Jens Staal

Sensing of Viral Infection and Activation of Innate Immunity by Toll-Like Receptor 3

SUMMARY Toll-like receptors (TLRs) form a major group of transmembrane receptors that are involved in the detection of invading pathogens. Double-stranded RNA is a marker for viral infection that is recognized by TLR3. TLR3 triggering activates specific signaling pathways that culminate in the activation of NF-κB and IRF3 transcription factors, as well as apoptosis, enabling the host to mount an effective innate immune response through the induction of cytokines, chemokines, and other proinflammatory mediators. In this review, we describe the paradoxical role of TLR3 in innate immunity against different viruses and in viral pathogenesis but also the evidence for TLR3 as a “danger” receptor in nonviral diseases. We also discuss the structure and cellular localization of TLR3, as well as the complex signaling and regulatory events that contribute to TLR3-mediated immune responses.

T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1

The paracaspase mucosa‐associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF‐κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF‐κB activation by cleaving the NF‐κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T‐cell receptors (TCR) activation, as well as overexpression of the oncogenic API2–MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c‐jun N‐terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR‐induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

ABA is required for Leptosphaeria maculans resistance via ABI1-and ABI4-dependent signaling

Abscisic acid (ABA) is a defense hormone with influence on callose-dependent and -independent resistance against Leptosphaeria maculans acting in the RLMcol pathway. ABA-deficient and -insensitive mutants in Ler-0 background (abal-3 and abil-1) displayed susceptibility to L. maculans, along with a significantly decreased level of callose depositions, whereas abi2-1 and abi3-1 remained resistant, together with the abi5-1 mutant of Ws-0 background. Suppressor mutants of abil-1 confirmed that the L. maculans-susceptible response was due to the dominant negative nature of the abil-1 mutant. Highly induced camalexin levels made ABA mutants in Col-0 background (aba2-1, aba3-1, and abi4-1) appear resistant, but displayed enhanced susceptibility as double mutants with pad3-1, impaired in camalexin biosynthesis. beta-Aminobutyric acid (BABA) pretreatment of Ler-0 contributed to an elevated level of endogenous ABA after L. maculans inoculation. Comparisons between (RLM1co1)pad3 and rlmlLerpad3 showed that ABA and BABA enhancement of callose deposition requires induction from RLM1col. ABII, but not ABI2, was found to be involved in a feedback mechanism that modulates RLM1co, expression. Genetic analysis showed further that this feedback occurs upstream of ABI4 and that components downstream of ABI4 modulate ABIJ activity. ABA and BABA treatments of the L. maculans-susceptible callose synthase mutant pmr4 showed that ABA also induces a callose-independent resistance. Similar treatments enhanced callose depositions and induced resistance to L. maculans in oilseed rape, and BABA-induced resistance was found to be independent of salicylic acid.

Early Responses in the Arabidopsis-Verticillium longisporum Pathosystem Are Dependent on NDR1, JA- and ET-Associated Signals via Cytosolic NPR1 and RFO1

The responses of Arabidopsis accessions and characterized genotypes were used to explore components in the early defense responses to the soilborne fungus Verticillium longisporum. V. longisporum susceptibility was found to be a complex trait, in which different disease phenotypes, such as stunting, altered flowering time, weight loss, and chlorosis were perceived differently across genotypes. A Bay-0 x Shahdara recombinant inbred line population was used to identify two loci on chromosomes 2 and 3 of Bay-0 origin that caused enhanced chlorosis after V. longisporum challenge. Furthermore, the observation that a mutation in RFO1 in Col-0 resulted in susceptibility whereas the natural rfo1 allele in Ty-0 showed a high degree of resistance to the pathogen supports the hypothesis that several resistance quantitative trait loci reside among Arabidopsis accessions. Analysis of mutants impaired in known pathogen response pathways revealed an enhanced susceptibility in ein2-1, ein4-1, ein6-1, esa1-1, and pad1-1, but not in other jasmonic acid (JA)-, ethylene (ET)-, or camalexin-deficient mutants, suggesting that V. longisporum resistance is regulated via a hitherto unknown JA- and ET-associated pathway. Pretreatments with the ET precursor 1-aminocyclo-propane-1-carboxylic acid (ACC) or methyl jasmonate (MeJA) caused enhanced resistance to V. longisporum. Mutants in the salicylic acid (SA) pathway (eds1-1, NahG, npr1-3, pad4-1, and sid2-1) did not show enhanced susceptibility to V. longisporum. In contrast, the more severe npr1-1 allele displayed enhanced V. longisporum susceptibility and decreased responses to ACC or MeJA pretreatments. This shows that cytosolic NPR1, in addition to SA responses, is required for JA- and ET-mediated V. longisporum resistance. Expression of the SA-dependent PR-1 and PR-2 and the ET-dependent PR-4 were increased 7 days postinoculation with V. longisporum. This indicates increased levels of SA and ET in response to V. longisporum inoculation. The R-gene signaling mutant ndr1-1 was found to be susceptible to V. longisporum, which could be complemented by ACC or MeJA pretreatments, in contrast to the rfo1 T-DNA mutant, which remained susceptible, suggesting that RFO1 (Fusarium oxysporum resistance) and NDR1 (nonrace specific disease resistance 1) activate two distinct signaling pathways for V. longisporum resistance.

RLM3, a TIR domain encoding gene involved in broad-range immunity of Arabidopsis to necrotrophic fungal pathogens

Here, we describe the rapid cloning of a plant gene, Leptosphaeria maculans 3 (RLM3(Col)), which encodes a putative Toll interleukin-1 receptor-nucleotide binding (TIR-NB) class protein, which is involved in defence against the fungal pathogen L. maculans and against three other necrotrophic fungi. We have, through microarray-based case control bulk segregant comparisons of transcriptomes in pools of Col-0 x An-1 progeny, identified the absence of a locus that causes susceptibility in An-1. The significance of this locus on chromosome 4 for L. maculans resistance was supported by PCR-based mapping, and denoted resistance to RLM3(Col). Differential susceptible phenotypes in four independent T-DNA insertion lines support the hypothesis that At4g16990 is required for RLM3(Col) function. The mutants in RLM3(Col) also exhibited an enhanced susceptibility to Botrytis cinerea, Alternaria brassicicola and Alternaria brassicae. Complementations of An-1 and T-DNA mutants using overexpression of a short transcript lacking the NB-ARC domain, or a genomic clone, restored resistance to all necrotrophic fungi. The elevated expression of RLM3(Col) on B. cinerea-susceptible mutants further suggested convergence in signalling and gene regulation between defence against B. cinerea and L. maculans. In the case of L. maculans, RLM3(Col) is required for efficient callose deposition downstream of RLM1(Col).

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.

MALT1 cleaves the E3 ubiquitin ligase HOIL‐1 in activated T cells, generating a dominant negative inhibitor of LUBAC‐induced NF‐κB signaling

Human paracaspase 1 (PCASP1), better known as mucosa associated lymphoid tissue lymphoma translocation 1 (MALT1), plays a key role in immunity and inflammation by regulating gene expression in lymphocytes and other immune cell types. Deregulated MALT1 activity has been implicated in autoimmunity, immunodeficiency and certain types of lymphoma. As a scaffold MALT1 assembles downstream signaling proteins for nuclear factor‐κB (NF‐κB) activation, while its proteolytic activity further enhances NF‐κB activation by cleaving NF‐κB inhibitory proteins. MALT1 also processes and inactivates a number of mRNA destabilizing proteins, which further fine‐tunes gene expression. MALT1 protease inhibitors are currently developed for therapeutic targeting. Here we show that T cell activation, as well as overexpression of the oncogenic fusion protein API2–MALT1, induces the MALT1‐mediated cleavage of haem‐oxidized IRP2 ubiquitin ligase 1 (HOIL‐1). In addition, to acting as a K48‐polyubiquitin specific E3 ubiquitin ligase for different substrates, HOIL‐1 co‐operates in a catalytic‐independent manner with the E3 ubiquitin ligase HOIL‐1L interacting protein (HOIP) as part of the linear ubiquitin chain assembly complex (LUBAC). Intriguingly, cleavage of HOIL‐1 does not directly abolish its ability to support HOIP‐induced NF‐κB signaling, which is still mediated by the N‐terminal cleavage fragment, but generates a C‐terminal fragment with LUBAC inhibitory properties. We propose that MALT1‐mediated HOIL‐1 cleavage provides a gain‐of‐function mechanism that is involved in the negative feedback regulation of NF‐κB signaling.

Pharmacological inhibition of MALT1 protease activity protects mice in a mouse model of multiple sclerosis.

BackgroundThe paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is crucial for lymphocyte activation through signaling to the transcription factor NF-κB. Besides functioning as a scaffold signaling protein, MALT1 also acts as a cysteine protease that specifically cleaves a number of substrates and contributes to specific T cell receptor-induced gene expression. Recently, small molecule inhibitors of MALT1 proteolytic activity were identified and shown to have promising anticancer properties in subtypes of B cell lymphoma. However, information on the therapeutic potential of small compound inhibitors that target MALT1 protease activity in autoimmunity is still lacking.MethodsThe present study aimed to elucidate whether MALT1 protease inhibitors are also useful in the treatment of lymphocyte-mediated autoimmune pathologies such as multiple sclerosis (MS). For this, we studied the therapeutic potential of a recently identified inhibitor of MALT1 protease activity, the phenothiazine derivative mepazine, in the context of experimental autoimmune encephalomyelitis (EAE), the main animal model for MS.ResultsWe demonstrate that administration of mepazine prophylactically or after disease onset, can attenuate EAE. Importantly, while complete absence of MALT1 affects the differentiation of regulatory T (Treg) cells in vivo, the MALT1 protease inhibitor mepazine did not affect Treg development.ConclusionsAltogether, these data indicate that small molecule inhibitors of MALT1 not only hold great promise for the treatment of B cell lymphomas but also for autoimmune disorders such as MS.

The multifaceted role of the E3 ubiquitin ligase HOIL-1: beyond linear ubiquitination

Ubiquitination controls and fine‐tunes many signaling processes driving immunity, inflammation, and cancer. The E3 ubiquitin ligase HOIL‐1 (heme‐oxidized IRP2 ubiquitin ligase‐1) is increasingly implicated in different signaling pathways and plays a vital role in immune regulation. HOIL‐1 co operates with the E3 ubiquitin ligase HOIP (HOIL‐1 interacting protein) to modify specific nuclear factor‐κB (NF‐κB) signaling proteins with linear M1‐linked polyubiquitin chains. In addition, through its ability to also add K48‐linked polyubiquitin chains to specific substrates, HOIL‐1 has been linked with antiviral signaling, iron and xenobiotic metabolism, cell death, and cancer. HOIL‐1 deficiency in humans leads to myopathy, amylopectinosis, auto‐inflammation, and immunodeficiency associated with an increased frequency of bacterial infections. HOIL‐1‐deficient mice exhibit amylopectin‐like deposits in the myocardium, pathogen‐specific immunodeficiency, but minimal signs of hyper‐inflammation. This review summarizes current knowledge on the mechanism of action of HOIL‐1 and highlights recent advances regarding its role in health and disease.

Cleavage by MALT1 induces cytosolic release of A20

The MALT1 paracaspase has arginine-directed proteolytic activity. A20 is a dual ubiquitin-editing enzyme involved in termination of NF-κB signaling. Upon T- or B-cell receptor engagement human (h) A20 is cleaved by MALT1 after arginine 439, yielding an N-terminal fragment (hA20p50) and a C-terminal one (hA20p37). The hA20p50 fragment has never been detected directly, thus limiting insight into the functional consequences of MALT1-mediated cleavage of A20. Here, various antibodies were tested, including newly generated hA20p50 and hA20p37 specific antibodies, leading to detection of the hA20p50 fragment produced after MALT1-mediated cleavage of ectopically expressed as well as endogenous A20 proteins. The properties of both A20 fragments, generated upon co-expression with a constitutively active MALT1 protein, were further studied by sub-cellular fractionation and fluorescence microscopy. In contrast to full-length A20 which is particulate and insoluble, we found hA20p50 to be soluble and readily released into the cytosol whereas hA20p37 was partially soluble, thus suggesting loss of compartmentalization as a possible mechanism for MALT1-mediated dampening of A20 function.