|Coordinate||60,778,032 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||Smith-Magenis syndrome chromosome region, candidate 8 homolog (human)|
|Chromosomal Location||60,777,524-60,788,287 bp (+)|
|MGI Phenotype||PHENOTYPE: Mouse embryonic fibroblasts homozygous for a knock-out allele show impaired autophagy induction, a reduced autophagic flux, and abnormal expression of lysosomal enzymes. [provided by MGI curators]|
|Amino Acid Change||Isoleucine changed to Threonine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000055926] [ENSMUSP00000099728]|
AA Change: I2T
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
AA Change: I2T
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Meta Mutation Damage Score||0.8472|
|Is this an essential gene?||Non Essential (E-score: 0.000)|
|Candidate Explorer Status||CE: excellent candidate; Verification probability: 0.978; ML prob: 0.974; human score: 4.5|
Linkage Analysis Data
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice|
|Last Updated||2020-02-04 4:44 PM by External Program|
|Record Created||2015-02-02 1:17 PM by Emre Turer|
The patriot phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1418, some of which showed susceptibility to dextran sodium sulfate (DSS)-induced colitis at 7 (Figure 1) and 10 days (Figure 2) after DSS exposure (1); weight loss is used to measure DSS susceptibility. Some mice also showed increased frequencies of B1b cells (Figure 3), CD11c+ dendritic cells (Figure 4), and macrophages (Figure 5), all in the peripheral blood. Increased TNF production in response to the TLR9 ligand CpG was observed using gene-based superpedigree analysis of pedigrees R1418 and R3960 (Figure 6) (1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 93 mutations. All of the above anomalies were linked by continuous variable mapping to a mutation in Smcr8: a T to C transition at base pair 60,778,032 (v38) on chromosome 11, or base pair 508 in the GenBank genomic region NC_000077 encoding Smcr8. The strongest association was found with a recessive model of inheritance to the B1b cell frequency, wherein three variant homozygotes departed phenotypically from 11 homozygous reference mice and 15 heterozygous mice with a P value of 7.998 x 10-8 (Figure 7).
The mutation corresponds to residue 508 in the mRNA sequence NM_001085440 within exon 1 of 2 total exons.
The mutated nucleotide is indicated in red. The mutation results in an isoleucine (I) to threonine (T) substitution at position 2 (I2T) in the SMCR8 protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).
The causative mutation in Smcr8 for the DSS day 7 phenotype was validated by CRISPR-mediated replacement of the Smcr8patriot allele (Figure 8; [DSS Day 7; P = 7.353 x 10-8]). The B1 cell, DC, and macrophage phenotypes failed to validate.
|Illustration of Mutations in
Gene & Protein
Smcr8 encodes SMCR8 (Smith-Magenis syndrome chromosome region, candidate 8). SMCR8 is a homolog of the DENN module, which is GDP-GTP exchange factor for Rab GTPases (2). The DENN module and SMCR8 both have an N-terminal longin (alternatively, u-DENN) domain, a DENN domain, and a d-DENN domain (Figure 9). The longin domain mediates interaction with GTPases (3) and has a PAS domain-like fold similar to ligand-binding domains (4). The central DENN domain has an α/β three-layered sandwich domain with a central sheet of 5-strands and β−α units arranged similar to the topology of a minimal version of the P-loop NTPase α/β domain (2). The d-DENN domain is an all-α helical domain.
TANK-Binding Kinase 1 (TBK1; see the record for pioneer)-mediated SMCR8 phosphorylation at Ser402 and Thr796 activates SMCR8, putatively increasing the GDP exchange rate toward the small GTPase RAB39B (5). SMCR8 is also putatively phosphorylated by AMPK, mTORC1, and ULK1 (5-7); the functional significance of AMPK-, mTORC1-, and ULK1-mediated SMCR8 phosphorylation is unknown.
Smcr8 generates a 935-amino acid and a 785-amino acid protein isoform (8). The first exon of Smcr8 is similar between the two variants, with exon 1 encompassing the entire shorter protein isoform and 84% of the longer protein isoform. The function of the shorter SMCR8 isoform is unknown.
The patriot mutation results in an isoleucine (I) to threonine (T) substitution at position 2 (I2T) in the SMCR8 protein; amino acid 2 is within an undefined region of the canonical SMCR8 protein.
Autophagy is a intracellular recycling and degradation process in which cytoplasmic proteins or organelles are engulfed into double-membrane vesicles called autophagosomes (Figure 10). The autophagosomes subsequently fuse with lysosomes to form autolysosomes, which are primed for degradation. Autophagy removes aggregates of misfolded proteins and defective organelles as well as provides energy and recycles cell components.
SMCR8 interacts with C9ORF72 and WDR41 (see the record for gogi) to form the SWC (SMCR8-WDR41-C9ORF72) tripartite complex, which functions as a GDP-GTP exchange factor for the small GTPases RAB8A and RAB39B that function in vesicle trafficking and autophagy (Figure 10) (10-14). After TBK1-mediated phosphorylation of SMCR8, the SWC complex interacts with the ULK1 autophagy initiation complex (ULK1/FIP200/autophagy-related protein 13 [ATG13]/ATG101) via C9ORF72 binding (11;13;14). The interaction between the SWC complex and the ULK1 complex regulates the expression and activity of ULK1 (11;12). CRISPR/Cas9-mediated knockout of SMCR8 or C9ORF72 in HeLa cells resulted in enlarged lysosome vesicles in the knockout cells, while SMCR8 knockout alone showed accumulation of lysosomes and lysosomal enzymes as well as impaired autophagy induction (10;11;13-15). Mouse embryonic fibroblasts from Smcr8-deficient mice exhibited abnormal autophagy as well as a block in lysosomal degradation (MGI; accessed August 17, 2017).
The mTOR-associated signaling pathway regulates cell growth, size, metabolism, and growth factor signaling by stimulating protein synthesis (16). When there are sufficient nutrients, mTOR signaling is active allowing for protein synthesis and an increase in cell size (17-19). In contrast, when nutrient levels decrease or in conditions of cell stress, protein synthesis is inhibited with a concomitant decrease in cell size and cell proliferation (17;18). mTOR can be incorporated into both the mTORC1 and mTORC2 complex. mTORC1 signaling in response to changes in amino acid availability is a lysosome-dependent process. When mTORC1 is activated upon raptor binding to mTOR, it phosphorylates several targets, including S6 kinase 1 (S6K1) and 4E-binding protein 1 (4E-BP1) (20;21). S6K1, in addition to S6K2, is a kinase that phosphorylates S6, a component of the small (40S) ribosomal subunit (19). Autophagy is initiated upon inhibition of mTORC1, resulting in formation of an active ULK1 complex (22). SMCR8-deficient cells showed increased phosphorylation of S6, indicating that SMCR8 negatively regulates mTORC1 signaling (10).
SMCR8 can also modulate the expression of several autophagic and lysosomal genes (e.g., ULK1 and WIPI2) independent of the SWC complex (12). SMCR8 is a putative STRaND (shuttling transcriptional regulators and non-DNA binding) protein. STRaND proteins translocate from the cytoplasm to the nucleus to control gene expression through association with transcription factors (23). The method by which SMCR8 translocates to the nucleus, and the transcription factors SMCR8 putatively associates with, are unknown.
Smith-Magenis syndrome is caused (in 90% of cases) by a 3.7-Mb interstitial deletion in chromosome 17p11.2; SMCR8 is within the 17p11.2 region that is deleted (24;25). Patients with Smith-Magenis syndrome exhibit several phenotypes, including brachycephaly, prognathism, growth retardation, behavioral problems, mental retardation, hypotonia, speech delay, small ears, conductive hearing loss, esotropia, dental enamel dysplasia, and prominent premaxilla (24;26).
Similar to patriot mice (see “Phenotypic Description”), Smcr8-/- and SMCR8-mutant (Smcr8I2T/I2T) showed increased TNF production in response to the TLR9 ligand CpG (1). The Smcr8-/- and Smcr8I2T/I2T mice also showed increased TNF responses and IL-6 production in response to stimulation with TLR7 and TLR3 ligands; TNF production was normal in response to TLR2 or TLR4 stimulation. Smcr8-/- and Smcr8I2T/I2T mice also showed splenomegaly and lymphadenopathy at 9 and 12 months of age (1). The mice showed normal numbers of macrophages, monocytes, neutrophils, B cells, and T cells in the peripheral blood; however, the percentages of naïve CD4+ and CD8+ T cells was reduced and the percentages of activated CD4+ and CD8+ T cells was increased (1). The mice also showed increased plasma levels of the cytokine IL-12p40 compared to wild-type mice (1). Smcr8-/- macrophages showed an accumulation of putative lysosomes. The Smcr8-/- and Smcr8I2T/I2T mice also showed susceptibility to DSS treatment (1). Another Smcr8-/- mouse model exhibited autoimmunity and increased lysosomal exocytosis in macrophages (8).
Toll-like receptors (TLRs) play an essential role in the innate immune response as key sensors of invading microorganisms by recognizing conserved molecular motifs found in many different pathogens, including bacteria, fungi, protozoa and viruses. TLR signaling initiates a cascade of signaling events involving various kinases, adaptors and ubiquitin ligases, ultimately leading to transcriptional activation of cytokine and other genes through the transcription factors NF-κB, AP-1, interferon responsive factor (IRF)-3, and IRF-7. The endosomal TLRs recognize exogenous nucleic acids: double-stranded DNA unmethylated at CpG motifs (TLR9), single-stranded (ss) RNA viruses (TLR7 and TLR8) and double-stranded RNA (dsRNA; TLR3) (Figure 11). Plasmacytoid dendritic cell recognition of some ssRNA viruses via TLR7 requires the transport of cytosolic viral replication intermediates into lysosomes by autophagy (27), a process by which cells engulf parts of their own cytoplasm to eliminate foreign material or recycle various molecules. Proteolytic cleavage of TLR7 and TLR9 within their respective ectodomains occurs in the endolysosome (28;29). Although full length and cleaved forms of TLR9 are capable of binding ligand, only the cleaved form can recruit MyD88 and lead to signaling. The cleavage mechanism has been postulated to restrict receptor activation to endolysosomal compartments and prevent responses to self-nucleic acids (28). Once activated, TLR9 signaling requires the adapter MyD88 and, like other MyD88-dependent TLRs, recruits IL-1R-associated kinase 1 (IRAK1), IRAK4 and tumor necrosis factor receptor-associated factor 6 (TRAF6), leading to NF-κB and MAP kinase activation (30). MyD88, together with TRAF6 and IRAK4, has also been shown to bind interferon regulatory factor 7 (IRF7) directly in order to stimulate IFN-α production (31;32).
Loss of SMCR8 function results in aberrant activation of endosomal TLRs. The defects in TLR9-associated signaling observed in the patriot mice is proposed to be caused by defects in the SWC complex due to loss of SMCR8-assocated function (1). Loss of SWC complex function causes defects in lysosome and phagosome maturation, resulting in protracted TLR stimulation. The colitis phenotype observed in the patriot mice is putatively caused by defects in endosomal TLR signaling; the endosomal TLRs are required for protection in colitis.
Patriot genotyping is performed by amplifying the region containing the mutation using PCR, followed by sequencing of the amplified region to detect the single nucleotide transition.
R14180061_PCR_F: 5’- AGGTCCCAAACCTTGAGAGTCTGC-3’
R14180061_PCR_R: 5’- CTGGTAATCCACCGACATAATCCGC-3’
R14180061_SEQ_F: 5’- AGAGTCTGCGGCAATTCG-3’
R14180061_SEQ_R: 5’- ACCTGCTCAGAGAACTCGG-3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C hold
The following sequence of 489 nucleotides is amplified (NCBI RefSeq: NC_000077, chromosome 11):
aggtcccaaa ccttgagagt ctgcggcaat tcgagttctt cgttgtgtga cagggcagtt
taggatcccc ggaagtgcaa agaaggccgc aagaacttcg ttttcgcatc cagaggcctg
actctccctc cgaccaaccc tacattattt tccatttcct ctcaatgtgc ttgccatatt
tcaggaaata tgatcagcgc ccctgatgtg gtggccttca ccaaggaaga tgaatacgag
gaagaacctt acaatgagcc cgctttgcct gaggagtact cagtccctct ctttccttat
gccagccagg gggcaaaccc ctggtctaaa ctgtctgggg ccaagttctc cagggacttc
atcctcattt ccgagttctc tgagcaggtg ggaccccagc ccttgcttac catccccaat
gacaccaaag tttttggcac ttttgatctt aattacttct ctttgcggat tatgtcggtg
Primer binding sites are underlined and the sequencing primer is highlighted; the mutated T is shown in red text (Chr. + strand, T>C).
1. McAlpine, W., Sun, L., Wang, K. W., Liu, A., Jain, R., San Miguel, M., Wang, J., Zhang, Z., Hayse, B., McAlpine, S. G., Choi, J. H., Zhong, X., Ludwig, S., Russell, J., Zhan, X., Choi, M., Li, X., Tang, M., Moresco, E. M. Y., Beutler, B., and Turer, E. (2018) Excessive Endosomal TLR Signaling Causes Inflammatory Disease in Mice with Defective SMCR8-WDR41-C9ORF72 Complex Function. Proc Natl Acad Sci U S A. 115, E11523-E11531.
2. Zhang, D., Iyer, L. M., He, F., and Aravind, L. (2012) Discovery of Novel DENN Proteins: Implications for the Evolution of Eukaryotic Intracellular Membrane Structures and Human Disease. Front Genet. 3, 283.
3. Schlenker, O., Hendricks, A., Sinning, I., and Wild, K. (2006) The Structure of the Mammalian Signal Recognition Particle (SRP) Receptor as Prototype for the Interaction of Small GTPases with Longin Domains. J Biol Chem. 281, 8898-8906.
4. Aravind, L., Mazumder, R., Vasudevan, S., and Koonin, E. V. (2002) Trends in Protein Evolution Inferred from Sequence and Structure Analysis. Curr Opin Struct Biol. 12, 392-399.
5. Sellier, C., Campanari, M. L., Julie Corbier, C., Gaucherot, A., Kolb-Cheynel, I., Oulad-Abdelghani, M., Ruffenach, F., Page, A., Ciura, S., Kabashi, E., and Charlet-Berguerand, N. (2016) Loss of C9ORF72 Impairs Autophagy and Synergizes with polyQ Ataxin-2 to Induce Motor Neuron Dysfunction and Cell Death. EMBO J. 35, 1276-1297.
6. Hsu, P. P., Kang, S. A., Rameseder, J., Zhang, Y., Ottina, K. A., Lim, D., Peterson, T. R., Choi, Y., Gray, N. S., Yaffe, M. B., Marto, J. A., and Sabatini, D. M. (2011) The mTOR-Regulated Phosphoproteome Reveals a Mechanism of mTORC1-Mediated Inhibition of Growth Factor Signaling. Science. 332, 1317-1322.
7. Hoffman, N. J., Parker, B. L., Chaudhuri, R., Fisher-Wellman, K. H., Kleinert, M., Humphrey, S. J., Yang, P., Holliday, M., Trefely, S., Fazakerley, D. J., Stockli, J., Burchfield, J. G., Jensen, T. E., Jothi, R., Kiens, B., Wojtaszewski, J. F., Richter, E. A., and James, D. E. (2015) Global Phosphoproteomic Analysis of Human Skeletal Muscle Reveals a Network of Exercise-Regulated Kinases and AMPK Substrates. Cell Metab. 22, 922-935.
8. Zhang, Y., Burberry, A., Wang, J. Y., Sandoe, J., Ghosh, S., Udeshi, N. D., Svinkina, T., Mordes, D. A., Mok, J., Charlton, M., Li, Q. Z., Carr, S. A., and Eggan, K. (2018) The C9orf72-Interacting Protein Smcr8 is a Negative Regulator of Autoimmunity and Lysosomal Exocytosis. Genes Dev. 32, 929-943.
9. Bi, W., Yan, J., Stankiewicz, P., Park, S. S., Walz, K., Boerkoel, C. F., Potocki, L., Shaffer, L. G., Devriendt, K., Nowaczyk, M. J., Inoue, K., and Lupski, J. R. (2002) Genes in a Refined Smith-Magenis Syndrome Critical Deletion Interval on Chromosome 17p11.2 and the Syntenic Region of the Mouse. Genome Res. 12, 713-728.
10. Amick, J., Roczniak-Ferguson, A., and Ferguson, S. M. (2016) C9orf72 Binds SMCR8, Localizes to Lysosomes, and Regulates mTORC1 Signaling. Mol Biol Cell. 27, 3040-3051.
11. Yang, M., Liang, C., Swaminathan, K., Herrlinger, S., Lai, F., Shiekhattar, R., and Chen, J. F. (2016) A C9ORF72/SMCR8-Containing Complex Regulates ULK1 and Plays a Dual Role in Autophagy. Sci Adv. 2, e1601167.
12. Jung, J., Nayak, A., Schaeffer, V., Starzetz, T., Kirsch, A. K., Muller, S., Dikic, I., Mittelbronn, M., and Behrends, C. (2017) Multiplex Image-Based Autophagy RNAi Screening Identifies SMCR8 as ULK1 Kinase Activity and Gene Expression Regulator. Elife. 6, 10.7554/eLife.23063.
13. Sellier, C., Campanari, M. L., Julie Corbier, C., Gaucherot, A., Kolb-Cheynel, I., Oulad-Abdelghani, M., Ruffenach, F., Page, A., Ciura, S., Kabashi, E., and Charlet-Berguerand, N. (2016) Loss of C9ORF72 Impairs Autophagy and Synergizes with polyQ Ataxin-2 to Induce Motor Neuron Dysfunction and Cell Death. EMBO J. 35, 1276-1297.
14. Sullivan, P. M., Zhou, X., Robins, A. M., Paushter, D. H., Kim, D., Smolka, M. B., and Hu, F. (2016) The ALS/FTLD Associated Protein C9orf72 Associates with SMCR8 and WDR41 to Regulate the Autophagy-Lysosome Pathway. Acta Neuropathol Commun. 4, 51-016-0324-5.
15. Ugolino, J., Ji, Y. J., Conchina, K., Chu, J., Nirujogi, R. S., Pandey, A., Brady, N. R., Hamacher-Brady, A., and Wang, J. (2016) Loss of C9orf72 Enhances Autophagic Activity Via Deregulated mTOR and TFEB Signaling. PLoS Genet. 12, e1006443.
16. Baba, M., Hong, S. B., Sharma, N., Warren, M. B., Nickerson, M. L., Iwamatsu, A., Esposito, D., Gillette, W. K., Hopkins, R. F.,3rd, Hartley, J. L., Furihata, M., Oishi, S., Zhen, W., Burke, T. R.,Jr, Linehan, W. M., Schmidt, L. S., and Zbar, B. (2006) Folliculin Encoded by the BHD Gene Interacts with a Binding Protein, FNIP1, and AMPK, and is Involved in AMPK and mTOR Signaling. Proc Natl Acad Sci U S A. 103, 15552-15557.
17. Fernandez, D., and Perl, A. (2010) MTOR Signaling: A Central Pathway to Pathogenesis in Systemic Lupus Erythematosus? Discov Med. 9, 173-178.
18. Lee, D. F., and Hung, M. C. (2007) All Roads Lead to mTOR: Integrating Inflammation and Tumor Angiogenesis. Cell Cycle. 6, 3011-3014.
19. Wang, X., and Proud, C. G. (2006) The mTOR Pathway in the Control of Protein Synthesis. Physiology (Bethesda). 21, 362-369.
20. Mills, R. E., and Jameson, J. M. (2009) T Cell Dependence on mTOR Signaling. Cell Cycle. 8, 545-548.
22. Kim, J., Kundu, M., Viollet, B., and Guan, K. L. (2011) AMPK and mTOR Regulate Autophagy through Direct Phosphorylation of Ulk1. Nat Cell Biol. 13, 132-141.
23. Lu, M., Muers, M. R., and Lu, X. (2016) Introducing STRaNDs: Shuttling Transcriptional Regulators that are Non-DNA Binding. Nat Rev Mol Cell Biol. 17, 523-532.
24. Smith, A. C., McGavran, L., Robinson, J., Waldstein, G., Macfarlane, J., Zonona, J., Reiss, J., Lahr, M., Allen, L., and Magenis, E. (1986) Interstitial Deletion of (17)(p11.2p11.2) in Nine Patients. Am J Med Genet. 24, 393-414.
25. Greenberg, F., Guzzetta, V., Montes de Oca-Luna, R., Magenis, R. E., Smith, A. C., Richter, S. F., Kondo, I., Dobyns, W. B., Patel, P. I., and Lupski, J. R. (1991) Molecular Analysis of the Smith-Magenis Syndrome: A Possible Contiguous-Gene Syndrome Associated with Del(17)(p11.2). Am J Hum Genet. 49, 1207-1218.
26. Patil, S. R., and Bartley, J. A. (1984) Interstitial Deletion of the Short Arm of Chromosome 17. Hum Genet. 67, 237-238.
27. Lee, H. K., Lund, J. M., Ramanathan, B., Mizushima, N., and Iwasaki, A. (2007) Autophagy-Dependent Viral Recognition by Plasmacytoid Dendritic Cells. Science. 315, 1398-1401.
28. Ewald, S. E., Lee, B. L., Lau, L., Wickliffe, K. E., Shi, G. P., Chapman, H. A., and Barton, G. M. (2008) The Ectodomain of Toll-Like Receptor 9 is Cleaved to Generate a Functional Receptor. Nature. 456, 658-662.
29. Park, B., Brinkmann, M. M., Spooner, E., Lee, C. C., Kim, Y. M., and Ploegh, H. L. (2008) Proteolytic Cleavage in an Endolysosomal Compartment is Required for Activation of Toll-Like Receptor 9. Nat Immunol. 9, 1407-1414.
30. Hemmi, H., Kaisho, T., Takeuchi, O., Sato, S., Sanjo, H., Hoshino, K., Horiuchi, T., Tomizawa, H., Takeda, K., and Akira, S. (2002) Small Anti-Viral Compounds Activate Immune Cells Via the TLR7 MyD88- Dependent Signaling Pathway. Nat Immunol. 3, 196-200.
31. Kawai, T., Sato, S., Ishii, K. J., Coban, C., Hemmi, H., Yamamoto, M., Terai, K., Matsuda, M., Inoue, J., Uematsu, S., Takeuchi, O., and Akira, S. (2004) Interferon-Alpha Induction through Toll-Like Receptors Involves a Direct Interaction of IRF7 with MyD88 and TRAF6. Nat Immunol. 5, 1061-1068.
|Science Writers||Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||William McAlpine, Emre Turer, and Bruce Beutler|