The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.

We have developed a microfluidic flow cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow cell channels consist of two parallel gold surfaces mounted face to face with a thin spacer and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow cell was found to bind a maximum of 1 μg proteoliposomes/cm(2), and a minimum proteoliposome concentration required for saturation of the flow cell was determined to be 500 μg/mL. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow cell, and the force acting on the proteoliposomes during flow cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single-digest protocol. The sequential protocol was found to detect ~65% more unique membrane-associated protein (p < 0.001, n = 6) based on peptide analysis with LC-MS/MS, compared to a single-digest protocol. Thus, the flow cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.

Several important drug targets, e.g., ion channels and G protein–coupled receptors, are extremely difficult to approach with current antibody technologies. To address these targets classes, we explored kinetically controlled proteases as structural dynamics–sensitive druggability probes in native-state and disease-relevant proteins. By using low–Reynolds number flows, such that a single or a few protease incisions are made, we could identify antibody binding sites (epitopes) that were translated into short-sequence antigens for antibody production. We obtained molecular-level information of the epitope-paratope region and could produce high-affinity antibodies with programmed pharmacological function against difficult-to-drug targets. We demonstrate the first stimulus-selective monoclonal antibodies targeting the transient receptor potential vanilloid 1 (TRPV1) channel, a clinically validated pain target widely considered undruggable with antibodies, and apoptosis-inducing antibodies selectively mediating cytotoxicity in KRAS-mutated cells. It is our hope that this platform will widen the scope of antibody therapeutics for the benefit of patients.

Even though gain, loss, or modulation of ion channel function is implicated in many diseases, both rare and common, the development of new pharmaceuticals targeting this class has been disappointing, where it has been a major problem to obtain correlated structural and functional information. Here, we present a microfluidic method in which the ion channel TRPV1, contained in proteoliposomes or in excised patches, was exposed to limited trypsin proteolysis. Cleaved-off peptides were identified by MS, and electrophysiological properties were recorded by patch clamp. Thus, the structure-function relationship was evaluated by correlating changes in function with removal of structural elements. Using this approach, we pinpointed regions of TRPV1 that affect channel properties upon their removal, causing changes in current amplitude, single-channel conductance, and EC50 value toward its agonist, capsaicin. We have provided a fast "shotgun" method for chemical truncation of a membrane protein, which allows for functional assessments of various peptide regions.

BackgroundHuman Thioredoxin 1 (Trx1) was initially discovered as Adult T cell Leukemia Derived Factor (ADF) that is capable of inducing IL-2R/Tac (CD25) in T and NK cells.1 Serum Trx1 levels have been correlated to incidence and severity of disease in melanoma patients2 and elevated serum Trx1 levels have been reported in hepatocellular carcinoma3 and pancreatic cancer.4 In contrast to extracellular Trx1, the role of intracellular Trx1 in redox biology is extensively studied (reviewed in 5). The role of Trx1 as a cytokine in regulating immune cells is less well explored. In order to understand the role of Trx1 in cancer immunology and evaluate the potential of anti-Trx1 monoclonal antibody (mAb) as a therapeutic, anti-Trx1 mAbs were generated and evaluated.Methods mAbs against full length human Trx1 were generated in mice using standard immunization protocol. The antibodies were tested for their binding affinity to human Trx1 and cross-reactivity with mouse Trx1 using sandwich ELISA method. mAbs were screened for their ability to neutralize hTrx1 in a functional assay where naïve human CD4+ cells were polarized to differentiate into T regulatory cells (Tregs). Effects of the mAbs in vivo were tested in a mouse syngeneic tumor model as well as a tumor model in humanized mice.ResultsFive unique mAbs were extensively characterized in in vitro and in vivo models. All five mAbs bound hTrx1 with high affinity with an EC50 of < 1 nM in a sandwich ELISA assay. To develop a screening assay for anti-Trx mAbs, the ability of exogenously added Trx1 to increase the level of expression of CD25 on human naïve peripheral CD4+ cells activated with anti-CD3 and anti-CD28 antibodies and polarized with TGFb1 and IL-2 was assessed. Trx1 increases the level of expression of CD25 on CD4+CD25+ cells as well as CD4+CD25+FOXP3+ cells. In this Treg polarization assay, four of the five anti-Trx mAbs showed a statistically highly significant effect in reducing the% CD4+CD25+ as well as CD4+CD25+FOXP3+ cells within viable cells. In addition, there was a dramatic reduction in the expression levels of CD25 and FOXP3 in the respective cell populations. The ability of these antibodies to modulate immune cells within the tumor microenvironment and have an anti-tumor effect in a syngeneic model (using a mouse cross-reactive antibody) and a humanized mouse tumor model was investigated. Results from these studies will be presented.ConclusionsWe describe a novel immunomodulator pathway in tumor immunology.AcknowledgementsWe acknowledge Proteogenix for production of Trx mAbs, Aquila Biomedical for screening Trx mAbs in Treg polarization assay and Champions Oncology for running tumor models.ReferencesYutaka et al. (1989). EMBO J 8(3):757–765.Wang et al. (2015). OncoImmunology 4(9):e1027471.Miyazake et al. (1998). Biotherapy 11:277–288.Nakamura et al. (2000). Cancer Detect Prev 24:53–60.Collet JF, Messens J. (2010). Antioxidants & Redox Signaling 13(8):1205–1216.Mougiakakos et al. (2011). Blood 17(3):857–861.