Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery

IL-38 Ameliorates Skin Inflammation and Limits IL-17 Production from γδ T Cells

Summary: Interleukin-38 (IL-38) is a cytokine of the IL-1 family with a role in chronic inflammation. However, its main cellular targets and receptors remain obscure. IL-38 is highly expressed in the skin and downregulated in psoriasis patients. We report an investigation in cellular targets of IL-38 during the progression of imiquimod-induced psoriasis. In this model, IL-38 knockout (IL-38 KO) mice show delayed disease resolution with exacerbated IL-17-mediated inflammation, which is reversed by the administration of mature IL-38 or γδ T cell-receptor-blocking antibodies. Mechanistically, X-linked IL-1 receptor accessory protein-like 1 (IL1RAPL1) is upregulated upon γδ T cell activation to feedforward-amplify IL-17 production and is required for IL-38 to suppress γδ T cell IL-17 production. Accordingly, psoriatic IL1RAPL1 KO mice show reduced inflammation and IL-17 production by γδ T cells. Our findings indicate a role for IL-38 in the regulation of γδ T cell activation through IL1RAPL1, with consequences for auto-inflammatory disease. : Han et al. report that genetic depletion of IL-38 in mice delays the resolution of imiquimod-induced psoriasis by increasing the production of the inflammatory cytokine IL-17A by skin-infiltrating T cells. Depleting these T cells or the receptor that is targeted by IL-38 reduces psoriatic skin inflammation. Keywords: IL-38, IL1RAPL1, IL-17, γδ T cells, psoriasis, inflammatio

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

Molecular and Biochemical Characterization of α-Glucosidase and α-Mannosidase and Their Clustered Genes from the Thermoacidophilic Archaeon Picrophilus torridus

The genes encoding a putative α-glucosidase (aglA) and an α-mannosidase (manA) appear to be physically clustered in the genome of the extreme acidophile Picrophilus torridus, a situation not found previously in any other organism possessing aglA or manA homologs. While archaeal α-glucosidases have been described, no α-mannosidase enzymes from the archaeal kingdom have been reported previously. Transcription start site mapping and Northern blot analysis revealed that despite their colinear orientation and the small intergenic space, the genes are independently transcribed, both producing leaderless mRNA. aglA and manA were cloned and overexpressed in Escherichia coli, and the purified recombinant enzymes were characterized with respect to their physicochemical and biochemical properties. AglA displayed strict substrate specificity and hydrolyzed maltose, as well as longer α-1,4-linked maltooligosaccharides. ManA, on the other hand, hydrolyzed all possible linkage types of α-glycosidically linked mannose disaccharides and was able to hydrolyze α3,α6-mannopentaose, which represents the core structure of many triantennary N-linked carbohydrates in glycoproteins. The probable physiological role of the two enzymes in the utilization of exogenous glycoproteins and/or in the turnover of the organism's own glycoproteins is discussed

Disrupting the LC3 interaction region (LIR) binding of selective autophagy receptors sensitizes AML cell lines to cytarabine

Short linear motifs (SLiMs) located in disordered regions of multidomain proteins are important for the organization of protein–protein interaction networks. By dynamic association with their binding partners, SLiMs enable assembly of multiprotein complexes, pivotal for the regulation of various aspects of cell biology in higher organisms. Despite their importance, there is a paucity of molecular tools to study SLiMs of endogenous proteins in live cells. LC3 interacting regions (LIRs), being quintessential for orchestrating diverse stages of autophagy, are a prominent example of SLiMs and mediate binding to the ubiquitin-like LC3/GABARAP family of proteins. The role of LIRs ranges from the posttranslational processing of their binding partners at early stages of autophagy to the binding of selective autophagy receptors (SARs) to the autophagosome. In order to generate tools to study LIRs in cells, we engineered high affinity binders of LIR motifs of three archetypical SARs: OPTN, p62, and NDP52. In an array of in vitro and cellular assays, the engineered binders were shown to have greatly improved affinity and specificity when compared with the endogenous LC3/GABARAP family of proteins, thus providing a unique possibility for modulating LIR interactions in living systems. We exploited these novel tools to study the impact of LIR inhibition on the fitness and the responsiveness to cytarabine treatment of THP-1 cells – a model for studying acute myeloid leukemia (AML). Our results demonstrate that inhibition of LIR of a single autophagy receptor is insufficient to sensitize the cells to cytarabine, while simultaneous inhibition of three LIR motifs in three distinct SARs reduces the IC50 of the chemotherapeutic

Apoptosis-induced tolerance. Role of the novel cytokine IL-38

Introduction: Necrotic cell death triggers inflammation, whereas apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction and are produced by necrotic cells to induce sterile inflammation. Release of IL-1 family proteins from apoptotic cells to regulate inflammation has not been described. The novel cytokine IL-38 shares homology with IL-1 family receptor antagonists and was therefore proposed as a negative regulator of IL-1 family receptor signaling. Methods: IL-38 concentrations were measured by ELISA. Apoptotictumor cell conditioned medium (ACM) was used to stimulate human primary immune cells. The response to ACM was determined as cytokine release (Cytometric Bead Array) as well as transcription factor activity (Luciferase reporter assays). N-terminal processing of IL-38 was determined by mass spectrometry. Binding of IL-38 to its putative receptors was determined by receptor binding assays. The pathophysiological function of IL-38 was analyzed using the imiquimod (IMQ)-induced psoriasis mouse model in IL-38-deficient animals compared to WT controls. Statistical analysis was performed using ANOVA with Bonferroni’s correction. Results: We show that IL-38 is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing IL-17-producing T cell activation. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1). However, we show higher affinity binding of mature IL-38. Likewise, we confirmed the previously reported low-affinity binding of the IL-38 precursor to IL1R1 and show an increased affinity of mature IL-38 to this receptor. Functionally, the IL-38 precursor induced an increase in IL-6 production by human macrophages, whereas truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. Ongoing studies show selective secretion of IL-38 from dying cells challenged with tolerogenic chemotherapy, but not with immunogenic chemotherapy. Moreover, strengthening the role of IL-38 as a tolerogenic factor, IL-38-deficient mice subjected to IMQ-induced psoriasis show an increased IL-17-mediated immune response and a strong delay in the restoration of skin architecture. Conclusion: We identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion. IL-38 limits cytokine production in macrophages antagonizing the IL1-RAPL1/JNK/AP1 pathway and subsequently preserving a low T cell IL-17 production. We propose apoptotic cell-derived IL-38 as the counter-regulatory equivalent of necrotic cell-derived alarmins of the IL-1 family, which might be relevant in resolution of inflammation, autoimmunity, and cancer