Peter Liu

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.

Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd−/− mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd−/− mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Microwave Assisted Proteomics

State-of-the-art proteomic analysis has recently undergone a rapid evolution; with more high-throughput analytical instrumentation and informatic tools available, sample preparation is becoming one of the rate-limiting steps in protein characterization workflows. Recently several protocols have appeared in the literature that employ microwave irradiation as a tool for the preparation of biological samples for subsequent mass spectrometric characterization. Techniques for microwave-assisted bio-catalyzed reactions (including sample reduction and alkylation, enzymatic and chemical digestion, removal and analysis of post-translational modifications and characterization of enzymes and protein-interaction sites) are described. This review summarizes the various approaches undertaken, instrumentation employed, and reduction in overall experimental time observed when microwave assistance is applied.

GsdmD p30 elicited by caspase-11 during pyroptosis forms pores in membranes.

Significance Pyroptosis is a form of cell death that is critical for eliminating innate immune cells infected with intracellular bacteria. Microbial products such as lipopolysaccharide, which is a component of Gram-negative bacteria, trigger activation of the inflammatory caspases 1, 4, 5, and 11. These proteases cleave the cytoplasmic protein Gasdermin-D into two pieces, p20 and p30. The p30 fragment is cytotoxic when liberated from the p20 fragment. Our work suggests that p30 induces pyroptosis by associating with cell membranes and forming pores that perturb vital electrochemical gradients. The resulting imbalance causes the cell to lyse and release intracellular components that can alert other immune cells to the threat of infection. Gasdermin-D (GsdmD) is a critical mediator of innate immune defense because its cleavage by the inflammatory caspases 1, 4, 5, and 11 yields an N-terminal p30 fragment that induces pyroptosis, a death program important for the elimination of intracellular bacteria. Precisely how GsdmD p30 triggers pyroptosis has not been established. Here we show that human GsdmD p30 forms functional pores within membranes. When liberated from the corresponding C-terminal GsdmD p20 fragment in the presence of liposomes, GsdmD p30 localized to the lipid bilayer, whereas p20 remained in the aqueous environment. Within liposomes, p30 existed as higher-order oligomers and formed ring-like structures that were visualized by negative stain electron microscopy. These structures appeared within minutes of GsdmD cleavage and released Ca2+ from preloaded liposomes. Consistent with GsdmD p30 favoring association with membranes, p30 was only detected in the membrane-containing fraction of immortalized macrophages after caspase-11 activation by lipopolysaccharide. We found that the mouse I105N/human I104N mutation, which has been shown to prevent macrophage pyroptosis, attenuated both cell killing by p30 in a 293T transient overexpression system and membrane permeabilization in vitro, suggesting that the mutants are actually hypomorphs, but must be above certain concentration to exhibit activity. Collectively, our data suggest that GsdmD p30 kills cells by forming pores that compromise the integrity of the cell membrane.

Ubiquitin ligase RNF146 regulates tankyrase and Axin to promote Wnt signaling.

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Global defects in collagen secretion in a Mia3/TANGO1 knockout mouse

Mia3’s contribution to protein secretion is broader than previously realized—its absence impairs collagen deposition and normal development of cartilage and bone.

Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7.

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

Host cell protein testing by ELISAs and the use of orthogonal methods

Host cell proteins (HCPs) are among the process‐related impurities monitored during recombinant protein pharmaceutical process development. The challenges of HCP detection include (1) low levels of residual HCPs present in large excess of product protein, (2) the assay must measure a large number of different protein analytes, and (3) the population of HCP species may change during process development. Suitable methods for measuring process‐related impurities are needed to support process development, process validation, and control system testing. A multi‐analyte enzyme‐linked immunosorbent assay (ELISA) is the workhorse method for HCP testing due to its high throughput, sensitivity and selectivity. However, as the anti‐HCP antibodies, the critical reagents for HCP ELISA, do not comprehensively recognize all the HCP species, it is especially important to ensure that weak and non‐immunoreactive HCPs are not overlooked by the ELISA. In some cases limited amount of antibodies to HCP species or antigen excess causes dilution‐dependent non‐linearity with multi‐product HCP ELISA. In our experience, correct interpretation of assay data can lead to isolation and identification of co‐purifying HCP with the product in some cases. Moreover, even if the antibodies for a particular HCP are present in the reagent, the corresponding HCP may not be readily detected in the ELISA due to antibody/antigen binding conditions and availability of HCP epitopes. This report reviews the use of the HCP ELISA, discusses its limitations, and demonstrates the importance of orthogonal methods, including mass spectrometry, to complement the platform HCP ELISA for support of process development. In addition, risk and impact assessment for low‐level HCPs is also outlined, with consideration of clinical information. Biotechnol. Bioeng. 2014;111: 2367–2379.

Metabolic and transcriptional profiling reveals pyruvate dehydrogenase kinase 4 as a mediator of epithelial-mesenchymal transition and drug resistance in tumor cells.

BackgroundAccumulating preclinical and clinical evidence implicates epithelial-mesenchymal transition (EMT) in acquired resistance to anticancer drugs; however, mechanisms by which the mesenchymal state determines drug resistance remain unknown.ResultsTo explore a potential role for altered cellular metabolism in EMT and associated drug resistance, we analyzed the metabolome and transcriptome of three lung cancer cell lines that were rendered drug resistant following experimental induction of EMT. This analysis revealed evidence of metabolic rewiring during EMT that diverts glucose to the TCA cycle. Such rewiring was at least partially mediated by the reduced expression of pyruvate dehydrogenase kinase 4 (PDK4), which serves as a gatekeeper of the TCA cycle by inactivating pyruvate dehydrogenase (PDH). Overexpression of PDK4 partially blocked TGFβ-induced EMT; conversely, PDK4 inhibition via RNAi-mediated knockdown was sufficient to drive EMT and promoted erlotinib resistance in EGFR mutant lung cancer cells. We identified a novel interaction between PDK4 and apoptosis-inducing factor (AIF), an inner mitochondrial protein that appears to play a role in mediating this resistance. In addition, analysis of human tumor samples revealed PDK4-low as a predictor of poor prognosis in lung cancer and that PDK4 expression is dramatically downregulated in most tumor types.ConclusionsTogether, these findings implicate PDK4 as a critical metabolic regulator of EMT and associated drug resistance.

Applications of Microwave-Assisted Proteomics in Biotechnology

Biotechnology has recently celebrated 30 years both as a science and as a multi-billion dollar industry. One application of biotechnology is to use human genetic information to discover, develop, manufacture, and commercialize biotherapeutics. Recombinant proteins can be produced in large quantities at high purity. High-throughput proteomic analysis is at the heart of the biotechnology research and development process, and the industry is constantly striving to streamline and automate the analytical processes involved. Microwave-assisted proteomics has recently emerged as a tool for increasing the bio-catalysis of several processes including tryptic digestions lipase selectivities, identification of metal-catalyzed oxidation sites on proteins, identification of protein N- and C-termini and enzyme catalyzed N-linked deglycosylation. Here, we explore the above mentioned methods, and describe our experiences evaluating microwave-technology for other common proteomic protocols including: removal of N-terminal pyroglutamyl for antibody characterization, beta elimination and Michael addition for identification of phosphorylation sites on recombinant proteins and enzyme mediated O-linked deglycosylation.

Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration

Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 ± 77, 20 ± 3, 288 ± 86 and 322 ± 82 ng/ml (mean ± standard deviation; n ≥ 10), respectively. Anti-NRP1B, a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1B antibody. In rats and monkeys receiving single injections of anti-NRP1B at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1B, respectively. In addition to the soluble NRP1 isoforms, for the first time, a ~120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by Western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1B treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1B induced membrane NRP1 shedding.