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MEDICAL SCIENCES

Correction for “Combined TRPC3 and TRPC6 blockade by selective small-molecule or genetic deletion inhibits pathological cardiac hypertrophy,” by Kinya Seo, Peter P. Rainer, Virginia Shalkey Hahn, Dong-ik Lee, Su-Hyun Jo, Asger Andersen, Ting Liu, Xiaoping Xu, Robert N. Willette, John J. Lepore, Joseph P. Marino, Jr., Lutz Birnbaumer, Christine G. Schnackenberg, and David A. Kass, which appeared in issue 4, January 28, 2014, of Proc Natl Acad Sci USA (111:1551–1556; first published January 22, 2014; 10.1073/pnas.1308963111). The authors note that on page 1555, left column, fourth full paragraph, line 2 “example 17” should instead appear as “example 15.”

ECOLOGY

Correction for “Tackling soil diversity with the assembly of large, complex metagenomes,” by Adina Chuang Howe, Janet K. Jansson, Stephanie A. Malfatti, Susannah G. Tringe, James M. Tiedje, and C. Titus Brown, which appeared in issue 13, April 1, 2014, of Proc Natl Acad Sci USA (111:4904–4909; first published March 14, 2014; 10.1073/pnas.1402564111). The authors note that the accession number 4504979.3 (Iowa corn) should instead appear as 4504797.3 (Iowa corn). www.pnas.org/cgi/doi/10.1073/pnas.1405719111

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Correction for “Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity,” by William B. Messer, Ruklanthi de Alwis, Boyd L. Yount, Scott R. Royal, Jeremy P. Huynh, Scott A. Smith, James E. Crowe, Jr., Benjamin J. Doranz, Kristen M. Kahle, Jennifer M. Pfaff, Laura J. White, Carlos A. Sariol, Aravinda M. de Silva, and Ralph S. Baric, which appeared in issue 5, February 4, 2014, of Proc Natl Acad Sci USA (111:1939–1944; first published January 2, 2014; 10.1073/pnas.1317350111). The authors note that the following statement should be added to the Acknowledgments: “This research was also supported by the Sunlin and Priscilla Chou Foundation (W.B.M).”

Correction for “Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival,” by Shengyi Sun, Guojun Shi, Xuemei Han, Adam B. Francisco, Yewei Ji, Nuno Mendonça, Xiaojing Liu, Jason W. Locasale, Kenneth W. Simpson, Gerald E. Duhamel, Sander Kersten, John R. Yates III, Qiaoming Long, and Ling Qi, which appeared in issue 5, February 4, 2014, of Proc Natl Acad Sci USA (111:E582–E591; first published January 22, 2014; 10.1073/pnas.1318114111). The authors note that on page E590, left column, first paragraph, line 1, “JAX 004781” should instead appear as “JAX 004682.” www.pnas.org/cgi/doi/10.1073/pnas.1405563111

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PNAS | April 22, 2014 | vol. 111 | no. 16 | 6115

Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival Shengyi Suna,1, Guojun Shib,c,1, Xuemei Hand, Adam B. Franciscoe, Yewei Jib, Nuno Mendonçaf, Xiaojing Liub, Jason W. Locasaleb, Kenneth W. Simpsong, Gerald E. Duhamelh, Sander Kerstenb,f, John R. Yates IIId, Qiaoming Longe,i,2, and Ling Qia,b,2 a Graduate Program in Biochemistry, Molecular and Cell Biology, bDivision of Nutritional Sciences, and eDepartment of Animal Science, Cornell University, Ithaca, NY 14853; cShanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital Shanghai Jiaotong University School of Medicine, Shanghai 200025, China; dDepartment of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037; fNutrition Metabolism and Genomics Group, Wageningen University, 6703HD, Wageningen, The Netherlands; Departments of gClinical Science and hBiomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and iLaboratory Animal Research Center, Medical College of Soochow University, Suzhou 215006, China

Edited by Iva Greenwald, Columbia University, New York, NY, and approved January 2, 2014 (received for review September 26, 2013)

Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involved in endoplasmic reticulum-associated degradation (ERAD). Sel1L’s physiological importance in mammalian ERAD, however, remains to be established. Here, using the inducible Sel1L knockout mouse and cell models, we show that Sel1L is indispensable for Hrd1 stability, ER homeostasis, and survival. Acute loss of Sel1L leads to premature death in adult mice within 3 wk with profound pancreatic atrophy. Contrary to current belief, our data show that mammalian Sel1L is required for Hrd1 stability and ERAD function both in vitro and in vivo. Sel1L deficiency disturbs ER homeostasis, activates ER stress, attenuates translation, and promotes cell death. Serendipitously, using a biochemical approach coupled with mass spectrometry, we found that Sel1L deficiency causes the aggregation of both small and large ribosomal subunits. Thus, Sel1L is an indispensable component of the mammalian Hrd1 ERAD complex and ER homeostasis, which is essential for protein translation, pancreatic function, and cellular and organismal survival.

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inducible ERAD-deficient models exocrine pancreatic insufficiency stress granule ER dilation ERAD tuning

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Among several key E3 ligases that have been identified so far, hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) is a principle ER-resident E3 ligase and forms a complex with an ER-resident single-transmembrane protein Hrd3 in yeast or Suppressor/Enhancer of Lin-12-like (Sel1L) in mammals, responsible for the degradation of a subset of misfolded proteins in the ER (13–19). The Hrd1–Hrd3 complex was first discovered in yeast, by the Hampton group, to be responsible for the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (13, 14) and in C. elegens by the Greenwald group through genetic interactions with Notch (20, 21). Recent studies from several groups have elegantly demonstrated that Sel1L is an integral part of the mammalian Hrd1 ERAD complex and is necessary for the ERAD process for a subset of model substrates (15–19) and endogenous substrates, including luminal hedgehog (22), transmembrane CD147 (23), and ATF6 (24). However, although Hrd3p determines the stability of Hrd1p in yeast (4, 14), knockdown of Sel1L seems to have negligible effect on the steady-state level of Hrd1 protein in cultured mammalian cells (15, 25, 26). Moreover, a recent proteomics study showed that Hrd1-mediated degradation of model substrates may proceed in a Sel1L-dependent or -independent manner, depending on substrate topology or accessibility of specific E3

P

rotein misfolding and aggregation in the endoplasmic reticulum (ER) contributes significantly to the etiology and pathogenesis of many devastating diseases, including α1-antitrypsin deficiency, type-1 diabetes, Creuzfeld–Jacob disease, and cystic fibrosis (1). ER-associated degradation (ERAD) targets misfolded secretory and membrane proteins in the ER for proteasomal degradation (2–4), and the unfolded protein response (UPR) senses ER stress signals and initiates global changes in transcription and translation (5, 6). These two are the key qualitycontrol systems in the cell to maintain ER homeostasis and adjust ER capacity in response to environmental cues. In yeast, although cells tolerate the loss of each pathway, loss of both pathways leads to synthetic lethality (7, 8), suggesting that these two pathways function in a cooperative but interdependent manner. In mammals, the relationships between the two systems are much more complicated in part because of increased complexities within the UPR and ERAD systems. At least three major branches of UPR and five major ERAD complexes have been identified to date. Moreover, studies have suggested that different cell types in mammals have different burdens and tolerance to ER misfolded proteins, and hence different dependency and requirements for UPR and ERAD for survival. How various cell types maintain ER homeostasis remains an open and challenging question. Animal models are needed to directly address physiological significance of the ERAD and UPR in a cell typespecific manner. Several animal models defective in UPR have been characterized to date; however, studies of ERAD mouse models have been limited (9–12). E582–E591 | PNAS | Published online January 22, 2014

Significance This study provides insights into the physiological role of Sel1L, an adaptor protein for the ubiquitin ligase Hrd1 in endoplasmic reticulum-associated degradation (ERAD). Using both animal and cell models, this study provides unequivocal evidence for an indispensable role of Sel1L in Hrd1 stabilization, mammalian ERAD, endoplasmic reticulum homeostasis, protein translation, and cellular and organismal survival. Moreover, generation of inducible knockout mouse and cell models deficient in both Sel1L and Hrd1 provides an unprecedented opportunity to elucidate the functional importance of this key branch of ERAD in vivo and to identify its physiological substrates. Author contributions: S.S., G.S., and L.Q. designed research; S.S., G.S., X.H., Y.J., X.L., and S.K. performed research; A.B.F. and Q.L. contributed new reagents/analytic tools; S.S., G.S., X.H., Y.J., N.M., X.L., J.W.L., K.W.S., G.E.D., S.K., J.R.Y., and L.Q. analyzed data; and S.S. and L.Q. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE52929). 1

S.S. and G.S. contributed equally to this work.

2

To whom correspondence may be addressed. E-mail: [email protected] or ql39@cornell. edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1318114111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1318114111

animals progressively lost body weight starting around day 8. The mice became runted and moribund with reduced body temperature and died within 2–3 wk (Fig. 1 D–F), despite higher food intake, normal blood glucose levels, energy expenditure, and physical activity (Fig. 1 G and H and Fig. S1C). The observations that Sel1LIKO mice lost body weight despite higher food intake and normal metabolic parameters prompted the speculation that Sel1LIKO mice may suffer from maldigestion and malabsorption. To directly test this theory, we gave mice an oral bolus of lipid. Indeed, serum triglyceride level was significantly lower in Sel1LIKO mice than that of WT mice (Fig. 1I). Moreover, fecal fat contents of Sel1LIKO mice were higher than those of WT littermates (Fig. 1J). Both evidence points to the diagnosis of maldigestion and malabsorption. Thus, acute Sel1L deficiency leads to early lethality and nutrient malabsorption in adult mice. Pancreatic Atrophy in the Pancreas of Sel1LIKO Mice. At day 8, visual and histological examinations revealed no apparent abnormalities in tissues such as small intestine, lung, liver, heart, and skeletal muscle (Fig. S2). However, that was not the case for the pancreas. Unlike the pancreas of WT mice, which had a firm consistency and uniform yellow opaque color, the pancreas of Sel1LIKO mice was diffusely dark red, soft, and weighed about half of that of the WT mice, a change consistent with severe pancreatic atrophy (Fig. 2 A and B). Histologically, dramatic morphological degenerative changes were noted in the exocrine pancreas of Sel1LIKO mice, including dramatic reduction of eosinophilic cytoplasmic secretory zymogen granules, increased basophilia-marked anisokaryosis, and binucleated cells (Fig. 2C). A few lymphocytes and neutrophils were frequently detected in the interstitium of the exocrine pancreas of Sel1LIKO mice, a change interpreted as mild pancreatitis (Fig. S3A). In line with reduced eosinophilic staining in the H&E-stained tissue sections, the protein levels of two major components of acinar cell zymogen granules, pancreatic α-amylase and lipase, were significantly reduced in the pancreas of Sel1LIKO mice (Fig. 2D). The reduction of pancreatic α-amylase was further confirmed by immunohisto-

tributed in many tissues, with the strongest expression in the pancreas (Fig. 1A). To study the role of Sel1L in vivo, we generated tamoxifen-inducible knockout mice (IKO) f/f;ERCre+ (Sel1LIKO) with f/f;ERCre− (WT) littermates as a control cohort (Fig. 1 B and C). Mice were born at an expected Mendelian ratio (Fig. S1A) and grew normally in the first 16-wk of life following weaning at 3 wk of age (Fig. S1B). To acutely induce Sel1L deficiency, adult mice were injected daily with tamoxifen for 3 d (from day 0 to day 2). Day 0 was defined as the day with the first tamoxifen injection. Following tamoxifen injection, Sel1LIKO

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CELL BIOLOGY

Premature Lethality of Sel1LIKO Mice. Sel1L is ubiquitously dis-

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Fig. 1. Early lethality and nutrient malabsorption in Sel1LIKO mice. (A) Western blot analysis of Sel1L in different tissues from WT mice. Gastroc, gastrocnemius; BM, bone marrow; WAT/BAT, white/brown adipose tissue; HSP90, a loading control. (B) Diagram illustrating the generation of Sel1Lflox/+ animals. Gray boxes, exons. The loxP sites flank the exon 6. (C) Diagram illustrating the generation of Sel1Lflox/flox;ERCre mice. ERCre, estrogen-receptor-controlled Cre. (D) Body weight change after three daily injections of tamoxifen in adult mice. Arrows point to three consecutive tamoxifen injection. WT, n = 20; IKO, n = 29. (E) Surviving curve of WT and IKO mice after tamoxifen injection. WT n = 16; IKO n = 25. ***P < 0.001 by the log-rank (Mantel–Cox) test. (F) Rectal body temperature at day 13 (n = 5). (G) Food intake after tamoxifen injection (n = 5). (H) Blood glucose levels (ad libitum) after tamoxifen injection (n = 5). (I) Serum Triglyceride (TG) levels before and at 1.5 h postlipid gavage in WT and IKO mice. n = 5 each. (J) Oil-red O staining of fecal smear from WT and IKO mice. Nonstained and stained fecal smear slides from 2 mo high-fat diet (HFD) -fed mice were used as negative and positive controls, respectively. Representative pictures of four mice each group shown. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by Student t test, except for E. Representative data of at least two experiments shown.

Sun et al.

PNAS PLUS

ligases (15). Degradation of ER-transmembrane proteins can be Sel1L–Hrd1-independent because of functional redundancy among the ERAD complexes (27). Finally, pointing to a dispensable role of Sel1L in ER homeostasis in vivo, knockdown of Sel1L in cultured cells fails to induce UPR (28, 29) and deletion of Hrd3/Sel1 in the fly has no effect on eye size (30). Thus, how Sel1L regulates ERAD and ER homeostasis in vivo remains unclear. Nonetheless, studies have implicated Sel1L in various cellular processes, including tumorigenesis of various cancer types (31), stem cell differentiation (32), pancreatic epithelial cell differentiation (33), and retrotranslocation of cholera toxin to the cytosol (26). Variants in the Sel1L gene have been identified in human patients with autoimmune thyroid diseases (34), in canines with progressive early-onset cerebellar ataxia (35), and in humans with Alzheimer’s disease (36). However, our ability to dissect physiological roles of Sel1L has been limited because of the embryonic lethality of the Sel1L-deficient mice (11). To circumvent this problem, we have generated and characterized inducible Sel1L-deficient mouse and cell models to permit an assessment of Sel1L function in adult animals and immortalized mouse embryonic fibroblasts (MEFs). Here our data demonstrate an indispensable role of Sel1L in mammalian ERAD and ER homeostasis and illustrate pathophysiological consequences associated with acute Sel1L deficiency.

PNAS | Published online January 22, 2014 | E583

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Fig. 2. Exocrine pancreatic insufficiency and increased cell death in the pancreas of Sel1LIKO mice. Experiments were performed at day 13. (A) Picture of WT and IKO pancreas from male mice. Each picture represents five mice each group. (B) H&E images showing pancreatic atrophy in IKO mice. (C) H&E images showing reduced eosinophilic zymogen staining, different nuclei sizes (arrowheads), and binucleated cells. (D) Western blot analysis of various pancreatic enzymes with quantitation shown below. Asterisk indicates the nonspecific band. Each lane represents one mouse. (E) Immunohistochemistry of amylase and Sel1L. n = 3–5 each group. Asterisk denotes an islet. Note Sel1L deletion is largely limited in the exocrine pancreas. (F) Enzymatic activities in WT and IKO pancreas. n = 5 each group. (G) qPCR analysis of genes in the pancreas. n = 5 each. Amy2a5, amylase 2a5; Pnlip, pancreatic lipase. (H) Ingenuity analysis showing top 25 significantly changed functional annotations between WT and IKO pancreas (day 13, n = 4 mice each) with a threshold of fold change >1.5 or

Sel1L is indispensable for mammalian endoplasmic reticulum-associated degradation, endoplasmic reticulum homeostasis, and survival.

Suppressor/Enhancer of Lin-12-like (Sel1L) is an adaptor protein for the E3 ligase hydroxymethylglutaryl reductase degradation protein 1 (Hrd1) involv...
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