Cleavage of Toll-like receptor 3 by cathepsins B and H is essential for signaling

Toll-like receptor (TLR) 3 is an endosomal TLR that mediates immune responses against viral infections upon activation by its ligand double-stranded RNA, a replication intermediate of most viruses. TLR3 is expressed widely in the body and activates both the innate and adaptive immune systems. However, little is known about how TLR3 intracellular trafficking and maturation are regulated. Here we show that newly synthesized endogenous TLR3 is transported through the ER and Golgi apparatus to endosomes, where it is rapidly cleaved. TLR3 protein expression is up-regulated by its own ligand, leading to the accumulation of its cleaved form. In agreement with its proposed role as a transporter, UNC93B1 expression is required for TLR3 cleavage and signaling. Furthermore, TLR3 signaling and cleavage are sensitive to cathepsin inhibition. Cleavage occurs between aa 252 and 346, and results in a functional receptor that signals upon activation. A truncated form of TLR3 lacking the N-terminal 345 aa also signals from acidic compartments in response to ligand activation. Screening of the human cathepsin family by RNA interference identified cathepsins B and H as key mediators of TLR3 processing. Taken together, our data indicate that TLR3 proteolytic processing is essential for its function, and suggest a mechanism of tight control of TLR3 signaling and thus immunity.     

Bone morphogenetic protein signaling is essential for terminal differentiation of the intestinal secretory cell lineage

BACKGROUND & AIMS: Bone morphogenetic proteins (Bmps) are morphogens known to play key roles in gastrointestinal development and pathology. Most Bmps are produced primarily by the mesenchymal compartment and activate their signaling pathways following a paracrine or autocrine route. The aim of this study was to investigate the role of epithelial Bmp signaling in intestinal morphogenesis and maintenance of adult epithelial cell functions. METHODS: With the use of tissue-specific gene ablation, we generated mice lacking the Bmp receptor type IA (Bmpr1a) exclusively in the intestinal epithelium. Bmpr1a mutant and control mice were sacrificed for histology, immunofluorescence, Western blot analysis, electron microscopy, and quantitative polymerase chain reaction. RESULTS: As well as showing increased proliferation and altered intestinal epithelial morphology, Bmpr1a mutant mice revealed that epithelial Bmp signaling is associated with impaired terminal differentiation of cells from the secretory lineage but not with the determination of cell fate. Loss of Bmp signaling exclusively in the epithelial compartment is not sufficient for the initiation of the de novo crypt phenomenon associated with juvenile polyposis syndrome. CONCLUSIONS: Epithelial Bmp signaling plays an important role in the terminal differentiation of the intestinal secretory cell lineage but not in de novo crypt formation. These findings emphasize the importance of delineating the contribution of the stroma vs the epithelium in gastrointestinal physiology and pathology.     

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.     

Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3

Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.Ubiquitin-dependent protein degradation plays a pivotal role in almost all biological processes, as the timely and selective removal of regulatory proteins is essential in many signaling pathways (1, 2). Ubiquitin molecules can form topologically distinct ubiquitin chains that can serve as signals for different pathways (3). Among them, ubiquitin chains linked through lysine 63 (K63) have been associated with endocytosis and were shown to be required for the efficient endocytic degradation of plasma membrane cargos (4, 5). In eukaryotes, ubiquitinated membrane proteins are transported into the vacuole/lysosome for degradation by resident proteases, depending on the function of the endosomal sorting complex required for transport (ESCRT) machinery. Ubiquitinated cargos are recognized and transported to late endosomes through the function of ESCRT-0, ESCRT-I, and ESCRT-II, and are subsequently sequestered to the intraluminal vesicles (ILVs) of the multivesicular body (MVB) by ESCRT-III (6, 7). Plants lack homologs of ESCRT-0 (8, 9), and it is suggested that ubiquitin binding proteins such as the target of Myb (TOM)-LIKEs (TOLs) take over its function (10).Ubiquitination of plasma membrane proteins depends on the activity of the ubiquitin conjugating machinery that creates an isopeptide bond between the C-terminal glycine of ubiquitin and a lysine of the substrate proteins or another ubiquitin molecule (11). Deubiquitinating enzymes (DUBs) can counteract the E3 ligase activity, in that they hydrolyze ubiquitin chains. In contrast to earlier assumptions that DUBs play merely a housekeeping role, it has been shown that DUBs can also be actively involved in the regulation of their target proteins (12, 13). The Arabidopsis genome encodes for at least 48 DUBs, although most of their molecular and biological functions are yet poorly understood (14). Whereas ubiquitinating enzymes interact specifically with their substrates (11), DUBs also can hydrolyze free ubiquitin chains unattached to target proteins (12, 15), and in most cases, do not require specific interactions with the substrate proteins. The elucidation of the spatiotemporal regulation of DUBs is therefore essential for a better understanding of the molecular mechanisms of DUB function.Associated molecule with the SH3 domain of STAM (AMSH) is a metalloprotease DUB that was first identified as an interactor of the signaling molecule and ESCRT-0 component signal transduction adaptor molecule (STAM) in mammals (16). AMSH genes are conserved in higher eukaryotes and are essential for growth and development. Knockout of AMSH in mice causes postnatal lethality and neurodegenerative aberrations (17), and mutations in human AMSH were associated with an infant neurodegenerative disease (18), indicating its essential function in mammals. In our previous work, we conducted a genetic analysis of AMSH genes in Arabidopsis, named AMSH1, AMSH2, and AMSH3. We have shown that knockdown of AMSH1 causes altered pathogen response, and that the knockout of AMSH3 is lethal in plants, leading to growth arrest in the early stages of development (19–21).AMSH proteins were shown to interact with ESCRT-III subunits and were implicated in endocytic protein degradation (21–26). Human AMSH and the Mpr1/Pad1 N-terminal (MPN)+ domain of Arabidopsis AMSH show specificity toward K63-linked ubiquitin chains (20–22), further supporting their function in endocytosis. Defects in AMSH function impair a number of intracellular trafficking events, including degradation of endocytosis cargos (22, 25, 27), vacuolar transport, and vacuole biogenesis (20), as well as defects in autophagic degradation (18, 20, 21). Although AMSH function in intracellular trafficking and protein degradation is well established, the molecular framework surrounding its function is not yet completely understood.With the aim of elucidating the molecular basis of AMSH regulation, we screened for interactors of Arabidopsis AMSH proteins and found a homolog of human apoptosis-linked gene-2 interacting protein X (ALIX) and budding yeast bypass of C kinase 1 (BCK1)-like resistance to osmotic shock 1p (Bro1p) as a direct interactor of AMSH3. ALIX is a conserved protein in eukaryotes that was implicated in cytokinesis, ILV, and exosome biogenesis and endosomal sorting (28). Human ALIX was also suggested to play a role during viral infection and budding (29–31). Mammalian ALIX, yeast Bro1p, and their Arabidopsis homolog were all shown to interact with ESCRT-III via the charged multivesicular body protein 4/sucrose nonfermenting 7p (CHMP4/Snf7p) subunit (32–35). Bro1p was shown to interact also with the endosome-associated DUB degradation of alpha 4p (Doa4p) and to be essential for recruiting Doa4p to late endosomes (36). Doa4p belongs to the ubiquitin-specific protease (UBP) family of DUBs and is structurally unrelated to AMSH. The involvement of ALIX/Bro1p in the regulation of other DUBs during endosomal sorting has not yet been reported. Our data show that ALIX is essential in Arabidopsis and that it is important for the degradation of ubiquitinated proteins, vacuole, and MVB biogenesis, as well as for the localization of AMSH3 to endosomes.     

A critical link between Toll-like receptor 3 and type II interferon signaling pathways in antiviral innate immunity

A conundrum of innate antiviral immunity is how nucleic acid-sensing Toll-like receptors (TLRs) and RIG-I/MDA5 receptors cooperate during virus infection. The conventional wisdom has been that the activation of these receptor pathways evokes type I IFN (IFN) responses. Here, we provide evidence for a critical role of a Toll-like receptor 3 (TLR3)-dependent type II IFN signaling pathway in antiviral innate immune response against Coxsackievirus group B serotype 3 (CVB3), a member of the positive-stranded RNA virus family picornaviridae and most prevalent virus associated with chronic dilated cardiomyopathy. TLR3-deficient mice show a vulnerability to CVB3, accompanied by acute myocarditis, whereas transgenic expression of TLR3 endows even type I IFN signal-deficient mice resistance to CVB3 and other types of viruses, provided that type II IFN signaling remains intact. Taken together, our results indicate a critical cooperation of the RIG-I/MDA5-type I IFN and the TLR3-type II IFN signaling axes for efficient innate antiviral immune responses.     

Control of hepatitis B virus replication by interferons and Toll-like receptor signaling pathways

Hepatitis B virus (HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon (IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors (TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.     

慢性乙型肝炎与Toll样受体2的关系

目的探讨CHB与Toll样受体2（TLR2）的相关性。方法采用Elivision二步法检测CHB、慢性重型肝炎患者及健康对照组肝组织TLR2的表达。采用直接免疫荧光流式细胞术检测TLR2在肝癌细胞株HepG2、HepG2-X及HepG2．2．15上表达的平均荧光强度（MFI）及阳性细胞率。结果TLR2在CHB及慢性重型肝炎肝组织上的表达明显高于健康对照组，差异有统计学意义，P〈0．01，主要表达于肝细胞质及部分胞膜。在CHB中，TLR2的表达强度与肝组织炎症活动度分级（G）呈显著正相关（r=0．597，P〈0．01），各级炎症患者肝组织TLR2阳性表达强度患者的血清TBil值，差异有统计学意义（P〈0．05）。在慢性乙型重型肝炎患者中，TLR2主要呈小灶性表达。TLR2在肝癌细胞株HepG2．2．15上表达的平均荧光强度为10．7±2．8，阳性细胞率为16．3％±7．0％；HepG2的平均荧光强度为1．0±0．3，阳性细胞率为0．4％±0．1％，两组比较t值分别为11．92和7．92，P值均〈0．0l。而HepG2和HepG2-X之间，差异无统计学意义。结论CHB患者肝组织中TLR2的表达明显升高，与肝组织的炎症活动密切相关。     

Glucosyltransferase activity of Clostridium difficile Toxin B is essential for disease pathogenesis

Clostridium difficile TcdB harbors a glucosyltransferase that targets host Rho GTPases. However, the role of the enzyme activity in the induction of host intestinal disease has not been demonstrated. In this study, we established a mouse acute intestinal disease model by cecum injection of wild type and glucosyltransferase-deficient TcdB and a chronic model by delivering toxin intraluminally via engineered surrogate host Bacillus megaterium. We demonstrated, for the first time, that the glucosyltransferase activity of TcdB is essential for inducing disease symptoms and intestinal pathological responses that resemble human disease, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.     

Toll样受体信号传导在关节炎发病中的作用

最近的研究证据显示：Toll样受体（TLR）是固有免疫的关键性识别结构。TLR的活化启动了炎症性细胞因子、趋化因子、组织破坏性酶和Ⅰ型干扰素的产生;同时,TLR的信号传导可以通过上调抗原递呈细胞的共刺激分子在适应性免疫系统的活化和发展中扮演重要作用。鉴于TLR信号传导在连接固有免疫和适应免疫中具有重要作用,可以推测TLR信号传导的失调可能与自身免疫性疾病的发生有关。本文将就由对应配体激活的TLR信号转导通路进行一些总结,最后讨论TLR传导通路在类风湿性关节炎致病机制中的作用。     

Toll样受体7及9在系统性红斑狼疮患者外周血B淋巴细胞内的表达及意义

目的 了解Toll样受体(TLR)7及9在系统性红斑狼疮(SLE)患者外周血B淋巴细胞内的表达情况及其意义.方法 流式细胞术检测50例SLE患者及30名健康人外周血B淋巴细胞内TLR7及9的表达水平.并将其与有关临床及实验窜指标进行相关性分析.结果 SLE患者B淋巴细胞内TLR7+及9+细胞所占比例均高于对照组.TLR7与患者红细胞沉降率(ESR)、SLE疾病活动指数(SLEDAI)呈正相关,与C3呈负相关.TLR9与患者尿蛋白、SLEDAI呈正相关.结论 TLR7及9在SLE患者B淋巴细胞内表达上调,且与病情有一定的相关性.     

High-frequency HTR3B variant associated with major depression dramatically augments the signaling of the human 5-HT3AB receptor

The 5-hydroxytryptamine-3 (5-HT3) receptor mediates the fast excitatory neurotransmission of serotonin and is known to mediate the nausea/emesis induced by radio/chemotherapy and anesthetics. A polymorphism encoding the variation Y129S in the 5-HT3B subunit exists in high frequency in the general population and has been shown to be inversely correlated to the incidence of major depression in women. We show that 5-HT3AB(Y129S) receptors exhibit a substantially increased maximal response to serotonin compared with WT receptors in two fluorescence-based cellular assays. In electrophysiological recordings, the deactivation and desensitization kinetics of the 5-HT3AB(Y129S) receptor are 20- and 10-fold slower, respectively, than those of the WT receptor. Single-channel measurements reveal a 7-fold-increased mean open time of 5-HT3AB(Y129S) receptors compared with WT receptors. The augmented signaling displayed by 5-HT3AB(Y129S) receptors may confer protection against the development of depression. The variant also may influence the development and/or treatment of nausea and other disorders involving 5-HT3 receptors. Thus, the impact of the high-frequency variant 5-HT3B(Y129S) on 5-HT3AB receptor signaling calls for a search for additional phenotypes, and the variant may thus aid in establishing the role of the 5-HT3AB receptor in pathophysiology.     

Alcohol,nutrition and liver cancer:Role of Toll-like receptor signaling

This article reviews the evidence that ties the development of hepatocellular carcinoma (HCC) to the natural immune pro-inflammatory response to chronic liver disease, with a focus on the role of Toll-like receptor (TLR) signaling as the mechanism of liver stem cell/progenitor transformation to HCC. Two exemplary models of this phenomenon are reviewed in detail. One model applies chronic ethanol/lipopolysaccharide feeding to the activated TLR4 signaling pathway. The other applies chronic feeding of a carcin...     

Evaluation of the colorectal cancer risk conferred by rare UNC5C alleles

AIM:To evaluate the risk associated with variants of the UNC5C gene recently suspected to predispose to familial colorectal cancer(CRC).METHODS:We screened patients with familial CRC forms as well as patients with sporadic CRCs.In a first time,we analyzed exon 11 of the UNC5C gene in 120unrelated patients with suspected hereditary CRC,58patients with suspected Lynch-associated cancer or polyposis,and 132 index cases of Lynch syndrome families with a characterized mutation in a DNA mismatch repair(MMR).Next,1023 patients with sporadic CRC and1121 healthy individuals were screened for the variants identified in patients with familial cancer.RESULTS:Of 120 patients with familial CRC of unknown etiology,one carried the previously reported mis-sense mutation p.Arg603Cys(R603C)and another exhibited the unreported variant of unknown significance p.Thr617Ile(T617I).The p.Ala628Lys(A628K)mutation previously described as the main UNC5C risk variant for familial CRC was not detected in any cases of familial CRC of unknown etiology,but was present in a patient with familial gastric cancer and in two Lynch syndrome patients in co-occurrence with MMR mutations.A statistically non-significant increase in cancer risk was identified in familial CRC and/or other Lynchassociated cancers(1/178 patients vs 2/1121 healthy controls,OR=3.2,95%CI:0.29-35.05,P=0.348)and in sporadic CRCs(4/1023 patients vs 2/1121 healthy controls,OR=2.2,95%CI:0.40-12.02,P=0.364).CONCLUSION:We confirm that UNC5C mutations are very rare in familial and sporadic CRCs,but further investigations are needed to justify routine UNC5C testing for diagnostic purposes.     

Adipokinetic hormone signaling through the gonadotropin-releasing hormone receptor modulates egg-laying in Caenorhabditis elegans

In mammals, hypothalamic gonadotropin-releasing hormone (GnRH) is a neuropeptide that stimulates the release of gonadotropins from the anterior pituitary. The existence of a putative functional equivalent of this reproduction axis in protostomian invertebrates has been a matter of debate. In this study, the ligand for the GnRH receptor in the nematode Caenorhabditis elegans (Ce-GnRHR) was found using a bioinformatics approach. The peptide and its precursor are reminiscent of both insect adipokinetic hormones and GnRH-preprohormone precursors from tunicates and higher vertebrates. We cloned the AKH-GnRH-like preprohormone and the Ce-GnRHR and expressed the GPCR in HEK293T cells. The GnRHR was activated by the C. elegans AKH-GnRH-like peptide (EC₅₀ = 150 nM) and by Drosophila AKH and other nematode AKH-GnRHs that we found in EST databases. Analogous to both insect AKH receptor and vertebrate GnRH receptor signaling, Ce-AKH-GnRH activated its receptor through a Gα_q protein with Ca²⁺ as a second messenger. Gene silencing of Ce-GnRHR, Ce-AKH-GnRH, or both resulted in a delay in the egg-laying process, comparable to a delay in puberty in mammals lacking a normal dose of GnRH peptide or with a mutated GnRH precursor or receptor gene. The present data support the view that the AKH-GnRH signaling system probably arose very early in metazoan evolution and that its role in reproduction might have been developed before the divergence of protostomians and deuterostomians.     

Vitamin B1 biosynthesis in plants requires the essential iron sulfur cluster protein, THIC

Vitamin B1 (thiamin) is an essential compound in all organisms acting as a cofactor in key metabolic reactions and has furthermore been implicated in responses to DNA damage and pathogen attack in plants. Despite the fact that it was discovered almost a century ago and deficiency is a widespread health problem, much remains to be deciphered about its biosynthesis. The vitamin is composed of a thiazole and pyrimidine heterocycle, which can be synthesized by prokaryotes, fungi, and plants. Plants are the major source of the vitamin in the human diet, yet little is known about the biosynthesis of the compound therein. In particular, it has never been verified whether the pyrimidine heterocycle is derived from purine biosynthesis through the action of the THIC protein as in bacteria, rather than vitamin B6 and histidine as demonstrated for fungi. Here, we identify a homolog of THIC in Arabidopsis and demonstrate its essentiality not only for vitamin B1 biosynthesis, but also plant viability. This step takes place in the chloroplast and appears to be regulated at several levels, including through the presence of a riboswitch in the 3'-untranslated region of THIC. Strong evidence is provided for the involvement of an iron-sulfur cluster in the remarkable chemical rearrangement reaction catalyzed by the THIC protein for which there is no chemical precedent. The results suggest that vitamin B1 biosynthesis in plants is in fact more similar to prokaryotic counterparts and that the THIC protein is likely to be the key regulatory protein in the pathway.     

Chemokine receptor CXCR3 and its ligands CXCL9 and CXCL10 are required for the development of murine cerebral malaria

Cerebral malaria is a significant cause of global mortality, causing an estimated two million deaths per year, mainly in children. The pathogenesis of this disease remains incompletely understood. Chemokines have been implicated in the development of cerebral malaria, and the IFN-inducible CXCR3 chemokine ligand IP-10 (CXCL10) was recently found to be the only serum biomarker that predicted cerebral malaria mortality in Ghanaian children. We show that the CXCR3 chemokine ligands IP-10 and Mig (CXCL9) were highly induced in the brains of mice with murine cerebral malaria caused by Plasmodium berghei ANKA. Mice deficient in CXCR3 were markedly protected against cerebral malaria and had far fewer T cells in the brain compared with wild-type mice. In competitive transfer experiments, CXCR3-deficient CD8(+) T cells were 7-fold less efficient at migrating into the infected brains than wild-type CD8(+) T cells. Adoptive transfer of wild-type CD8(+) effector T cells restored susceptibility of CXCR3-deficient mice to cerebral malaria and also restored brain proinflammatory cytokine and chemokine production and recruitment of T cells, independent of CXCR3. Mice deficient in IP-10 or Mig were both partially protected against cerebral malaria mortality when infected with P. berghei ANKA. Brain immunohistochemistry revealed Mig staining of endothelial cells, whereas IP-10 staining was mainly found in neurons. These data demonstrate that CXCR3 on CD8(+) T cells is required for T cell recruitment into the brain and the development of murine cerebral malaria and suggest that the CXCR3 ligands Mig and IP-10 play distinct, nonredundant roles in the pathogenesis of this disease.