Extracellular Domain Of Protein Research Articles

Abstract LB034: Analysis of PD-L1 IHC tests using NIST SRM 1934-traceable reference materials: A new paradigm for development of predictive IHC biomarkers

Abstract BACKGROUND. The challenges in accurate patient stratification for immune checkpoint inhibitors have been compounded by the fact that the FDA-cleared PD-L1 IHC tests are analytic ‘black boxes'. Relatively basic analytic parameters such as lower limit of detection and analytic dynamic range are unknown to both developers of assays as well as end users. The recent development of standardized PD-L1 immunohistochemistry (IHC) reference materials enables quantitative test characterizations that were not previously possible. METHODS. We surveyed 41 PD-L1 testing laboratories in North America and Europe, quantitatively defining each of the PD-L1 tests' analytic performance in terms of lower limit of detection and dynamic range. All four commercial PD-L1 kits were assessed by multiple laboratories. A variety of laboratory-developed tests (LDTs) we also assessed. The reference materials incorporated defined concentrations of PD-L1 peptide (intracellular domain) or recombinant extracellular domain protein, traceable to NIST Standard Reference Material 1934. Each laboratory received a slide with 10 separate PD-L1 calibrator concentrations: 2,200 - 600,000 molecules of PD-L1 extracellular domain or 34,000 - 2,200,000 molecules of PD-L1 intracellular domain. The calibrator concentrations ranged from those that are below the lower limit of detection to others that yield maximal staining. RESULTS. The data obtained with the four PD-L1 kits (VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, DAKO PD-L1 IHC 28-8 pharmDx and DAKO PD-L1 IHC 22C3 pharmDx assays) revealed that the lower limits of detection (PD-L1 molecules per cell equivalent) are approximately: 50,000 - 180,000 (SP263), 800,000 - 1,200,000 (SP142), 220,000 - 360,000 (28-8), and 200,000 - 400,000 (22C3). The dynamic ranges for all of these tests are generally narrow, spanning less than a log concentration of PD-L1. The SP263 and SP142 assays showed no overlap of their analytic response curves. This means that a maximal stain intensity with SP263 kit can be associated with zero staining with the SP142 kit. Consequently, it is not possible to compensate for the variability in analytic sensitivity between these two tests by adjusting the percent positive cell cutoff. The 28-8 was more sensitive than, but statistically indistinguishable from the 22C3 assay. Laboratory-developed tests (LDTs) using these and other primary antibodies have their own unique analytic performance characteristics. CONCLUSIONS. The PD-L1 reference materials enable precise definitions of analytic test performance and linking them with clinical management thresholds. Therefore, this tool finds its most important implementation at the stage of development of new IHC predictive biomarkers in clinical trials as well as at the stage of methodology transfer to clinical IHC laboratories. Furthermore, our results also help define more precisely the possibility for assay interchangeability and to what degree the assays may be harmonized. Citation Format: Emina E. Torlakovic, Seshi Sompuram, Kodela Vani, Steve Bogen. Analysis of PD-L1 IHC tests using NIST SRM 1934-traceable reference materials: A new paradigm for development of predictive IHC biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB034.

Protein Family Content Uncovers Lineage Relationships and Bacterial Pathway Maintenance Mechanisms in DPANN Archaea.

DPANN are small-celled archaea that are generally predicted to be symbionts, and in some cases are known episymbionts of other archaea. As the monophyly of the DPANN remains uncertain, we hypothesized that proteome content could reveal relationships among DPANN lineages, constrain genetic overlap with bacteria, and illustrate how organisms with hybrid bacterial and archaeal protein sets might function. We tested this hypothesis using protein family content that was defined in part using 3,197 genomes including 569 newly reconstructed genomes. Protein family content clearly separates the final set of 390 DPANN genomes from other archaea, paralleling the separation of Candidate Phyla Radiation (CPR) bacteria from all other bacteria. This separation is partly driven by hypothetical proteins, some of which may be symbiosis-related. Pacearchaeota with the most limited predicted metabolic capacities have Form II/III and III-like Rubisco, suggesting metabolisms based on scavenged nucleotides. Intriguingly, the Pacearchaeota and Woesearchaeota with the smallest genomes also tend to encode large extracellular murein-like lytic transglycosylase domain proteins that may bind and degrade components of bacterial cell walls, indicating that some might be episymbionts of bacteria. The pathway for biosynthesis of bacterial isoprenoids is widespread in Woesearchaeota genomes and is encoded in proximity to genes involved in bacterial fatty acids synthesis. Surprisingly, in some DPANN genomes we identified a pathway for synthesis of queuosine, an unusual nucleotide in tRNAs of bacteria. Other bacterial systems are predicted to be involved in protein refolding. For example, many DPANN have the complete bacterial DnaK-DnaJ-GrpE system and many Woesearchaeota and Pacearchaeota possess bacterial group I chaperones. Thus, many DPANN appear to have mechanisms to ensure efficient protein folding of both archaeal and laterally acquired bacterial proteins.

An mRNA-based vaccine candidate against SARS-CoV-2 elicits stable immuno-response with single dose

D614G genotype of SARS-CoV-2 virus is highly infectious and responsible for almost all infection for 2nd wave. However, there are currently no reports with D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine with D614G variant and characterization in animal model. We have used special mRNA-architecture and formulation that provides suitable response of the product. The surface plasmon resonance (SPR) data with spike protein (S) revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice neutralized SARS-CoV-2-pseudoviruses in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, single-dose immunization protected mice-lungs from homotypic-pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The data suggested great promise for immediate translation of the technology to the clinic.

Structural Basis of Dystroglycan Function and the Pathogenesis of Muscular Dystrophy

Dystroglycan (DG) is a widely expressed high-affinity extracellular matrix receptor that requires extensive glycosylation and additional post-translational processing. In skeletal muscle, DG is part of the dystrophin-glycoprotein complex, which is embedded in the plasma membrane and establishes a continuous link between laminin-G-like (LG) domains of extracellular matrix-resident proteins (laminin, agrin, and perlecan) and the cytoskeleton. Extracellular matrix proteins that contain LG domains bind to α-DG via a novel heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n called matriglycan, which is synthesized by the bifunctional enzyme known as Like-acetylglucosaminyltransferase (LARGE). Abnormalities in the post-translational processing of DG lead to an absence or reduction of the matriglycan modification on α-DG, and thereby lead to various forms of glycosylation-deficient muscular dystrophy (secondary dystroglycanopathies). We have used a multidisciplinary approach to provide the first atomic-level insights into the structure of a unique protein-carbohydrate interaction that is essential for the binding of α-DG to the extracellular matrix and proper skeletal muscle function. We first purified LARGE and synthesized matriglycan chains of defined lengths in sufficient quantities to perform both biochemical and structural studies. Using NMR spectroscopy, we found that matriglycan bound to laminin-α2 LG4,5 with high affinity (KD=0.23 µM) and that this binding was calcium dependent. Next, we initiated a collaboration with Erhard Hohenester, who had previously crystallized laminin-α2 LG4,5. Dr. Hohenester soaked matriglycan into crystals of LG4,5 to produce a high-resolution crystal structure of LG4,5 bound to matriglycan. This analysis revealed that the LG4 domain is a Ca2+-dependent lectin with specificity for GlcA-β1,3-Xyl disaccharides, and that a single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. This chelating binding mode is unprecedented among animal lectins and accounts for the high affinity of this protein-carbohydrate interaction. Finally, we used two novel exoglycosidases to provide direct evidence that native α-DG contains unmodified matriglycan and numerous GlcA-Xyl repeats. The multiple tandem repeats in matriglycan are predicted to increase the apparent affinity of the protein for LG domains by favoring rapid rebinding after dissociation. Our results represent a major conceptual advancement regarding a functional carbohydrate and shed light on the mechanism of protein-carbohydrate interactions underlying dystroglycanopathies.

Periplasmic expression of EGFRvIII extracellular domain as a fusion protein in Escherichia coli NiCo21(DE3)

Epidermal growth factor receptor variant III is a mutant variant of EGFR that has a deletion on its DNA encoding extracellular ligan-binding domain. EGFRvIII is only found in cancer cells and not in normal cells, make it an ideal target as a biomarker for antibody-based cancer therapy. This study performed the expression and characterization of EGFRvIII extracellular domain conjugated with a blue fluorescent protein (BFP) as a fusion protein in Escherichia coli periplasmic space. Endoxylanase signal peptide was employed to guide the recombinant protein through the membrane. IPTG as inducer was added into expression medium with various concentrations of 0; 0.1; 0.25; 0.5; 1 mM, followed by periplasmic extraction using the hypertonic solution. Total proteins and periplasmic proteins were characterized using SDS-PAGE and slot blot analyses. Extracellular domain of EGFRvIII-BFP fusion protein was detected using confocal fluorescence microscopy. This study showed that the ∼43 kDa target protein was successfully expressed on E. coli NiCo21(DE3) periplasmic space with optimum IPTG concentration of 0.1 mM and and showed a blue fluorescence color under the microscope..

HER2-Targeted Immunotherapy and Combined Protocols Showed Promising Antiproliferative Effects in Feline Mammary Carcinoma Cell-Based Models.

Simple SummaryMammary tumors are common in cats, presenting an aggressive behavior with high tumor recurrence. Therefore, new and efficient therapeutic protocols are urgent. Monoclonal antibodies (mAbs; ADC) are widely used in human breast cancer therapy, inhibiting the HER2 dimerization and leading to cell apoptosis. Furthermore, drug combinations, with tyrosine kinase inhibitors (TKi) are valuable in patients’ therapeutic protocols. In this study, two mAbs, and an ADC, as well as combined protocols between mAbs and mAbs plus lapatinib (TKi) were tested to address if the drugs could be used as new therapeutic options in feline mammary tumors. All the compounds and the combined treatments revealed valuable antiproliferative effects, and a conserved cell death mechanism, by apoptosis, in the feline cell lines, where the mutations found in the extracellular domain of the HER2 suggest no immunotherapy resistance.Feline mammary carcinoma (FMC) is a highly prevalent tumor, showing aggressive clinicopathological features, with HER2-positive being the most frequent subtype. While, in human breast cancer, the use of anti-HER2 monoclonal antibodies (mAbs) is common, acting by blocking the extracellular domain (ECD) of the HER2 protein and by inducing cell apoptosis, scarce information is available on use these immunoagents in FMC. Thus, the antiproliferative effects of two mAbs (trastuzumab and pertuzumab), of an antibody–drug conjugate compound (T-DM1) and of combined treatments with a tyrosine kinase inhibitor (lapatinib) were evaluated on three FMC cell lines (CAT-MT, FMCm and FMCp). In parallel, the DNA sequence of the her2 ECD (subdomains II and IV) was analyzed in 40 clinical samples of FMC, in order to identify mutations, which can lead to antibody resistance or be used as prognostic biomarkers. Results obtained revealed a strong antiproliferative effect in all feline cell lines, and a synergistic response was observed when combined therapies were performed. Additionally, the mutations found were not described as inducing resistance to therapy in breast cancer patients. Altogether, our results suggested that anti-HER2 mAbs could become useful in the treatment of FMC, particularly, if combined with lapatinib, since drug-resistance seems to be rare.

Structural Basis of VSIG3: The Ligand for VISTA.

B7 family members and their receptors play key roles in regulating T cell responses, and constitute very attractive targets for developing immunotherapeutic drugs. V-Set and Immunoglobulin domain containing 3 (VSIG3), a ligand for the novel B7 family immune checkpoint V-domain immunoglobulin suppressor of T cell activation (VISTA), can significantly inhibit T cell functions. Inhibitors targeting the VISTA/VSIG3 pathway are of great significance in tumor immunology. Here, we show the crystal structure of the extracellular domain (ECD) of the human VSIG3 protein at 2.64 angstrom resolution, and we produce recombinant human VSIG-3 ECD in both CHO cells and E. coli. Furthermore, we demonstrated the interaction of VISTA and VSIG3 by coimmunoprecipitation (Co-IP). Based on protein-protein docking for VISTA and VSIG3, we report a small molecule inhibitor of VSIG3 K284-3046 and evaluate its biological activities in vitro. This study was the first to reveal the crystal structure of VSIG3, and provides the structural basis for designing antibodies or compounds for the unique VSIG3/VISTA coinhibitory pathway in the treatment of cancers, autoimmune diseases and may be beneficial of designing vaccines.

Autoantibodies Targeting Intracellular and Extracellular Proteins in Autoimmunity.

Detecting autoantibodies provides foundational information for the diagnosis of most autoimmune diseases. An important pathophysiological distinction is whether autoantibodies are directed against extracellular or intracellular proteins. Autoantibodies targeting extracellular domains of proteins, such as membrane receptors, channels or secreted molecules are often directly pathogenic, whereby autoantibody binding to the autoantigen disrupts the normal function of a critical protein or pathway, and/or triggers antibody-dependent cell surface complement killing. By comparison, autoantibodies directed against intracellular proteins are recognized as useful diagnostic biomarkers of abnormal autoimmune activity, but the link between antigenicity and pathogenicity is less straightforward. Because intracellular autoantigens are generally inaccessible to autoantibody binding, for the most part, they do not directly contribute to pathogenesis. In a few diseases, autoantibodies to intracellular targets cause damage indirectly by immune complex formation, immune activation, and other processes. In this review, the general features of and differences between autoimmune diseases segregated on the basis of intracellular or extracellular autoantigens are explored using over twenty examples. Expression profiles of autoantigens in relation to the tissues targeted by autoimmune disease and the temporal appearance of autoantibodies before clinical diagnosis often correlate with whether the respective autoantibodies mostly recognize either intracellular or extracellular autoantigens. In addition, current therapeutic strategies are discussed from this vantage point. One drug, rituximab, depletes CD20+ B-cells and is highly effective for autoimmune disorders associated with autoantibodies against extracellular autoantigens. In contrast, diseases associated with autoantibodies directed predominately against intracellular autoantigens show much more complex immune cell involvement, such as T-cell mediated tissue damage, and require different strategies for optimal therapeutic benefit. Understanding the clinical ramifications of autoimmunity derived by autoantibodies against either intracellular or extracellular autoantigens, or a spectrum of both, has practical implications for guiding drug development, generating monitoring tools, stratification of patient interventions, and designing trials based on predictive autoantibody profiles for autoimmune diseases.

Effects of competitive physical activity on serum irisin levels and bone turnover markers

BackgroundIrisin, a myokine, is a polypeptide derived from the cleavage of the extracellular domain of fibronectin domain-containing protein 5, a receptor that is present on different tissues (skeletal muscle, pericardium, myocardium, and brain), whose functions are not yet fully defined.PurposeThe main aim of our study was to evaluate the effect of competitive physical activity on serum irisin levels and bone turnover markers.MethodsFifteen male footballers and an equal number of subjects of the same age and gender, but with a predominantly sedentary lifestyle, had their serum levels of irisin and bone turnover markers measured. Bone mineral status was evaluated in both groups by quantitative bone ultrasound of the calcaneus. In addition, only in footballers, biochemical analyses were repeated after 3 months.ResultsWe did not observe significant differences in the serum levels of calcium, phosphorus, and parathyroid hormone between the two groups. The footballers had significantly higher quantitative bone ultrasound, 25-OH vitamin D, and creatinine values than the controls. There were also no significant differences in the bone alkaline phosphatase, carboxy-terminal telopeptide of type I collagen, osteoprotegerin, sclerostin or Dkk-1 values, while the irisin levels (+ 89%, p < 0.001) and RANKL were significantly higher in the footballers compared to those in the controls.ConclusionOur study shows that footballers have significantly higher serum irisin values than the general population. Irisin could be the "trait d’union" between bone health and physical activity.

Abstract PD11-09: Vadis trial: Phase II trial of nelipepimut-s peptide vaccine in women with DCIS of the breast

Abstract Background: Peptide cancer vaccines may be most effective when used in earlier stage cancers or pre-cancers where systemic and tumor microenvironmental immune suppression are less profound. Nelipepimut-S (NPS) plus granulocyte-macrophage colony-stimulating factor (GM-CSF) is a vaccine comprised of a human leukocyte antigen (HLA) restricted peptide from the extracellular domain of the HER2 protein (E75) combined with GM-CSF. We have completed a randomized phase II trial of preoperative vaccination with NPS+GM-CSF vs. GM-CSF alone with the primary outcome being NPS-specific cytotoxic T lymphocyte (CTL) responses. Methods: HLA-A2 positive, DCIS patients were enrolled and randomized to either NPS+GM-CSF vs GM-CSF alone. The patients received two vaccinations prior to surgery at 2-week intervals. The number of NPS-specific CTL was measured at specified intervals (pre-vaccination, time of surgery, 1 month (+/- 7 days) post-op, and 3 months (+/- 7 days) post-op) using a flow cytometry-based dextramer assay. Differences in NPS-specific CTL responses between the two groups and between baseline pre-vaccination and 1-month post-op were analyzed using either a two-sample t-test or Wilcoxon rank sum test, when appropriate. The incidence and severity of adverse events, graded according to Common Terminology Criteria for Adverse Events (CTCAE) version (v) 4.03, were recorded and compared between treatment groups. Results: 45 patients were registered; 7 withdrew consent, 1 opted for surgery at an external facility, 20 were ineligible due to negative HLA-A2, and 4 failed screening for other reasons, leaving 13 patients enrolled. The 13 patients were randomized (2:1) into treatment groups, with nine patients receiving NPS+GM-CSF and four patients receiving GM-CSF alone. The two groups were well-matched for age; however, the GM-CSF alone group had higher percentages of African American (50% vs. 22%) and Hispanic (25% vs. 11%) patients as compared to the NPS+GM-CSF group. In general, vaccination was well-tolerated with similar treatment-related toxicity profiles in the NPS+GM-CSF vs GM-GSF groups (Grade 1 - 93.3% vs. 89.3%, Grade 2 - 6.7% vs. 10.7%, respectively). The mean NPS-specific CTL% in the NPS+GM-CSF group at 1-month post-op was double that of the GM-CSF alone group (0.10 +/- 0.12% vs. 0.05 +/- 0.08, p=0.70). In addition, between baseline pre-vaccination and 1-month post-op, the NPS+GM-CSF group experienced an 11-fold increase in percentage of NPS-specific CTL (0.01 +/- 0.02% vs. 0.11 +/- 0.12%) as compared to only a 2.25-fold increase of NPS-specific CTL in the GM-CSF alone group (0.04 +/- 0.07% vs. 0.09 +/- 0.15%). Conclusions: NPS+GM-CSF is safe and well-tolerated when given preoperatively to patients with DCIS. In HLA-A2 positive patients with DCIS, a single inoculation with NPS+GM-CSF can induce in vivo immunity and a continued antigen-specific T-cell response one month post-surgery. This data provides support for further testing of NPS+GM-CSF in the neoadjuvant and adjuvant settings in an attempt to prevent invasive recurrence in DCIS. Citation Format: Anne E O'Shea, Guy T Clifton, Na Qiao, Brandy Heckman-Stoddard, Malgorzata Wojtowicz, Eileen Dimond, Isabelle Bedrosian, Diane Weber, Alex Husband, Ricardo Pastorello, Lana Vornik, George Peoples, Elizabeth A Mittendorf. Vadis trial: Phase II trial of nelipepimut-s peptide vaccine in women with DCIS of the breast [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD11-09.

Elicitation of Highly Pathogenic Avian Influenza H5N1 M2e and HA2-Specific Humoral and Cell-Mediated Immune Response in Chicken Following Immunization With Recombinant M2e-HA2 Fusion Protein.

The study was aimed to evaluate the elicitation of highly pathogenic avian influenza (HPAI) virus (AIV) M2e and HA2-specific immunity in chicken to develop broad protective influenza vaccine against HPAI H5N1. Based on the analysis of Indian AIV H5N1 sequences, the conserved regions of extracellular domain of M2 protein (M2e) and HA2 were identified. Synthetic gene construct coding for M2e and two immunodominant HA2 conserved regions was designed and synthesized after codon optimization. The fusion recombinant protein (~38 kDa) was expressed in a prokaryotic system and characterized by Western blotting with anti-His antibody and anti-AIV polyclonal chicken serum. The M2e–HA2 fusion protein was found to be highly reactive with known AIV-positive and -negative chicken sera by ELISA. Two groups of specific pathogen-free (SPF) chickens were immunized (i/m) with M2e synthetic peptide and M2e–HA2 recombinant protein along with one control group with booster on the 14th day and 28th day with the same dose and route. Pre-immunization sera and whole blood were collected on day 0 followed by 3, 7, 14, 21, and 28 days and 2 weeks after the second booster (42 day). Lymphocyte proliferation assay by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) method revealed that the stimulation index (SI) was increased gradually from days 0 to 14 in the immunized group (p < 0.05) than that in control chicken. Toll-like receptor (TLR) mRNA analysis by RT-qPCR showed maximum upregulation in the M2e–HA2-vaccinated group compared to M2e- and sham-vaccinated groups. M2e–HA2 recombinant protein-based indirect ELISA revealed that M2e–HA2 recombinant fusion protein has induced strong M2e and HA2-specific antibody responses from 7 days post-primary immunization, and then the titer gradually increased after booster dose. Similarly, M2e peptide ELISA revealed that M2e–HA2 recombinant fusion protein elicited M2e-specific antibody from day 14 onward. In contrast, no antibody response was detected in the chicken immunized with synthetic peptide M2e alone or control group. Findings of this study will be very useful in future development of broad protective H5N1 influenza vaccine targeting M2e and HA2.

Analysis of plasma HER2 copy number in cell-free DNA of breast cancer patients: a comparison with HER2 extracellular domain protein level in serum

HER2 (human epidermal growth factor receptor 2) status has been evaluated in breast cancer (BC) tissues by immunohistochemistry or in situ hybridization. We evaluated HER2 copy number (CN) assay in plasma cell-free DNA (cfDNA) from blood samples and compared it with protein measurements of HER2 extracellular domain (ECD) in serum. Serum HER2-ECD levels were measured by chemi-luminescence immunoassay using anti-HER2 monoclonal antibodies. Analyses were performed on 120 cases of primary BC, 30 cases of metastatic BC and 34 cases treated by neoadjuvant chemotherapy (NAC). This study was approved by Medical Research Review Advancement No. 1857 for Kumamoto University. There was a positive correlation between HER2-CN ratios and HER2-ECD levels, in primary (n = 54) and metastatic (n = 30) HER2-positive BC (P = 0.003 and P < 0.001, respectively). HER2-ECD levels were significantly higher in patients with a larger number of metastatic sites (P = 0.02). The usefulness of HER2 levels in discriminating primary and metastatic HER2-positive BC evaluated by ROC curve analysis was better in the HER2-ECD assay than in the HER2-CN assay. In 34 patients who received NAC, there was a small decrease in HER2-CN ratios between before and after NAC (P = 0.10), while there was an obvious decrease in HER2-ECD levels between before and after NAC (P < 0.001). Compared to HER2-ECD levels, the clinical usefulness of HER2-CN ratio was somewhat inferior. Improved measurement methods and further examination of the association with long-term prognosis and the response to anti-HER2 treatment analyzed by HER2-CN and HER2-ECD assay are required.

Structural Studies of the Neuronal Apoptotic Complex of the Prongf-P75NTR-Sortilin

One of the prominent causes of neurodegenerative diseases is the dysregulation of the p75 neurotrophin receptor (p75NTR)-mediated signaling. Mature nerve growth factor (NGF) binds to p75NTR and the co-receptor tyrosine kinase a (TrkA) to maintain the neuronal proliferation, whereas the precursor of NGF (proNGF) interacts with p75NTR and sortilin, leading to neuronal apoptosis. The imbalance of the two signaling activities has strongly associated with Alzheimer's Disease and neurodegeneration. To elucidate the molecular mechanism of the proNGF-p75NTR-sortilin complex formation, we generated the extracellular domains of the target proteins and characterized the protein bindings in vitro. The extracellular domain complex will show the key structural motifs for protein-protein interactions, since the extracellular domains drive the complex formation. We used the Sf9 insect cell to perform heterologous overexpression, maintaining the simple post-glycosylation for membrane proteins. Previous study showed that sortilin forms a dimer in an acidic environment. However, negative-stain electron microscopy showed that the ternary complex of the proNGF-p75NTR-sortilin was formed in a dimeric assembly and was not sensitive to the environmental pH. This is likely that the binding interface may not be via the interaction between sortilins. To unravel the mechanistic view and identify the key residues in the binding interface, we are pursuing single-particle electron cryogenic microscopy (cryo-EM) to determine a high-resolution structure of the ternary complex. The result will provide a structural insight into the development of new medical interventions for treating neurodegenerative disorders.

Analysis of Target Molecules towards Anti-cancer Therapeutic Antibodies

Target molecules of existing anti-cancer therapeutic monoclonal antibodies (mAbs) are divided into 1) receptor-type tyrosine kinases, such as human epidermal growth factor receptor (HER) family, 2) differentiation antigens, such as CD20 (Rituxan target), 3) angiogenesis-related molecules, and 4) immune checkpoint molecules (PD-1, etc.). We have recently reported a novel therapy targeting lymphangiogenesis, but not angiogenesis, using an anti-LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) mAb. At present, many transporters are not considered to be target molecules for the cancer therapy; however, our study strongly suggested that the inhibition of cancer metabolism by mAbs against amino acid transporters will play a significant role in future cancer therapies. Most anti-cancer therapeutic mAbs bind cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAbs recognizing epitopes on the extracellular domains of native and non-denatured proteins. We concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAbs. We introduce our efforts to develop seeds for therapeutic mAbs using whole cancer cells and transfectants as the immunogen. As many target candidates in the future are multi-pass membrane proteins, such as 12-pass amino acid transporter proteins belonging to the solute carrier (SLC) family, and their possible immunogenic extracellular regions are small, the production of specific mAbs is highly difficult. In this review, we summarize the successful preparation and characterization of mAbs recognizing the extracellular domain of oncoproteins, including transporters.

Approaches towards Influenza Vaccine

Influenza viruses still constitute a true public ill health today. The influenza an epidemic was isolated for the primary time in 1931, and therefore the first attempts to develop a vaccine against the virus began soon afterwards. Additionally to causing seasonal epidemics, influenza viruses can cause pandemics randomly intervals, which are very hard to predict. Vaccination is that the best way of preventing the spread of influenza infection. However, seasonal vaccination is ineffective against pandemic influenza viruses due to antigenic differences, and it takes approximately six months from isolation of a replacement virus to develop an efficient vaccine. To deal with the emergence of latest circulating strains, but also the emergence of resistant strains to classic antivirals, it's necessary to develop new antiviral approaches. One among the possible ways to fight the emergence of pandemics could also be by employing a new sort of vaccine, with an extended and broad spectrum of action. The extracellular domain of the matrix protein 2 (M2e) of Influenza an epidemic may be a conservative region, and a beautiful target for a universal influenza vaccine.

Synthesis and functional studies of self-adjuvanting multicomponent anti-HER2 cancer vaccines.

Breast cancer is the leading cause of cancer-related deaths among women worldwide. Human epidermal growth factor receptor 2 (HER2) overexpression is significantly associated with higher breast tumor proliferation rates. MFCH401, a 9-mer specific peptide fragment (DTILWKDIF) in the extracellular domain of the HER2 protein, is an attractive epitope for developing anti-HER2 cancer vaccines. However, the inherent low immunogenicity of MFCH401 limits its application. Herein, to induce a stronger and more durable immune response, a self-adjuvanting MFCH401-conjugated multiple-component anti-HER2 cancer vaccine was designed and synthesized by incorporating MFCH401 with lipopeptide Pam3CSK4 and a helper T cell epitope derived from tetanus toxoid P2 via an iterative condensation reaction. In vivo immunological evaluation demonstrated that the tricomponent anti-HER2 vaccine induced stronger humoral and cellular immune responses than the two-component conjugates. In addition, the induced antibodies effectively bound to HER2-overexpressing human BT474 cells. Our data clearly indicated that the MFCH401-based tricomponent anti-HER2 cancer vaccine could effectively enhance the immunogenicity of MFCH401. Structure–activity relationship analysis demonstrated that Pam3CSK4 confers better immunostimulatory activity than the helper T cell epitope P2 when conjugated with MFCH401.

Diseases related to Notch glycosylation

The Notch receptors are a family of transmembrane proteins that mediate direct cell-cell interactions and control numerous cell-fate specifications in humans. The extracellular domains of mammalian Notch proteins contain 29-36 tandem epidermal growth factor-like (EGF) repeats, most of which have O-linked glycan modifications: O-glucose added by POGLUT1, O-fucose added by POFUT1 and elongated by Fringe enzymes, and O-GlcNAc added by EOGT. The extracellular domain is also N-glycosylated. Mutations in the glycosyltransferases modifying Notch have been identified in several diseases, including Dowling-Degos Disease (haploinsufficiency of POFUT1 or POGLUT1), a form of limb-girdle muscular dystrophy (autosomal recessive mutations in POGLUT1), Spondylocostal Dysostosis 3 (autosomal recessive mutations in LFNG), Adams-Oliver syndrome (autosomal recessive mutations in EOGT), and some cancers (amplification, gain or loss-of-function of POFUT1, Fringe enzymes, POGLUT1, MGAT3). Here we review the characteristics of these diseases and potential molecular mechanisms.

Exploration of the Potential Role for Aβ in Delivery of Extracellular Copper to Ctr1.

Amyloid beta (Aβ) peptides are notorious for their involvement in Alzheimer's disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood-brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aβ peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aβ peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aβ1-16 and Aβ4-16 peptides as well-established non-aggregating models of major Aβ species in healthy and AD brains, and the Ctr1-14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aβ peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aβ1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aβ4-16/Ctr1-14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aβ species released Cu(I) to Ctr1-14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aβ model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aβ1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aβ4-16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aβ peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.

The Secreted Tyrosine Kinase Vlk Is Essential for Normal Platelet Activation and Thrombus Formation

Tyrosine phosphorylation of proteins secreted into the extracellular space has been observed in cell cultures and in vivo, yet little is known about the role that phosphorylation of extracellular proteins serves in modulating cell function. An important reason for the gap in our knowledge of the functional significance of extracellular protein phosphorylation has been the delay in identifying extracellular kinases. Within the last decade, however, bioinformatic strategies to identify kinases with signal peptides, coupled with biochemical approaches to characterize kinases in the secretory pathway, have described several kinases that phosphorylate secretory pathway and extracellular substrates. Of the known kinases containing signal sequences, Fam20B and VLK have been identified in platelets. VLK has been identified as a broadly expressed secretory pathway tyrosine kinase secreted from platelets in an activation dependent manner. Its role in platelet function, however, has not been previously studied. To understand the contribution of tyrosine phosphorylation of secreted factors and extracellular domains of transmembrane proteins in platelet function and thrombus formation, we generated mice whose platelets lacked VLK. Mice with megakaryocyte/platelet-specific VLK deficiency (Vlk-cKO) exhibited normal platelet abundance, volume and morphology, and tail clip bleeding times, but showed dramatic changes in platelet function in vitro and in vivo. In vivo, platelet accumulation was reduced by 90% in Vlk-cKO mice compared to control (Vlkf/f) littermates (P = 0.02) following laser-induced injury of cremaster arterioles (Figure). Likewise, fibrin generation was reduced in mice lacking platelet VLK by 62% (P = 0.009). In vitro, evaluation of resting and thrombin-stimulated VLK-deficient platelets demonstrated a significant decrease of several tyrosine phosphobands compared to control. Platelet function testing of VLK-deficient platelets (Figure) showed decreased platelet aggregation in response to stimulation with 100 µM AYPGKF, a PAR4 agonist, (Vlkf/f: 70+5.1%; Vlk-cKO: 23+8.0%) or 4 µg/mL collagen (Vlkf/f: 53+2.5%; Vlk-cKO: 27.5+2.9%). Dense and α-granule release in response to AYPGKF were also significantly decreased in platelets in which VLK had been silenced. In contrast, Vlk-cKO platelets aggregated normally in response to either 10 µM, 40 µM, or 100 µM ADP, and the aggregation defect in response to low doses of AYPGKF was reversed by subthreshold concentrations (2.5 µM) of ADP. Furthermore, stimulation with high-dose 150 µM AYPGFK or 5 U/ml thrombin resulted in comparable platelet function and ATP secretion in control and Vlk-cKO platelets respectively, ruling out a storage pool defect. Taken together, these results suggest that a dense granule secretion defect contributes to the decrease in platelet aggregation observed in platelets in which VLK is absent. In human platelets, tyrosines phosphorylated in secreted and extracellular domains of transmembrane proteins implicated in the regulation of platelet function were identified by mass spectroscopy analysis. Extracellular proteins or proteins with phosphosites that mapped to extracellular domains included ectonucleoside triphosphate diphosphodydrolase 6 [ENTPD6], platelet basic protein, integrin αIIß, and multimerin-1. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus dependent platelet aggregation and thrombus formation, and provide the first evidence that secreted kinases contribute to platelet function. Disclosures De Ceunynck: Sanofi: Current Employment. Dilks:PlateletBiogenesis: Current Employment. Peters:PlateletBiogenesis: Current Employment. Noetzli:Anylam: Current Employment. Rosen:Keros Therapeutics: Membership on an entity's Board of Directors or advisory committees. Italiano:PlateletBioGenesis: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding. Flaumenhaft:STRM.Bio: Membership on an entity's Board of Directors or advisory committees; PlateletDiagnostics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties, Research Funding; QuercisPharma: Membership on an entity's Board of Directors or advisory committees.

Molecular interaction of an antagonistic amylin analog with the extracellular domain of receptor activity-modifying protein 2 assessed by fluorescence polarization

The peptide hormone amylin receptor is a complex of the calcitonin receptor (CTR) and an accessory protein called receptor activity-modifying proteins (RAMPs). The soluble extracellular domain (ECD) of CTR is an important binding site of peptide hormone calcitonin. RAMPs also have an ECD and the association of CTR ECD with RAMP ECD enhances the affinity of peptide hormone amylin. However, the mechanism of how RAMP ECD association enhances amylin affinity remains elusive. Here, we report evidence supporting direct molecular interaction between an antagonistic amylin analog AC413 and RAMP2 ECD. We measured FITC-labeled peptide affinity for purified receptor ECD using fluorescence polarization (FP). We first found that RAMP2 ECD addition to maltose-binding protein (MBP)-tagged CTR ECD and an engineered MBP-tagged RAMP2 ECD-CTR ECD fusion protein (MBP-RAMP2-CTR ECD fusion) enhanced AC413 affinity. This suggests that these recombinant ECD systems represent functional amylin receptors. Interestingly, AC413 C-terminal residue Tyr25 (Y25) to Pro mutation eliminated its selective affinity for the MBP-RAMP2-CTR ECD fusion suggesting the critical role of the AC413 C-terminal residue in amylin receptor selectivity. Our structural model of the RAMP2 ECD:CTR ECD complex predicted molecular interaction of AC413 C-terminal residue Y25 with RAMP2 Glu101 (E101). Our FP peptide-binding assay showed that the RAMP2 E101A mutation of MBP-RAMP2-CTR ECD fusion decreased AC413 affinity by 7-fold, while the affinity of AC413 with the Y25P mutation was minimally changed. Consistently, AC413 binding affinity for the MBP-free RAMP2-CTR ECD fusion protein was also markedly decreased by the RAMP2 E101A mutation, while the affinity of AC413 with the Y25P mutation was moderately decreased. Together, our results support the molecular interaction between the AC413 C-terminal residue Y25 and RAMP2 E101 expanding our understanding of how the accessory protein RAMP2 enhances affinity of peptide hormone amylin for its receptor.