|Coordinate||119,529,030 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Gene Name||membrane-bound transcription factor peptidase, site 1|
|Synonym(s)||subtilisin/kexin isozyme-1, SKI-1, site-1 protease, S1P, 0610038M03Rik|
|Chromosomal Location||119,508,156-119,558,761 bp (-)|
|MGI Phenotype||Mice homozygous for a gene trap allele die prior to implantation. Mice homozygous for an ENU-induced allele exhibit hypopigmentation, reduced female fertility, altered lipid homeostasis, and increased susceptibility to induced colitis.|
|Amino Acid Change||Tyrosine changed to Cysteine|
|Institutional Source||Beutler Lab|
Y496C in Ensembl: ENSMUSP00000095965 (fasta)
|Gene Model||not available|
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||Autosomal Recessive|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Embryos, gDNA|
|Last Updated||2016-11-10 12:03 PM by Anne Murray|
The woodrat (wrt) phenotype was identified as a visible recessive variant among G3 mice homozygous for mutations induced by ENU (1). Homozygous woodrat mice exhibit coat hypopigmentation beginning on postnatal day 8, which progresses with time so that by 4 weeks of age wrt/wrt coats are composed of both white and black hairs, varying in pigment content along the length of each hair shaft. Adult wrt/wrt mice have a more homogeneous coat composed of hairs with a white base and a grey/brown tip (Figure 1). Studies confirmed that the wrt mutation led to the observed progressive hypopigmentation (2).
Compound heterozygosity for a null mutation (a gene trap allele) and the wrt allele (Mbtps1wrt/trap) results in embryonic lethality, while 53% and 54% of the expected numbers of Mbtps1+/trap or Mbtps1wrt/wrt mice, respectively, survive to term (2). Because a single copy of the gene (Mbtps1+/trap) is associated with diminished survival to term and causes no visible phenotype, while Mbtps1wrt/wrt is associated with about the same impairment of survival to term but does cause a visible phenotype, it is inferred that the wrt allele encodes a protein with ≤50% of wild type activity. In addition, it appears that the survival threshold likely falls between 25% and 50% of wild type enzyme activity levels. It was determined that the wrt allele causes maternal-zygotic effect lethality (i.e. homozygous females can only give birth to offspring carrying at least one wild-type copy of the Mbtps1 locus) (2). Further examination determined that death and resorption of maternal-zygotic mutant embryos (homozygote embryos derived from homozygote mothers) occurs before the eighth day of gestation (2).
When challenged with 2% DSS in drinking water (DSS-induced Colitis Screen), wrt homozygotes develop severe and sometimes fatal colitis, as measured by weight loss relative to wild type controls (Figure 2A). DSS-induced weight loss is similar in wrt/wrt and wild type mice when they are also treated with antibiotics, indicating that the enhanced susceptibility of wrt mice to DSS-induced colitis depends on commensal flora. In wrt homozygotes, nine days of 2% DSS treatment causes bleeding in the colon, and a shortening and thickening of the colon indicative of edema and inflammation, which were not observed in wild type mice (Figure 2B). Histological analysis demonstrates leukocyte recruitment and crypt loss in the colons of DSS-treated wrt/wrt animals. In addition, wrt/wrt mice have increased levels of interleukin (IL)-1β and IL-6 in epithelium and lamina propria both before and after seven days of DSS administration, as measured in the supernatants of distal colonic explants after 24 hours in culture. The gut epithelial barrier was found to be intact in naïve wrt/wrt mice.
The unfolded protein response (UPR) was investigated in wrt homozygous and heterozygous mice by determining the protein levels of glucose-regulated protein (GRP)78 (also known as BiP and Hspa5) and GRP94. Colons of wild type mice, wrt heterozygotes, and wrt homozygotes showed similar levels of GRP78 and GRP94 before DSS treatment, but wrt homozygotes had significantly lower levels of both proteins after three days of DSS administration. Interestingly, in wrt heterozygotes, GRP78 and GRP94 protein levels were intermediate between wild type and homozygous mutant levels. GRP78 and GRP94 mRNA expression levels are reduced in colonic epithelial cells from wrt mice even before DSS treatment. Using chimeras made by reciprocal bone marrow transplantation between wild type and wrt mice, both DSS-induced colitis and reduced colon levels of GRP78 and GRP94 proteins were found to be conferred by non-hematopoietic wrt mutant cells. Induction of the UPR by intraperitoneal injection of tunicamycin is markedly more toxic to wrt mice, with 10/11 dying within 30 hours of treatment, compared to wild type mice, where only 1/11 died during the same time. Tunicamycin treatment causes colitis in wrt, but not wild type mice.
Lymphocytic choromeningitis virus (LCMV) is a pathogenic arenavirus, and it depends on S1P to cleave its envelope glycoprotein. Without S1P, the virus cannot replicate (3). Following infection with a persistant variant of LCMV Armstrong strain, LCMV clone 13 (LCMV Cl13), woodrat animals were found to resist persistent infection. The LCMV Cl13 strain exhibit extended persistence in wild-type animals, LCMV Cl13 was not detected in the woodrat animals at 3 weeks post-infection (3). Examination of T cell number and activity as well as antibody responses in the woodrat animals determined that the animals had reduced immune responses compared to wild-type animals (Figure 3). To understand the protective effect of the mutation, the animals were supplemented with a 5% cholesterol diet and subsequently exposed to LCMV Cl13. Although cholesterol has been shown to be essential for the viral life cycle and host immune responses (4-6), cholesterol repletion did not revert woodrat resistance. Examination of interferon (IFN)alpha levels did not find a significant increase in the woodrat animals, indicating that IFN production was not leading to woodrat resistance. Examination of bone marrow cells found that woodrat dendritic cells (DCs) and their monocyte precursors were resistant to infection by LCMV Cl13; the splenic DC reaction to LCMV Cl13 was equivalent to wild-type (Figure 4). The differential phenotype between the bone marrow and splenic DCs indicates that there might be differences in the expression of Mbtps1. Indeed, examination of Mbtps1 expression found that there are decreased levels in the bone marrow; target genes of Mbtps1 were also decreased in the woodrat bone marrow. Examination of LCMV Cl13 viral replication in woodrat bone marrow-derived macrophages and fibroblasts showed that there is no difference in viral growth kinetics when compared to wild-type (3). In contrast, viral replication was almost completely null in the bone marrow-derived DCs. Adoptive transfer of woodrat bone marrow-derived DCs to wild-type hosts conferred resistance of persistent viral infection (3) (Figure 5B).
|Nature of Mutation|
The wrt mutation was mapped to Chromosome 8, and corresponds to an A to G transition at position 2041 of the Mbtps1 transcript, in exon 13 of 24 total exons.
The mutated nucleotide is indicated in red lettering, and results in a tyrosine to cysteine change at residue 496 of the Site 1 protease (S1P).
Mbtps1 encodes the Site 1 protease (S1P), also known as subtilisin-kexin-isoenzyme 1 (SKI-1), which was simultaneously discovered by two groups as the enzyme responsible for the Site 1 cleavage of sterol regulatory element binding proteins (SREBPs), and as a proprotein convertase with specificity for nonbasic amino acids (7;8). The family of proprotein convertases (PCs) comprise nine mammalian serine proteases that convert secretory precursor proteins into active forms by proteolysis at selected sites [reviewed in (9)]. Seven of the nine known PCs are specific for basic amino acids and cleave at single or paired basic residues. In contrast, only two known PCs, S1P and proprotein convertase subtilisin kexin 9 (PCSK9), are specific for nonbasic amino acids. S1P recognizes the motif (R/K)-X-(L/I/V)-Z↓, where X is any amino acid, and Z is any amino acid except Pro, Cys, Glu, Asp or Val (7;10-12). Mouse S1P is 98% identical to that found in rat, and 96% identical to human S1P, with 100% identity among the three catalytic domains (7).
S1P is a type I transmembrane protein with its N-terminal proteolytic domain projecting into the ER lumen. It contains 1052 amino acids, comprising a signal peptide (amino acids 1-17), a prosegment (amino acids 18-186), and a catalytic protease domain (amino acids 218-414) (Figure 6) (7). Asp, His and Ser residues, known as the catalytic triad, make up the active site of S1P, as is characteristic of all serine proteases; they are located at positions 218, 249, and 414, respectively (7). Asn338 of S1P forms part of an oxyanion hole, another characteristic of serine proteases, which serves to stabilize the transition state and thereby lower the activation energy of the peptide cleavage reaction. Following the catalytic domain, where all seven basic amino acid-specific PCs contain a P-domain, S1P has an extended C-terminal region with unknown function (amino acids 415-1052). This region contains a conserved growth factor/cytokine receptor family motif (amino acids 849-861), followed by a putative 24-amino acid hydrophobic transmembrane segment, and a highly basic C-terminal tail that may regulate cellular localization (7;13). S1P may be alternatively spliced into forms lacking amino acids 430-483 or 858-901, regions found within the extended C-terminal tail (7). Notably, amino acids 858-901, encoded by exon 21, encompass part of the growth factor/cytokine receptor family motif. Deletion of exon 21 results in abrogation of prosegment processing, retention in the endoplasmic reticulum (ER), and abrogation of protease activity towards a substrate, suggesting that the motif may be important for the proper folding of the protein that allows its processing (14). S1P functions most efficiently near neutral pH and at 2-3 mM Ca2+ (11;15).
The prosegments of many peptidases, including PCs, act both as intramolecular chaperones and inhibitors of the associated protease. Enzyme activation typically requires release of the prosegment in an organelle-specific manner. Likewise, S1P is a zymogen activated by autocatalytic cleavage of its prosegment in the ER. First, a signal peptidase removes the signal peptide by cleavage at the sequence LVVLLC17↓GKKHLG resulting in S1P-A (amino acids 18-1052), which is inactive (11;12). Then, sequential autocatalytic cleavages at R134SLK137↓YA and R183RLL186↓RA occur to generate the active S1P-B (amino acids 138-1052) and S1P-C (amino acids 187-1052), respectively, plus the major ~24 kDa and 14 kDa prosegment cleavage fragments (11;12). These fragments are further processed into 10- and 8-kDa products. Mutational analysis demonstrates that Arg130 and Arg134 are critical for autocatalytic processing of the prosegment, as well as for the subsequent efficient exit of S1P from the ER (14). Once cleaved, the 14 kDa prosegment fragment reportedly remains tightly bound to S1P, unlike the prosegments of other PCs, which are released by acidic pH and high Ca2+ concentrations found in the trans-Golgi network (16). In addition, only the full length 24-kDa prosegment, but none of the smaller fragments, is inhibitory for S1P protease activity towards a synthetic peptide substrate (11). Based on these findings, the 14 kDa prosegment fragment has been hypothesized to serve a chaperone-like function.
S1P exists as both cell surface transmembrane and extracellularly shed protein forms (7;8). Shedding occurs through cleavage at the sequence KHQKLL953↓SIDL, a non-canonical S1P recognition sequence in which the two leucine residues adjacent to the cleavage site are critical (14). Evidence suggests that S1P catalyzes its own cleavage and shedding, but whether proteolysis occurs in cis or in trans is unknown. Using cell surface biotinylation followed by immunoprecipitation, S1P has been localized at the cell membrane in transiently transfected HEK293 cells. In addition, soluble S1P accumulates in the cell media, which may be blocked by treatment with brefeldin-A, suggesting that S1P is cleaved and shed from the plasma membrane (14). However, it remains possible that S1P is secreted from cells, as S1P mutants lacking the transmembrane and cytosolic domains fail to be cleaved at the L953 shedding site but are still detected in the cell media (14;15).
The wrt mutation predicts the amino acid substitution Y496C, located within the extended C-terminal tail. Protein expression and catalytic activity of the mutant protein have not been tested. Based on the wrt phenotype, the mutant protein is hypothesized to possess ≤50% of wild type protein activity, but the nature of its dysfunction is unknown (see Phenotypic Description).
Northern blot analysis of mRNA from adult rat demonstrates that Mbtps1 is expressed in most tissues, including brain (striatum hippocampus, hypothalamus), liver, thymus, spleen, kidney, and heart (7). It is also highly expressed in adrenal, anterior pituitary, and thyroid glands. A similar widespread expression pattern is detected in two-day-old rat by in situ hybridization, with additional expression in skin, muscles, cardiac muscles, bones, teeth, and intestine (7).
Full length and processed forms of the membrane-anchored S1P localizes to the endoplasmic reticulum (ER) and Golgi membranes (7;11;12;17). The short C-terminal sequence after the transmembrane domain is oriented in the cytosol, with the bulk of the protein oriented in the lumen (Figure 3). S1P has been found associated with other cytoplasmic compartments that may represent lysosomes and/or endosomes (7). Treatment of cells with brefeldin-A does not alter the production of the prosegment fragments, and S1P-B is endo H-sensitive, indicating that S1P prosegment cleavage occurs in the ER (11;12). S1P-C is endo H-resistant, indicating a localization in the Golgi. An active, soluble form of S1P generated by cleavage after Leu953 can be shed into the culture medium of cells expressing the complete protein (11;12). The 24 kDa prosegement fragment may also be shed into the culture medium (12).
PCs play a central role in processing a wide range of protein precursors, including hormones, growth factors, transcription factors, bacterial toxins and viral glycoproteins. Consequently, they have been implicated in multiple disease states, participating in the pathogenesis of dyslipidemias and atherosclerosis (13), Alzheimer’s disease (18), tumorigenesis (19), and several bacterial and viral infections (20-23).
Since its discovery in 1998, S1P has been shown to cleave diverse substrates. A group of these substrate proteins are activated from a latent state through regulated intramembrane proteolysis (RIP), a process by which transmembrane proteins are cleaved within a membrane-spanning helix to release cytosolic domains that enter the nucleus (or other cellular compartments) to regulate transcription (or other functions) (24). RIP typically requires an initial cleavage in the extracytoplasmic domain of the substrate before the intramembrane proteolytic event. S1P and the Site 2 protease (S2P) function sequentially to carry out RIP for an expanding list of proteins, with S1P performing the initial extracytoplasmic cleavage (typically in the Golgi), and S2P mediating intramembrane cleavage. RIP substrates for S1P and S2P include the SREBPs (8), and CREB/ATF (cAMP response element binding/activating transcription factor) family proteins including ATF6, CREBH, CREB4, OASIS, and Luman, all of which are transcription factors of the basic leucine zipper family (25-29) (see below). Another major group of S1P substrates consists of the surface glycoproteins of viruses, which includes those of the Arenaviruses lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus, and the Bunyavirus Crimean-Congo hemorrhagic fever virus (20;23;30). S1P has been shown to cleave pro-brain derived neurotrophic factor (BDNF) in vitro (7).
RIP was first found to occur during the activation of SREBPs, now the paradigm for this type of processing (Figure 7). SREBPs are transcriptional activators of genes responsible for the biosynthesis and uptake of cholesterol and unsaturated fatty acids. SREBPs are bound to the ER membrane and nuclear envelope in a hairpin orientation, with the N and C termini projecting into the cytosol [reviewed in (31)]. The N-terminal segment is a basic helix-loop-helix-leucine zipper transcription factor; the C-terminal segment forms a complex with the membrane-anchored regulatory protein SREBP-cleavage activating protein (SCAP). Between the cytosolic N and C termini is a segment of ~80 amino acids consisting of two membrane-spanning helices separated by a ~30 amino acid ER luminal loop. S1P initiates the processing of SREBP-1 and SREBP-2 by cleaving this loop at the sequence RSVL↓S (32). S2P subsequently cleaves the N-terminal segment within its transmembrane region, releasing it for entry into the nucleus (33;34). When sterols accumulate in cells, SREBPs remain membrane-anchored in a complex with the sterol-sensing SCAP protein within the ER. Retention in the ER requires either Insig-1 or Insig-2, which bind to the sterol-sensing domain of SCAP (35;36). Thus, cleavage of SREBPs by S1P is blocked, and gene transcription declines. When cellular demand for sterols increases, Insig proteins release SCAP, which then transport SREBPs to the Golgi apparatus via coatamer protein complex II (COPII) vesicles, where the SREBPs encounter active S1P and are cleaved to an active state (37-41).
More recent studies have identified S1P and S2P as the proteases mediating RIP for several ER stress-responsive CREB/ATF transcription factors. ATF6 is an ER-resident transcription factor activated in response to ER stress, which is perceived by cells as an excess of unfolded or misfolded proteins in the ER and triggers the unfolded protein response (UPR). The UPR facilitates the folding, processing, export, and degradation of misfolded proteins in the ER. ATF6 is a type II transmembrane protein with a basic leucine zipper domain in the cytosol and a stress-sensing domain in the ER lumen (42). Under normal conditions, ATF6 is retained in the ER through interaction with the chaperone GRP78 (43). The accumulation of unfolded or misfolded proteins in the ER results in release of ATF6 and its translocation to the Golgi in COPII vesicles, where it is cleaved by S1P and S2P similarly to SREBPs (28;44;45). The cleaved cytosolic domain traffics to the nucleus, where it binds to sequences known as ER stress responsive elements (ERSE), and activates transcription of UPR target genes including the chaperones GRP78, GRP94, CCAAT/enhancer-binding protein homologous protein (CHOP), and X-box binding protein 1 (XBP1) (Figure 4) (46). In a similar manner, CREBH, CREB4, Luman, and OASIS are activated in response to ER stress by S1P- and S2P-mediated RIP (25;27;29;47).
The physiological function of S1P has been investigated in mice with a targeted deletion of S1P. Homozygous null mutations of S1P result in embryonic lethality before day 4 due to abnormal epiblast formation and subsequent embryo implantation (48;49). However, an inducible liver-specific knockout of S1P is viable, and was used to study the requirement for S1P in the processing of SREBPs in the liver (48). The Mbtps1 gene was flanked with loxP sites, and mice homozygous for the floxed allele and heterozygous for a transgene encoding the Cre recombinase under control of the interferon (IFN)-inducible liver-specific MX1 promoter were generated. Intraperitoneal injection of poly I:C was used to induce IFN, activating Cre recombination and inactivation of S1P in livers. These animals exhibited reduced levels of nuclear SREBPs, and 74% and 64% reductions of cholesterol and fatty acid biosynthesis, respectively, in hepatocytes. Low density lipoprotein (LDL) receptor mRNA and LDL receptor-mediated clearance of plasma LDL declined by 50%. However, plasma cholesterol levels fell, suggesting that LDL production was reduced. The merely partial effects of conditional deletion of S1P on SREBP activation and lipoprotein biosynthesis were unexpected, as S1P function was presumed to be nonredundant. The data suggest that another protease may substitute for S1P in the liver. Alternatively, the 77-90% efficiency of Cre-mediated recombination may not be sufficient to abolish S1P function.
Inflammatory bowel disease (IBD) is a chronically recurring inflammatory disorder of the intestine, with causative contributions from genetic, environmental and immunological factors (OMIM #266600). The clinical appearance of IBD is heterogeneous, and can include diarrhea, abdominal pain, rectal bleeding, fever, weight loss, and signs of malnutrition (50). Several recent reports provide evidence for a causal role for ER stress in IBD. For example, a mutation of the Muc2 gene (see record for Schlendrian), which encodes the highly glycosylated mucin-2 protein that constitutes the majority of the intestinal mucus barrier, leads to ER stress and spontaneous colitis in mice (51). In these animals, ER stress was attributed to the aberrant oligomerization and accumulation of mucin-2. Mice lacking IRE1β, an ER stress sensor expressed in intestinal epithelial cells, are more susceptible to DSS-induced colitis relative to wild type mice (52). These mice exhibit increased levels of the chaperone GRP78 in the colonic mucosa, indicative of ER stress. Mice lacking XBP1, a UPR response protein activated by IRE1, in intestinal epithelial cells display a loss of secretory Paneth cells and goblet cells in the intestinal epithelia, as well as spontaneous inflammation in the ileum (53). Elevated levels of GRP78 and CHOP, and IRE1 hyperactivation in the small intestine were also detected in these mice. In addition, single nucleotide polymorphisms (SNPs) in the XBP1 locus have been correlated with IBD in humans (53).
The wrt phenotype provides the first evidence that the ATF6-mediated UPR, initiated by S1P, is required for prevention of IBD and maintenance of intestinal homeostasis. Other ER stress-induced S1P substrates, including CREBH, CREB4, Luman, and OASIS, may also play a role in the prevention of IBD. In contrast to XBP1 and IRE1 mutant mice, which have higher basal levels of ER stress response proteins such as GRP78 and CHOP relative to wild type mice, wrt mice have normal basal protein levels of both GRP78 and GRP94 which fail to be upregulated in response to ER stress. Mouse embryonic fibroblasts from ATF6α knockout mice similarly display normal basal levels of ER chaperones including GRP78 and GRP94, but fail to induce them upon ER stress (54). These data support the finding that ATF6 is required for full IRE1α-dependent induction of UPR genes, but IRE1α is not required for ATF6-dependent transcriptional activation of target genes (55). In the absence of IRE1 or XBP1, compensatory activation of the ATF6-mediated UPR may result in elevated levels of GRP78 and CHOP.
The precise mechanism by which ER stress causes colitis remains under investigation, but evidence suggests it involves the induction of both inflammatory and apoptotic signaling. While the goal of the UPR is to adapt to a changing environment and restore normal ER function, when adaptation fails and homeostasis is not restored, ER-mediated pathways alert the cell (and possibly neighboring cells) by activating NF-κB, which induces a variety of genes involved in host defense. In the case of excessive and prolonged ER stress, apoptotic signaling pathways are activated in order to rid the body of the dysfunctional cells [reviewed in (56)]. Thus, while UPR signaling initially combats apoptosis by attempting to repair damage, it may later promote it if homeostasis is not reestablished. ER stress is associated with a variety of diseases, including neurodegeneration, ischemia/reperfusion injury, heart disease, and diabetes (56).
Activation of PERK, IRE1α, and ATF6α by ER stress has been shown to initiate apoptotic signaling (56). However, ATF6α-deficient cells exhibit increased apoptosis upon ER stress (54). It has been proposed that this increased apoptosis may be due to a compromised ability of ATF6α-deficient cells to adapt to ER stress by downregulating UPR signaling and thereby prevent eventual apoptosis (52). The prolonged ER stress-induced elevation of CHOP, which has been shown to induce apoptosis (57) and spliced XBP1 levels in cells lacking ATF6α supports this hypothesis. Likewise, a failure in adaptation to ER stress in the colons of wrt mice may, through aberrant prolonged activation of ATF6-independent UPR signaling, result in activation of apoptotic signaling pathways. Death of intestinal epithelial cells may cause disruption of the gut epithelial barrier and permit lumenal antigens to penetrate into subepithelial tissues leading to inflammation.
ER stress in the intestine and colon may also directly cause inflammation, without a disruption of the intestinal barrier. Kaser et al. report that even minor deficiencies in XBP1 expression within intestinal epithelial cells lead to spontaneous enteritis, while leaving the intestinal barrier largely intact (53). In addition, XBP1 deficiency in a small intestinal cell line promotes a proinflammatory state in which TNFα and flagellin activate elevated JNK/SAPK signaling in the absence of microbes and cytokines (53). Such signaling may in turn contribute to the increased apoptosis of Paneth, goblet and intestinal epithelial cells observed in XBP1 mutant mice. A deficiency of ATF6 signaling may directly promote a proinflammatory state in woodrat colons, a hypothesis supported by the finding that woodrat mice display increased levels of IL-1β and IL-6 in epithelium and lamina propria even before DSS administration, suggestive of a low level of constitutive inflammation.
|Primers||Primers cannot be located by automatic search.|
Woodrat 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. The same primers are used for PCR amplification and for sequencing. The woodrat mutation destroys an Afl III restriction enzyme site in the Mbtps1 genomic sequence, and genotyping may also be performed by PCR amplification of the region containing the mutation followed by Afl III restriction enzyme digestion.
woodrat(F): 5’- ACACCCTCAGTGCCAGCCAGGAC -3’
woodrat(R): 5’- ACAAAGTCTGGGCCACGTCACACAG -3’
1) 94°C 2:00
2) 94°C 0:30
3) 60°C 0:30
4) 72°C 0:30
5) repeat steps (2-4) 34X
6) 72°C 3:00
7) 4°C ∞
The following sequence of 300 nucleotides (from Genbank genomic region NC_000074 for linear genomic sequence of Mbtps1) is amplified:
29541 acaccctcag tgccagccag gacaccctac aagcaggcct
29581 ttagtgtgca gccccactgt gctacctgtg cccggcactc tctgatgtgg tctacagtaa
29641 cactctcttc ttgtttcagc ctgagtccta gctacatcga cctgactgag tgtccctaca
29701 tgtggcccta ctgctcccag cctatctact atggaggaat gccaacaatc gttaatgtca
29761 ccatcctcaa tggcatgggc gtcacaggaa gaattgtgga taaggtgaaa cttctctgtg
29821 tgacgtggcc cagactttgt
Primer binding sites are underlined; the Afl III site destroyed by the woodrat mutation is highlighted in gray; the mutated A is indicated in red lettering.
Instead of sequencing, digest PCR reactions with Afl III. Run on 2% agarose gel with heterozygous and C57BL/6J controls.
Products: woodrat allele- 300 bp; wild type allele- 158 bp, 142 bp.
1. Brandl, K., Rutschmann, S., Li, X., Du, X., Xiao, N., Schnabl, B., Brenner, D. A., and Beutler, B. (2009) Enhanced Sensitivity to DSS Colitis Caused by a Hypomorphic Mbtps1 Mutation Disrupting the ATF6-Driven Unfolded Protein Response. Proc. Natl. Acad. Sci. U. S. A.. 106, 3300-3305.
2. Rutschmann, S., Crozat, K., Li, X., Du, X., Hanselman, J. C., Shigeoka, A. A., Brandl, K., Popkin, D. L., McKay, D. B., Xia, Y., Moresco, E. M., and Beutler, B. (2012) Hypopigmentation and Maternal-Zygotic Embryonic Lethality Caused by a Hypomorphic mbtps1 Mutation in Mice. G3 (Bethesda). 2, 499-504.
3. Popkin, D. L., Teijaro, J. R., Sullivan, B. M., Urata, S., Rutschmann, S., de la Torre, J. C., Kunz, S., Beutler, B., and Oldstone, M. (2011) Hypomorphic Mutation in the Site-1 Protease Mbtps1 Endows Resistance to Persistent Viral Infection in a Cell-Specific Manner. Cell. Host Microbe. 9, 212-222.
4. Bukrinsky, M., and Sviridov, D. (2006) Human Immunodeficiency Virus Infection and Macrophage Cholesterol Metabolism. J. Leukoc. Biol.. 80, 1044-1051.
5. Riethmuller, J., Riehle, A., Grassme, H., and Gulbins, E. (2006) Membrane Rafts in Host-Pathogen Interactions. Biochim. Biophys. Acta. 1758, 2139-2147.
6. Rojek, J. M., Perez, M., and Kunz, S. (2008) Cellular Entry of Lymphocytic Choriomeningitis Virus. J. Virol.. 82, 1505-1517.
7. Seidah, N. G., Mowla, S. J., Hamelin, J., Mamarbachi, A. M., Benjannet, S., Toure, B. B., Basak, A., Munzer, J. S., Marcinkiewicz, J., Zhong, M., Barale, J. C., Lazure, C., Murphy, R. A., Chretien, M., and Marcinkiewicz, M. (1999) Mammalian subtilisin/kexin Isozyme SKI-1: A Widely Expressed Proprotein Convertase with a Unique Cleavage Specificity and Cellular Localization. Proc. Natl. Acad. Sci. U. S. A.. 96, 1321-1326.
8. Sakai, J., Rawson, R. B., Espenshade, P. J., Cheng, D., Seegmiller, A. C., Goldstein, J. L., and Brown, M. S. (1998) Molecular Identification of the Sterol-Regulated Luminal Protease that Cleaves SREBPs and Controls Lipid Composition of Animal Cells. Mol. Cell. 2, 505-514.
9. Scamuffa, N., Calvo, F., Chretien, M., Seidah, N. G., and Khatib, A. M. (2006) Proprotein Convertases: Lessons from Knockouts. FASEB J.. 20, 1954-1963.
10. Pasquato, A., Pullikotil, P., Asselin, M. C., Vacatello, M., Paolillo, L., Ghezzo, F., Basso, F., Di Bello, C., Dettin, M., and Seidah, N. G. (2006) The Proprotein Convertase SKI-1/S1P. in Vitro Analysis of Lassa Virus Glycoprotein-Derived Substrates and Ex Vivo Validation of Irreversible Peptide Inhibitors. J. Biol. Chem.. 281, 23471-23481.
11. Toure, B. B., Munzer, J. S., Basak, A., Benjannet, S., Rochemont, J., Lazure, C., Chretien, M., and Seidah, N. G. (2000) Biosynthesis and Enzymatic Characterization of Human SKI-1/S1P and the Processing of its Inhibitory Prosegment. J. Biol. Chem.. 275, 2349-2358.
12. Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999) Autocatalytic Processing of Site-1 Protease Removes Propeptide and Permits Cleavage of Sterol Regulatory Element-Binding Proteins. J. Biol. Chem.. 274, 22795-22804.
13. Seidah, N. G., and Prat, A. (2007) The Proprotein Convertases are Potential Targets in the Treatment of Dyslipidemia. J. Mol. Med. (Berl). 85, 685-696.
14. Elagoz, A., Benjannet, S., Mammarbassi, A., Wickham, L., and Seidah, N. G. (2002) Biosynthesis and Cellular Trafficking of the Convertase SKI-1/S1P: Ectodomain Shedding Requires SKI-1 Activity. J. Biol. Chem.. 277, 11265-11275.
15. Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999) Secreted Site-1 Protease Cleaves Peptides Corresponding to Luminal Loop of Sterol Regulatory Element-Binding Proteins. J. Biol. Chem.. 274, 22805-22812.
16. Anderson, E. D., VanSlyke, J. K., Thulin, C. D., Jean, F., and Thomas, G. (1997) Activation of the Furin Endoprotease is a Multiple-Step Process: Requirements for Acidification and Internal Propeptide Cleavage. EMBO J.. 16, 1508-1518.
17. Pullikotil, P., Benjannet, S., Mayne, J., and Seidah, N. G. (2007) The Proprotein Convertase SKI-1/S1P: Alternate Translation and Subcellular Localization. J. Biol. Chem.. 282, 27402-27413.
18. Creemers, J. W., Ines Dominguez, D., Plets, E., Serneels, L., Taylor, N. A., Multhaup, G., Craessaerts, K., Annaert, W., and De Strooper, B. (2001) Processing of Beta-Secretase by Furin and Other Members of the Proprotein Convertase Family. J. Biol. Chem.. 276, 4211-4217.
19. Bassi, D. E., Fu, J., Lopez de Cicco, R., and Klein-Szanto, A. J. (2005) Proprotein Convertases: "Master Switches" in the Regulation of Tumor Growth and Progression. Mol. Carcinog.. 44, 151-161.
20. Lenz, O., ter Meulen, J., Klenk, H. D., Seidah, N. G., and Garten, W. (2001) The Lassa Virus Glycoprotein Precursor GP-C is Proteolytically Processed by Subtilase SKI-1/S1P. Proc. Natl. Acad. Sci. U. S. A.. 98, 12701-12705.
21. Gordon, V. M., Klimpel, K. R., Arora, N., Henderson, M. A., and Leppla, S. H. (1995) Proteolytic Activation of Bacterial Toxins by Eukaryotic Cells is Performed by Furin and by Additional Cellular Proteases. Infect. Immun.. 63, 82-87.
22. Moulard, M., Hallenberger, S., Garten, W., and Klenk, H. D. (1999) Processing and Routage of HIV Glycoproteins by Furin to the Cell Surface. Virus Res.. 60, 55-65.
23. Vincent, M. J., Sanchez, A. J., Erickson, B. R., Basak, A., Chretien, M., Seidah, N. G., and Nichol, S. T. (2003) Crimean-Congo Hemorrhagic Fever Virus Glycoprotein Proteolytic Processing by Subtilase SKI-1. J. Virol.. 77, 8640-8649.
24. Ehrmann, M., and Clausen, T. (2004) Proteolysis as a Regulatory Mechanism. Annu. Rev. Genet.. 38, 709-724.
25. Stirling, J., and O'hare, P. (2006) CREB4, a Transmembrane bZip Transcription Factor and Potential New Substrate for Regulation and Cleavage by S1P. Mol. Biol. Cell. 17, 413-426.
26. Raggo, C., Rapin, N., Stirling, J., Gobeil, P., Smith-Windsor, E., O'Hare, P., and Misra, V. (2002) Luman, the Cellular Counterpart of Herpes Simplex Virus VP16, is Processed by Regulated Intramembrane Proteolysis. Mol. Cell. Biol.. 22, 5639-5649.
27. Zhang, K., Shen, X., Wu, J., Sakaki, K., Saunders, T., Rutkowski, D. T., Back, S. H., and Kaufman, R. J. (2006) Endoplasmic Reticulum Stress Activates Cleavage of CREBH to Induce a Systemic Inflammatory Response. Cell. 124, 587-599.
28. Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) ER Stress Induces Cleavage of Membrane-Bound ATF6 by the Same Proteases that Process SREBPs. Mol. Cell. 6, 1355-1364.
29. Murakami, T., Kondo, S., Ogata, M., Kanemoto, S., Saito, A., Wanaka, A., and Imaizumi, K. (2006) Cleavage of the Membrane-Bound Transcription Factor OASIS in Response to Endoplasmic Reticulum Stress. J. Neurochem.. 96, 1090-1100.
30. Beyer, W. R., Popplau, D., Garten, W., von Laer, D., and Lenz, O. (2003) Endoproteolytic Processing of the Lymphocytic Choriomeningitis Virus Glycoprotein by the Subtilase SKI-1/S1P. J. Virol.. 77, 2866-2872.
31. Rawson, R. B. (2003) The SREBP Pathway--Insights from Insigs and Insects. Nat. Rev. Mol. Cell Biol.. 4, 631-640.
32. Duncan, E. A., Brown, M. S., Goldstein, J. L., and Sakai, J. (1997) Cleavage Site for Sterol-Regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-Binding Protein-2. J. Biol. Chem.. 272, 12778-12785.
33. Duncan, E. A., Dave, U. P., Sakai, J., Goldstein, J. L., and Brown, M. S. (1998) Second-Site Cleavage in Sterol Regulatory Element-Binding Protein Occurs at Transmembrane Junction as Determined by Cysteine Panning. J. Biol. Chem.. 273, 17801-17809.
34. Rawson, R. B., Zelenski, N. G., Nijhawan, D., Ye, J., Sakai, J., Hasan, M. T., Chang, T. Y., Brown, M. S., and Goldstein, J. L. (1997) Complementation Cloning of S2P, a Gene Encoding a Putative Metalloprotease Required for Intramembrane Cleavage of SREBPs. Mol. Cell. 1, 47-57.
35. Yang, T., Espenshade, P. J., Wright, M. E., Yabe, D., Gong, Y., Aebersold, R., Goldstein, J. L., and Brown, M. S. (2002) Crucial Step in Cholesterol Homeostasis: Sterols Promote Binding of SCAP to INSIG-1, a Membrane Protein that Facilitates Retention of SREBPs in ER. Cell. 110, 489-500.
36. Yabe, D., Brown, M. S., and Goldstein, J. L. (2002) Insig-2, a Second Endoplasmic Reticulum Protein that Binds SCAP and Blocks Export of Sterol Regulatory Element-Binding Proteins. Proc. Natl. Acad. Sci. U. S. A.. 99, 12753-12758.
37. DeBose-Boyd, R. A., Brown, M. S., Li, W. P., Nohturfft, A., Goldstein, J. L., and Espenshade, P. J. (1999) Transport-Dependent Proteolysis of SREBP: Relocation of Site-1 Protease from Golgi to ER Obviates the Need for SREBP Transport to Golgi. Cell. 99, 703-712.
38. Nohturfft, A., DeBose-Boyd, R. A., Scheek, S., Goldstein, J. L., and Brown, M. S. (1999) Sterols Regulate Cycling of SREBP Cleavage-Activating Protein (SCAP) between Endoplasmic Reticulum and Golgi. Proc. Natl. Acad. Sci. U. S. A.. 96, 11235-11240.
39. Nohturfft, A., Yabe, D., Goldstein, J. L., Brown, M. S., and Espenshade, P. J. (2000) Regulated Step in Cholesterol Feedback Localized to Budding of SCAP from ER Membranes. Cell. 102, 315-323.
40. Sakai, J., Nohturfft, A., Goldstein, J. L., and Brown, M. S. (1998) Cleavage of Sterol Regulatory Element-Binding Proteins (SREBPs) at Site-1 Requires Interaction with SREBP Cleavage-Activating Protein. Evidence from in Vivo Competition Studies. J. Biol. Chem.. 273, 5785-5793.
41. Espenshade, P. J., Li, W. P., and Yabe, D. (2002) Sterols Block Binding of COPII Proteins to SCAP, Thereby Controlling SCAP Sorting in ER. Proc. Natl. Acad. Sci. U. S. A.. 99, 11694-11699.
42. Haze, K., Yoshida, H., Yanagi, H., Yura, T., and Mori, K. (1999) Mammalian Transcription Factor ATF6 is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress. Mol. Biol. Cell. 10, 3787-3799.
43. Shen, J., Chen, X., Hendershot, L., and Prywes, R. (2002) ER Stress Regulation of ATF6 Localization by Dissociation of BiP/GRP78 Binding and Unmasking of Golgi Localization Signals. Dev. Cell.. 3, 99-111.
44. Chen, X., Shen, J., and Prywes, R. (2002) The Luminal Domain of ATF6 Senses Endoplasmic Reticulum (ER) Stress and Causes Translocation of ATF6 from the ER to the Golgi. J. Biol. Chem.. 277, 13045-13052.
45. Nadanaka, S., Yoshida, H., Kano, F., Murata, M., and Mori, K. (2004) Activation of Mammalian Unfolded Protein Response is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum. Mol. Biol. Cell. 15, 2537-2548.
46. Todd, D. J., Lee, A. H., and Glimcher, L. H. (2008) The Endoplasmic Reticulum Stress Response in Immunity and Autoimmunity. Nat. Rev. Immunol.. 8, 663-674.
47. Liang, G., Audas, T. E., Li, Y., Cockram, G. P., Dean, J. D., Martyn, A. C., Kokame, K., and Lu, R. (2006) Luman/CREB3 Induces Transcription of the Endoplasmic Reticulum (ER) Stress Response Protein Herp through an ER Stress Response Element. Mol. Cell. Biol.. 26, 7999-8010.
48. Yang, J., Goldstein, J. L., Hammer, R. E., Moon, Y. A., Brown, M. S., and Horton, J. D. (2001) Decreased Lipid Synthesis in Livers of Mice with Disrupted Site-1 Protease Gene. Proc. Natl. Acad. Sci. U. S. A.. 98, 13607-13612.
49. Mitchell, K. J., Pinson, K. I., Kelly, O. G., Brennan, J., Zupicich, J., Scherz, P., Leighton, P. A., Goodrich, L. V., Lu, X., Avery, B. J., Tate, P., Dill, K., Pangilinan, E., Wakenight, P., Tessier-Lavigne, M., and Skarnes, W. C. (2001) Functional Analysis of Secreted and Transmembrane Proteins Critical to Mouse Development. Nat. Genet.. 28, 241-249.
51. Heazlewood, C. K., Cook, M. C., Eri, R., Price, G. R., Tauro, S. B., Taupin, D., Thornton, D. J., Png, C. W., Crockford, T. L., Cornall, R. J., Adams, R., Kato, M., Nelms, K. A., Hong, N. A., Florin, T. H., Goodnow, C. C., and McGuckin, M. A. (2008) Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis. PLoS Med.. 5, e54.
52. Bertolotti, A., Wang, X., Novoa, I., Jungreis, R., Schlessinger, K., Cho, J. H., West, A. B., and Ron, D. (2001) Increased Sensitivity to Dextran Sodium Sulfate Colitis in IRE1beta-Deficient Mice. J. Clin. Invest.. 107, 585-593.
53. Kaser, A., Lee, A. H., Franke, A., Glickman, J. N., Zeissig, S., Tilg, H., Nieuwenhuis, E. E., Higgins, D. E., Schreiber, S., Glimcher, L. H., and Blumberg, R. S. (2008) XBP1 Links ER Stress to Intestinal Inflammation and Confers Genetic Risk for Human Inflammatory Bowel Disease. Cell. 134, 743-756.
54. Wu, J., Rutkowski, D. T., Dubois, M., Swathirajan, J., Saunders, T., Wang, J., Song, B., Yau, G. D., and Kaufman, R. J. (2007) ATF6alpha Optimizes Long-Term Endoplasmic Reticulum Function to Protect Cells from Chronic Stress. Dev. Cell.. 13, 351-364.
55. Lee, K., Tirasophon, W., Shen, X., Michalak, M., Prywes, R., Okada, T., Yoshida, H., Mori, K., and Kaufman, R. J. (2002) IRE1-Mediated Unconventional mRNA Splicing and S2P-Mediated ATF6 Cleavage Merge to Regulate XBP1 in Signaling the Unfolded Protein Response. Genes Dev.. 16, 452-466.
56. Kim, I., Xu, W., and Reed, J. C. (2008) Cell Death and Endoplasmic Reticulum Stress: Disease Relevance and Therapeutic Opportunities. Nat. Rev. Drug Discov.. 7, 1013-1030.
|Science Writers||Eva Marie Y. Moresco, Anne Murray|
|Illustrators||Diantha La Vine|
|Authors||Katharina Brandl, Sophie Rutschmann, Karine Crozat, Michael Berger, Xin Du, Bruce Beutler|