Triple-fusion protein (TriFu): A potent, targeted, enzyme-like inhibitor of all three complement activation pathways.

The introduction of a therapeutic anti-C5 antibody into clinical practice in 2007 inspired a surge into the development of complement-targeted therapies. This has led to the recent approval of a C3 inhibitory peptide, an antibody directed against C1s and a full pipeline of several complement inhibitors in preclinical and clinical development. However, no inhibitor is available that efficiently inhibits all three complement initiation pathways and targets host cell surface markers as well as complement opsonins. To overcome this, we engineered a novel fusion protein combining selected domains of the three natural complement regulatory proteins decay accelerating factor, factor H and complement receptor 1. Such a triple fusion complement inhibitor (TriFu) was recombinantly expressed and purified alongside multiple variants and its building blocks. We analyzed these proteins for ligand binding affinity and decay acceleration activity by surface plasmon resonance. Additionally, we tested complement inhibition in several in vitro/ex vivo assays using standard classical and alternative pathway restricted hemolysis assays next to hemolysis assays with paroxysmal nocturnal hemoglobinuria erythrocytes. A novel in vitro model of the alternative pathway disease C3 glomerulopathy was established to evaluate the potential of the inhibitors to stop C3 deposition on endothelial cells. Next to the novel engineered triple fusion variants which inactivate complement convertases in an enzyme-like fashion, stoichiometric complement inhibitors targeting C3, C5, factor B, and factor D were tested as comparators. The triple fusion approach yielded a potent complement inhibitor that efficiently inhibits all three complement initiation pathways while targeting to surface markers.

Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.

DNA ends alter the molecular composition and localization of Ku multicomponent complexes.

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Cell-specific effects on surface α7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein.

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH-EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH-EP1 and human embryonic kidney (HEK) cells measured by α-bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH-EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH-EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co-transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half-life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.

Parallel comparison of three methodologies for measuring functional C1-inhibitor in Hereditary angioedema patients.

Hereditary angioedema (HAE) types I and II are characterized by functional C1 inhibitor (fC1-INH) deficiency which results in bradykinin overproduction. Sensitive, specific and robust methods to quantitate fC1-INH in human samples are required for diagnosing HAE and/or to measure pharmacodynamic activity of C1-INH drugs in clinical studies. To date, three methods have been reported in literature to measure fC1-INH: conventional chromogenic assay measuring residual C1-esterase activity, and immunoassays based on functional binding to either activated complement C1s or Factor XIIa/kallikrein. We used three qualified/validated fit-for purpose methods to quantitate fC1-INH in human plasma and to conduct a parallel comparison for diagnostic purposes and as a read-out for pharmacodynamic activity. Sensitivity and specificity were determined from the Receiver Operator Characteristics (ROC) curve analysis of the three fC1-INH methods through testing of fifty healthy control vs. HAE plasma samples. fC1-INH profile of fifteen HAE subjects, who underwent different treatment regimen in a cross-over Shire C1-INH clinical study, was analyzed in these three methods in parallel. A correlation analysis performed between these methods using data generated from clinical samples showed that profiles obtained from different fC1-INH methods matched for individual HAE subjects. Our findings suggest that functional binding immunoassay methods serve as reliable alternates for conventional chromogenic method to quantitate fC1-INH in human plasma samples with a better dynamic range of detection and ease of use. Of the two immunoassays used in this study, FXIIa-binding method gave better sensitivity, specificity, and correlation to the chromogenic method as a diagnostic method to distinguish HAE samples from healthy controls.

A first of its kind quantitative functional C1-esterase inhibitor lateral flow assay for hereditary angioedema point-of-care diagnostic testing.

Hereditary Angioedema (HAE) is a rare, autosomal dominant disease caused by mutations in SERPING1 gene leading to dysfunction/deficiency of C1-esterase inhibitor (C1-INH) protein and subsequent dysregulation of the contact system and bradykinin overproduction. As functional C1-INH (fC1-INH) levels are reduced in HAE types I and II (HAE-I/II), a specific, sensitive and accessible rapid diagnostic method to quantitate fC1-INH is crucial in diagnosing HAE-I/II. Previously, we developed/validated methods to detect fC1-INH levels in human plasma based on functional binding to C1s or FXIIa for C1-INH-based therapies. Quantitative fC1-INH immunoassay methods were converted to the Lateral flow assay (LFA) platform after identifying the best reagent/s pair. The assay was developed and optimized as a first of its kind LFA method for quantifying fC1-INH in human plasma to aid HAE point-of-care diagnosis. Receiver operating characteristic analysis was performed using normal control and HAE subject plasma samples to calculate area-under-curve and a cut-off point to distinguish normal versus HAE subject samples. LFA data was correlated with the conventional diagnostic assay for fC1-INH in HAE plasma samples and profiles matched for individual subjects. Here, we demonstrate a proof-of-concept for the quantitative fC1-INH LFA using normal and HAE plasma samples. We propose that the method could be used as a point-of-care test to diagnose HAE in a variety of settings, such as, a hospital or physician's office, at home or in an ambulance.

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

GTS-21 has cell-specific anti-inflammatory effects independent of α7 nicotinic acetylcholine receptors.

α7 Nicotinic acetylcholine receptors (nAChRs) reportedly reduce inflammation by blocking effects of the important pro-inflammatory transcription factor, nuclear factor kappa-light chain-enhancer of B cells (NFκB). The α7 nAChR partial agonist GTS-21 reduces secretion of pro-inflammatory cytokines including interleukin-6 (IL6) and tumor-necrosis factor (TNF) in models of endotoxemia and sepsis, and its anti-inflammatory effects are widely ascribed to α7 nAChR activation. However, mechanistic details of α7 nAChR involvement in GTS-21 effects on inflammatory pathways remain unclear. Here, we investigate how GTS-21 acts in two cell systems including the non-immune rat pituitary cell line GH4C1 expressing an NFκB-driven reporter gene and cytokine secretion by ex vivo cultures of primary mouse macrophages activated by lipopolysaccharide (LPS). GTS-21 does not change TNF-stimulated NFκB signaling in GH4C1 cells expressing rat α7 nAChRs, suggesting that GTS-21 requires additional unidentified factors besides α7 nAChR expression to allow anti-inflammatory effects in these cells. In contrast, GTS-21 dose-dependently suppresses LPS-induced IL6 and TNF secretion in primary mouse macrophages endogenously expressing α7 nAChRs. GTS-21 also blocks TNF-induced phosphorylation of NFκB inhibitor alpha (IκBα), an important intermediary in NFκB signaling. However, α7 antagonists methyllycaconitine and α-bungarotoxin only partially reverse GTS-21 blockade of IL6 and TNF secretion. Further, GTS-21 significantly inhibited LPS-induced IL6 and TNF secretion in macrophages isolated from knockout mice lacking α7 nAChRs. These data indicate that even though a discrete component of the anti-inflammatory effects of GTS-21 requires expression of α7 nAChRs in macrophages, GTS-21 also has anti-inflammatory effects independent of these receptors depending on the cellular context.

Tandem Affinity Purification and Mass Spectrometry (TAP-MS) for the Analysis of Protein Complexes.

Affinity purification followed by mass spectrometry has become the technique of choice to identify binding partners in biochemical complexes isolated from a physiologic cellular context. In this report we detail our protocol for tandem affinity purification (TAP) primarily based on the use of the FLAG and HA peptide epitopes, with a particular emphasis on factors affecting yield and specificity, as well as steps to implement an automated version of the TAP procedure. © 2019 by John Wiley & Sons, Inc.

Evaluating Commercially Available Antibodies for Rat α7 Nicotinic Acetylcholine Receptors.

Alpha7 nicotinic acetylcholine receptors (α7 nAChRs) are important drug targets in neurological disorders and inflammation, making their detection and localization by validated antibodies highly desirable. However, tests in knockout animals raised questions about specificity of antibodies to mouse α7 nAChRs. To date, methods for validating antibodies for rat or human α7 nAChR have not been reported. We developed a gel-shift assay for western blots using GH4C1 cells expressing either native rat receptors or α7 nAChR-green fluorescent protein (GFP) chimeras to evaluate seven commercially available α7 nAChR antibodies. Blots with anti-GFP antibody detected GFP or α7 nAChR-GFP expressed in GH4C1 cells, and 125I-α-bungarotoxin binding and RNA analysis demonstrated α7 nAChR expression. Validated samples were used to evaluate α7 nAChR antibodies by western blot and immunofluorescence studies. These methods confirmed that two of seven α7 nAChR antibodies identify gel-shifts for α7 nAChR/nAChR-GFP but only one antibody demonstrated low background and significant immunofluorescence differences between wild-type and α7 nAChR expressing GH4C1 cells. However, that polyclonal antibody displayed lot-to-lot variability. Our findings suggest that careful validation methods are required for all α7 nAChR receptor species and antibody lots and that the gel-shift assay may allow for relatively rapid antibody screening.