Intracellular C-terminal Region Research Articles

C-terminus of PIEZO1 governs Ca2+ influx and intracellular ERK1/2 signaling pathway in mechanotransduction

Cells sense and respond to extracellular mechanical stress through mechanotransduction receptors and ion channels, which regulate cellular behaviors such as cell proliferation and differentiation. Among them, PIEZO1, piezo-type mechanosensitive ion channel component 1, has recently been highlighted as a mechanosensitive ion channel in various cell types including mesenchymal stem cells. We previously reported that PIEZO1 is essential for ERK1/2 phosphorylation and osteoblast differentiation in bone marrow-derived mesenchymal stem cells (BMSCs), induced by hydrostatic pressure loading and treatment with the PIEZO1-specific activator Yoda1. However, the molecular mechanism underlying how PIEZO1 induces mechanotransduction remains unclear. In this study, we investigated that the role of the C-terminus in regulating extracellular Ca2+ influx and activating the ERK1/2 signaling pathway. We observed the activation of Fluo-4 AM in the Yoda1-stimulated human BMSC line UE7T-13, but not in a calcium-depleted cell culture medium. Similarly, Western blotting analysis revealed that Yoda1 treatment induced ERK1/2 phosphorylation, but this induction was not observed in calcium-depleted cell culture medium. To investigate the functional role of the C-terminus of PIEZO1, we generated HEK293 cells stably expressing the full-length mouse PIEZO1 (PIEZO1-FL) and a deletion-type PIEZO1 lacking the C-terminal intracellular region containing the R-Ras-binding domain (PIEZO1-ΔR-Ras). We found that Yoda1 treatment predominantly activated Flou-4 AM and ERK1/2 in PIEZO1-FL-trasfected cells but neither in PIEZO1-ΔR-Ras-transfected cells nor control cells. Our results indicate that the C-terminus of PIEZO1, which contains the R-Ras binding domain, plays an essential role in Ca2+ influx and activation of the ERK1/2 signaling pathway, suggesting that this domain is crucial for the mechanotransduction of osteoblastic differentiation in BMSCs.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site.

The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory β and γ subunits. The γ1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BKα is indispensable in BK channel modulation by the γ1 subunit. Notably, synthetic peptide mimics of the γ1-3 subunits' C-terminal positively charged regions caused 30-50 mV shifts in BKα channel voltage-gating toward the hyperpolarization direction. The cationic cell-penetrating HIV-1 Tat peptide exerted a similar BK channel-activating effect. The BK channel-activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the γ subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the γ1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the γ1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary γ subunits.

Critical contribution of the intracellular C-terminal region to TRESK channel activity is revealed by the epithelial Na+ current ratio method

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology, and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in a low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function.

TRP channel responsible for the responses to collision, agitation, and shear in Chlamydomonas.

Swimming microorganisms show various responses to mechanical stimuli. Chlamydomonas cells temporarily swim backward on collision with an obstacle. Beat frequency of cilia increases when agitation is applied by voltexing. Cilia are detached on application of shear with a Dounce-type homogenizer. In this study, we show that a knockout mutant of TRP11, a TRP channel of Chlamydomonas, is defective in all these mechanoresponses. To mimic the mechanical stimulus on collision, we caught a single cell on a glass pipette and tapped the anterior cilia-bearing end of the cell with a micro-glass rod while recording the ciliary motility. On tapping the cell, the bending pattern of cilia changed from the forward mode to the reverse mode. The knockout mutant of TRP11 was deficient in showing this response. The response rarely occurred when the speed of the rod was slower than 100 µm/s, which roughly coincides with the swimming velocity. In addition, the bending pattern conversion hardly occurred when the rod stopped moving 1 µm or more apart from the anterior end of the cell. These findings are consistent with the localization of TRP11 in the proximal region of cilia and the idea that the avoiding reaction on collision is brought about by the activation of TRP11 through the deformation of the proximal region of cilia. TRP11 structure predicted by AlphaFold2 shows conservation of the typical TRP channel structure including pre-S1, S1-S4, S4-S5 linker, S5-pore helix-S6, and TRP helix, while bearing long but poorly resolved sequences of extracellular S1-S2 linker and intracellular C-terminal region.

Klotho protein and atherosclerotic cardiovascular diseases: prolonging the thread of life

To date, the most widely used biomarkers are natriuretic peptides and cardiac troponins. Many other biomarkers have also been identified, but only a few of them have found application in actual clinical practice. This review focuses on the Klotho protein and its role in cardiovascular diseases.
 In 1997, a gene that slows down the aging process was identified. It was named Klotho (Greek, ώ, spinning; Latin, Clotho; English, Klotho) in honor of the goddess of ancient Greek mythology, spinning the thread of life and being the personification of the steady, calm course of fate. Mice with an insertional mutation in the region of the Klotho gene promoter were characterized by premature aging processes. Three families of Klotho are known: -Klotho, -Klotho, and -Klotho, and the most studied is -Klotho. The Klotho protein consists of a large extracellular domain and a short C-terminal intracellular region. The extracellular domain consists of two repeat sequences called KL1 and KL2. -Klotho and -Klotho contain the KL1 and KL2 domains, respectively, and -Klotho contains only the KL1 domain. The Klotho gene in humans is located on chromosome 13q12 and consists of five exons. The Klotho protein is mainly expressed in the distal convoluted tubules of the kidneys and the vascular epithelial plexus of the cerebral ventricles. At lower concentrations, the Klotho gene is also found in other organs and tissues, particularly in the heart.
 Many studies have demonstrated the protective role of the Klotho protein in cardiovascular pathology. The pleiotropic properties of this protein are reflected in the diversity and interaction of cardioprotective mechanisms. The regulation of the concentrations of the Klotho protein in the blood and its expression in heart cells with the help of drugs can play a significant role in cellular metabolism and represent a promising target for the treatment of heart and vascular pathologies.

Signal Transduction of C-Terminal Phosphorylation Regions for Equine Luteinizing Hormone/Chorionic Gonadotropin Receptor (eLH/CGR).

This study aimed to investigate the signal transduction of phosphorylation sites at the carboxyl (C)-terminal region of equine luteinizing hormone/chorionic gonadotropin receptor (eLH/ CGR). The eLH/CGR has a large extracellular domain of glycoprotein hormone receptors within the G protein-coupled receptors. We constructed a mutant (eLH/CGR-t656) of eLH/ CGR, in which the C-terminal cytoplasmic tail was truncated at the Phe656 residue, through polymerase chain reaction. The eLH/CGR-t656 removed 14 potential phosphorylation sites in the intracellular C-terminal region. The plasmids were transfected into Chinese hamster ovary (CHO)-K1 and PathHunter Parental cells expressing β-arrestin, and agonist-induced cAMP responsiveness was analyzed. In CHO-K1 cells, those expressing eLH/CGR-t656 were lower than those expressing eLH/CGR wild-type (eLH/CGR-wt). The EC50 of the eLH/ CGR-t656 mutant was approximately 72.2% of the expression observed in eLH/CGR-wt. The maximal response in eLH/CGR-t656 also decreased to approximately 43% of that observed in eLH/CGR-wt. However, in PathHunter Parental cells, cAMP activity and maximal response of the eLH/CGR-t656 mutant were approximately 173.5% and 100.8%, respectively, of that of eLH/CGR-wt. These results provide evidence that the signal transduction of C-terminal phosphorylation in eLH/CGR plays a pivotal role in CHO-K1 cells. The cAMP level was recovered in PathHunter Parental cells expressing β-arrestin. We suggest that the signal transduction of the C-terminal region phosphorylation sites is remarkably different depending on the cells expressing β-arrestin in CHO-K1 cells.

Eys +/- ; lrp5 +/- Zebrafish Reveals Lrp5 Can Be the Receptor of Retinol in the Visual Cycle.

SummaryVision is essential for vertebrates including humans. Sustained vision is accomplished by retinoid metabolism, the “visual cycle,” where all-trans retinol (atROL) is incorporated into the retinal pigment epithelium (RPE) from photoreceptors presumably through decade-long missing receptor(s). Here, we show that the LDL-related receptor-5 (Lrp5) protein is linked to the retinol binding protein 1a (Rbp1a), the transporter of atROL in the visual cycle, by generating and analyzing the digenic eyes shut homolog+/-; lrp5+/− zebrafish, the same form of gene defect detected in a human case of inherited retinal degeneration. Global gene expression analysis followed by genetic study clarified that rbp1a played a role downstream of lrp5. Rbp1a protein was colocalized with Lrp5 protein at microvilli of RPE cells. Furthermore, Rbp1a directly bound to the C-terminal intracellular region of Lrp5 in vitro. Collectively, these results strongly suggest that Lrp5 is a potent candidate of the receptor of atROL in the visual cycle.

Structural basis for distinct quality control mechanisms of GABAB receptor during evolution.

Cell surface trafficking of many G protein-coupled receptors is tightly regulated. Among them, the mandatory heterodimer GABAB receptor for the main inhibitory neurotransmitter, γ-aminobutyric acid (GABA), is a model. In mammals, its cell surface trafficking is highly controlled by an endoplasmic reticulum retention signal in the C-terminal intracellular region of the GB1 subunit that is masked through a coiled-coil interaction with the GB2 subunit. Here, we investigate the molecular basis for the export of its homolog in Drosophila melanogaster that regulates the circadian rhythm and sleep. In contrast to mammals, the endoplasmic retention signal is carried by GB2, while GB1 reaches the cell surface alone. NMR analysis showed that the coiled-coil domain that controls GABAB heterodimer formation is structurally conserved between flies and mammals, despite specific features. These findings show the adaptation of a similar quality control system during evolution for maintaining the subunit composition of a functional heterodimeric receptor.

Rational Design of Monodispersed Mutants of Proteins by Identifying Aggregation Contact Sites Using Solubilizing Agents.

Suppression of protein aggregation is a subject of growing importance in the treatment of protein aggregation diseases, an urgent worldwide human health problem, and the production of therapeutic proteins, such as antibody drugs. We previously reported a method to identify compounds that suppress aggregation, based on screening using multiple terminal deletion mutants. We now present a method to determine the aggregation contact sites of proteins, using such solubilizing compounds, to design monodispersed mutants. We applied this strategy to the chemokine receptor-binding domain (CRBD) of FROUNT, which binds to the membrane-proximal C-terminal intracellular region of CCR2. Initially, the backbone NMR signals were assigned to a certain extent by available methods, and the putative locations of five α-helices were identified. Based on NMR chemical shift perturbations upon varying the protein concentrations, the first and third helices were found to contain the aggregation contact sites. The two helices are amphiphilic, and based on an NMR titration with 1,6-hexanediol, a CRBD solubilizing compound, the contact sites were identified as the hydrophobic patches located on the hydrophilic sides of the two helices. Subsequently, we designed multiple mutants targeting amino acid residues on the contact sites. Based on their NMR spectra, a doubly mutated CRBD (L538E/P612S) was selected from the designed mutants, and its monodispersed nature was confirmed by other biophysical methods. We then assessed the CCR2-binding activities of the mutants. Our method is useful for the protein structural analyses, the treatment of protein aggregation diseases, and the improvement of therapeutic proteins.

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction

Heterozygous missense variants in KCNQ2, which encodes the potassium channel subunit Kv7.2, are among the most common genetic causes of severe neonatal-onset epileptic encephalopathy. Because about 20% of known severe Kv7.2 missense changes lie within the intracellular C-terminal region, improving understanding of the underlying pathogenic mechanisms is important. We analyzed the basis for the severe phenotypes of Kv7.2 A337T and A337G, variants in the C-terminal’s calmodulin (CaM)-binding Helix A. When expressed heterologously in mammalian cells, alone or in combination with wild type Kv7.2 or with wild type Kv7.2 and Kv7.3, both variants strongly suppressed channel currents. A337T channels expressed alone exhibited significantly reduced protein half-life and surface trafficking and co-immunoprecipitated less CaM. For both variants, increasing cellular phosphatidylinositol 4,5-bisphosphate (PIP2) by overexpression of PI(4)P5-kinase restored current densities. For both variants, the fraction of current suppressed by activation of M1 muscarinic receptors with 10 μM oxotremorine methiodide, which depletes PIP2, was less than for controls. During voltage-sensitive phosphatase-induced transient PIP2 depletion and resynthesize, potassium current inhibition and recovery kinetics were both markedly slowed. These results suggest that these variants may reduce currents by a mechanism not previously described: slowing of PIP2 migration between the bulk membrane and binding sites mediating channel electromechanical coupling. A novel Kv7.2/3-selective opener, SF0034, rescued current amplitudes. Our findings show that these two Helix A variants suppress channel current density strongly, consistent with their severe heterozygous phenotypes, implicate impairment of CaM and PIP2 regulation in KCNQ2 encephalopathy pathogenesis, and highlight the potential usefulness of selective Kv7 openers for this distinctive pathogenic mechanism and patient subgroup.

Analysis of <i>eys</i> <sup>+/-</sup>; <i>lrp5</i> <sup>+/-</sup> Zebrafish Reveals LRP5 as a Strong Candidate for the Receptor Of All- <i>Trans</i> Retinol in the Visual Cycle

Vision is essential for vertebrates including humans. Sustained vision is accomplished by the retinoid metabolism, ‘visual cycle’, where all-trans retinol (atROL) is incorporated into the retinal pigment epithelium (RPE) from photoreceptors presumably through decade-long missing receptor(s). Here, we show that LDL-related receptor-5 (LRP5) protein is linked to retinol binding protein 1 (RBP1), the transporter of atROL in the visual cycle, by generating and analyzing the digenic eyes shut homolog+/-; lrp5+/- zebrafish, the same form of gene defects emerged from a human case report of a candidate inherited retinal degeneration. Global gene expression analysis followed by genetic study clarified that rbp1 played a role downstream of lrp5. RBP1 protein was colocalized with LRP5 protein at microvilli of RPE cells. Furthermore, RBP1 directly bound to C-terminal intracellular region of LRP5 in vitro. Collectively, these results strongly suggest that LRP5 is a potent candidate of the receptor of atROL in the visual cycle.

Selectins: functional importance as cell adhesion molecule in immune system

Selectins are cell membrane glycoproteins that recognize specific glycoconjugates expressed on the surface of cells. Then, selectins adjust cell-cell interactions that are important in inflammation, hemostasis and cancer metastasis. Selectins mediate leukocyte calls to move into the site of inflammation through interactions with activated endothelial cells or endogenous selectin ligands expressed in high endothelial venules. Types of selectins are divided into L-selectin, E-selectin and P-selectin, which are called to CD62L, CD62E, and CD62P, respectively. Each selectin is composed of four regions; the C-type lectin region of N-terminal, the epidermal growth factor (EGF) region, the intracellular C-terminal region, and the hydrophobic transmembrane region. They have similar structures but differ in their binding specificities and tissue distributions. The selectin family commonly recognizes the sialyl Lewis X (sLeX) on carbohydrate structures. Although biological ligands bound to each selectin are different from each other, they commonly bind to P-selectin glycoprotein ligand-1 (PSGL-1) ligand. The PSGL-1 ligand is a glycoprotein promoting cell adhesion in inflammatory responses. If the absence of selectins and their ligands in humans and animals are, should lead to persistent infections and diseases. Selectin family must be considered as a key subject for drug discovery since they have various functions depending on the ligand which they bind to.

Methylosome protein 50 associates with the purinergic receptor P2X5 and is involved in osteoclast maturation.

Purinergic signaling plays important roles in bone. P2X5, a member of ligand-gated ion channel receptors, has been demonstrated to regulate osteoclast maturation. However, the molecular mechanism of P2X5-mediated osteoclast regulation remains unclear. Here, we identified methylosome protein 50 (MEP50), a critical cofactor of the protein arginine methyltransferase 5 (PRMT5), as a P2X5-associating molecule. RNAi-mediated knockdown of MEP50 results in decreased formation of mature osteoclasts. MEP50 associates with P2X5, and this association requires the C-terminal intracellular region of P2X5. Additionally, impaired maturation of P2X5-deficient osteoclasts could be restored by transduction of full-length P2X5, but not a C-terminal deletion mutant of P2X5. These results indicate that P2X5 associates with MEP50 and suggest a link between the PRMT5 complex and P2X5 signaling in osteoclast maturation.

Differential expression of PMCA2 mRNA isoforms in a cohort of Spanish patients with breast tumor types.

The present study examined the mRNA expression levels of different isoforms of the plasma membrane calcium ATPase 2 (PMCA2) gene generated by alternative splicing at the first intracellular loop (site A) and C-terminal region (site C) in 85 human breast cancer tumor and 69 adjacent non-tumor tissues. Associations were identified between the expression of PMCA2 splice isoforms and the following clinical variables: Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status, tumor size, staging and histological classification, and lymph node status. Transcripts including splice site A or splice site C were amplified by reverse transcription-quantitative polymerase chain reaction using PMCA2 isoform-specific primers. Tumor and adjacent tissues were determined to express the different PMCA2 splice isoforms 2w, 2× and 2z (site A), and 2b (site C). The mRNA levels for these variants indicated high biological variability, but increased expression was observed in breast tumor tissues, compared with in adjacent tissues. Significantly increased PMCA2×/b expression levels were detected in breast tumor tissues histologically classified as lobulillar, compared with in ductal-types breast tumor tissues (P<0.028). Furthermore, PMCA2z expression was significantly associated with PR status (P<0.024, compared with in PR-negative tumor tissues), and PMCA2w expression was significantly associated with ER status (P<0.048, increased in ER-positive tumor tissues, compared with ER-negative tumor tissues). Finally, PMCA2b was overexpressed in HER2-positive tumor tissues, compared with in HER2-negative tumor tissues (P<0.014). The data demonstrated the differential mRNA expression of a number of splice site A and C variants of PMCA2 in breast tumor and adjacent tissues, depending on tumor hormone receptor status and histological classification. In agreement with previous data, PMCA2b was overexpressed in HER2-positive tumor tissues, indicating that high mRNA levels of this variant could be a marker of poor prognosis.

Targeting the Prolactin Receptor Signaling Using an Antipsychotic Drug to Suppress Pancreatic Cancer

Pancreatic cancer (PCa) is deadly cancer with an abysmal five‐year survival rate of &lt; 6%. PCa has a bad prognosis and is associated with resistance to standard of care chemotherapeutic gemcitabine and high recurrence rate after surgery. Unfortunately, gemcitabine remains the drug of choice for metastatic pancreatic adenocarcinoma, with minor improvements in survival rate. Hence, there is a need for novel therapeutic interventions for the treatment of PCa. Here we report that prolactin receptor (PRLR) is overexpressed in both PCa tissues and in cell lines. When it is bound by the ligand prolactin (PRL), various downstream events such as JAK‐STAT and ERK‐MAPK signaling pathways are activated. In PCa cell lines, PRL treatment induced dose‐ and time‐dependent STAT3, and ERK1/2 phosphorylation. Moreover, while PRL did not affect PCa cell proliferation, it significantly enhanced pancosphere formation suggesting that PRL affects stem cells. We manipulated PRLR levels using the CRISPR/Cas9 system. This resulted in the lower colony and spheroid formation capacity, decreased migration in vitro as well as reduced tumor‐forming capacity in an orthotopic model of PCa in C57BL/6 mice. Based on these results, we propose that PRL signaling is a novel target for therapeutic intervention. Currently, there are no known small molecules targeting PRL‐PRLR signaling, and X‐ray crystal structure of the intracellular domain of PRLR is not available. Hence, we obtained a homology model for the C‐terminal intracellular region of the receptor and performed a virtual screening for small molecules in silico. In this, we demonstrated that penfluridol (a first‐generation antipsychotic drug) interacts with the JAK2 binding site of the receptor. Penfluridol is known for its long half‐life and is used in the treatment of chronic schizophrenia. We experimentally confirmed binding of penfluridol to PRLR in the cells by using the cellular thermal shift assay (CETSA). Penfluridol treatment decreased PRL‐induced STAT3 and ERK phosphorylation, spheroid formation as well as the migration of PCa cells. We observed a significant increase in autophagy based on increased levels of LC3B, p62 and enhanced expression of autophagy‐related genes ATG‐5, −7 and −12 after penfluridol treatment. Penfluridol treatment also significantly decreased tumor burden in orthotopic PCa model and induced the expression of autophagic related proteins. Taken together, these results suggest that PRL‐PRLR signaling is important for PCa growth and repurposing penfluridol to target PRL‐signaling an effective therapeutic targeting strategy for treating the disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A point mutation in the heavy chain complementarity-determining region 3 (HCDR3) significantly enhances the specificity of an anti-ROS1 antibody

The proto-oncogene tyrosine kinase ROS1 plays a key role in carcinogenesis through gene rearrangement to form a fusion protein with other genes, in which the C-terminal intracellular region of ROS1 participates. The possibility of wild type ROS1 overexpression through epigenetic regulation has been proposed. Here, we generated an antibody, 3B20, reactive to the N-terminal region of ROS1 to use it for the detection of wild type ROS1 in cancerous tissues. Using immunoblot and immunoprecipitation analyses, we found that 3B20 also reacted with heat shock proteins (Hsp)70s. Using homology searching, ROS1 and Hsp70s were found to share an identical amino acid sequence: DLGT. Using alanine mutagenesis of ROS1, the epitope was found to harbor this sequence. To modify the idiotope with the aim of selecting more specific antibodies, we introduced random mutations into the heavy chain complementarity-determining region 3 and successfully generated an antibody clone, 3B20-G1K, with a point mutation that only reacted with ROS1 in enzyme-linked immunosorbent assays, and in immunoblot and immunoprecipitation analysis. In immunohistochemical analysis using 3B20-G1K, ROS1 was found to be absent in normal lung tissues and was overexpressed in a case of lung adenocarcinoma.

Identification of the ADPR binding pocket in the NUDT9 homology domain of TRPM2.

Activation of the transient receptor potential melastatin 2 (TRPM2) channel occurs during the response to oxidative stress under physiological conditions as well as in pathological processes such as ischemia and diabetes. Accumulating evidence indicates that adenosine diphosphate ribose (ADPR) is the most important endogenous ligand of TRPM2. However, although it is known that ADPR binds to the NUDT9 homology (NUDT9-H) domain in the intracellular C-terminal region, the molecular mechanism underlying ADPR binding and activation of TRPM2 remains unknown. In this study, we generate a structural model of the NUDT9-H domain and identify the binding pocket for ADPR using induced docking and molecular dynamics simulation. We find a subset of 11 residues-H1346, T1347, T1349, L1379, G1389, S1391, E1409, D1431, R1433, L1484, and H1488-that are most likely to directly interact with ADPR. Results from mutagenesis and electrophysiology approaches support the predicted binding mechanism, indicating that ADPR binds tightly to the NUDT9-H domain, and suggest that the most significant interactions are the van der Waals forces with S1391 and L1484, polar solvation interaction with E1409, and electronic interactions (including π-π interactions) with H1346, T1347, Y1349, D1431, and H1488. These findings not only clarify the roles of a range of newly identified residues involved in ADPR binding in the TRPM2 channel, but also reveal the binding pocket for ADPR in the NUDT9-H domain, which should facilitate structure-based drug design for the TRPM2 channel.

Abstract 1310: Targetingprolactin signaling to suppress pancreatic cancer stem cells

Abstract Background: Pancreatic cancer (PCa) is a major cause of cancer related mortality in United States with &amp;lt; 6% survival rate. It is an aggressive and devastating disease, which is characterized by poor prognosis, invasiveness, rapid progression, profound resistance to drug treatment and recurrance after surgery. Presently, single agent based chemotherapy (e.g. Gemcitabine) is the major treatment for metastatic adenocarcinoma of pancreas but it has a tumor response rate of below 10%. Moreover combination therapy of gemcitabine and erlotinib only marginally improved survival rate. Hence, there is a dire need to identify novel ways to inhibit pancreatic cancer growth. Methods: PCa cells (MiaPaCa-2 and PanC-1) were grown to 70-80% of confluency and treated with and without prolactin (PRL) and JAK2, STAT3 and ERK phosphorylation in presence and absence of antipsychotic compound were evaluated by western blot. Growth of PCa lines (MiaPaCa-2, PanC-1, BxPC-3, AsPC-1) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, respectively. Pancosphere formation was used to identify effects on stem cells. Results: We have recently identified that the receptor for the pituitary hormone prolactin is overexpressed in pancreatic cancers, and in pancreatic cancer cell lines. When prolactin (PRL) binds its cognate receptor (PRLR), it induces various downstream events including the JAK-STAT and ERK MAPK pathways. In pancreatic cancer cell lines, we observe that PRL treatment induced dose- and time-dependent JAK2, STAT3, and ERK1/2 phosphorylation. Furthermore, there was an increase in the expression of cancer stem cell (CSC) markers DCLK1 (doublecortin calmodulin like kinase 1) and CD44. In addition, PRL-induced pancosphere formation further suggesting that PRL affects stem cells. Based on these data, we conclude that PRL signaling enhances stemness in pancreatic cancers, and therefore we decided to target it for therapeutic intervention. For this, we developed a homology model for the C-terminal intracellular region of the receptor and performed a virtual screening in silico with FDA approved drugs. One compound, a first generation antipsychotic drug diphenylbutylpiperidine, also called Penfluridol was found to interact with the region of the receptor that also binds site for JAK2. The compound has a long half-life, and is used in the treatment of chronic schizophrenia and similar psychotic disorders. We have further determined that Penfluridol inhibits PRL-induced STAT3 and ERK phosphorylation. In addition, the compound reduced proliferation, colony formation, and spheroid formation. Moreover, it induced cells to undergo autophagy by activating LC3B and increasing expression of autophagy-related genes ATG5, 7 and 12. Conclusions: PRL signaling through its cognate PRLR receptor is critical for aggressive pancreatic cancer behavior, and therefore may be an effective therapeutic strategy. Citation Format: Prasad Dandawate, Gaurav Kaushik, Dharmalingam Subramaniam, Prabhu Ramamoorthy, Scott J. Weir, Roy A. Jensen, Shrikant Anant. Targetingprolactin signaling to suppress pancreatic cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1310.

Analysis of the conformation and thermal stability of the high-affinity IgE Fc receptor β chain polymorphic proteins.

The high-affinity IgE Fc receptor (FcεRI) β chain acts as a signal amplifier through the immunoreceptor tyrosine-based activation motif in its C-terminal intracellular region. Polymorphisms in FcεRI β have been linked to atopy, asthma, and allergies. We investigated the secondary structure, conformation, and thermal stability of FcεRI β polymorphic (β-L172I, β-L174V, and β-E228G) proteins. Polymorphisms did not affect the secondary structure and conformation of FcεRI β. However, we calculated Gibbs free energy of unfolding (ΔGunf) and significant differences were observed in ΔGunf values between the wild-type FcεRI β (β-WT) and β-E228G. These results suggested that β-E228G affected the thermal stability of FcεRI β. The role of β-E228G in biological functions and its involvement in allergic reactions have not yet been elucidated in detail; therefore, differences in the thermal stability of β-E228G may affect the function of FcεRI β.