ERIS,an endoplasmic reticulum IFN stimulator,activates innate immune signaling through dimerization

We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non–self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.     

From the Cover: A plant-specific calreticulin is a key retention factor for a defective brassinosteroid receptor in the endoplasmic reticulum

Mammalian calreticulin (CRT) is a multifunctional Ca²⁺-binding protein involved in more than 40 cellular processes in various subcellular compartments, such as Ca²⁺ storage and protein folding in the endoplasmic reticulum (ER). CRT homologues were discovered in plants almost 15 years ago, and recent studies revealed that many plant species contain 2 or more CRTs that are members of 2 distinct families, the CRT1/2 family and the plant-specific CRT3 family. However, little is known about their physiological functions. Here we report ebs2 (EMS-mutagenized bri1 suppressor 2) as an allele-specific suppressor of bri1–9, a dwarf Arabidopsis mutant caused by retention of a defective brassinosteroid receptor in the ER. EBS2 encodes the Arabidopsis CRT3 that interacts with ER-localized bri1–9 in a glycan-dependent manner. Loss-of-function ebs2 mutations compromise ER retention of bri1–9 and suppress its dwarfism, whereas EBS2 over-expression enhances its dwarf phenotype. In contrast, mutations of 2 other CRTs or their membrane-localized homologues calnexins had little effect on bri1–9. A domain-swapping experiment revealed that the positively charged C-terminal tail of CRT3 is crucial for its “bri1–9-retainer” function. Our study revealed not only a functional role for a plant-specific CRT, but also functional diversity among the 3 Arabidopsis CRT paralogues.     

Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum

Canonical Wnt signaling is initiated by binding of Wnt proteins to members of the Frizzled family and subsequent complex formation with lipoprotein receptor-related proteins 5/6 (LRP5/6). Here, we show that LRP6 is palmitoylated on a juxtamembranous cysteine and that palmitoylation is required for exit from the endoplasmic reticulum (ER). We propose that palmitoylation serves to tilt the long, 23-residue transmembrane domain of LRP6 with respect to the plane of membrane to prevent a hydrophobic mismatch and subsequent recognition by the ER quality control. In support of this model, a palmitoylation-deficient LRP6 mutant could be rescued from ER retention by deletion of two to four residues in the transmembrane domain. Importantly, we found that palmitoylation-deficient LRP6 was retained in the ER by a completely novel monoubiquitination-dependent ER retention mechanism. Mutation of a specific lysine indeed abolished ubiquitination of palmitoylation-deficient LRP6 and led to a rescue from ER retention. Finally, at the cell surface, we found that interplay between palmitoylation and ubiquitination was necessary for efficient Wnt signaling.     

CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis

Chitin is a major component of fungal cell walls and serves as a microbe-associated molecular pattern (MAMP) for the detection of various potential pathogens in innate immune systems of both plants and animals. We recently showed that chitin elicitor-binding protein (CEBiP), plasma membrane glycoprotein with LysM motifs, functions as a cell surface receptor for chitin elicitor in rice. The predicted structure of CEBiP does not contain any intracellular domains, suggesting that an additional component(s) is required for signaling through the plasma membrane into the cytoplasm. Here, we identified a receptor-like kinase, designated CERK1, which is essential for chitin elicitor signaling in Arabidopsis. The KO mutants for CERK1 completely lost the ability to respond to the chitin elicitor, including MAPK activation, reactive oxygen species generation, and gene expression. Disease resistance of the KO mutant against an incompatible fungus, Alternaria brassicicola, was partly impaired. Complementation with the WT CERK1 gene showed cerk1 mutations were responsible for the mutant phenotypes. CERK1 is a plasma membrane protein containing three LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain with autophosphorylation/myelin basic protein kinase activity, suggesting that CERK1 plays a critical role in fungal MAMP perception in plants.     

Tunicamycin aggravates endoplasmic reticulum stress and airway inflammation via PERK-ATF4-CHOP signaling in a murine model of neutrophilic asthma

Introduction: Endoplasmic reticulum (ER) stress has been considered to be an important regulator of airway inflammation in the pathogenesis of bronchial asthma, but the mechanism of ER stress involved in neutrophilic asthma remain not fully understood. Methods: Tunicamycin is a mixture of homologous nucleoside antibiotics, which is used to induce ER stress. In the present study, Tunicamycin was administered to mouse bronchial epithelial cells and a neutrophilic asthma model (OVA_LPS-OVA mice), and ER stress indicators and inflammatory cytokines were measured by Western blotting and Elisa. Results: Tunicamycin not only induced ER stress in mouse bronchial epithelial cells, but also increased expression of inflammation indicators such as IL-6, IL-8, and TNF-α via PERK-ATF4-CHOP signaling. Additionally, the phosphorylation of PERK and the expression levels of ATF4 and CHOP proteins and inflammatory cytokines (IL-6, IL-8 and TNF-α) were elevated in the lung tissue of OVA_LPS-OVA mice. Administering tunicamycin further increased protein expression levels of ER stress indicators and inflammatory cytokines, and resulted in more severe asthma phenotypes in OVA_LPS-OVA mice, suggesting that PERK-ATF4-CHOP signaling is associated with airway inflammation in neutrophil-dominant asthma. Conclusions: These data support the emerging notion that regulation of ER stress could be strongly associated with the development of neutrophilic asthma.     

Congenital sucrase-isomaltase deficiency because of an accumulation of the mutant enzyme in the endoplasmic reticulum

BACKGROUND & AIMS: Congenital sucrase-isomaltase deficiency (CSID) is an autosomal recessive human disorder characterized by reduced activities of the brush border enzyme sucrase-isomaltase (SI). Here, we elucidate the pathogenesis of a new variant of CSID at the cellular and molecular level. METHODS: Assessment of the CSID phenotype was achieved by enzymatic activity measurements, biosynthetic labeling of intestinal biopsy specimens, immunoprecipitation of SI, and immunoelectronmicroscopy. The putative mutation was identified by sequencing of the SI cDNA isolated by RT-PCR from intestinal biopsy samples. The function of the mutation was verified by immunoprecipitation and confocal microscopy of transiently transfected cells. RESULTS: Biosynthetic labeling and immunoelectron microscopy reveal a predominant localization of SI in the endoplasmic reticulum (ER) similar to phenotype I of CSID. Unlike phenotype I, however, a partial conversion of SI to a complex glycosylated mature form takes place. The SI cDNA in this phenotype revealed 3 mutations, 2 of which, Val to Phe at residue 15 and Ala to Thr at residue 231, had no effect on the structure or function of SI. By contrast, the third mutation resulted in an exchange of leucine by proline at position 620 (L620P) and revealed in transfected COS cells structural features and subcellular localization similar to the phenotype identified in the patient's enterocytes. CONCLUSIONS: This is the first identification at the molecular and subcellular levels of a novel variant of CSID in which SI accumulates predominantly in the ER, and a minor proportion is further processed and transported to the apical membrane of enterocytes.     

Disease-associated mutations cause premature oligomerization of myelin proteolipid protein in the endoplasmic reticulum

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disease caused by mutations, deletions, or duplications of the proteolipid protein (PLP) gene. Mutant forms of PLP are retained in the endoplasmic reticulum (ER), and the resulting accumulation of mutant protein is thought to be a direct cause of oligodendrocyte cell death, which is the primary clinical feature of PMD. The molecular mechanisms underlying the toxicity of mutant PLP are however currently unknown. We report here that PMD-linked mutations of PLP are associated with the accelerated assembly of the protein into stable homooligomers that resemble mature, native PLP. Thus although WT PLP forms stable oligomers after an extended maturation period, most likely at the cell surface, mutant forms of PLP rapidly assemble into such oligomers at the ER. Using PLP mutants associated with diseases of varying severity, we show that the formation of stable oligomers correlates with the development of PMD. Based on these findings, we propose that the premature oligomerization of PLP in the ER of oligodendrocytes contributes to the pathology of PMD.     

A common mechanism of PLP/DM20 misfolding causes cysteine-mediated endoplasmic reticulum retention in oligodendrocytes and Pelizaeus-Merzbacher disease

A large number of mutations in the human PLP1 gene lead to abnormal myelination and oligodendrocyte death in Pelizaeus-Merzbacher disease (PMD). Here we show that a major subgroup of PMD mutations that map into the extracellular loop region of PLP/DM20 leads to the failure of oligodendrocytes to form the correct intramolecular disulfide bridges. This leads to abnormal protein cross-links and endoplasmic reticulum retention and activates the unfolded protein response. Importantly, surface expression of mutant PLP/DM20 can be restored and the unfolded protein response can be reverted by the removal of two cysteines. Thus, covalent protein cross-links emerge as a cause, rather than as a consequence, of endoplasmic reticulum retention.     

Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana. Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis, activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N- and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis. The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.Immune responses in plants generally start by receptor-mediated detection of nonself molecules that are conserved among different classes of microbes, both beneficial and pathogenic (1). These molecules often have essential functions in microbial fitness (2) and are known as microbe-associated molecular patterns (MAMPs). Upon their perception by the plant, MAMPs trigger basal immune responses (3), e.g., ethylene biosynthesis, production of reactive oxygen species, release of antimicrobial compounds (4), and in certain cases programmed cell death (2). Collectively, these responses contribute to resistance against nonadapted pathogens [MAMP-triggered immunity (MTI)].MAMPs of plant-infecting microbes have been described for bacteria, fungi, and oomycetes. Three characterized bacterial MAMPs are flagellin (5), EF-Tu (6), and peptidoglycan (7). Flagellin is the main protein of the bacterial flagellum, which is used by eubacteria for movement. A highly conserved fragment of 22 aa, named flg22 (5), is sufficient to activate MTI in Arabidopsis and other plant species. Elongation factor thermo unstable (EF-Tu) is an abundant and conserved bacterial protein that plays a central role in the elongation phase of protein synthesis. An 18-aa domain of EF-Tu, named elf18, is recognized as a MAMP in Brassicaceae species, but not in other tested plant families (6). Peptidoglycan (PGN), the third characterized bacterial MAMP, is a major structural component of most bacterial cell walls. PGN, consisting of strands of alternating N-acetylglucosamine and N-acetylmuramic acid residues, triggers immunity in Arabidopsis (7). An important fungal MAMP is chitin, a structural component of all fungal cell walls. Plants are able to recognize chitin, and fragments of 4–10 N-acetylglucosamine residues are the most potent inducers of defense (8). Recently, a second fungal MAMP was identified, a secreted polygalacturonase of Botrytis cinerea that triggers immunity in Arabidopsis (9).Four oomycete-derived MAMPs have been identified to date (10): (i) heptaglucoside fragments, originating from branched β-glucans that are major cell wall polysaccharides, and that trigger defense responses in many Fabaceous plants (11); (ii) glycoprotein 42, a calcium-dependent transglutaminase that functions in irreversible protein cross-linking and is abundant in Phytophthora cell walls, and a 13-aa peptide fragment thereof that elicit MTI responses in parsley (12) and potato (13); (iii) elicitins, secreted proteins with sterol-binding activity (14), which provoke necrosis in Nicotiana plants through induction of cell death (15); and (iv) the Phytophthora cellulose-binding elicitor lectin, which is thought to cause perturbation of the cell wall cellulose status, thereby triggering necrosis and MTI in tobacco and Arabidopsis (16, 17). Other groups of cell death-inducing proteins may also qualify as MAMPs based on their widespread occurrence among different pathogens (2), e.g., the Crinklers and the cytotoxic necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) (10).Two major NLP types are found in bacteria, fungi, and oomycetes (18, 19) and are known to cause rapid necrosis and ethylene production in many dicot, but not in monocot plant species (18, 20). Type 2 NLPs differ from type 1 by an additional conserved second cysteine bridge and putative calcium-binding domain (19). In Arabidopsis, cytotoxic NLPs were found to activate immunity-related gene expression, which strongly overlapped with that induced by flg22 (21, 22). However, it was suggested that immune responses resulted from cytotoxicity. Moreover, necrosis was only induced upon treatment with the complete NLP protein (23). In vitro, cytotoxic NLPs cause rapid leakage of dicot membrane-derived vesicles, suggesting a direct cytolytic activity (24). The immunogenic effect of NLPs was therefore suggested to result from direct cellular damage (24), or release of damage-associated molecular patterns (3).Several plant-infecting oomycetes have large expansions of NLPs in their genomes (25–27), suggesting that these proteins play an important role in the pathogen’s lifestyle. A clear virulence function was observed for NLP_Pcc of the rot bacterium Pectobacterium carotovorum (27). Also, individual deletion of two NLP genes in the fungus Verticillium dahliae resulted in reduced virulence on different host plants (28). Five other NLP genes in this fungus encode noncytotoxic proteins (29), a phenomenon that is also observed in oomycetes. When tested by transient expression in tobacco, necrosis was only induced by 1 out of 3 tested NLPs of Phytophthora infestans (30), 8 out of 33 NLPs of Phytophthora sojae (31), whereas not a single 1 of 10 NLPs of Hyaloperonospora arabidopsidis tested caused necrosis (26). In contrast to cytotoxic NLPs that are mainly expressed during necrotrophic stages of infection, noncytotoxic NLPs appear to be expressed early during infection (26, 30), suggesting they serve an, as-yet-unknown, function during penetration or initial colonization of the host.In our search for the biological function of noncytotoxic NLPs of H. arabidopsidis, transgenic HaNLP-expressing Arabidopsis plants were generated that were severely stunted. In this paper, we show that Arabidopsis responds to noncytotoxic HaNLPs and small peptide fragments thereof that are highly conserved in type 1 NLPs. The peptides activate ethylene production and other typical MAMP-triggered defense responses, but not tissue necrosis, indicating they act as a MAMP. NLPs are not restricted to a single class of microbes but present in a broad range of mostly plant-associated microbes (bacteria, fungi, and oomycetes) belonging to three kingdoms of life, making this a MAMP with an unprecedented broad taxonomic occurrence.     

Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling

Mitogen-activated protein kinase (MAPK)–mediated responses are in part regulated by the repertoire of MAPK substrates, which is still poorly elucidated in plants. Here, the in vivo enzyme–substrate interaction of the Arabidopsis thaliana MAP kinase, MPK6, with an ethylene response factor (ERF104) is shown by fluorescence resonance energy transfer. The interaction was rapidly lost in response to flagellin-derived flg22 peptide. This complex disruption requires not only MPK6 activity, which also affects ERF104 stability via phosphorylation, but also ethylene signaling. The latter points to a novel role of ethylene in substrate release, presumably allowing the liberated ERF104 to access target genes. Microarray data show enrichment of GCC motifs in the promoters of ERF104–up-regulated genes, many of which are stress related. ERF104 is a vital regulator of basal immunity, as altered expression in both erf104 and overexpressors led to more growth inhibition by flg22 and enhanced susceptibility to a non-adapted bacterial pathogen.     

A role of Histidine in the lamprey gonadotropin-releasing hormone receptor-1 (lGnRHR-1): Functional insight of diverse amino acid residues in the position of Tyr of the DRY motif in GnRHR from an ancestral type II receptor

The highly conserved DRY motif located at the end of the third transmembrane of G-protein-coupled receptors has been described as a key motif for several aspects of GPCR functions. However, in the case of the vertebrate gonadotropin-releasing hormone receptor (GnRHR), the amino acid in the third position in the DRY motif is variable. In the lamprey, a most basal vertebrate, the third amino acid of the “DRY” in lamprey (lGnRHR-1) is His, while it is most often His/Gln in the type II GnRHR. To investigate the functional significance of the substitution of DRY to DRH in the GnRHR-1, second messenger signaling, ligand binding and internalization of the wild-type and mutant lGnRH receptors were characterized with site-directed mutagenesis. Treatment of the DRE¹⁵¹ and DRS¹⁵¹ mutant receptors with lamprey GnRH-I significantly reduced inositol phosphate compared to wild-type (DRH¹⁵¹) and DRY¹⁵¹ receptors. The Log IC₅₀ of wild-type receptor (−9.554 ± 0.049) was similar to the Log IC₅₀ of DRE¹⁵¹, DRS¹⁵¹ and DRX¹⁵¹ mutants, yet these same mutants were shown to significantly reduce cell-surface expression. However, the DRY¹⁵¹ mutant compared to the wild-type receptor increased cell-surface expression, suggesting that the reduction of IP production was due to the level of the cell-surface expression of the mutant receptors. The rate of internalization of DRX¹⁵¹ (35.60%) was reduced compared to wild-type and other mutant receptors. These results suggest that His¹⁵¹ of the lamprey GnRH receptor-1 may play a critical role in the retention of a certain level of cell-surface expression for subsequent cellular second messenger events.