Rosario Maroto

TRPC1 forms the stretch-activated cation channel in vertebrate cells

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation, cell locomotion, muscle dystrophy and cardiac arrhythmias. However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp. The oocyte was chosen because it expresses the prototypical MscCa (≥107MscCa/oocyte) that is preserved in cytoskeleton-deficient membrane vesicles. We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (Mr 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1). Heterologous expression of the human TRPC1 resulted in a >1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels.

Revisiting TRPC1 and TRPC6 mechanosensitivity

This article addresses whether TRPC1 or TRPC6 is an essential component of a mammalian stretch-activated mechano-sensitive Ca2+ permeable cation channel (MscCa). We have transiently expressed TRPC1 and TRPC6 in African green monkey kidney (COS) or Chinese hamster ovary (CHO) cells and monitored the activity of the stretch-activated channels using a fast pressure clamp system. Although both TRPC1 and TRPC6 are highly expressed at the protein level, the amplitude of the mechano-sensitive current is not significantly altered by overexpression of these subunits. In conclusion, although several TRPC channel members, including TRPC1 and TRPC6, have been recently proposed to form MscCa in vertebrate cells, the functional expression of these TRPC subunits in heterologous systems remains problematic.

Brefeldin A block of integrin-dependent mechanosensitive ATP release from Xenopus oocytes reveals a novel mechanism of mechanotransduction.

Many animal cells release ATP into the extracellular medium, and often this release is mechanosensitive. However, the mechanisms underlying this release are not well understood. Using the luciferin-luciferase bioluminescent assay we demonstrate that a Xenopus oocyte releases ATP at a basal rate ∼0.01 fmol/s, and gentle mechanical stimulation can increase this to 50 fmol/s. Brefeldin A, nocodazole, and progesterone-induced- maturation block basal and mechanosensitive ATP release. These treatments share the common feature of disrupting the Golgi complex and vesicle trafficking to the cell surface and thereby block protein secretion and membrane protein insertion. We propose that ATP release occurs when protein transport vesicles enriched in ATP fuse with the plasma membrane. Collagenase, integrin-binding peptides, and cytochalasin D also block ATP release, indicating that extracellular, membrane and cytoskeletal elements are involved in the release process. Elevation of intracellular Ca2+ does not evoke ATP release but potentiates mechanosensitive ATP release. Our study indicates a novel mechanism of mechanotransduction that would allow cells to regulate membrane trafficking and protein transport/secretion in response to mechanical loading.

BCL-2-related protein Expression in apoptosis : Oxidative stress versus serum deprivation in PC12 cells

Abstract: Expression of the BCL‐2 protein family members, BAX, BAK, BAD, BCL‐xL, BCL‐xS, and BCL‐2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H2O2 treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H2O2‐induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose‐dependent increase in BAX, (b) a dose‐independent increase in BAK, and (c) a dose‐dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time‐dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis‐sparing medium (i.e., NGF‐supplemented serum‐free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL‐xL expression was increased by H2O2 but unaffected by serum deprivation or long‐term NGF treatment. Neither BCL‐2 nor BCL‐xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL‐xL is altered in a stimulus‐dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL‐2‐related proteins induced by H2O2 exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.

Mechanosensitive Ca(2+) permeant cation channels in human prostate tumor cells.

The acquisition of cell motility plays a critical role in the spread of prostate cancer (PC), therefore, identifying a sensitive step that regulates PC cell migration should provide a promising target to block PC metastasis. Here, we report that a mechanosensitive Ca2+-permeable cation channel (MscCa) is expressed in the highly migratory/invasive human PC cell line, PC-3 and that inhibition of MscCa by Gd3+ or GsMTx-4 blocks PC-3 cell migration and associated elevations in [Ca2+]i. Genetic suppression or overexpression of specific members of the canonical transient receptor potential Ca2+ channel family (TRPC1 and TRPC3) also inhibit PC-3 cell migration, but they do so by mechanisms other that altering MscCa activity. Although LNCaP cells are nonmigratory, they also express relatively large MscCa currents, indicating that MscCa expression alone cannot confer motility on PC cells. MscCa in both cell lines show similar conductance and ion selectivity and both are functionally coupled via Ca2+ influx to a small Ca2+-activated K+ channel. However, MscCa in PC-3 and LNCaP cell patches show markedly different gating dynamics—while PC-3 cells typically express a sustained, non-inactivating MscCa current, LNCaP cells express a mechanically-fragile, rapidly inactivating MscCa current. Moreover, mechanical forces applied to the patch, can induce an irreversible transition from the transient to the sustained MscCa gating mode. Given that cancer cells experience increasing compressive and shear forces within a growing tumor, a similar shift in channel gating in situ would have significant effects on Ca2+ signaling that may play a role in tumor progression.

The role of CD4 in regulating homeostasis of T helper cells

Intrathymic T cell selection and peripheral activation of mature T cells are crucial for self-recognition and the general immune response to viral, bacterial, and tumor antigens. The T cell coreceptors, CD4 and CD8, contribute to the regulation of these processes. The importance of interactions between CD4 and moleculesencoded by the class II major histocompatibility complex (MHC) for thymic T cell selection has been clearly established, however, the role of CD4-MHC class II interactions in T helper (Th) cell differentiation, in the maintenance of homeostasis in the peripheral immune system, and in the generation of memoryTh cells is largely unclear. Here, we present evidence for a role of CD4 in controlling homeostasis in the peripheral immune system. We also demonstrate the importance of CD4-MHC class II interactions in inducing these previously not recognized functions of CD4.

Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses

ABSTRACT Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or −80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at −80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound “peri-exosomal” proteins. In preparations stored at −80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

ATP suppresses activity in slowly adapting but not rapidly adapting mechanoreceptors in toad skin

To test the possible role of ATP in transducing or modulating touch sensation, an isolated skin–nerve preparation from the toad, Bufo marinus, perfused on the inner side, was used to examine the effects of ATP on slowly adapting (SA) and rapidly adapting (RA) mechanoreceptors, identified by ramp and hold indentation of the skin. ATP (1–50 mM) did not generate impulse activity in either SA or RA mechanoreceptors nor increase their responses to mechanical stimulation. Instead, ATP (≥5 mM) reversibly and selectively suppressed impulse discharge from SA mechanoreceptors. Our results indicate that while ATP may modulate the responses of specific touch receptors, it is not involved in transducing touch into nerve impulses.

Selective Affinity Enrichment of Nitrotyrosine-Containing Peptides for Quantitative Analysis in Complex Samples

Protein tyrosine nitration by oxidative and nitrate stress is important in the pathogenesis of many inflammatory or aging-related diseases. Mass spectrometry analysis of protein nitrotyrosine is very challenging because the non-nitrated peptides suppress the signals of the low-abundance nitrotyrosine (NT) peptides. No validated methods for enrichment of NT-peptides are currently available. Here we report an immunoaffinity enrichment of NT-peptides for proteomics analysis. The effectiveness of this approach was evaluated using nitrated protein standards and whole-cell lysates in vitro. A total of 1881 NT sites were identified from a nitrated whole-cell extract, indicating that this immunoaffinity-MS method is a valid approach for the enrichment of NT-peptides, and provides a significant advance for characterizing the nitrotyrosine proteome. We noted that this method had higher affinity to peptides with N-terminal nitrotyrosine relative to peptides with other nitrotyrosine locations, which raises the need for future study to develop a pan-specific nitrotyrosine antibody for unbiased, proteome-wide analysis of tyrosine nitration. We applied this method to quantify the changes in protein tyrosine nitration in mouse lungs after intranasal poly(I:C) treatment and quantified 237 NT sites. This result indicates that the immunoaffinity-MS method can be used for quantitative analysis of protein nitrotyrosines in complex samples.