Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells-a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

ABSTRACTMelioidosis is a serious infectious disease endemic in Southeast Asia, Northern Australia and has been increasingly reported in other tropical and subtropical regions in the world. Percutaneous inoculation through cuts and wounds on the skin is one of the major modes of natural transmission. Despite cuts in skin being a major route of entry, very little is known about how the causative bacterium Burkholderia pseudomallei initiates an infection at the skin and the disease manifestation at the skin known as cutaneous melioidosis. One key issue is the lack of suitable and relevant infection models. Employing an in vitro 2D keratinocyte cell culture, a 3D skin equivalent fibroblast-keratinocyte co-culture and ex vivo organ culture from human skin, we developed infection models utilizing surrogate model organism Burkholderia thailandensis to investigate Burkholderia-skin interactions. Collectively, these models show that the bacterial infection was largely limited at the wound's edge. Infection impedes wound closure, triggers inflammasome activation and cellular extrusion in the keratinocytes as a potential way to control bacterial infectious load at the skin. However, extensive infection over time could result in the epidermal layer being sloughed off, potentially contributing to formation of skin lesions.

MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.

Signals arising from bacterial infections are detected by pathogen recognition receptors (PRRs) and are transduced by specialized adapter proteins in mammalian cells. The Receptor-interacting-serine/threonine-protein kinase 2 (RIPK2 or RIP2) is such an adapter protein that is critical for signal propagation of the Nucleotide-binding-oligomerization-domain-containing proteins 1/2 (NOD1 and NOD2). Dysregulation of this signaling pathway leads to defects in bacterial detection and in some cases autoimmune diseases. Here, we show that the Caspase-activation-and-recruitment-domain (CARD) of RIP2 (RIP2-CARD) forms oligomeric structures upon stimulation by either NOD1-CARD or NOD2-2CARD. We reconstitute this complex, termed the RIPosome in vitro and solve the cryo-EM filament structure of the active RIP2-CARD complex at 4.1 Å resolution. The structure suggests potential mechanisms by which CARD domains from NOD1 and NOD2 initiate the oligomerization process of RIP2-CARD. Together with structure guided mutagenesis experiments at the CARD-CARD interfaces, we demonstrate molecular mechanisms how RIP2 is activated and self-propagating such signal.

Alteration of the PI3K/AKT/mTOR pathway is a common genomic abnormality detected in triple-negative breast cancer (TNBC). Everolimus acts synergistically with eribulin in TNBC cell lines and xenograft models. This phase I trial was designed to test the safety and tolerability of combining eribulin and everolimus in patients with metastatic TNBC.

Nucleotide-binding oligomerization domain (NBD), leucine-rich repeat (LRR) containing protein family (NLRs) are intracellular pattern recognition receptors that mediate innate immunity against infections. The endothelium is the first line of defense against blood-borne pathogens, but it is unclear which NLRs control endothelial cell (EC) intrinsic immunity. Here, we demonstrate that human ECs simultaneously activate NLRP1 and CARD8 inflammasomes in response to DPP8/9 inhibitor Val-boro-Pro (VbP). Enterovirus Coxsackie virus B3 (CVB3)-the most common cause of viral myocarditis-predominantly activates CARD8 in ECs in a manner that requires viral 2A and 3C protease cleavage at CARD8 p.G38 and proteasome function. Genetic deletion of CARD8 in ECs and human embryonic stem cell-derived cardiomyocytes (HCMs) attenuates CVB3-induced pyroptosis, inflammation, and viral propagation. Furthermore, using a stratified endothelial-cardiomyocyte co-culture system, we demonstrate that deleting CARD8 in ECs reduces CVB3 infection of the underlying cardiomyocytes. Our study uncovers the unique role of CARD8 inflammasome in endothelium-intrinsic anti-viral immunity.