity to proteasome inhibition ( 23 , 24 ). Because the NGLY1 null strain also carries the common SEL1L S780 allele, when we cross this strain with our CRISPR generated SEL1L variant strains, we generate heterozygous NGLY1 null flies with the same SEL1L genotypes to what we tested in the NGLY1 knockdown model: SEL1L (S/S, +/+) , SEL1L ( S/P ,+/+) , or SEL1L (S/S, +/DEL) . Heterozygous NGLY1 null Drosophila larvae develop smaller when exposed to proteasome inhibition, compared to NGLY1 wildtype and DMSO-treated heterozygous NGLY1 null controls ( 23 , 24 ). In previous studies, heterozygous NGLY1 null larvae, when exposed to 5μM BTZ, are significantly smaller than DMSO-treated heterozygous NGLY1 null larvae ( 24 ). We observed an equally strong decrease in larval size with the treatment of 5μM bortezomib in all heterozygous NGLY1 null larvae compared to DMSO controls, regardless of SEL1L genotype ( Fig 2A and S2 Data). SEL1L genotype does not impact the size defects induced by 5μM BTZ in NGLY1 heterozygous null larvae. Download figure Open in new tab Fig 2. SEL1L variants affect proteasome inhibition sensitivity. (A) NGLY1 +/- larvae are smaller when treated with 5μM BTZ, but there is no effect of SEL1L genotype (Larval size on DMSO: SEL1L (S/S, +/+) 23.59 ± 4.97, SEL1L (S/P, +/+) 19.37 ± 9.16, and SEL1L (S/S, +/DEL) 21.32 ± 6.73. Decreased larval size on BTZ: SEL1L (S/S, +/+) 9.19 ± 1.61, p<0.0001; SEL1L (S/P, +/+) 8.55 ± 2.04, p<0.0001; and SEL1L (S/S, +/DEL) 10.07 ± 1.14, p<0.01). (B) When treated with 1μM BTZ, the NGLY1 +/- larvae show a SEL1L genotype-dependent decrease in size. While SEL1L (S/S, +/+) showed no change between DMSO (19.65 ± 5.98) and BTZ (17.88 ± 4.32), SEL1L (S/P, +/+) (DMSO 21.03 ± 3.42, BTZ 16.45 ± 3.65, p<0.0001) and SEL1L (S/S, +/DEL) (DMSO 24.92 ± 3.39, BTZ 20.08 ± 2.69, p=0.001) genotypes were smaller when treated with BTZ compared to DMSO treated larvae. (C) NGLY1 WT larvae show no significant decrease in size with 1μM BTZ treatment, regardless of SEL1L genotype (Larval size on DMSO: SEL1L (S/S, +/+) 25.75 ± 6.91, SEL1L (S/P, +/+) 23.74 ± 6.94, and SEL1L (S/S, +/DEL) 26.42 ± 4.29; larval size on BTZ: SEL1L (S/S, +/+) 24.75 ± 5.63, SEL1L (S/P, +/+) 20.96 ± 7.38, and SEL1L (S/S, +/DEL) 25.70 ± 5.23). To determine whether there might be more subtle effects, we treated the heterozygous NGLY1 null larvae with a lower concentration of 1μM BTZ. The SEL1L (S/S, +/+) larvae showed no significant size differences between BTZ and DMSO treatments. However, SEL1L (S/P, +/+) (p<0.0001) and SEL1L (S/S, +/DEL) (p= 0.001) larvae had significant decreases in larval size with 1μM BTZ treatment compared to DMSO ( Fig 2B and S2 Data). This indicates that larval size in heterozygous NGLY1 null larvae carrying the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes are particularly sensitive to proteasome inhibition compared to SEL1L (S/S, +/+) . On an NGLY1 wildtype background, the different SEL1L genotypes showed no larval size changes with the 1μM BTZ treatment ( Fig 2C and S2 Data). SEL1L variants increase survival of NGLY1 +/- flies in response to proteasome inhibition To further examine the impact of the SEL1L variants on NRF1 signaling, we tested other phenotypes affected by NGLY1 deficiency and proteasome inhibition. When heterozygous NGLY1 null larvae were treated with 1μM BTZ, we observed a SEL1L variant specific effect on survival through eclosion to adulthood. The SEL1L (S/P, +/+) and the SEL1L (S/S, +/DEL) larvae showed higher survival at 83% (p<0.0001) and 96% (p<0.0001), respectively, compared to the SEL1L (S/S, +/+) flies at 59% survival ( Fig 3A and S3 Data). SEL1L (S/S, +/+) larvae treated with bortezomib showed high rates of non-eclosed and partially eclosed flies. The improved eclosion and survival rates of the SEL1L (S/P, +/+) and the SEL1L (S/S, +/DEL) genotypes suggests a protective effect of the SEL1L P780 and SEL1L Δ806-809 variants against NGLY1 deficiency and proteasome inhibition during larval development. Importantly, when NGLY1 wildtype larvae were raised on 1μM BTZ, we observed no lethality and nearly 100% eclosion of flies, regardless of SEL1L genotype, demonstrating that lethality to BTZ is NGLY1-dependent (S1 Fig and S3 Data). SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes provide near complete rescue of the heterozygous NGLY1 null larvae lethality induced by 1μM BTZ. Download figure Open in new tab Fig 3. SEL1L variants increase survival under proteasome inhibition. (A) When treated with 1μM BTZ, NGLY1 +/- larvae eclose at significantly higher rates when carrying the SEL1L (S/P, +/+) (83%, p<0.0001) and SEL1L (S/S, +/DEL) (96%, p<0.0001) genotypes compared to the SEL1L (S/S, +/+) genotype (59%). (B) When treated with 1μM BTZ, adult NGLY1+/- flies with the SEL1L (S/P, +/+) (p<0.0001) and SEL1L (S/S, +/DEL) (p<0.0001) genotypes live longer than the SEL1L (S/S, +/+) . Cox proportional hazard regression analysis. (C) There is no significant difference in survival between adult NGLY1 +/- flies treated with DMSO regardless of SEL1L genotype. To understand if the SEL1L variants impact NRF1 signaling in adult flies, we treated heterozygous NGLY1 null adult flies with bortezomib or DMSO and observed their long-term, adult survival. The adult flies were raised on DMSO during their larval stages and were either maintained on DMSO or BTZ food. SEL1L (S/S, +/+) adult flies showed significantly decreased survival time on BTZ compared to the SEL1L (S/P, +/+) (p<0.0001) and the SEL1L (S/S, +/DEL) (p<0.0001) genotypes ( Fig 3B and S3 Data). The increased survival times of the bortezomib-treated SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes indicate that these variants are protective against proteasome inhibition during adulthood. When treated with DMSO, heterozygous NGLY1 null adult flies displayed no significant differences in long-term survival, regardless of SEL1L variant genotypes ( Fig 3C and S3 Data). These experiments demonstrate that the sensitivity to proteasome inhibition in NGLY1 deficiency is modified by these SEL1L variants, both during larval development and in adulthood. These results suggest that these SEL1L variants impact proteasome sensitivity in an NGLY1-dependent manner. SEL1L effects on NRF1 signaling and proteasome gene expression In response to proteasome inhibition, NRF1 upregulates proteasome genes ( 21 ). To test for changes in NRF1 signaling in our SEL1L variant flies, we examined the expression of several proteasomal subunit genes: prosalpha6 , prosalpha3 , prosbeta2 , prosbeta4 , and prosbeta5 . These five genes were among other proteasome genes that we previously identified as downregulated in our NGLY1 deficiency fly model ( 7 ). We treated flies with BTZ to induce NRF1 signaling. When we compared expression of the proteasome genes, in whole flies, between NGLY1 wildtype and heterozygous NGLY1 null adults, we mostly observed no differences in gene expression within each condition and SEL1L genotype (S2 Fig A and S4 Data). We observed increases in proteasome gene expression with the treatment of BTZ compared to DMSO; however, we found no SEL1L variant specific differences in gene expression in either the NGLY1 wildtype or heterozygous NGLY1 null flies (S2 Fig B and S4 Data). While this is unexpected in light of the previous phenotypic data we present, it is possible that there are specific effects in different tissues that are missed when we examine expression in whole flies. To date, it is unknown which tissues contribute to the lethality in NGLY1 flies and more work is needed to determine which tissues are most impacted by proteasome inhibition. SEL1L P780 and SEL1L Δ806-809 variants enhance ERAD in an ER stress model SEL1L is an integral component of the Hrd1 ERAD complex, which retrotranslocates misfolded proteins from the ER lumen and ubiquitinates them for degradation by the proteasome ( 10 , 13 , 15 ). Although its role in ERAD remains unclear, NGLY1 has been shown to physically interact with proteins in the ERAD complex, including Derlin-1 and VCP ( 25 , 26 ). The deglycosylation of ERAD substrates by NGLY1 is thought to prepare misfolded glycoproteins for proteasomal degradation ( 5 , 27 , 28 ). We crossed a Drosophila eye model of ER stress with the SEL1L strains to determine the variant-specific effects on ERAD. In this model, a transgene carries an eye-specific GAL4 ( GMR-GAL4 ) that drives the overexpression of a mutant misfolded rhodopsin protein (encoded by UAS- Rh1 G69D ) that constitutively misfolds and induces degeneration in the developing larval eye disc ( 29 – 32 ). The misfolded rhodopsin protein leads to chronic ER stress in the eye disc, cell death, and a rough eye phenotype in adult flies. These eyes are also significantly smaller than wildtype eyes. This model is sensitive to changes in ERAD function and increased ERAD function is protective against eye degeneration ( 29 ). In larval eye discs, overexpressing Hrd1, the ERAD protein essential for transporting misfolded proteins out of the ER, nearly completely restored the normal appearance of the eye ( 29 ). Increasing Hrd1 levels enhances ERAD, helping to clear misfolded rhodopsin, which in turn prevents ER stress and protects against degeneration. Conversely, knockdown of ERAD components in this model leads to increased eye degeneration ( 29 ). Because SEL1L is a component of the Hrd1 ERAD complex, modifying SEL1L should similarly affect phenotypes in the ER stress eye model. We hypothesized that losing SEL1L would decrease ERAD and enhance the eye degeneration phenotype, leading to a smaller eye size. We expressed SEL1L RNAi in the eye discs to knockdown SEL1L in the ER stress model (and NGLY1 wildtype) and observed that the eyes were significantly smaller than in the flies without knockdown of SEL1L (p= 0.0016) ( Fig 4A and S5 Data). The reduction in eye size observed in this model following SEL1L knockdown is expected, given SEL1L’s established role in ERAD function. We next crossed the different SEL1L variants onto this model (and NGLY1 wildtype) to investigate their effects on eye size. Because the ER stress model strain carries the common SEL1L S780 allele, crossing our SEL1L variants onto this model provided the same SEL1L genotypes as previously described. We observed an increase in eye size of the SEL1L (S/P, +/+) (p<0.0001) and SEL1L (S/S, +/DEL) (p<0.0001) genotypes compared to the SEL1L (S/S, +/+) flies ( Fig 4B and S5 Data), opposite of what we observed with the RNAi knockdown experiment. Given that previous studies show that improvement in ERAD function increases eye size, this result suggests that the SEL1L P780 and SEL1L Δ806-809 alleles improve ERAD function in an NGLY1 wildtype background. Download figure Open in new tab Fig 4. SEL1L variants enhance ERAD in an NGLY1-dependent manner. (A) Knockdown of SEL1L reduces eye size in an ER stress model expressing a misfolded protein in the eye (Eye size of Control 12.38 ± 0.93, SEL1L RNAi 10.93 ± 0.65, p=0.0016). (B) In the ER stress model, with an NGLY1 wildtype background, SEL1L (S/P, +/+) (9.13 ± 0.49, p<0.0001) and SEL1L (S/S, +/DEL) (9.29 ± 0.69, p < 0.0001) genotypes significantly increase eye size compared to the SEL1L (S/S, +/+) genotype (8.36 ± 0.33). (C) Eye-specific knockdown of NGLY1 in the ER stress model results in no difference in eye size between the SEL1L (S/S, +/+) and SEL1L (S/P, +/+) genotypes. The SEL1L (S/S, +/DEL) genotype shows a significantly smaller eye size (p <0.05). (Eye size: SEL1L (S/S, +/+) 12.60 ± 0.50, SEL1L (S/P, +/+) 12.34 ± 0.89, and SEL1L (S/S, +/DEL) 12.16 ± 0.65). (D) Heterozygous loss of NGLY1 in the ER stress model results in no difference in eye size between the SEL1L (S/S, +/+) and SEL1L (S/P, +/+) genotypes. The SEL1L (S/S, +/DEL) genotype shows a significantly smaller eye size (p <0.0001). (Eye size: SEL1L (S/S, +/+) 10.58 ± 0.76, SEL1L (S/P, +/+) 10.88 ± 0.61, and SEL1L (S/S, +/DEL) 8.67 ± 0.54). We next tested whether ERAD is still improved by the SEL1L P780 and SEL1L Δ806-809 alleles when NGLY1 activity is reduced. When we knockdown NGLY1 in the eye of this ER stress model, eye size is no longer increased in the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes compared to the SEL1L (S/S, +/+) ( Fig 4C and S5 Data). This result suggests that the improvement in ERAD associated with the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes is dependent on NGLY1 activity. Heterozygous NGLY1 null flies also showed no improvement in ERAD in the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) genotypes compared to the SEL1L (S/S, +/+) genotype ( Fig 4D and S5 Data). Interestingly, with the loss of NGLY1, the SEL1L (S/S, +/DEL) genotype had smaller eyes than the SEL1L (S/S, +/+) genotype in both the NGLY1 knockdown (p<0.05) and heterozygous NGLY1 null (p<0.0001). We conclude that the SEL1L variants improve ERAD in an NGLY1-dependent manner, further supporting the role of NGLY1 in ERAD. In the absence or reduction of NGLY1, the SEL1L variants do not increase ERAD in this model. We also conclude that the increased survival observed with SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) variants in the NGLY1 deficiency Drosophila model are likely not attributed to improvements in ERAD of misfolded proteins. DISCUSSION In this study, we sought to understand how the SEL1L protein-coding variants identified in our Drosophila genetic screen affect NGLY1 deficiency lethality by investigating the ERAD and NRF1 signaling pathways. We conclude that the SEL1L variants are increasing survival in our NGLY1 deficiency models through the NRF1 signaling pathway. We observed that the SEL1L P780 and SEL1L Δ806-809 variants provide protection against proteasome inhibition during both larval development and in adult flies when NGLY1 was reduced. We propose that our SEL1L variants are more efficiently removing NRF1 from the ER, thereby increasing NRF1 activation by the remaining NGLY1 and resisting proteasome inhibition ( Fig 5 ). Download figure Open in new tab Fig 5. Proposed model of how SEL1L variants interact with NGLY1. (A) Under normal conditions, inactive, glycosylated NRF1 (orange oval) is removed from the ER lumen by ERAD (SEL1L) where it can be deglycosylated and activated (yellow oval) by NGLY1 and elicit a robust proteasome response. (B) In NGLY1 deficiency, less NGLY1 is available to activate NRF1, resulting in a decrease of proteasome response. (C) In NGLY1 deficiency with the SEL1L (S/P, +/+) and SEL1L (S/S, +/DEL) variants, enhanced ERAD results in an increased amount of NRF1 removed from the ER. This allows for increased NRF1 activation and increased proteasome response, despite reduced NGLY1. The results from our ERAD experiments suggest that the SEL1L variants enhance ERAD in an NGLY1-dependent manner. While ERAD is a separate pathway from NRF1 signaling, the pathways are inextricably linked. The Lehrbach and Ruvkin NRF1 screen identified loss-of-function mutations in SEL1L that decreased NRF1 signaling and previous work has shown that ERAD complex components are responsible for NRF1 removal from the ER ( 17 – 19 , 33 ). If ERAD is enhanced by SEL1L P780 and SEL1L Δ806-809 variants on an NGLY1 deficiency background, we hypothesize that, in these genotypes, more NRF1 can be translocated from the ER. This increased NRF1 removal from the ER is likely compensating for the reduction in NGLY1 and elicits a more robust proteasome bounce-back response, despite the reduction in NGLY1 ( Fig 5 ). These results point to ERAD and NRF1 signaling as being potential therapeutic targets for NGLY1 deficiency patients. While there are still no therapeutic treatments for NGLY1 deficiency patients, there are two ongoing clinical trials, including an intracerebroventricular (ICV) NGLY1 gene replacement therapy ( 34 ) and a GlcNac supplementation trial to treat alacrima, or reduced tear production ( 35 ). Further study is needed to investigate how enhancing ERAD and NRF1 signaling affect the proteasome bounce-back response in NGLY1 deficiency patients who already suffer from proteasome dysfunction ( 36 ). Most current therapeutics that target ERAD only aim to decrease its function ( 37 – 39 ), however, enhancing ERAD is possible in cells and animal models through increased expression of ERAD complex proteins ( 40 , 41 ). In addition to SEL1L, our previous modifier screen has also identified other genes involved in ERAD or ER function, including TMEM259 , TMTC2 , and ERMP1 ( 9 ). Interestingly, for both TMCT2 and ERMP1 , our GWAS independently hit two separate fly orthologs of these genes ( 9 ). ERAD components and NRF1 are strong candidates for modifier genes of NGLY1 deficiency patients and may be good drug targets for further development. METHODS Fly stocks and maintenance Stocks were maintained on standard agar-dextrose-yeast medium and standard Archon Scientific glucose fly food at 25°C on a 12-h light/dark cycle. SEL1L CRISPR strains were created by WellGenetics, Inc ( www.wellgenetics.com ) and alleles were verified by Sanger sequencing (S3 Fig). The following stocks were obtained from the Bloomington Drosophila Stock Center (Bloomington, IN): Tubulin-GAL4 (BDSC #5138), UAS-pngl-RNAi (BDSC #54853), attp2 (BDSC #36303), and attp40 (BDSC #36304). The Tubulin -GAL80 strain was provided by Dr. Carl Thummel (University of Utah). The SEL1L-RNAi strain (v1161) was obtained from the Vienna Drosophila Resource Center ( 42 ). The NGLY1 null allele carries an early stop codon in the NGLY1 gene, and was previously characterized and generously provided by Perlara, PBC ( 23 , 24 ). The NGLY1 null allele is homozygous lethal and the stock is maintained with the CyO balancer. The “ER stress model” contains GMR-GAL4 and UAS-Rh1G69D on the second chromosome and has been previously described ( 30 – 32 , 43 ). The endogenous SEL1L in this strain is homozygous for the SEL1L P780 allele. We backcrossed the SEL1L S780 allele from our CRISPR generated SEL1L S780 strain into the ER stress model for 20 generations to create the desired homozygous SEL1L S780 genotype and verified genotype through sequencing. Proteasome sensitivity larval size assay Proteasome sensitivity larval size assays were performed as previously described ( 24 ). Standard Drosophila food was melted and cooled to 60°C prior to the addition of DMSO or bortezomib. NGLY1 +/- females were pre-mated with males from a CRISPR SEL1L variant strain for 24 hours. Mated females were placed on food containing DMSO and allowed to lay eggs for approximately 8 hours. Adult flies were removed and after four days of development, 3 rd instar larvae were transferred to vials containing food with DMSO or bortezomib. After two days on bortezomib containing food, larvae were genotyped using the presence of GFP. The CyO balancer also carries a GFP marker. Larvae were confirmed to lack the balancer chromosome by checking that they were also GFP negative. Larvae were imaged at 2.5X magnification using a Leica EC3 camera. Larval size was quantified using ImageJ as previously described ( 23 , 24 ). Survival assays NGLY1 knockdown (KD) eclosion survival: Virgin females from the CRISPR SEL1L variant strains were fed yeast overnight and then crossed with males from the donor strain UAS- NGLY1RNAi/Cyo,Tubulin-GAL80; Tubulin-GAL4/TM3,Sb. Progeny were collected and scored for the four balancer classes: CyO, Sb, double balanced, or no balancers, with the no balancer flies being the NGLY1 KD. This cross should produce the expected 1:1:1:1 ratio of the four genotypes. Given that there is always a very low level of lethality associated with each balancer, the largest balancer class was considered the closest to the expected number. We scored at least 200 flies per cross. Males and females were combined for a single count. To calculate the proportion of NGLY1 KD flies by generating a ratio of NGLY1 knockdown/largest balancer class as previously described ( 9 ). For long-term survival, flies were collected, placed in a vial and flipped into fresh food every 2-3 days. Lifespan was measured as days post-eclosion. Vials were checked daily for dead flies and recorded. Eye imaging and quantification Adult female flies aged 3-5 days were collected under CO2 anesthesia then frozen at −80°C for later imaging. Eyes were imaged at 3x magnification using the Leica EC3 Camera. Eye area was measured as previously described ( 30 – 32 , 44 ) Proteasome gene RT-qPCR Changes in proteasome subunit gene expression were measured using RT-qPCR. Flies with appropriate SEL1L and NGLY1 genotypes were crossed as previously described. 24 hours after eclosion, male flies were collected and placed on either 0.2% DMSO or 3μM BTZ for 12 hours or 24 hours. Immediately after drug treatments, RNA was extracted from 8-10 whole-body flies using a Direct-zol RNA Miniprep (Zymo Research R2061) using TRIzol Reagent (ThermoFisher Cat # 15596026) and including the DNAse step. RNA was converted to cDNA using a ProtoScript® II First Strand cDNA Synthesis Kit (NEB Cat # E6560L). RT-qPCR was performed using a QuantStudio 3 96-well 0.2 ml block instrument and PowerUp SYBR Green Master Mix (ThermoFisher Cat #A25741). If available, we used primers from the FlyPrimerBank ( 45 ) located at http://www.flyrnai.org/flyprimerbank . Other primers were designed using Primer3Plus ( 46 ) located at www.primer3plus.com . All primer sequences listed in S4 Data. FUNDING This work was supported by NIGMS R35 GM124780, a grant from the Grace Science Foundation, and a gift from the Might Family to CYC. TKT was supported by NCATS NIH TL1TR002540 and ASHG Human Genetics Scholars Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supporting Information S1 Data. NGLY1 ubiquitous knockdown fly counts and long-term survival. S2 Data. Larval size assay measurements. S3 Data. Fly counts for eclosion on BTZ and survival data. S1 Fig. Wildtype NGLY1 larvae are unaffected by proteasome inhibition. S2 Fig. Proteasome gene expression changes in response to BTZ. S4 Data. qPCR results and primers used. S5 Data. Fly eye measurements with ER stress model. S3 Fig. Sequencing of CRISPR fly lines confirm appropriate SEL1L variants. REFERENCES 1. ↵ Freeze HH . Understanding Human Glycosylation Disorders: Biochemistry Leads the Charge . J Biol Chem . 2013 Mar 8; 288 ( 10 ): 6936 – 45 . OpenUrl Abstract / FREE Full Text 2. ↵ Enns GM , Shashi V , Bainbridge M , Gambello MJ , Zahir FR , Bast T , et al. Mutations in NGLY1 cause an inherited disorder of the endoplasmic reticulum–associated degradation pathway . Genetics in Medicine . 2014 Oct ; 16 ( 10 ): 751 – 8 . OpenUrl CrossRef PubMed 3. ↵ Suzuki T , Huang C , Fujihira H . The cytoplasmic peptide:N-glycanase (NGLY1); structure, expression and cellular functions . Gene . 2016 Feb 10; 577 ( 1 ): 1 – 7 . OpenUrl CrossRef PubMed 4. ↵ Hosomi A , Fujita M , Tomioka A , Kaji H , Suzuki T . Identification of PNGase-dependent ERAD substrates in Saccharomyces cerevisiae . Biochem J . 2016 Oct 1; 473 ( 19 ): 3001 – 12 . OpenUrl Abstract / FREE Full Text 5. ↵ Hirsch C , Blom D , Ploegh HL . A role for N-glycanase in the cytosolic turnover of glycoproteins . EMBO J . 2003 Mar 3; 22 ( 5 ): 1036 – 46 . OpenUrl Abstract / FREE Full Text 6. Kario E , Tirosh B , Ploegh HL , Navon A . N-Linked Glycosylation Does Not Impair Proteasomal Degradation but Affects Class I Major Histocompatibility Complex Presentation . J Biol Chem . 2008 Jan 4; 283 ( 1 ): 244 – 54 . OpenUrl Abstract / FREE Full Text 7. ↵ Owings KG , Lowry JB , Bi Y , Might M , Chow CY . Transcriptome and functional analysis in a Drosophila model of NGLY1 deficiency provides insight into therapeutic approaches . Hum Mol Genet . 2018 Mar 15; 27 ( 6 ): 1055 – 66 . OpenUrl CrossRef PubMed 8. ↵ Caglayan AO , Comu S , Baranoski JF , Parman Y , Kaymakçalan H , Akgumus GT , et al. NGLY1 Mutation Causes Neuromotor Impairment, Intellectual Disability, and Neuropathy . Eur J Med Genet . 2015 Jan ; 58 ( 1 ): 39 – 43 . OpenUrl CrossRef PubMed 9. ↵ Talsness DM , Owings KG , Coelho E , Mercenne G , Pleinis JM , Partha R , et al. A Drosophila screen identifies NKCC1 as a modifier of NGLY1 deficiency . Bellen HJ , Wittkopp PJ , Tiemeyer M , editors. eLife . 2020 Dec 14; 9 : e57831 . OpenUrl CrossRef PubMed 10. ↵ Sun S , Shi G , Han X , Francisco AB , Ji Y , Mendonça N , et al. Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival . PNAS . 2014 Feb 4; 111 ( 5 ): E582 – 91 . OpenUrl Abstract / FREE Full Text 11. Mehnert M , Sommermeyer F , Berger M , Kumar Lakshmipathy S , Gauss R , Aebi M , et al. The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins . MBoC . 2015 Jan 15; 26 ( 2 ): 185 – 94 . OpenUrl CrossRef PubMed 12. ↵ Jeong H , Sim HJ , Song EK , Lee H , Ha SC , Jun Y , et al. Crystal structure of SEL1L: Insight into the roles of SLR motifs in ERAD pathway . Sci Rep . 2016 Feb 9; 6 ( 1 ): 20261 . OpenUrl CrossRef PubMed 13. ↵ Mueller B , Lilley BN , Ploegh HL . SEL1L, the homologue of yeast Hrd3p, is involved in protein dislocation from the mammalian ER . J Cell Biol . 2006 Oct 23; 175 ( 2 ): 261 – 70 . OpenUrl Abstract / FREE Full Text 14. Mueller B , Klemm EJ , Spooner E , Claessen JH , Ploegh HL . SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins . PNAS . 2008 Aug 26; 105 ( 34 ): 12325 – 30 . OpenUrl Abstract / FREE Full Text 15. ↵ Iida Y , Fujimori T , Okawa K , Nagata K , Wada I , Hosokawa N . SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates . J Biol Chem . 2011 May 13; 286 ( 19 ): 16929 – 39 . OpenUrl Abstract / FREE Full Text 16. ↵ Hosokawa N , Wada I . Association of the SEL1L protein transmembrane domain with HRD1 ubiquitin ligase regulates ERAD-L . The FEBS Journal . 2016 ; 283 ( 1 ): 157 – 72 . OpenUrl CrossRef PubMed 17. ↵ Lehrbach NJ , Ruvkun G. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1 . Dillin A , editor. eLife . 2016 Aug 16; 5 : e17721 . OpenUrl CrossRef PubMed 18. ↵ Tomlin FM , Gerling-Driessen UIM , Liu YC , Flynn RA , Vangala JR , Lentz CS , et al. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity . ACS Cent Sci . 2017 Nov 22; 3 ( 11 ): 1143 – 55 . OpenUrl CrossRef PubMed 19. ↵ Radhakrishnan SK, den Besten W, Deshaies RJ. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition . Brown MS , editor. eLife . 2014 Jan 21; 3 : e01856 . OpenUrl CrossRef PubMed 20. ↵ Koizumi S , Irie T , Hirayama S , Sakurai Y , Yashiroda H , Naguro I , et al. The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction . Dikic I , editor. eLife . 2016 Aug 16; 5 : e18357 . OpenUrl CrossRef PubMed 21. ↵ Radhakrishnan SK , Lee CS , Young P , Beskow A , Chan JY , Deshaies RJ . Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells . Molecular Cell . 2010 Apr 9; 38 ( 1 ): 17 – 28 . OpenUrl CrossRef PubMed Web of Science 22. ↵ Mackay TFC , Richards S , Stone EA , Barbadilla A , Ayroles JF , Zhu D , et al. The Drosophila melanogaster Genetic Reference Panel . Nature . 2012 Feb ; 482 ( 7384 ): 173 – 8 . OpenUrl CrossRef PubMed Web of Science 23. ↵ Rodriguez TP , Mast JD , Hartl T , Lee T , Sand P , Perlstein EO . Defects in the Neuroendocrine Axis Contribute to Global Development Delay in a Drosophila Model of NGLY1 Deficiency . G3: Genes, Genomes, Genetics . 2018 Jul 1; 8 ( 7 ): 2193 – 204 . OpenUrl CrossRef PubMed 24. ↵ Hope KA , Berman AR , Peterson RT , Chow CY . An in vivo drug repurposing screen and transcriptional analyses reveals the serotonin pathway and GSK3 as major therapeutic targets for NGLY1 deficiency . PLOS Genetics . 2022 Feb 6; 18 ( 6 ): e1010228 . OpenUrl CrossRef 25. ↵ Katiyar S , Joshi S , Lennarz WJ . The retrotranslocation protein Derlin-1 binds peptide:N- glycanase to the endoplasmic reticulum . Molecular Biology of the Cell . 2005 ; 16 ( 10 ): 4584 – 94 . OpenUrl Abstract / FREE Full Text 26. ↵ McNEILL H , Knebel A , Arthur JSC , Cuenda A , Cohen P . A novel UBA and UBX domain protein that binds polyubiquitin and VCP and is a substrate for SAPKs . Biochem J . 2004 Dec 1; 384 ( 2 ): 391 – 400 . OpenUrl Abstract / FREE Full Text 27. ↵ Bebök Z , Mazzochi C , King SA , Hong JS , Sorscher EJ . The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary . J Biol Chem . 1998 Nov 6; 273 ( 45 ): 29873 – 8 . OpenUrl Abstract / FREE Full Text 28. ↵ Misaghi S , Pacold ME , Blom D , Ploegh HL , Korbel GA . Using a Small Molecule Inhibitor of Peptide: N-Glycanase to Probe Its Role in Glycoprotein Turnover . Chemistry & Biology . 2004 Dec 1; 11 ( 12 ): 1677 – 87 . OpenUrl CrossRef PubMed Web of Science 29. ↵ Kang MJ , Ryoo HD . Suppression of retinal degeneration in Drosophila by stimulation of ER- associated degradation . Proc Natl Acad Sci U S A . 2009 Oct 6; 106 ( 40 ): 17043 – 8 . OpenUrl Abstract / FREE Full Text 30. ↵ Chow CY , Kelsey KJP , Wolfner MF , Clark AG . Candidate genetic modifiers of retinitis pigmentosa identified by exploiting natural variation in Drosophila . Hum Mol Genet . 2016 Feb 15; 25 ( 4 ): 651 – 9 . OpenUrl CrossRef PubMed 31. Palu RAS , Chow CY. Baldspot/ELOVL6 is a conserved modifier of disease and the ER stress response . Lin JH , editor. PLoS Genet . 2018 Aug 6; 14 ( 8 ): e1007557 . OpenUrl CrossRef PubMed 32. ↵ Palu RAS , Dalton HM , Chow CY . Decoupling of Apoptosis from Activation of the ER Stress Response by the Drosophila Metallopeptidase superdeath . Genetics . 2020 Apr 1; 214 ( 4 ): 913 – 25 . OpenUrl Abstract / FREE Full Text 33. ↵ Steffen J , Seeger M , Koch A , Krüger E . Proteasomal Degradation Is Transcriptionally Controlled by TCF11 via an ERAD-Dependent Feedback Loop . Molecular Cell . 2010 Oct 8; 40 ( 1 ): 147 – 58 . OpenUrl CrossRef PubMed Web of Science 34. ↵ Grace Science , LLC. A Phase 1/2/3 Open-label, Single Arm, Dose-finding Study to Investigate Long-term Safety, Tolerability and Efficacy of GS-100, an Adeno-associated Virus Serotype 9 (AAV9) Vector-mediated Gene Transfer of Human NGLY1, in Patients With NGLY1 Deficiency [Internet] . clinicaltrials.gov ; 2024 Jun [cited 2025 Jan 31]. Report No.: NCT06199531 . Available from: https://clinicaltrials.gov/study/NCT06199531 35. ↵ Morava-Kozicz E. A Phase II Randomized, Multicenter, Double-Blind, Placebo-Controlled Study Evaluating Effect Of GlcNAc On Tear Production In Individuals With NGLY1-CDDG [Internet] . clinicaltrials.gov ; 2025 Jan [cited 2025 Jan 31]. Report No.: NCT05402345 . Available from: https://clinicaltrials.gov/study/NCT05402345 36. ↵ Yoshida Y , Asahina M , Murakami A , Kawawaki J , Yoshida M , Fujinawa R , et al. Loss of peptide:N-glycanase causes proteasome dysfunction mediated by a sugar-recognizing ubiquitin ligase . PNAS [Internet ]. 2021 Jul 6 [cited 2021 Jul 6]; 118 ( 27 ). Available from: https://www.pnas.org/content/118/27/e2102902118 37. ↵ Li X , Zhang K , Li Z . Unfolded protein response in cancer: the Physician’s perspective . Journal of Hematology & Oncology . 2011 Feb 23; 4 ( 1 ): 8 . OpenUrl CrossRef PubMed 38. Harbut MB , Patel BA , Yeung BKS , McNamara CW , Bright AT , Ballard J , et al. Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design . Proc Natl Acad Sci U S A . 2012 Dec 26; 109 ( 52 ): 21486 – 91 . OpenUrl Abstract / FREE Full Text 39. ↵ Kim H , Bhattacharya A , Qi L . Endoplasmic reticulum quality control in cancer: Friend or foe . Seminars in Cancer Biology . 2015 Aug 1; 33 : 25 – 33 . OpenUrl CrossRef PubMed 40. ↵ Belmont PJ , Chen WJ , San Pedro MN , Thuerauf DJ , Lowe NG , Gude N , et al. Roles for ER- associated Degradation (ERAD) and the Novel ER Stress Response Gene, Derlin-3, in the Ischemic Heart . Circ Res. 2010 Feb 5; 106 ( 2 ): 307 – 16 . OpenUrl Abstract / FREE Full Text 41. ↵ Doroudgar S , Völkers M , Thuerauf DJ , Khan M , Mohsin S , Respress JL , et al. Hrd1 and ER- Associated Protein Degradation, ERAD, Are Critical Elements of the Adaptive ER Stress Response in Cardiac Myocytes . Circ Res . 2015 Aug 28; 117 ( 6 ): 536 – 46 . OpenUrl Abstract / FREE Full Text 42. ↵ Dietzl G , Chen D , Schnorrer F , Su KC , Barinova Y , Fellner M , et al. A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila . Nature . 2007 Jul ; 448 ( 7150 ): 151 – 6 . OpenUrl CrossRef PubMed Web of Science 43. ↵ Ryoo HD , Domingos PM , Kang MJ , Steller H. Unfolded protein response in a Drosophila model for retinal degeneration . The EMBO Journal [Internet] . 2006 Dec 14 [cited 2024 Dec 6]; Available from: https://www.embopress.org/doi/10.1038/sj.emboj.7601477 44. ↵ Dalton HM , Viswanatha R , Jr RB , Zuno JS , Berman AR , Rushforth R , et al. A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress . PLOS Genetics . 2022 Sep 27; 18 ( 9 ): e1010430 . OpenUrl CrossRef PubMed 45. ↵ Hu Y , Sopko R , Foos M , Kelley C , Flockhart I , Ammeux N , et al. FlyPrimerBank: An Online Database for Drosophila melanogaster Gene Expression Analysis and Knockdown Evaluation of RNAi Reagents . G3 Genes|Genomes|Genetics . 2013 Sep 1; 3 ( 9 ): 1607 – 16 . OpenUrl CrossRef 46. ↵ Untergasser A , Cutcutache I , Koressaar T , Ye J , Faircloth BC , Remm M , et al. Primer3--new capabilities and interfaces . Nucleic Acids Res . 2012 Aug ; 40 ( 15 ): e115 . OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted March 11, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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