C-terminal Amino Acid Sequence Research Articles

A-042 Assessment of Known and Newly Discovered IGF-1 Variants by a High-throughput Clinical Reference Laboratory

Abstract Background The top-down approach to quantitate intact insulin-like growth factor 1 (IGF-1) by liquid chromatography coupled to high-resolution, accurate-mass, mass spectrometry (LC-HRAM-MS) is a highly specific and robust technique. The method also provides a unique opportunity to study point mutations represented as amino acid sequence variants in IGF-1. While physical consequences of most variants remain unknown, those in or close to the binding region of the IGF-1 protein (positions 30-62) may be pathogenic, while those at the C-terminus are likely benign. In addition, information about the presence of IGF-1 variants is critical for accurate estimation of growth hormone levels. In the current study, our goal was to assess the presence of new and known variants by targeted screening of a clinical population. Methods LC-HRAM-MS was used for the detection of the wild type (WT) IGF-1 and its variants in specimens submitted for IGF-1 testing. In total, 2,013,503 specimens were screened. A novel variant detection and identification (NVDI) approach was employed that uses variant group m/z, isotopic peak index (IPi), and relative retention time (rRT) to characterize IGF-1 variants. Deidentified specimens were analyzed with tandem mass spectrometry (MS/MS) and DNA sequencing to confirm variants. Results In the specimens that were analyzed, 0.44% had IGF-1 variants and 98% of these had amino acid substitutions in the C-terminal region. We identified 8 variants represented in the Exome Aggregation Consortium (ExAC) database (A67T, A70T, P66A, A67S, S34N, A38V, R36Q, and T4M), 2 previously reported variants (V44M and A67V), and 11 new variants (Y31H, S33P, R50Q, R56K, T41I, A62T, S34G, N26K, V44L, G7A, and R36W). The underlined variants listed were discovered in the current study and are first reported here. In total, 16 variants had amino acid substitutions in or close to the binding region of the IGF-1 protein, and these were found in 0.4% of patients with variants in the clinical population. Conclusions In the study population, almost all patients with IGF-1 variants had point mutations affecting the C-terminal amino acid sequence. Although these variants are likely benign, they could affect patient care if IGF-1 levels were underestimated by being based purely on WT concentrations. Pathogenic or possibly pathogenic variants occurred in a small but important fraction of the study population. The discovery of novel variants further validates this platform as a discovery engine for IGF-1 variants. We expect more variants to be discovered with more patients and broader criteria. Information about the presence of IGF-1 variants is critical for proper estimation of growth hormone levels. Patients with variants should have their physician informed to properly assess the patient’s condition. This collaboration between clinical laboratories and attending physicians may result in the characterization and better understanding of the role and effect of IGF-1 variants on growth disorders.

μ-Opioid receptor transcriptional variants in the murine forebrain and spinal cord

Oprm1, the gene encoding the μ-opioid receptor, has multiple reported transcripts, with a variable 3′ region and many alternative sequences encoding the C-terminus of the protein. The functional implications of this variability remain mostly unexplored, though a recurring notion is that it could be exploited by developing selective ligands with improved clinical profiles. Here, we comprehensively examined Oprm1 transcriptional variants in the murine central nervous system, using long-read RNAseq as well as spatial and single-cell transcriptomics. The results were validated with RNAscope in situ hybridization. We found a mismatch between transcripts annotated in the mouse genome (GRCm38/mm10) and the RNA-seq results. Sequencing data indicated that the primary Oprm1 transcript has a 3′ terminus located on chr10:6,860,027, which is ∼ 9.5 kilobases downstream of the longest annotated exon 4 end. Long-read sequencing confirmed that the final Oprm1 exon included a 10.2 kilobase long 3′ untranslated region, and the presence of the long variant was unambiguously confirmed using RNAscope in situ hybridization in the thalamus, striatum, cortex and spinal cord. Conversely, expression of the Oprm1 reference transcript or alternative transcripts of the Oprm1 gene was absent or close to the detection limit. Thus, the primary transcript of the Oprm1 mouse gene is a variant with a long 3′ untranslated region, which is homologous to the human OPRM1 primary transcript and encodes the same conserved C-terminal amino acid sequence.

Efficient expression and purification of tag-free recombinant human procalcitonin (hPCT) with precise sequence in E.coli

We present an efficient method for expression and purification of recombinant human procalcitonin (hPCT) in E. coli T7 express LysY/Iq cells, ensuring precise N- and C-terminal amino acid sequences. Our method involves fusing codon-optimized cDNA with two distinct tag sequences: eXact tag and chitin binding domain (CBD) tag. To purify the protein, we employ a two-step affinity chromatography process. Firstly, we utilize the N-terminal Profinity eXact tag and purify the protein through Profinity eXact-affinity column chromatography using a resin on which a mutant subtilisin protease was immobilized. The eXact tag was removed by adding NaF to activate the enzyme. Subsequently, the digested sample containing C-terminal CBD tag is directly loaded for the second step of chitin affinity chromatography. Elution is achieved through dithiothreitol (DTT)-catalyzed self-cleavage of the intein sequence from the fusion protein. As a result, the target protein is selectively recovered in the flow-through, completely tag-free, with a purity exceeding 95%. To ensure high purity and eliminate potential contaminants, we effectively remove E. coli host DNA and endotoxins through a combination of streptomycin sulfate, Triton X-114, and ammonium sulfate treatment. The exceptional level of purity obtained eliminates the need for further purification steps in most applications. This highly purified hPCT can be used as a calibrator in procalcitonin or calcitonin immunoassays. Notably, our approach effectively manages small peptides that are prone to degradation by E. coli host proteases, offering a robust solution for various research and application requirements.

Characterising the structural and cellular role of immunoglobulin C-terminal lysine in secretory pathways.

Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.

Optimization of an alum-anchored clinical HIV vaccine candidate

In the ongoing effort to develop a vaccine against HIV, vaccine approaches that promote strong germinal center (GC) responses may be critical to enable the selection and affinity maturation of rare B cell clones capable of evolving to produce broadly neutralizing antibodies. We previously demonstrated an approach for enhancing GC responses and overall humoral immunity elicited by alum-adjuvanted protein immunization via the use of phosphoserine (pSer) peptide-tagged immunogens that stably anchor to alum particles via ligand exchange with the alum particle surface. Here, using a clinically relevant stabilized HIV Env trimer termed MD39, we systematically evaluated the impact of several parameters relevant to pSer tag composition and trimer immunogen design to optimize this approach, including phosphate valency, amino acid sequence of the trimer C-terminus used for pSer tag conjugation, and structure of the pSer tag. We also tested the impact of co-administering a potent saponin/monophosphoryl lipid A (MPLA) nanoparticle co-adjuvant with alum-bound trimers. We identified MD39 trimer sequences bearing an optimized positively-charged C-terminal amino acid sequence, which, when conjugated to a pSer tag with four phosphates and a polypeptide spacer, bound very tightly to alum particles while retaining a native Env-like antigenicity profile. This optimized pSer-trimer design elicited robust antigen-specific GC B cell and serum IgG responses in mice. Through this optimization, we present a favorable MD39-pSer immunogen construct for clinical translation.

Pigment-dispersing factor is present in circadian clock neurons of pea aphids and may mediate photoperiodic signalling to insulin-producing cells.

The neuropeptide pigment-dispersing factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing.

Interaction of C-terminal p53 isoforms depends strongly upon DNA sequence and topology

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and β isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53β and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms

AbstractMutant calreticulin (CALR) proteins resulting from a −1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

Light and prey influence the abundances of two rhodopsins in the dinoflagellate Oxyrrhis marina

Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina. The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii. The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii. Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled.

MO034: Novel MUC1 variant identified by massively parallel sequencing explains interstitial kidney disease in a large Dutch family

Abstract BACKGROUND AND AIMS Chronic kidney disease (CKD) can be caused by a wide variety of systemic and renal disorders. In approximately 20% of patients, the cause of CKD remains unknown. Previous studies have shown that massively parallel sequencing (MPS) can be a valuable diagnostic tool in patients with unexplained CKD. Here, we describe a large family with kidney failure of unknown origin, in which we aimed to identify the primary renal disease diagnosis with MPS in a routine healthcare setting. METHOD We conducted MPS in a large family in the Netherlands including six generations with documented kidney failure at young age. Affected individuals had a bland sediment and kidney biopsy revealed interstitial fibrosis in several family members. Whole exome sequencing followed by variant filtering and analysis based on the CKD-Y (CKD-young) gene panel (v18.1, 141 genes) was performed by the Genome Diagnostics Section of the University Medical Center Utrecht. RESULTS MPS revealed a novel frameshift variant [c.326_350dup, p.(Ser119Profs*119), NM_001 204 286.1] in MUC1, which co-segregated with the renal phenotype in the family. This variant is positioned before the first variable number of tandem repeat (VNTR) domain of MUC1 and leads to a + 1 base frameshift in the open reading frame of the MUC1 mRNA. The predicted C-terminal amino-acid sequence of the resulting mutant protein is the same as the previously reported pathogenic cytosine insertions in the VNTR. Variants in MUC1 are associated with autosomal dominant tubulointerstitial kidney disease (ADTKD), which matches the clinical phenotype in this family. Immunohistochemical analysis in the index patient showed MUC1fs deposition in kidney tissue, confirming the diagnosis of ADTKD-MUC1. CONCLUSION We report a novel frameshift mutation in MUC1 identified with MPS. This study highlights that MUC1 variants positioned before the first VNTR region can lead to ADTKD and that genetic testing in patients with suspected ADTKD-MUC1 should also include regions outside of the VNTR. Additionally, this study confirms the relevance of MPS as a tool to screen adult patients with early-onset kidney failure.

Development of a Therapeutic Peptide for Cachexia Suggests a Platform Approach for Drug-like Peptides.

During the development of a melanocortin (MC) peptide drug to treat the condition of cachexia (a hypermetabolic state producing lean body mass wasting), we were confronted with the need for peptide transport across the blood-brain barrier (BBB): the MC-4 receptors (MC4Rs) for metabolic rate control are located in the hypothalamus, i.e., behind the BBB. Using the term "peptides with BBB transport", we screened the medical literature like a peptide library. This revealed numerous "hits"-peptides with BBB transport and/or oral activity. We noted several features common to most peptides in this class, including a dipeptide sequence of nonpolar residues, primary structure cyclization (whole or partial), and a Pro-aromatic motif usually within the cyclized region. Based on this, we designed an MC4R antagonist peptide, TCMCB07, that successfully treated many forms of cachexia. As part of our pharmacokinetic characterization of TCMCB07, we discovered that hepatobiliary extraction from blood accounted for a majority of the circulating peptide's excretion. Further screening of the literature revealed that TCMCB07 is a member of a long-forgotten peptide class, showing active transport by a multi-specific bile salt carrier. Bile salt transport peptides have predictable pharmacokinetics, including BBB transport, but rapid hepatic clearance inhibited their development as drugs. TCMCB07 shares the general characteristics of the bile salt peptide class but with a much longer half-life of hours, not minutes. A change in its C-terminal amino acid sequence slows hepatic clearance. This modification is transferable to other peptides in this class, suggesting a platform approach for producing drug-like peptides.

Genetic Diversity, Repeat Motifs, and Natural Selection at the C-Terminal Knob-Associated Histidine Rich Protein (KAHRP) of Plasmodium falciparum Clinical Samples from Saudi Arabia.

Background Malaria is still a public health problem in Saudi Arabia specifically in the Jazan region. Plasmodium falciparum knob-associated histidine-rich proteins (PfKAHRPs) play an important role in cerebral malaria pathophysiology as well as pathogenesis of P. falciparum infections. The repeat region of PfKAHRP C-terminal interaction domain has been found to bind to the infected red blood cells and the vascular endothelium. Thus, this study aimed to assess the allelic variations, genetic diversity, and natural selection acting at the C-terminal PfKAHRP between parasite isolates from Saudi Arabia. Materials and Methods The PfKHARP C-terminal interaction domain was successfully PCR-amplified and sequence data from 441 clinical isolates from Saudi Arabia were obtained. The DnaSP v5.10 software was used to determine the genetic diversity, polymorphism, haplotype, and natural selection. Haplotype network analysis was constructed by using the median-joining method in the NETWORK version 5.0.0.1 software. Results Alignment and analysis of 441 C-terminal PfKAHRP-deduced amino acid sequences identified 5 genotypes (I–V) based on the decapeptide repeat arrangements (TKEASTSKEA, TKEASTSKGA, TKEASTTEGA, and TKEASTSKRA). Among the repeat types, Type I (49.43%, 218/441) was the most abundant in Saudi Arabia, followed by Type II (48.29%, 213/441). Overall, the nucleotide diversity in the PfKHARP C-terminal region was found to be low in Saudi Arabia (π = 0.00142); however, natural selection tests indicated positive selection (dN-dS = 1.64, P < 0.05) which was due to the variations within the repeat motifs. Genealogical relationship haplotype network of PfKAHRP from 4 different countries (i.e., Saudi Arabia, Iran, Burundi, and India) revealed 1 major shared haplotype cluster (H_1) with samples representative from all 4 countries (Saudi Arabia; n = 441, Burundi; n = 4, Iran; n = 13, and India; n = 1). Conclusion Since this is the first study to report on genetic diversity of C-terminal PfKAHRP interaction domain and the repeat motifs from clinical samples in Saudi Arabia, it will contribute towards the rational design of antiadhesion drug therapies for P. falciparum malaria.

Demonstration of Equivalence of Generic Glatiramer Acetate and Copaxone®.

The objective of the current work was to demonstrate the equivalence of Mylan’s glatiramer acetate (GA) to that of the reference product Copaxone® (COP) using the four criteria for active pharmaceutical ingredient sameness as established by the US Food and Drug Administration (FDA). The reaction scheme used to produce Mylan’s glatiramer acetate (MGA) was compared with that of COP, determined from publicly available literature. Comparative analyses of MGA and COP were performed for physicochemical properties such as amino acid composition and molecular weight distributions. Spectroscopic fingerprints were obtained using circular dichroism spectroscopy. Structural signatures for polymerization and depolymerization including total diethylamine (DEA) content, relative proportions of DEA-adducted amino acids, and N-and C-terminal amino acid sequences were probed with an array of highly sensitive analytical methods. Biological activity of the products was assessed using validated murine Experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. MGA is produced using the same fundamental reaction scheme as COP and was shown to have equivalent physicochemical properties and composition. Analyses of multiple structural signatures demonstrated equivalence of MGA and COP with regard to polymerization, depolymerization, and propagational shift. Examination of the impact on prevention and treatment of EAE demonstrated equivalence of MGA and COP with respect to both activity and toxicity, and thereby provided confirmatory evidence of sameness. A rigorous, multi-pronged comparison of MGA and COP produced using an equivalent fundamental reaction scheme demonstrated equivalent physicochemical properties, structural signatures for polymerization and depolymerization, and biological activity as evidenced by comparable effects in EAE. These studies demonstrate the equivalence of MGA and COP, establishing active ingredient sameness by the US Food and Drug Administration (FDA) criteria for GA, and provide compelling evidence that the FDA-approved generic MGA can be substituted for COP for the treatment of patients with relapsing-remitting MS.

Inhibition of Vasoactive Intestinal Peptide Signaling with More Potent Inhibitors Augments T-Cell Activation and Prolongs Survival in Leukemic Mice

Introduction: Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide with immunosuppressive effects on T cells. Inhibition of VIP receptor (VIP-R) signaling by VIPhyb, a first-generation VIP-R antagonist, not only enhances T-cell activation and proliferation in vitro but also improves T cell dependent anti-tumor response in mouse models of acute myeloid leukemia (AML) and T lymphoblastic leukemia (Li et al. 2016; Petersen, Li, and Waller 2017). The goal of the project is to develop more potent VIP-R antagonists that generate a significantly more robust anti-tumor response in mouse models of AML, when compared to VIPhyb and validate a screening method to test the efficacy of novel peptides in activating human T cells in vitro. In this study, we report, for the first time, the activity of novel VIP-R antagonists on the activation profile of human T cells.Methods: We utilized in-silico-based modeling to identify 10 novel VIP-R antagonists from a library of 300 peptide sequences predicted to have increased binding affinity to VIP receptors VPAC1 and VPAC2 when compared to VIP or VIPhyb (Table 1). The library was generated from peptide sequences that contain the six charged N-terminal residues of the neurotensin present in VIPhyb with two or more amino acid substitutions within the C-terminal amino acid sequence of VIP. The ability of these peptides was tested in vitro using T cells from multiple healthy human donors activated using anti-CD3 monoclonal antibody coated plates. Activation status was assessed by flow cytometry of CD69, OX40, PD1, Tim3 and Lag3 expression relative to control cultures without added peptides. Potency of the novel antagonists in vivo was tested in a mouse AML model, by treating C1498- bearing mice with subcutaneous administration of VIP, VIPhyb, scrambled peptide (SCRAM1) or the second-generation VIP-R antagonists (labeled as ‘ANT‘) from day 6-12 after tumor implantation.Results: Inhibiting VIP-R signaling in human T cells using second-generation VIP-R antagonists ANT008, ANT308 and ANT195 showed approximately 1.5-to-2-fold increase in CD69, OX40, Tim3, Lag3 and OX40 expression in CD4+ T cells following 24-hour of drug exposure compared to control cultures (Figure 1A). A smaller effect of VIP-R antagonists on activation of CD8+ subsets was observed (Figure 1B). Among the peptides, ANT195 was superior to ANT008 and ANT308 which shows potency even at 1μM compared to 3μM for ANT008 and ANT308. However, significant increase in CD69 expression was observed in both CD4+ and CD8+ T cells in cultures treated with ANT308 (Figure 1 A&B, *p<0.05). Viability of the T cells was not affected by incubation with the queried peptides (Data not shown). These data corresponded to in vivo activity of the novel VIP-R antagonists such as ANT308 and ANT195 which rendered 40% of mice leukemia-free at day 60 compared to only 5% long-term survival with VIPhyb (Figure 2). Another candidate, ANT300, increased median survival time (MST) by up to 47 days compared to MST of 34 days with VIPhyb (Figure 2).Conclusions: Here, we report a simple and robust in vitro method to screen for immune activity potential of novel second-generation VIP-R antagonists using human T cells. Preliminary screen shows VIP-R antagonists augment activation of both CD4+ and CD8+ T cells. Our results indicate that ANT308 and ANT195 are more potent VIP-R antagonists with enhanced activity in vitro (human) and in vivo (mouse) than VIPhyb and ANT008, which demonstrate lower predicted binding affinities to VPAC1 and VPAC2. Our study supports the hypothesis that higher predicted binding affinity to VPAC1 and/or VPAC2 is associated with enhanced activity in stimulating human T cells and promoting anti-leukemia activity in mice. Further mechanistic studies on how inhibition of VIP-R signaling augments T cell activation and function are underway. These novel antagonists can lead to peptide-based immunotherapy for the treatment of various liquid cancers. Clinical development of this novel concept will require appropriate pre-clinical pharmacokinetic and toxicology studies. [Display omitted] DisclosuresWaller: Cambium Oncology: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Verastem Oncology: Consultancy, Research Funding.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2–7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0–7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

Complex CSF3R Protein Isoform Interactions Dictate Cytokine Responsiveness

CSF3R, the receptors for granulocyte colony stimulating factor, is a critical regulator of neutrophil production. Multiple CSF3R mRNA transcripts have been identified and are annotated in Genbank. The expression and function of the different CSF3R proteins have not been fully elucidated. We generated antibodies specific for two of the identified and annotated isoforms, V3 and V4. CSF3R-V4 is a truncated variant of V1 with a unique C-terminal 34 amino acids and this variant confers enhanced growth signals. Changes in the ratio of V1:V4 isoforms have been implicated in chemotherapy resistance and relapse of AML. CSF3R-V3 is a variant of V1 with a 27 amino acid insertion between two conserved domains in the cytoplasmic portion of the receptor involved in JAK/STAT activation, termed the box 1 and box 2. CSF3R-V3 produces reduced proliferative signaling in response to G-CSF. When V3 is co-expressed with V1, proliferative signaling is reduced in a concentration dependent manner. In order to generate custom rabbit polyclonal antibodies specific for CSF3R-V3 and CSF3R-V4 we used either a peptide that corresponds to a unique amino acid sequence present only in CSF3R-V3 or a peptide specific for a portion of the C-terminal amino acid sequence unique to the CSF3R-V4 isoform conjugated to an immunogenic carrier protein. These immunogens both produced robust immune responses, and the polyclonal antibodies were subsequently purified from bulk sera. Immunoblot analysis of lysates from Ba/F3 cells expressing CSF3R-V1 (V1), CSF3R-V3 (V3), or CSF3R-V4 (V4) demonstrated that both the custom generated anti-CSF3R-V3 and anti-CSF3R-V4 antibodies were very specific, recognizing only the appropriate CSF3R receptor isoform. All three CSF3R splice variants are recognized by commercially available anti-CSF3R (clone LMM741 to CD114), while the anti-CSF3R-V4 custom antibody and the custom anti-CSF3R-V3 antibody recognizes only the CSF3R-V4 and CSF3R-V3 isoforms, respectively. We next sought to detect the CSF3R receptor isoforms in primary human cells. Using our custom antibodies, we detected for the first time, both the CSF3R-V3 and CSF3R-V4 receptor forms in primary neutrophils isolated from healthy donors. Each of the CSF3R isoforms produce unique signaling, and we hypothesized that the observed differences in G-CSF-dependent signaling is produced by the expression level of each receptor isoform via both homodimerization and by heterodimerization of the receptor splice variant proteins. To investigate the potential for heterodimerization of the CSF3R-V1 with the V3 and V4 isoforms, we generated a CSF3R-V1 with a c-terminal epitope tag and co-expressed this construct with both CSF3R-V3 or CSF3R-V4. Immunoprecipitation with an antibody to the epitope tag (recognizing the V1 variant) followed by immunoblotting with the custom anti-V3 or anti-V4 antibodies demonstrated that both CSF3R-V3 and CSF3R-V4 co-immunoprecipitated with CSF3R-V1, in agreement with our hypothesis that the splice variants form receptor heterodimers. Of note, the CSF3R receptor heterodimers are detected even in the absence of G-CSF, thus demonstrating that CSF3R exist as a preformed receptor dimer in an inactive state. In conclusion, we have generated antibodies that specifically detect the CSF3R-V3 and the CSF3R-V4 receptor proteins. These are the first studies to demonstrate the expression of the CSF3R splice variants at the protein level, in both cell lines and primary human cells. In addition, these are the first studies to demonstrate the formation of heterodimers of the CSF3R splice variants, providing a mechanism for the observed alteration in ligand-dependent signaling produced under conditions of altered splice variant expression. Disclosures Avalos: Juno: Membership on an entity's Board of Directors or advisory committees; Best Practice-Br Med J: Patents &amp; Royalties: receives royalties from a coauthored article on evaluation of neutropenia.

Expression of protein kinase HIPK2 is subject to a quality control mechanism that acts during translation and requires its kinase activity to prevent degradation of nascent HIPK2

HIPK2 is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in a broad spectrum of biological processes. We have previously reported that the expression of HIPK2 is auto-regulated by a mechanism that depends on the activity of its kinase domain, leading to decreased expression of kinase-dead versus wild-type HIPK2. We have now explored this mechanism in more detail. Differential expression of wild-type and kinase-dead HIPK2 is dependent on sequences located in the C-terminal part of HIPK2, but is only observed when this part of HIPK2 is translated together with the defective kinase domain. On their own, both the defective kinase domain and the C-terminal amino acid sequences are expressed at normal levels and independently of kinase activity. Insertion of a 2A-ribosomal skipping sequence into the HIPK2 coding sequence revealed that the differential expression of wild-type and kinase-dead HIPK2 is caused by degradation of nascent kinase-dead HIPK2. Because HIPK2 is constitutively active and auto-activates its kinase domain already during its translation we speculate that the regulatory mechanism discovered here serves as a quality control mechanism that leads to degradation of nascent kinase molecules with defective kinase domains. Overall our work provides insight into a novel auto-regulatory mechanism of HIPK2 expression, thereby adding a new layer of control to the regulation of HIPK2.

Initial In Vitro and In Vivo Evaluation of a Novel CCK2R Targeting Peptide Analog Labeled with Lutetium-177.

Targeting of cholecystokinin-2 receptor (CCK2R) expressing tumors using radiolabeled minigastrin (MG) analogs is hampered by rapid digestion of the linear peptide in vivo. In this study, a new MG analog stabilized against enzymatic degradation was investigated in preclinical studies to characterize the metabolites formed in vivo. The new MG analog DOTA-DGlu-Pro-Tyr-Gly-Trp-(N-Me)Nle-Asp-1Nal-NH2 comprising site-specific amino acid substitutions in position 2, 6 and 8 and different possible metabolites thereof were synthesized. The receptor interaction of the peptide and selected metabolites was evaluated in a CCK2R-expressing cell line. The enzymatic stability of the 177Lu-labeled peptide analog was evaluated in vitro in different media as well as in BALB/c mice up to 1 h after injection and the metabolites were identified based on radio-HPLC analysis. The new radiopeptide showed a highly increased stability in vivo with >56% intact radiopeptide in the blood of BALB/c mice 1 h after injection. High CCK2R affinity and cell uptake was confirmed only for the intact peptide, whereas enzymatic cleavage within the receptor specific C-terminal amino acid sequence resulted in complete loss of affinity and cell uptake. A favorable biodistribution profile was observed in BALB/c mice with low background activity, preferential renal excretion and prolonged uptake in CCK2R-expressing tissues. The novel stabilized MG analog shows high potential for diagnostic and therapeutic use. The radiometabolites characterized give new insights into the enzymatic degradation in vivo.

Role of Arginine Methylation in Alternative Polyadenylation of VEGFR-1 (Flt-1) pre-mRNA.

Mature mRNA is generated by the 3ʹ end cleavage and polyadenylation of its precursor pre-mRNA. Eukaryotic genes frequently have multiple polyadenylation sites, resulting in mRNA isoforms with different 3ʹ-UTR lengths that often encode different C-terminal amino acid sequences. It is well-known that this form of post-transcriptional modification, termed alternative polyadenylation, can affect mRNA stability, localization, translation, and nuclear export. We focus on the alternative polyadenylation of pre-mRNA for vascular endothelial growth factor receptor-1 (VEGFR-1), the receptor for VEGF. VEGFR-1 is a transmembrane protein with a tyrosine kinase in the intracellular region. Secreted forms of VEGFR-1 (sVEGFR-1) are also produced from the same gene by alternative polyadenylation, and sVEGFR-1 has a function opposite to that of VEGFR-1 because it acts as a decoy receptor for VEGF. However, the mechanism that regulates the production of sVEGFR-1 by alternative polyadenylation remains poorly understood. In this review, we introduce and discuss the mechanism of alternative polyadenylation of VEGFR-1 mediated by protein arginine methylation.