RIP death domain structural interactions implicated in TNF-mediated proliferation and survival

Death domain (DD)-containing proteins are involved in both apoptosis and survival/proliferation signaling induced by activated death receptors. Here, a phylogenetic and structural analysis was performed to highlight differences in DD domains and their key regulatory interaction sites. The phylogenetic analysis shows that receptor DDs are more conserved than DDs in adaptors. Adaptor DDs can be subdivided into those that activate or inhibit apoptosis. Modeling of six homotypic DD interactions involved in the TNF signaling pathway implicates that the DD of RIP (Receptor interacting protein kinase 1) is capable of interacting with the DD of TRADD (TNFR1-associated death domain protein) in two different, exclusive ways: one that subsequently recruits CRADD (apoptosis/inflammation) and another that recruits NFkappaB (survival/proliferation).     

In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein

Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long alpha-helix of residues 30-44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure-function studies of plant LRR proteins.     

Consensus design of a NOD receptor leucine rich repeat domain with binding affinity for a muramyl dipeptide,a bacterial cell wall fragment

Repeat proteins have recently emerged as especially well‐suited alternative binding scaffolds due to their modular architecture and biophysical properties. Here we present the design of a scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Consensus sequence design has emerged as a protein design tool to create de novo proteins that capture sequence‐structure relationships and interactions present in nature. The multiple sequence alignment of 311 individual LRRs, which are the putative ligand‐recognition domain in NOD proteins, resulted in a consensus sequence protein containing two internal and N‐ and C‐capping repeats named CLRR2. CLRR2 protein is a stable, monomeric, and cysteine free scaffold that without any affinity maturation displays micromolar binding to muramyl dipeptide, a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand.     

The NB‐LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance

Plant resistance proteins of the class of nucleotide‐binding and leucine‐rich repeat domain proteins (NB‐LRRs) are immune sensors which recognize pathogen‐derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB‐LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR‐independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR‐Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo‐ and hetero‐complexes and interact through their coiled‐coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR‐Pia, neither RGA4 nor RGA5 is re‐localized to the nucleus. These results establish a model for the interaction of hetero‐pairs of NB‐LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.     

DuaJ—like蛋白研究进展

DuaJ-like蛋白由N-端保守的J区域、富含Gly和Phe区域、富含Cys区域和C-端低同源区域组成。J功能域能调节HSP70分子伴侣的ATPase活性,C-端不保守区域能调节与多肽的关系。真核细胞中存在着多种结构不同的DuaJ-like蛋白,但都含有一个J功能域。DuaJ-like蛋白通过J功能域调节HSP70功能而参与蛋白的折叠、装配和运输过程。     

Autoproteolysis of PIDD marks the bifurcation between pro-death caspase-2 and pro-survival NF-kappaB pathway

Upon DNA damage, a complex called the PIDDosome is formed and either signals NF-kappaB activation and thus cell survival or alternatively triggers caspase-2 activation and apoptosis. PIDD (p53-induced protein with a death domain) is constitutively processed giving rise to a 48-kDa N-terminal fragment containing the leucine-rich repeats (LRRs, PIDD-N) and a 51-kDa C-terminal fragment containing the death domain (DD, PIDD-C). The latter undergoes further cleavage resulting in a 37-kDa fragment (PIDD-CC). Here we show that processing occurs at S446 (generating PIDD-C) and S588 (generating PIDD-CC) by an auto-processing mechanism similar to that found in the nuclear pore protein Nup98/96 and inteins. Auto-cleavage of PIDD determines the outcome of the downstream signaling events. Whereas initially formed PIDD-C mediates the activation of NF-kappaB via the recruitment of RIP1 and NEMO, subsequent formation of PIDD-CC causes caspase-2 activation and thus cell death. A non-cleavable PIDD mutant is unable to translocate from the cytoplasm to the nucleus and loses both activities. In this way, auto-proteolysis of PIDD might participate in the orchestration of the DNA damage-induced life and death signaling pathways.     

Fire and death: the pyrin domain joins the death-domain superfamily

Apoptosis and inflammation are important cellular processes that are highly regulated through specific protein-protein interactions (PPI). Proteins involved in these signaling cascades often carry PPI domains that belong to the death-domain superfamily. This includes the structurally well-characterized Death Domain (DD), the Death Effector Domain (DED) and the Caspase Recruitment Domain (CARD) subfamilies. Recently, a fourth member of the DD superfamily was identified, the Pyrin Domain (PYD). Based on sequence alignments, homology to other domains occurring in death-signalling pathways, and secondary-structure prediction, the PYD was predicted to have an overall fold similar to other DD superfamily members. Just recently, NMR structures of two PYDs have been determined. The PYD structures not only revealed the DD superfamily fold as previously predicted, but also distinct features that are characteristic exclusively for this subfamily. This review summarizes recent findings and developments regarding structural aspects of the DD superfamily, with a special emphasis on the PPIs of the DD superfamily.     

Functional Integration of the Conserved Domains of Shoc2 Scaffold

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.     

Upon intracellular processing, the C-terminal death domain-containing fragment of the p53-inducible PIDD/LRDD protein translocates to the nucleoli and interacts with nucleolin

The p53-inducible and death domain-containing PIDD/LRDD protein has been described as an adaptor protein, which forms large protein complexes with RAIDD, another death domain-containing protein, leading to recruitment, and activation of the initiator caspase-2, and p53-mediated apoptosis. Here, we describe in further detail the proteolytic processing of PIDD/LRDD that occurs in healthy cells before induction of apoptosis. We could demonstrate that the C-terminal fragment containing the PIDD death domain shuttles into the nucleoli. This translocation is mediated by or leads to the interaction of the PIDD death domain with nucleolin, a protein important for rRNA processing within nucleoli and possibly involved in the DNA damage response. Ectopically expressed LRDD and endogenous nucleolin co-localized within the nucleoli, and overexpression of both full-length LRDD and the LRDD death domain sensitized cells for UV-induced apoptosis. When expressed alone, the PIDD/LRDD death domain tended to form large filamentous structures resembling so-called death filaments. The functional consequences of the identified PIDD/nucleolin interaction remain to be elucidated, but may be related to a recently discovered new role for PIDD in the activation of NF-kappaB upon genotoxic stress.     

高等植物的LRR蛋白:结构与功能

ＬＲＲ结构存在于细胞定位和功能上各不相同的多种蛋白质中,与蛋白质之间的相互作用和细胞内的信号传递过程密切相关。植物中含ＬＲＲ的蛋白主要有类受体蛋白激酶、抗病基因编码的蛋白和多聚半乳糖醛酸酶抑制蛋白等,它们分别在细胞的生长发育、抗病反应等过程中发挥着重要作用,其相似的ＬＲＲ结构为从分子水平上研究这些蛋白的作用机制提供了结构基础。     

Resistance proteins: molecular switches of plant defence

Specificity of the plant innate immune system is often conferred by resistance (R) proteins. Most R proteins contain leucine-rich repeats (LRRs), a central nucleotide-binding site (NBS) and a variable amino-terminal domain. The LRRs are mainly involved in recognition, whereas the amino-terminal domain determines signalling specificity. The NBS forms part of a nucleotide binding (NB)-ARC domain that presumably functions as a molecular switch. The conserved nature of NB-ARC proteins makes it possible to map mutations of R protein residues onto the crystal structures of related NB-ARC proteins, providing hypotheses for the functional roles of these residues. A functional model emerges in which the LRRs control the molecular state of the NB-ARC domain. Pathogen recognition triggers nucleotide-dependent conformational changes that might induce oligomerisation, thereby providing a scaffold for activation of downstream signalling components.     

The Nod-like receptor (NLR) family: a tale of similarities and differences

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.     

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain (DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-kappaB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone 15N NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha2-alpha3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha2-alpha3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha2-alpha3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.     

Identification of a basic surface area of the FADD death effector domain critical for apoptotic signaling

Death effector domains (DEDs) are protein-protein interaction domains found in the death inducing signaling complex (DISC). Performing a structure-based alignment of all DED sequences we identified a region of high diversity in alpha-helix 3 and propose a classification of DEDs into class I DEDs typically containing a stretch of basic residues in the alpha-helix 3 region whereas DEDs of class II do not. Functional assays using mutants of Fas-associated death domain revealed that this basic region influences binding and recruitment of caspase-8 and cellular FLICE inhibitor protein to the DISC.     

Plant disease resistance protein signaling: NBS-LRR proteins and their partners

Most plant disease resistance (R) proteins contain a series of leucine-rich repeats (LRRs), a nucleotide-binding site (NBS), and a putative amino-terminal signaling domain. They are termed NBS-LRR proteins. The LRRs of a wide variety of proteins from many organisms serve as protein interaction platforms, and as regulatory modules of protein activation. Genetically, the LRRs of plant R proteins are determinants of response specificity, and their action can lead to plant cell death in the form of the familiar hypersensitive response (HR). A total of 149 R genes are potentially expressed in the Arabidopsis genome, and plant cells must deal with the difficult task of assembling many of the proteins encoded by these genes into functional signaling complexes. Eukaryotic cells utilize several strategies to deal with this problem. First, proteins are spatially restricted to their sub-cellular site of function, thus improving the probability that they will interact with their proper partners. Second, these interactions are architecturally organized to avoid inappropriate signaling events and to maintain the fidelity and efficiency of the response when it is initiated. Recent results provide new insights into how the signaling potential of R proteins might be created, managed and held in check until specific stimulation following infection. Nevertheless, the roles of the R protein partners in these regulatory events that have been defined to date are unclear.     

Comparative structural analysis of the binding domain of follicle stimulating hormone receptor

Proteins with leucine-rich repeats (LRRs) specialize in mediating protein-protein interactions. The hormone binding portion of the receptor for follicle stimulating hormone (FSH) is an LRR protein by sequence, and the crystal structure of this domain from human FSH receptor in a complex with FSH shows that it does indeed have an LRR structure. It differs from other LRR domains, however, in being an all-beta protein composed of highly irregular repeats and having only slight overall curvature. Despite these distinctions and a superficial resemblance to beta-helical proteins, the binding domain of FSH receptor clearly is an LRR protein. The structure does consist of two parts with distinctively different curvatures. Comparison with the structures of other LRR-containing proteins shows a correlation between curvature and main-chain hydrogen bonding pattern of the parallel beta-sheet. The hormone-binding site is located at the concave surface of the receptor structure, a feature common to proteins with LRR motifs. Analysis of the ligand-binding site of LRR-containing proteins reveals that they generally utilize extensive interface area and a large number of charged residues to facilitate high-affinity protein-protein interactions.