Interleukin 37 (IL-37) and IL-1R8 (SIGIRR or TIR8) are anti-inflammatory orphan members of the IL-1 ligand family and IL-1 receptor family, respectively. Here we demonstrate formation and function of the endogenous ligand-receptor complex IL-37-IL-1R8-IL-18Rα. The tripartite complex assembled rapidly on the surface of peripheral blood mononuclear cells upon stimulation with lipopolysaccharide. Silencing of IL-1R8 or IL-18Rα impaired the anti-inflammatory activity of IL-37. Whereas mice with transgenic expression of IL-37 (IL-37tg mice) with intact IL-1R8 were protected from endotoxemia, IL-1R8-deficient IL-37tg mice were not. Proteomic and transcriptomic investigations revealed that IL-37 used IL-1R8 to harness the anti-inflammatory properties of the signaling molecules Mer, PTEN, STAT3 and p62(dok) and to inhibit the kinases Fyn and TAK1 and the transcription factor NF-κB, as well as mitogen-activated protein kinases. Furthermore, IL-37-IL-1R8 exerted a pseudo-starvational effect on the metabolic checkpoint kinase mTOR. IL-37 thus bound to IL-18Rα and exploited IL-1R8 to activate a multifaceted intracellular anti-inflammatory program.

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterial and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDSinsoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.Ataxin-3 is a 42-kDa multi-domain protein consisting of an N-terminal Josephin domain, two ubiquitin-interacting motifs, which are situated next to a polymorphous C-terminal polyglutamine (poly(Q)) 3 tract (1, 2). Ataxin-3 functions as a de-ubiquitinating enzyme (3, 4) and binds polyubiquitin chains through the ubiquitin-interacting motifs (5-8). Expansion of the poly(Q) tract beyond 45 residues causes spinocerebellar ataxia type 3 (SCA3) also known as Machado-Joseph disease (9, 10). Similar dynamic expansion of poly(Q) tracts within various other proteins causes a further eight autosomally dominant neurodegenerative diseases, collectively termed poly(Q) diseases (2, 11).Several key observations have led to the conclusion that poly(Q) diseases originate via a toxic gain of function, mediated by poly(Q) tract expansion. Firstly, the manifestation of each poly(Q) disease is directly reliant on a threshold length of consecutive glutamine residues. In all of the diseases, except for SCA6, this threshold is remarkably similar with a tract length in excess of 40 residues associated with disease onset (2).Secondly, there is a non-linear correlation between an increasing glutamine tract length and an earlier age of disease onset (12). Thirdly, different proteins involved in each of the various poly(Q) diseases share no sequence homology except the presence of the glutamine tract (13).Various studies have suggested that this toxic gain of function is causally linked to aberrant protein aggregation mediated by the extended poly(Q) tract (14). In vitro studies have shown that poly(Q) peptides, fragments, and proteins can form amyloid-like fibrillar aggregates and that the aggregation rate increases with increasing glutamine tract length (15)(16)(17)(18)(19). Various types of ...

SummaryThe yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

The conserved TREX-2 transcription-export complex integrates transcription and processing of many actively-transcribed nascent mRNAs with the recruitment of export factors at nuclear pores and also contributes to transcriptional memory and genomic stability. We report the crystal structure of the Sac3–Thp1–Sem1 segment of Saccharomyces cerevisiae TREX-2 that interfaces with the gene expression machinery. Sac3–Thp1–Sem1 forms a novel PCI-domain complex characterized by the juxtaposition of Sac3 and Thp1 winged helix domains, forming a platform that mediates nucleic acid binding. Structure-guided mutations underline the essential requirement of the Thp1–Sac3 interaction for mRNA binding and for the coupling of transcription and processing with mRNP assembly and export. These results provide insight into how newly synthesized transcripts are efficiently transferred from TREX-2 to the principal mRNA export factor and, identify how Sem1 stabilizes PCI domain-containing proteins and promotes complex assembly.

No abstract

Malaria sporozoites are deposited into the skin by mosquitoes and infect hepatocytes. The molecular basis of how Plasmodium falciparum sporozoites migrate through host cells is poorly understood, and direct evidence of its importance in vivo is lacking. Here, we generated traversal-deficient sporozoites by genetic disruption of sporozoite microneme protein essential for cell traversal (PfSPECT) or perforin-like protein 1 (PfPLP1). Loss of either gene did not affect P. falciparum growth in erythrocytes, in contrast with a previous report that PfPLP1 is essential for merozoite egress. However, although traversal-deficient sporozoites could invade hepatocytes in vitro, they could not establish normal liver infection in humanized mice. This is in contrast with NF54 sporozoites, which infected the humanized mice and developed into exoerythrocytic forms. This study demonstrates that SPECT and perforin-like protein 1 (PLP1) are critical for transcellular migration by P. falciparum sporozoites and demonstrates the importance of cell traversal for liver infection by this human pathogen.

Polyglutamine proteins that cause neurodegenerative disease are known to form proteinaceous aggregates, such as nuclear inclusions, in the neurons of affected patients. Although polyglutamine proteins have been shown to form fibrillar aggregates in a variety of contexts, the mechanisms underlying the aberrant conformational changes and aggregation are still not well understood. In this study, we have investigated the hypothesis that polyglutamine expansion in the protein ataxin-3 destabilizes the native protein, leading to the accumulation of a partially unfolded, aggregation-prone intermediate. To examine the relationship between polyglutamine length and native state stability, we produced and analyzed three ataxin-3 variants containing 15, 28, and 50 residues in their respective glutamine tracts. At pH 7.4 and 37°C, Atax3(Q50), which lies within the pathological range, formed fibrils significantly faster than the other proteins. Somewhat surprisingly, we observed no difference in the acid-induced equilibrium and kinetic un/folding transitions of all three proteins, which indicates that the stability of the native conformation was not affected by polyglutamine tract extension. This has led us to reconsider the mechanisms and factors involved in ataxin-3 misfolding, and we have developed a new model for the aggregation process in which the pathways of un/folding and misfolding are distinct and separate. Furthermore, given that native state stability is unaffected by polyglutamine length, we consider the possible role and influence of other factors in the fibrillization of ataxin-3.