Nucleic Acid Programmable Protein Arrays Research Articles

Discovering Protein-Protein Interactions Using Nucleic Acid Programmable Protein Arrays.

We have developed a protocol enabling the study of protein-protein interactions (PPIs) at the proteome level using in vitro-synthesized proteins. Assay preparation requires molecular cloning of the query gene into a vector that supports in vitro transcription/translation (IVTT) and appends a HaloTag to the query protein of interest. In parallel, protein microarrays are prepared by printing plasmids encoding glutathione S-transferase (GST)-tagged target proteins onto a carrier matrix/glass slide coated with antibody directed against GST. At the time of the experiment, the query protein and the target protein are produced separately through IVTT. The query protein is then applied to nucleic acid programmable protein arrays (NAPPA) that display thousands of freshly produced target proteins captured by anti-GST antibody. Interactions between the query and immobilized target proteins are detected through addition of a fluorophore-labeled HaloTag ligand. Our protocol allows the elucidation of PPIs in a high-throughput fashion using proteins produced in vitro, obviating the scientific challenges, high cost, and laborious work, as well as concerns about protein stability, which are usually present in protocols using conventional protein arrays. © 2017 by John Wiley & Sons, Inc.

Identification of Antibody Against SNRPB, Small Nuclear Ribonucleoprotein-Associated Proteins B and B', as an Autoantibody Marker in Crohn's Disease using an Immunoproteomics Approach.

Current non-invasive biomarkers for Crohn's disease are limited in their utility. Progress in identifying individual autoantigens and autoantibodies in Crohn's disease has been challenging due to limitations of available immunoassays. Our aim was to identify autoantibodies associated with Crohn's disease that may be useful in diagnosis and management using an innovative protein array technology, namely nucleic acid programmable protein arrays [NAPPA]. Serum samples of 96 patients with established Crohn's disease and 96 healthy controls were included and evenly split into discovery and validation sets randomly. Autoantibodies of both IgG and IgA classes were profiled against ~1900 human proteins in the discovery set on NAPPA. Autoantibodies discovered to be Crohn's disease-specific were further validated in the independent validation set by enzyme-linked immunosorbent assay. Overall, reactivity of IgG autoantibodies was stronger than that of IgA autoantibodies; however, IgA autoantibodies showed greater differential reactivity between cases and controls. Four IgA autoantibodies against SNRPB, PRPH, PTTG1 and SNAI1 were newly identified with sensitivities above 15% at 95% specificity, among which anti-SNRPB-IgA had the highest sensitivity of 24.0%. Autoantibodies associated with specific disease subtypes were also found. As one of the first studies to use immunoproteomics for the identification of autoantibodies in Crohn's disease, our results support the utility of NAPPA in implementing future expanded studies with better coverage of the human proteome and microbial proteomes relevant to Crohn's disease and identifying antibody markers that may have clinical impact in diagnosis and management.

Multiplexed Nucleic Acid Programmable Protein Arrays.

Rationale: Cell-free protein microarrays display naturally-folded proteins based on just-in-time in situ synthesis, and have made important contributions to basic and translational research. However, the risk of spot-to-spot cross-talk from protein diffusion during expression has limited the feature density of these arrays.Methods: In this work, we developed the Multiplexed Nucleic Acid Programmable Protein Array (M-NAPPA), which significantly increases the number of displayed proteins by multiplexing as many as five different gene plasmids within a printed spot.Results: Even when proteins of different sizes were displayed within the same feature, they were readily detected using protein-specific antibodies. Protein-protein interactions and serological antibody assays using human viral proteome microarrays demonstrated that comparable hits were detected by M-NAPPA and non-multiplexed NAPPA arrays. An ultra-high density proteome microarray displaying > 16k proteins on a single microscope slide was produced by combining M-NAPPA with a photolithography-based silicon nano-well platform. Finally, four new tuberculosis-related antigens in guinea pigs vaccinated with Bacillus Calmette-Guerin (BCG) were identified with M-NAPPA and validated with ELISA.Conclusion: All data demonstrate that multiplexing features on a protein microarray offer a cost-effective fabrication approach and have the potential to facilitate high throughput translational research.

Tracking the Antibody Immunome in Type 1 Diabetes Using Protein Arrays.

We performed an unbiased proteome-scale profiling of humoral autoimmunity in recent-onset type 1 diabetes (T1D) patients and nondiabetic controls against ∼10 000 human proteins using a Nucleic Acid Programmable Protein Array (NAPPA) platform, complemented by a knowledge-based selection of proteins from genes enriched in human pancreas. Although the global response was similar between cases and controls, we identified and then validated six specific novel T1D-associated autoantibodies (AAbs) with sensitivities that ranged from 16 to 27% at 95% specificity. These included AAbs against PTPRN2, MLH1, MTIF3, PPIL2, NUP50 (from NAPPA screening), and QRFPR (by targeted ELISA). Immunohistochemistry demonstrated that NUP50 protein behaved differently in islet cells, where it stained both nucleus and cytoplasm, compared with only nuclear staining in exocrine pancreas. Conversely, PPIL2 staining was absent in islet cells, despite its presence in exocrine cells. The combination of anti-PTPRN2, -MLH1, -PPIL2, and -QRFPR had an AUC of 0.74 and 37.5% sensitivity at 95% specificity. These data indicate that these markers behave independently and support the use of unbiased screening to find biomarkers because the majority was not predicted based on predicted abundance. Our study enriches the knowledge of the "autoantibody-ome" in unprecedented breadth and width.

BioSpotlight / Citations

BioTechniquesVol. 61, No. 2 BioSpotlight / CitationsOpen AccessBioSpotlight / CitationsPatrick C.H. Lo & Nathan S. BlowPatrick C.H. LoSearch for more papers by this author & Nathan S. BlowSearch for more papers by this authorPublished Online:16 Mar 2018https://doi.org/10.2144/000114439AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit A reality check for dsRNAsA number of popular methods and commercial kits for single-step isolation of high-quality RNA use a concentrated solution of guanidinium thiocyanate (GuSCN)–a powerful chaotropic agent—to denature cellular proteins and inactivate RNases. Besides acting as a robust protein denaturant, a less well-known effect of concentrated GuSCN solutions is to promote DNA—DNA, RNA—DNA, and RNA—RNA hybridization. In this issue of BioTechniques, Triin Mölder and Mart Speek at the Tallinn University of Technology provide evidence suggesting that use of concentrated GuSCN during isolation of cellular RNA causes the formation of double-stranded RNAs (dsRNAs) from complementary single-stranded RNAs (ssRNAs). The authors demonstrated that incubation in a concentrated GuSCN solution of low concentrations of two in vitro—transcribed RNAs with a region of complementarity catalyzed their hybridization into dsRNA, in the absence or presence of total cellular RNA. This suggests the need to carefully verify that any putative novel dsRNAs identified from RNA samples isolated by GuSCN extraction are not actually artifacts caused by this effect.See “Accelerated RNA—RNA hybridization by concentrated guanidinium thiocyanate solution in single-step RNA isolation”Indenting multiple cellsStudies of the response of cells to mechanical force transduction have relied on instruments such as atomic force microscopes (AFMs) or cytoindentors, but these tools can only indent single cells one-by-one, which makes the collection of data from a sufficient number of cells a tedious and time-consuming affair. To overcome this problem, researchers at McGill University led by Claire Brown and Sabrina Leslie describe in this month's issue their development of a high-throughput, parallelized cytoindentor for the simultaneous local compression of multiple live cells. They modified their previously described convex lens—induced confinement (CLiC) set-up by adhering a film patterned with microposts to a microscope lens. This lens is then gradually lowered onto multiple cells maintained in a stage-top incubator to indent micrometer-sized areas on each cell while allowing imaging of those cells. By functionalizing the micropost arrays with an extracellular matrix protein such as fibronectin, it was also possible to pull on the cells. This novel parallelized cytoindentor will greatly facilitate studies of mechanobiology in living cells.See “Parallelized cytoindentation using convex micropatterned surfaces”Protein interactions: limiting diffusion on arraysProtein arrays are important tools for those exploring protein—protein interactions on a large scale. These arrays can be spotted with purified proteins, or cell-free protein synthesis can be performed on the arrays. Protein synthesis approaches such as the nucleic acid programmable protein array (NAPPA) streamline the building of large arrays, but they also produce large, diffuse protein spots that limit array density. Yazaki et al. solve this problem with their newly devised HaloTag- NAPPA technology, which produces higher-density protein spotting. To accomplish this, they exchanged the C-terminal GST fusion used in the original NAPPA for a HaloTag fusion that can be irreversibly bound to ligands co-spotted on the array. HaloTag-NAPPA captured synthesized proteins with significantly less diffusion compared to GST fusion proteins. To test their method, the authors spotted 12,000 Arabidopsis clones onto arrays, synthesized proteins, and then examined protein— protein interactions with 38 plant transcription factors. The new approach identified novel interactions with high sensitivity and low background.Yazaki, J. et al. 2016. Mapping transcription factor interactome networks using HaloTag protein arrays. Proc. Natl. Acad. Sci. USA. DOI: 10.1073/pnas.1603229113Gene expression profiling: it's all about locationProfiling gene expression is a powerful approach for understanding basic biology and human disease. Now, Stahl et al. introduce a new method that could significantly expand the scope of gene expression profiling by adding spatial resolution within tissue samples. In “spatial transcriptomics,” histological samples are placed onto slides with reverse transcription primers possessing positional barcodes immobilized on the slide surface. Following permeabilization, reverse transcription reagents are placed onto the tissues, where cDNA is synthesized and left coupled to the oligonucleotides on the slide. Once the tissue is enzymatically removed, the cDNAs are collected and sequenced, akin to traditional gene expression profiling. After sequencing, the locations of cDNA transcripts can be mapped back to the tissue using the incorporated positional barcodes. By adding only a few additional steps, scientists will now be able to obtain spatial information by transcriptional profiling, thus greatly enhancing gene expression data sets.Stahl, P.L., et al. 2016. Visualization and analysis of gene expression in tissue sections by spatial transcriptomics. Science. 353(6294):78-82.FiguresReferencesRelatedDetails Vol. 61, No. 2 Follow us on social media for the latest updates Metrics History Published online 16 March 2018 Published in print August 2016 Information© 2016 Author(s)PDF download

Mapping transcription factor interactome networks using HaloTag protein arrays

Protein microarrays enable investigation of diverse biochemical properties for thousands of proteins in a single experiment, an unparalleled capacity. Using a high-density system called HaloTag nucleic acid programmable protein array (HaloTag-NAPPA), we created high-density protein arrays comprising 12,000 Arabidopsis ORFs. We used these arrays to query protein-protein interactions for a set of 38 transcription factors and transcriptional regulators (TFs) that function in diverse plant hormone regulatory pathways. The resulting transcription factor interactome network, TF-NAPPA, contains thousands of novel interactions. Validation in a benchmarked in vitro pull-down assay revealed that a random subset of TF-NAPPA validated at the same rate of 64% as a positive reference set of literature-curated interactions. Moreover, using a bimolecular fluorescence complementation (BiFC) assay, we confirmed in planta several interactions of biological interest and determined the interaction localizations for seven pairs. The application of HaloTag-NAPPA technology to plant hormone signaling pathways allowed the identification of many novel transcription factor-protein interactions and led to the development of a proteome-wide plant hormone TF interactome network.

Immunoproteomics technologies in the discovery of autoantigens in autoimmune diseases.

Proteomics technologies are often used for the identification of protein targets of the immune system. Here, we discuss the immunoproteomics technologies used for the discovery of autoantigens in autoimmune diseases where immune system dysregulation plays a central role in disease onset and progression. These autoantigens and associated autoantibodies can be used as potential biomarkers for disease diagnostics, prognostics and predicting/monitoring drug responsiveness (theranostics). Here, we compare a variety of methods such as mass spectrometry (MS)-based [serological proteome analysis (SERPA), antibody mediated identification of antigens (AMIDA), circulating immune complexome (CIC) analysis, surface enhanced laser desorption/ionization-time of flight (SELDI-TOF)], nucleic acid based serological analysis of antigens by recombinant cDNA expression cloning (SEREX), phage immunoprecipitation sequencing (PhIP-seq) and array-based immunoscreening (proteomic microarrays), luciferase immunoprecipitation systems (LIPS), nucleic acid programmable protein array (NAPPA) methods. We also review the relevance of immunoproteomic data generated in the last 10 years, with a focus on the aforementioned MS based methods.

Comparative Study of Autoantibody Responses between Lung Adenocarcinoma and Benign Pulmonary Nodules

IntroductionThe reduction in lung cancer mortality associated with computed tomography (CT) screening has led to its increased use and a concomitant increase in the detection of benign pulmonary nodules. Many individuals found to have benign nodules undergo unnecessary, costly, and invasive procedures. Therefore, there is a need for companion diagnostics that stratify individuals with pulmonary nodules into high-risk or low-risk groups. Lung cancers can trigger host immune responses and elicit antibodies against tumor antigens. The identification of these autoantibodies (AAbs) and their corresponding antigens may expand our knowledge of cancer immunity, leading to early diagnosis or even benefiting immunotherapy. Previous studies were performed mostly in the context of comparing cancers and healthy (smoker) controls. We have performed one of the first studies to understand humoral immune response in patients with cancer, patients with benign nodules, and healthy smokers. MethodsWe first profiled seroreactivity to 10,000 full-length human proteins in 40 patients with early-stage lung cancer and 40 smoker controls by using nucleic acid programmable protein arrays to identify candidate cancer-specific AAbs. Enzyme-linked immunosorbent assays of promising candidates were performed on 137 patients with lung cancer and 127 smoker controls, as well as on 170 subjects with benign pulmonary nodules. ResultsFrom protein microarray screening experiments using a discovery set of 40 patients and 40 smoker controls, 17 antigens showing higher reactivity in lung cancer cases relative to the controls were subsequently selected for evaluation in a large sample set (n = 264) by using enzyme-linked immunosorbent assay. A five-AAb classifier (tetratricopeptide repeat domain 14 [TTC14], B-Raf proto-oncogene, serine/threonine kinase [BRAF], actin like 6B [ACTL6B], MORC family CW-type zinc finger 2 [MORC2], and cancer/testis antigen 1B [CTAG1B]) that can differentiate lung cancers from smoker controls with a sensitivity of 30% at 89% specificity was developed. We further tested AAb responses in subjects with CT-positive benign nodules (n = 170), and developed a five-AAb panel (keratin 8, type II, TTC14, Kruppel-like factor 8, BRAF, and tousled like kinase 1) with a sensitivity of 30% at 88% specificity. Interestingly, messenger RNA levels of six AAb targets (TTC14, BRAF, MORC family CW-type zinc finger 2, cancer/testis antigen 1B, keratin 8, type II, and tousled like kinase 1) were also found to increase in lung adenocarcinoma tissues based on The Cancer Genome Atlas data set. ConclusionWe discovered AAbs associated with lung adenocaricnoma that have the potential to differentiate cancer from CT-positive benign diseases. We believe that these antibodies warrant future validation using a larger sample set and/or longitudinal samples individually or as a panel. They could potentially be part of companion molecular diagnostic modalities that will benefit subjects undergoing CT screening for lung cancer.

Abstract B20: Targeting Olig2 co-regulators for malignant glioma therapy

Abstract The transcription factor OLIG2 is expressed within the highly tumorigenic sub-population of stem-like cells in malignant glioma and required for tumor formation in orthotopic grafts of human glioma as well as genetically defined murine glioma models. Our work (recently confirmed and extended by Suva et al., 2014) suggests that small molecule OLIG2 antagonist could serve as targeted therapy for high grade gliomas – either as a mono therapeutic or as an adjuvant to conventional radiotherapy or cytotoxic drugs. However, transcription factors in general are considered to be unattractive targets for drug development because their interactions with DNA and co-regulatory proteins involve large and complex surface area contacts. Our recent data suggests that OLIG2 interacts with distinct partner proteins based on its phosphorylation state, which in turn regulates its pro-mitogenic functions. Here we employed two independent proteomics screens (mass spectrometry and Nucleic Acid Programmable Protein Arrays, NAPPA) to identify OLIG2 co-regulators as possible surrogate targets to suppress OLIG2 function. These screens led to identification of two novel OLIG2 partner proteins: HDAC1 and proto-oncogene FYN kinase. OLIG2 is known to be a negative regulator of gene expression and histone deacetylation is required for suppression of gene expression. We demonstrate that OLIG2 functions as an HDAC1 carrier, delivering this enzyme to specific target genes (e.g., p21) and non-histone proteins like p53. HDACs are ubiquitously expressed and are known to be redundant, but we show that ablation of HDAC1 alone (using shRNA) significantly decreases the proliferation of glioma initiating cells with minimal impact on normal neural stem cells. In addition, we show that FYN kinase interacts with OLIG2 and phosphorylates it at tyrosine residues. Ablation of FYN decreases phosphorylation of OLIG2 at tyrosine residues and affects OLIG2-mediated expression of oncogenic target genes like EGFR and WNT5a. This work provides two potential co-regulators of OLIG2 which could serve as druggable surrogate targets to block OLIG2 function in malignant glioma. Further work will evaluate the efficacy of HDAC1 and FYN-specific inhibitor as single agents or as adjuvant therapy to radiation and temozolomide in orthotopic patient-derived xenograft models. Targeting factors regulating OLIG2-mediated tumor growth will have practical overtones for targeted therapies for malignant gliomas. Citation Format: Robert Kupp, Costanza Lo Cascio, Femina Rauf, Dimphna Meijer, Charles D. Stiles, Nader Sanai, Joshua LaBaer, Shwetal V. Mehta. Targeting Olig2 co-regulators for malignant glioma therapy. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B20.

EPID-32PROTEIN-SPECIFIC ANTIBODY RESPONSES TO VARICELLA AND CYTOMEGALOVIRUS IN GLIOMA PATIENTS COMPARED TO CONTROLS

An etiologic role of neurotropic viruses in glioma is supported by epidemiologic and clinical observations. Glioma patients consistently report a lower history of clinical responses to varicella (VZV, chickenpox and shingles). Pathologic and clinical evidence for an active role of Cytomegalovirus (CMV) in gliomagenesis is also accumulating, though the subject remains controversial. We hypothesized that the neurotropic location of viral latency and subsequent patient-restricted response to these infections may impact efficacy of immune response against glioma, affecting incidence of the disease as well as survival in those afflicted. We explored the viral protein-restricted response against 99 VZV and 156 CMV protein antigens using Nucleic Acid Programmable Protein Arrays (NAPPA) in serum samples from 45 glioma patients and 45 controls from the University of California, San Francisco Adult Glioma Study (AGS). The proportion of agreement between the NAPPA positivity for CMV and VZV reactivity with traditional ELISA whole virus serological assay was 86% and 95%, respectively (Cohen's Kappa 0.72 and 0.9, respectively). Cases were more likely to have complex reactions to 5 or more antigens of VZV (p = 0.03). Cases reacted more strongly to specific proteins including Orf12, Orf14, and Orf49 (p < 0.03), and antigen recognition was strikingly stronger among cases for Orf23 and Orf9, a viral capsid and tegument protein, respectively. Multivariable analysis pointed towards significant differences between cases and controls for Orf9, Orf37, and Orf44. For CMV, protein reactivity was also stronger in cases for the specific proteins UL25, TRS1, and UL48a, also capsid and tegument proteins. Ongoing studies will assess (1) anti-VZV antigens in prediagnostic sera from glioma cases and matched controls in the PLCO cohort study and (2) the role of antibody reactions in survival of glioma patients within AGS. Specific differences in reactivity to these Herpesviruses may promote strategies for glioma prevention and therapies.

Nonenzymatic Protein Acetylation Detected by NAPPA Protein Arrays.

Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here, we address the possibility that nonenzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the -7 to -3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation.

NAPPA as a Real New Method for Protein Microarray Generation.

Nucleic Acid Programmable Protein Arrays (NAPPA) have emerged as a powerful and innovative technology for the screening of biomarkers and the study of protein-protein interactions, among others possible applications. The principal advantages are the high specificity and sensitivity that this platform offers. Moreover, compared to conventional protein microarrays, NAPPA technology avoids the necessity of protein purification, which is expensive and time-consuming, by substituting expression in situ with an in vitro transcription/translation kit. In summary, NAPPA arrays have been broadly employed in different studies improving knowledge about diseases and responses to treatments. Here, we review the principal advances and applications performed using this platform during the last years.

High-throughput identification of proteins with AMPylation using self-assembled human protein (NAPPA) microarrays.

AMPylation (adenylylation) has been recognized as an important post-translational modification that is used by pathogens to regulate host cellular proteins and their associated signaling pathways. AMPylation has potential functions in various cellular processes, and it is widely conserved across both prokaryotes and eukaryotes. However, despite the identification of many AMPylators, relatively few candidate substrates of AMPylation are known. This is changing with the recent development of a robust and reliable method for identifying new substrates using protein microarrays, which can markedly expand the list of potential substrates. Here we describe procedures for detecting AMPylated and auto-AMPylated proteins in a sensitive, high-throughput and nonradioactive manner. The approach uses high-density protein microarrays fabricated using nucleic acid programmable protein array (NAPPA) technology, which enables the highly successful display of fresh recombinant human proteins in situ. The modification of target proteins is determined via copper-catalyzed azide-alkyne cycloaddition (CuAAC). The assay can be accomplished within 11 h.

Host-pathogen interaction profiling using self-assembling human protein arrays.

Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.

SpADS and SNAP-NAPPA Microarrays towards Biomarkers Identification in Humans: Background Subtraction in Mass Spectrometry with E.coli Cell Free Expression System

We present a useful approach towards for biomarkers identification in an innovative self-assembling protein microarray based on “Nucleic Acid Programmable Protein Array” (NAPPA) and SNAP tag coupled to E.coli cell free expression system. This approach prove capable to resolve the “background” problem associated to the above label free detection system for the identification of proteins and of protein-protein interaction in humans that could become used in clinical practice.

AMPylation of Rho GTPases Subverts Multiple Host Signaling Processes

Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules. Herein, we demonstrate that AMPylation of Rho GTPases by VopS is a multifaceted virulence mechanism that counters several host immunity strategies. Activation of NFκB, Erk, and JNK kinase signaling pathways were inhibited in a VopS-dependent manner during infection with Vibrio parahaemolyticus. Phosphorylation and degradation of IKBα were inhibited in the presence of VopS as was nuclear translocation of the NFκB subunit p65. AMPylation also prevented the generation of superoxide by the phagocytic NADPH oxidase complex, potentially by inhibiting the interaction of Rac and p67. Furthermore, the interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a nucleic acid programmable protein array and discovered that Rac1 binds to the protein C1QA, a protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted by AMPylation. We conclude that AMPylation of Rho Family GTPases by VopS results in diverse inhibitory consequences during infection beyond the most obvious phenotype, the collapse of the actin cytoskeleton.

Analysis of Autoantibody Profiles in Osteoarthritis Using Comprehensive Protein Array Concepts

Osteoarthritis (OA) is the most common rheumatic disease and one of the most disabling pathologies worldwide. To date, the diagnostic methods of OA are very limited, and there are no available medications capable of halting its characteristic cartilage degeneration. Therefore, there is a significant interest in new biomarkers useful for the early diagnosis, prognosis, and therapeutic monitoring. In the recent years, protein microarrays have emerged as a powerful proteomic tool to search for new biomarkers. In this study, we have used two concepts for generating protein arrays, antigen microarrays, and NAPPA (nucleic acid programmable protein arrays), to characterize differential autoantibody profiles in a set of 62 samples from OA, rheumatoid arthritis (RA), and healthy controls. An untargeted screen was performed on 3840 protein fragments spotted on planar antigen arrays, and 373 antigens were selected for validation on bead-based arrays. In the NAPPA approach, a targeted screening was performed on 80 preselected proteins. The autoantibody targeting CHST14 was validated by ELISA in the same set of patients. Altogether, nine and seven disease related autoantibody target candidates were identified, and this work demonstrates a combination of these two array concepts for biomarker discovery and their usefulness for characterizing disease-specific autoantibody profiles.

Abstract 874: Autoantibody biomarker discovery in basal-like breast cancer using nucleic acid programmable protein array

Abstract Background: Basal-like breast cancer (BLBC) is a rare aggressive subtype of breast cancer (∼10-20% of all BC) with high recurrence rates and poor prognosis. BLBC disproportionately affects African American and younger women, has a high growth rate and occurs at young ages. These factors render mammographic screening challenging, necessitating the development of new methods for BLBC's early detection. Proteins released from small early tumors may only be present intermittently and get diluted to tiny concentrations in the blood, making them difficult to use as biomarkers. However, they can induce an autoantibody (AAb) response, which can amplify the signal and persist in the blood even if the antigen is gone. Accordingly, we assessed whether circulating AAbs could act as early detection biomarkers of BLBC. We have developed a sero-proteomic technology, called Nucleic Acid Programmable Protein Arrays (NAPPA), which tests the immune responses to thousands of proteins simultaneously. Results: Plasma samples were from the Polish Breast Cancer Study. BLBC patients were classified by immunohistochemical staining of ER-, PR-, HER2-, and either EGFR+ or CK5/6+. Case and control plasma were probed against ∼10,000 full-length human proteins on NAPPA to discover informative antigens, which were selected based on the following statistical metrics: 1) sensitivity at 95% specificity; 2) area under receiver operating characteristic curve (AUC); 3) partial AUC above 95% specificity; 4) Welch's t test. We then identified candidate AAbs in a training study combining the 800 top antigens on a single array. These arrays were screened with an independent training set of plasma from 50 each of BLBC cases and healthy controls, and 30 other BC cases. Preliminary analyses suggest 5 candidate biomarkers with sensitivities ranging from 26-32% at 94% specificity. The antigen list will be refined using AUC, and the sensitivity and partial AUC at 95%, 90% and 80% specificity levels. Permutation-based false discovery rates will be used to assess statistical significance, and the antigens will be combined into a biomarker panel using a random forests algorithm. Finally, we will test the individual markers and the antigen panel in a blinded independent validation set of 50 cases, 50 controls and 30 other BC cases. These tests will use cutoffs and algorithm determined entirely from the training data. We are assessing whether the candidate biomarkers are specific for cancer vs. controls and BLBC vs. other molecular subtypes of breast cancer. Summary: This describes the first immunoproteomic screen for AAb biomarkers of BLBC. Using protein microarrays displaying more than 10,000 folded unique human proteins, controls, BLBC and non-BLBC cases were tested in independent discovery and training stages identifying AAbs with potential diagnostic value. Blinded validation studies are in progress. Citation Format: Jie Wang, Jonine D. Figueroa, Garrick Wallstrom, Joshua Sampson, Eliseo Mendoza Garcia, Jason Steel, Jin Park, Karen S. Anderson, Louise Brinton, Montserrat Garcia-Closas, Jolanta Lissowska, Mark E. Sherman, Ji Qiu, Joshua LaBaer. Autoantibody biomarker discovery in basal-like breast cancer using nucleic acid programmable protein array. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 874. doi:10.1158/1538-7445.AM2014-874

Abstract 887: Proteome scale identification of autoantibody biomarkers in colorectal cancer

Abstract It is widely appreciated that early detection is the key to making inroads in the fight against cancers. Despite the availability of endoscopic screening, compliance is poor. The diagnosis of CRC is often made in advanced stages, and the consequence is a dismal 50% five year survival across all CRC. There is urgent need for biomarkers with performance that can meet the need of early diagnosis. Like most cancers, colorectal cancers (CRCs) express tumor antigens that can induce humoral immune response. The identification of the autoantibodies against tumor antigens may facilitate early diagnosis of CRC. The goal for this pilot project was to demonstrate the feasibility of discovering autoantibody biomarkers for colorectal cancer using Nucleic Acid Programmable Protein Arrays (NAPPA). Autoantibodies in serum samples collected from 40 patients with DNA mismatch repair proficient colorectal cancer who had died of their cancer within 18 months of their blood draw and from 40 age-gender matched healthy controls were profiled on NAPPA arrays with randomly selected 2000 human protein. The fact that p53, a tumor antigen that has been previously reported to be related to various cancers including CRC, was one of the top candidates based on the statistical analysis demonstrates the validity of our discovery platform. Besides p53, thirty-one candidate antigens were identified to have sensitivity greater than or equal to 25% at 95% specificity and fifty-one candidate antigens had an area under the ROC curve (AUC) equal to or greater than 0.6. Validation of the combined 83 unique genes selected based on these two different data analysis methods in an independent sample set in a blind fashion is on-going. This pilot study using late stage cancer samples demonstrates the feasibility and power of an immunoproteomics study using protein arrays. This approach may rapidly lead to identification of markers that could have relevance in monitoring disease progression as well as in early cancer detection. Citation Format: Gokhan Demirkan, Leah Soderberg, Garrick Wallstrom, Emily Szeto, Kristi Barker, Jie Wang, Joshua LaBaer, Noralane M. Lindor, Ji Qiu. Proteome scale identification of autoantibody biomarkers in colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 887. doi:10.1158/1538-7445.AM2014-887

Abstract 2661: Patient-derived breast cancer targeting heavy chain antibodies

Abstract The emergence of tumor-targeting antibodies such as Trastuzumab, Rituximab and anti-CD47 antibody has highlighted the significant role of antibodies for effective cancer therapy. The most common approach to generate tumor specific antibodies is to select an established tumor specific antigen and generate antibodies to that target. The drawback with this approach is that it does not lead to identification of novel antigens or antigenic epitopes. Therefore, we developed a strategy that will lead to the identification of both tumor specific antibodies and novel tumor antigens. The approach uses the patient's immune system to guide us to define tumor specific antigens. In order to achieve this, we identified reactive germinal centers in the tumor draining sentinel lymph nodes from breast cancer patients. Immunohistochemical analysis of reactive germinal centers defined the B-cell regions for mRNA isolation and generation of cDNA molecules for the variable heavy chain segment (VH) of antibodies. The VH cDNA molecules were cloned into a mammalian expression vector to generate bivalent heavy chain antibodies with a C-terminus mouse Fc tag. At present, we have generated about 250 antigen-driven variable heavy chain clones from 27 breast cancer patient lymph node samples. We screened 46 heavy chain antibodies against a panel of breast cancer cell lines using immunofluorescence and identified 8 antibodies that showed strong and specific staining. Of these 8 antibodies, one of them (VH6-93) showed membrane staining preferentially on MDA-MB-231 cells in comparison to MCF7 cells. VH6-93 antibody also showed strong staining on conditionally reprogrammed tumor cells derived from a breast cancer patient with only moderate staining on normal breast epithelial cells derived from the same patient. VH6-93 also revealed preferential binding to breast tumor tissues as compared to normal ductal tissues evidenced by immunofluorescence and immunohistochemistry. To identify the target antigens, a multi-tiered approach was used, which included immunoprecipitation of target antigen from cell lysates followed by peptide sequencing with mass spectrometry as well as utilizing Nucleic Acid Programmable Protein Array (NAPPA) technology. A comprehensive analysis of two patient-derived antibodies has produced a panel of potential antigen targets, which are undergoing specificity and validation analysis. In summary, this methodology of using activated B-cells from sentinel lymph nodes is a unique way to enrich for tumor-specific antibody clones. The findings from this research will not only lead to tumor specific antibodies but will also reveal novel tumor specific antigens. Identification of these novel antigens can give rise to additional therapeutic strategies such as peptide or protein vaccination. Citation Format: Charan Kumar V. Devarakonda, Daniel Kita, Kathryn N. Phoenix, Dewey M. Magee, Kevin P. Claffey. Patient-derived breast cancer targeting heavy chain antibodies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2661. doi:10.1158/1538-7445.AM2014-2661