Robust immune responses are essential for eliminating pathogens, but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase, or cGAS, produces the second messenger cGAMP to initiate the STING pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double stranded (ds)DNA stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNAs to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNAs. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced anti-microbial immune responses.

Polo-like kinase 4 (Plk4) is a key regulator of centriole duplication, an event critical for the maintenance of genomic integrity. Here we showed that Plk4 relocalizes from the inner Cep192 ring to the outer Cep152 ring as newly recruited Cep152 assembles around the Cep192-encircled daughter centriole. Crystal structure analyses revealed that Cep192 - and Cep152-derived peptides bind the cryptic polo box (CPB) of Plk4 in opposite orientations and in a mutually exclusive manner. The Cep152-peptide bound to the CPB markedly better than the Cep192-peptide and effectively snatched the CPB away from a preformed CPB–Cep192-peptide complex. A cancer-associated Cep152 mutation impairing the Plk4 interaction induced defects in procentriole assembly and chromosome segregation. Thus, Plk4 is intricately regulated in time and space through ordered interactions with two distinct scaffolds, Cep192 and Cep152, and a failure in this process may lead to human cancer.

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CART cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ-and CD28-containing endodomain signaling chain. We tested STOP-CART cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CART cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CART cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies. T cells engineered with CARs, hybrid molecules linking antigen binding to T-cell signaling endodomains, have mediated potent and durable responses against both chronic and acute B cell leukemias 1-4. CART cell therapy, however, is frequently associated with lifethreatening side effects, including cytokine release syndrome (CRS) and neurotoxicity. The clinical development of CART cells against solid tumors has proven challenging, but there is great optimism that next-generation CART cells will bring benefit to a broader range of cancer patients 5. Indeed, it is now well understood that physical and immunometabolic barriers upregulated in the solid tumor microenvironment as well as prolonged exposure to antigens can impair T-cell function and drive T-cell exhaustion 6. Innovative engineering strategies, such as the expression of cytokines, chemokines, decoy molecules or stimulatory ligands, are being developed to overcome these obstacles and have shown favorable preclinical responses 6-8. Safety, however, remains an important barrier to clinical entry because most solid tumor antigens targeted to date are also found in healthy tissues, sometimes leading to serious on-target off-tumor toxicity 9. The ability to control CART cell activity on command could greatly accelerate the clinical development of cellular immunotherapies. The above considerations have driven the development of CART cell control and safety systems 5 , such as drug-inducible suicide switches 10,11 , coinhibitory receptor (i)CARs that upon engagement with specific antigens suppress effector function 12 and split-signaling CART cells that require co-engagement of two ligands for full T-cell activation 13. SUPRA (split, universal and programmable) CARs 14 and a variety of universal CARs 15,16 have been developed that require administration of an adaptor protein to link nontumor antigen binding CART cells to tumor cells. While these approaches offer the possibility to sequentially target different tumor antigens (for ...

Many bacteria, including Legionella pneumophila, rely on the type IV secretion system to translocate a repertoire of effector proteins into the hosts for their survival and growth. Type IV coupling protein (T4CP) is a hexameric ATPase that links translocating substrates to the transenvelope secretion conduit. Yet, how a large number of effector proteins are selectively recruited and processed by T4CPs remains enigmatic. DotL, the T4CP of L. pneumophila, contains an ATPase domain and a C-terminal extension whose function is unknown. Unlike T4CPs involved in plasmid DNA translocation, DotL appeared to function by forming a multiprotein complex with four other proteins. Here, we show that the C-terminal extension of DotL interacts with DotN, IcmS, IcmW and an additionally identified subunit LvgA, and that this pentameric assembly binds Legionella effector proteins. We determined the crystal structure of this assembly and built an architecture of the T4CP holocomplex by combining a homology model of the ATPase domain of DotL. The holocomplex is a hexamer of a bipartite structure composed of a membrane-proximal ATPase domain and a membrane-distal substrate-recognition assembly. The presented information demonstrates the architecture and functional dissection of the multiprotein T4CP complexes and provides important insights into their substrate recruitment and processing.

The cullin-RING ubiquitin ligases are multisubunit complexes that ubiquitinate various proteins. Six different cullins encoded by the human genome selectively pair with different adaptors and substrate receptors. It is presently poorly understood how cullin-2 (Cul2) and cullin-5 (Cul5) associate specifically with their adaptor elongin BC and a SOCS-box-containing substrate receptor. Here, crystallographic and mutational analyses of a quaternary complex between the N-terminal half of Cul5, elongin BC and SOCS2 are reported. Cul5 interacts extensively with elongin BC via residues that are highly conserved in Cul2 but not in other cullins. Cul5 also interacts with SOCS2, but via only two residues, Pro184 and Arg186, which are located in the C-terminal part of the SOCS box called the Cul5 box. Pro184 makes a ring-to-ring interaction with Trp53 of Cul5, which is substituted by alanine in Cul2. This interaction is shown to contribute significantly to the overall binding affinity between Cul5 and SOCS2-elongin BC. This study provides structural bases underlying the specificity of Cul5 and Cul2 for elongin BC and their preferential association with Cul5 or Cul2 box-containing substrate receptors.