Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.

Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin-like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3-interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C-specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.

Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells.

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.

SpyDisplay: A versatile phage display selection system using SpyTag/SpyCatcher technology.

Phage display is an established method for the in vitro selection of recombinant antibodies and other proteins or peptides from gene libraries. Here we describe SpyDisplay, a phage display method in which the display is achieved via SpyTag/SpyCatcher protein ligation instead of genetically fusing the displayed protein to a phage coat protein. In our implementation, SpyTagged antibody antigen-binding fragments (Fabs) are displayed via protein ligation on filamentous phages carrying SpyCatcher fused to the pIII coat protein. A library of genes encoding Fab antibodies was cloned in an expression vector containing an f1 replication origin, and SpyCatcher-pIII was separately expressed from a genomic locus in engineered E. coli. We demonstrate the functional, covalent display of Fab on phage, and rapidly isolate specific high-affinity clones via phage panning, confirming the robustness of this selection system. SpyTagged Fabs, the direct outcome of the panning campaign, are compatible with modular antibody assembly using prefabricated SpyCatcher modules and can be directly tested in diverse assays. Furthermore, SpyDisplay streamlines additional applications that have traditionally been challenging for phage display: we show that it can be applied to N-terminal display of the protein of interest and it enables display of cytoplasmically folding proteins exported to periplasm via the TAT pathway.

Periplasmic expression of SpyTagged antibody fragments enables rapid modular antibody assembly.

Antibodies are essential tools in research and diagnostics. Although antibody fragments typically obtained from in vitro selection can be rapidly produced in bacteria, the generation of full-length antibodies or the modification of antibodies with probes is time and labor intensive. Protein ligation such as SpyTag technology could covalently attach domains and labels to antibody fragments equipped with a SpyTag. However, we found that the established periplasmic expression of antibody fragments in E. coli led to quantitative cleavage of the SpyTag by the proteases Tsp and OmpT. Here we report successful periplasmic expression of SpyTagged Fab fragments and demonstrate the coupling to separately prepared SpyCatcher modules. We used this modular toolbox of SpyCatcher proteins to generate reagents for a variety of immunoassays and measured their performance in comparison with traditional reagents. Furthermore, we demonstrate surface immobilization, high-throughput screening of antibody libraries, and rapid prototyping of antibodies based on modular antibody assembly.

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.

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.

Tissue Cytokine IL-33 Modulates the Cytotoxic CD8 T Lymphocyte Activity During Nutrient Deprivation by Regulation of Lineage-Specific Differentiation Programs.

IL-1 family member IL-33 exerts a variety of immune activating and regulating properties and has recently been proposed as a prognostic biomarker for cancer diseases, although its precise role in tumor immunity is unclear. Here we analyzed in vitro conditions influencing the function of IL-33 as an alarmin and a co-factor for the activity of cytotoxic CD8+ T cells in order to explain the widely discussed promiscuous behavior of IL-33 in vivo. Circulating IL-33 detected in the serum of healthy human volunteers was biologically inactive. Additionally, bioactivity of exogenous recombinant IL-33 was significantly reduced in plasma, suggesting local effects of IL-33, and inactivation in blood. Limited availability of nutrients in tissue causes necrosis and thus favors release of IL-33, which-as described before-leads to a locally high expression of the cytokine. The harsh conditions however influence T cell fitness and their responsiveness to stimuli. Nutrient deprivation and pharmacological inhibition of mTOR mediated a distinctive phenotype characterized by expression of IL-33 receptor ST2L on isolated CD8+ T cells, downregulation of CD8, a transitional CD45RAlowROlow phenotype and high expression of secondary lymphoid organ chemokine receptor CCR7. Under nutrient deprivation, IL-33 inhibited an IL-12 induced increase in granzyme B protein expression and increased expression of GATA3 and FOXP3 mRNA. IL-33 enhanced the TCR-dependent activation of CD8+ T cells and co-stimulated the IL-12/TCR-dependent expression of IFNγ. Respectively, GATA3 and FOXP3 mRNA were not regulated during TCR-dependent activation. TCR-dependent stimulation of PBMC, but not LPS, initiated mRNA expression of soluble IL-33 decoy receptor sST2, a control mechanism limiting IL-33 bioactivity to avoid uncontrolled inflammation. Our findings contribute to the understanding of the compartment-specific activity of IL-33. Furthermore, we newly describe conditions, which promote an IL-33-dependent induction of pro- or anti-inflammatory activity in CD8+ T cells during nutrient deprivation.

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

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.

Tolerizing CTL by Sustained Hepatic PD-L1 Expression Provides a New Therapy Approach in Mouse Sepsis.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

Transposable modules generated by a single copy of insertion sequence ISPme1 and their influence on structure and evolution of natural plasmids of Paracoccus methylutens DM12.

We demonstrated that a single copy of insertion sequence ISPme1 can mobilize adjacent segments of genomic DNA of Paracoccus methylutens DM12, which leads to the generation of diverse transposable elements of various size and DNA contents. All elements (named transposable modules [TMos]) contain ISPme1 (placed at the 5' ends of the elements) and have variable 3'-end regions of between 0.5 and 5 kb. ISPme1 was shown to encode an outwardly oriented promoter, which may activate the transcription of genes transposed within TMos in evolutionarily distinct hosts. TMos may therefore be considered to be natural systems enabling gene capture, expression, and spread. However, unless these elements have been inserted into a highly conserved genetic context to enable a precise definition of their termini, it is extremely difficult or even impossible to identify them in bacterial genomes by in silico sequence analysis. We showed that TMos are present in the chromosome and plasmids of strain DM12. Sequence analysis of plasmid pMTH1 (32 kb) revealed that four TMos, previously identified with a trap vector, pMEC1, comprise 87% of its genome. Repeated TMos within pMTH1 may stimulate other structural rearrangements resulting from homologous recombination between long repeat sequences. This illustrates that TMos may play a significant role in shaping the structure of natural plasmids, which consequently may have a great impact on the evolution of plasmid genomes.

Interleukin-38 is released from apoptotic cells to limit inflammatory macrophage responses.

Different modes of cell death regulate immunity. Whereas necrotic (necroptotic, pyroptotic) cell death triggers inflammation, apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction. A number of IL-1 family cytokines are produced by necrotic cells to induce sterile inflammation. However, release of IL-1 family proteins from apoptotic cells to regulate inflammation was not described. Here we show that IL-38, a poorly characterized IL-1 family cytokine, 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 Th17 cell expansion at the expense of IL-10-producing T cells. 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, whereas the IL-38 precursor induced an increase in IL-6 production by human macrophages, truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. In conclusion, we identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion, which might be relevant in resolution of inflammation, autoimmunity, and cancer.

PIP3 induces the recycling of receptor tyrosine kinases.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

PLCß isoforms differ in their subcellular location and their CT-domain dependent interaction with Gαq.

Phospholipase C (PLC) ß isoforms are implicated in various physiological processes and pathologies. However, mechanistic insight into the localization and activation of each of the isoforms is limited. Therefore, it is crucial to gain more in-depth knowledge as to the regulation of the different isoforms. Here we describe the subcellular location of full-length PLCß isozymes and their C-terminal (CT) domains. Strikingly, we found isoforms PLCß1 and PLCß4 to be enriched at the plasma membrane, contrary to isoforms PLCß2 and PLCß3. We determined that the CT domain is an inhibitor of Gq-mediated increases in intracellular calcium, the potency of its effect being dependent upon the CT domain isoform used. Furthermore, ratiometric fluorescence resonance energy transfer (FRET) imaging was used to study the kinetics of the Gαq-CTßx interactions. By the use of recently developed tools, which enable the on-demand activation of Gαq, we could show that the interaction between constitutively active Gαq and PLCß3 prolongs the residence time of PLCß3 at the plasma membrane. These findings suggest that under physiological circumstances, PLCß3 and Gαq interact in a kiss-and-run fashion, likely due to the GTPase-activating activity of PLCß towards Gαq.

Protein translocation as a tool: The current rapamycin story.

In cell biology and pharmacology, small chemicals are mostly used as agonists and antagonists against receptors and enzymes. The immunosuppressant rapamycin can serve an entirely different purpose: if employed sensibly, it might function as an inducer of dimerization that is able to rapidly activate enzyme activity inside the intact cell. A number of very recent developments such as photoactivatable derivatives make rapamycin an even more attractive tool for basic science.

Switching heterotrimeric G protein subunits with a chemical dimerizer.

The selective manipulation of single intracellular-signaling events remains one of the key tasks when studying signaling networks. Here, we demonstrate for the first time the stimulation of FKBP fusions of various subunits of heterotrimeric G proteins by the simple addition of the chemical dimerizer rapamycin. Activation of constitutively active Gα(q), but not its GDP-bound form, leads to sustained oscillations of intracellular calcium and myo-inositol 1,4,5-trisphosphate (InsP(3)) levels in HEK cells, independent of the activation of endogenous Gα(q), in full agreement with the InsP(3)-Ca(2+) cross-coupling model of calcium oscillations. Rapamycin-induced translocation of wild-type Gα(s) to the plasma membrane results in elevated cAMP levels. Activation of rapamycin-inducible Gα(s) or Gß(1)γ(2) evokes extensive modulation of ATP-induced calcium transients. The results demonstrate that inducible heterotrimeric G protein subunits will provide ways for dissecting G protein-coupled receptor signaling.

Molecular and biochemical characterization of alpha-glucosidase and alpha-mannosidase and their clustered genes from the thermoacidophilic archaeon Picrophilus torridus.

The genes encoding a putative alpha-glucosidase (aglA) and an alpha-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 alpha-glucosidases have been described, no alpha-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 alpha-1,4-linked maltooligosaccharides. ManA, on the other hand, hydrolyzed all possible linkage types of alpha-glycosidically linked mannose disaccharides and was able to hydrolyze alpha3,alpha6-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.