STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.     

Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells

Mammalian cells play a dominant role in the industrial production of biopharmaceutical proteins. However, the productivity of producer cells is often hindered by a bottleneck in the saturated secretory pathway, where a sophisticated mechanism of vesicle trafficking is mediated by numerous proteins and their complexes, among which are the cross‐kingdom conserved SNAREs [soluble NSF (N‐ethylmaleimide‐sensitive factor) receptor]. The SNAREs assemble into complexes by means of four interactive α‐helices and, thus, trigger the fusion of transport vesicles with the respective target membranes. We report that the transgenic expression of exocytic SNAREs, which control the fusion of secretory vesicles to the plasma membrane, differentially impacts the secretory capacity of HEK‐293, HeLa, and CHO‐K1 cells. While other exocytic SNAREs have no effect or a negative effect, SNAP‐23 [synaptosome‐associated protein of 23 kDa] and VAMP8 [vesicle‐associated membrane protein 8] specifically increase the production of recombinant proteins when they are ectopically and stably expressed in mammalian cells. The targeted and effective intervention in the secretory capacity of SNARE proteins is a novel engineering strategy, which could lead to the development of new therapies by increasing the production of biopharmaceutical proteins or by boosting the secretion of cell implants in cell therapy initiatives. Biotechnol. Bioeng. 2011; 108:611–620. © 2010 Wiley Periodicals, Inc.     

36° step size of proton‐driven c‐ring rotation in FoF1‐ATP synthase

Synthesis of adenosine triphosphate ATP, the ‘biological energy currency’, is accomplished by F_oF₁‐ATP synthase. In the plasma membrane of Escherichia coli, proton‐driven rotation of a ring of 10 c subunits in the F_o motor powers catalysis in the F₁ motor. Although F₁ uses 120° stepping during ATP synthesis, models of F_o predict either an incremental rotation of c subunits in 36° steps or larger step sizes comprising several fast substeps. Using single‐molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36° sequential stepping mode of the c‐ring during ATP synthesis.     

Protein-protein interactions as a tool for site-specific labeling of proteins

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.     

Curcumin prevents high glucose damage in retinal pigment epithelial cells through ERK1/2-mediated activation of the Nrf2/HO-1 pathway

To study the effects of curcumin on human retinal pigment epithelial (RPE) cells exposed to high glucose (HG) insult, we performed in vitro studies on RPE cells cultured both in normal and HG conditions to assess the effects of curcumin on the cell viability, nuclear factor erythroid 2-related factor 2 (Nrf2) expression, HO-1 activity, and ERK1/2 expression. RPE cells exposed to HG insult were treated with curcumin. The cell viability, apoptosis, HO-1 activity, ERK, and Nrf2 expression were evaluated. The data indicated that treatment with curcumin caused a significant decrease in terms of apoptosis. Further, curcumin was able to induce HO-1 expression via Nrf2 activation and counteracts the damage elicited by HG. The present study demonstrated that curcumin provides protection against HG-induced damage in RPE cells through the activation of Nrf2/HO-1 signaling that involves the ERK pathway, suggesting that curcumin may have therapeutic value in the treatment of diabetic retinopathy.     

A surface plasmon resonance assay for characterisation and epitope mapping of anti‐GLP‐1 antibodies

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP‐1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP‐1 receptor agonists. Surface plasmon resonance (SPR) facilitates real‐time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme‐linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti‐GLP‐1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti‐GLP‐1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10³ to 4.54 × 10³ M^?1 s^?1 and dissociation rates of 3.56 × 10^?5 to 1.56 × 10^?3 s^?1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC_p < 0). Pair‐wise epitope mapping was performed on captured anti‐GLP‐1 antibodies followed by subsequent interaction with GLP‐1 (7‐36) and other anti‐GLP‐1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti‐GLP‐1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.     

A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca²⁺-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization.Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic.We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density.This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.     

CRISPR/Cas9介导的LATS1/2敲除抑制卵巢癌细胞ES-2增殖

Hippo信号通路在哺乳动物肝脏发育、动态平衡、再生和疾病中发挥非常重要的作用。大肿瘤抑制基因1/2（large tumor suppressor 1/2, LATS1/2）激酶是Hippo信号通路的关键激酶,可以磷酸化YES相关蛋白（yes-associated protein,YAP）,从而调节YAP的核质定位和降解。本文采用CRISPR/Cas9方法构建慢病毒介导的Last1/2基因敲除的载体,通过包装、感染和嘌呤霉素筛选,获得LATS1/2部分敲除的人卵巢癌ES-2和H08910细胞,免疫印迹方法检测LATS1/2表达明显减少。细胞增殖实验检测LATS1/2缺失明显抑制ES-2和HO8910细胞增殖。软琼脂克隆形成实验表明,LATS1/2缺失抑制卵巢癌ES-2细胞的克隆形成能力。细胞划痕和Transwell实验证明,LATS1/2缺失明显抑制卵巢癌ES-2细胞迁移。流式细胞检测发现,LATS1/2敲除促进卵巢癌ES-2细胞凋亡并影响细胞周期。裸鼠成瘤实验表明,LATS1/2缺失明显抑制体内肿瘤组织增殖。分子机制研究表明, LATS1/2敲除促进卵巢癌ES-2细胞中胶原I型α1（collagen type I α1,ColIα1）基因表达量增加,在卵巢癌ES-2细胞中同时敲除LATS1/2和COL1A1,可以促进细胞克隆形成。综上结果,人卵巢癌ES-2细胞中LATS1/2缺失能促进COL1A1表达增加, 从而抑制细胞增殖、转移和克隆形成,并影响细胞周期和促进细胞凋亡。     

Influence of MT7 toxin on the oligomerization state of the M1 muscarinic receptor1

Background information. The idea that GPCRs (G‐protein‐coupled receptors) may exist as homo‐ or hetero‐oligomers, although still controversial, is now widely accepted. Nevertheless, the functional roles of oligomerization are still unclear and gaining greater insight into the mechanisms underlying the dynamics of GPCR assembly and, in particular, assessing the effect of ligands on this process seems important. We chose to focus our present study on the effect of MT7 (muscarinic toxin 7), a highly selective allosteric peptide ligand, on the oligomerization state of the hM₁ (human M₁ muscarinic acetylcholine receptor subtype). Results. We analysed the hM₁ oligomerization state in membrane preparations or in live cells and observed the effect of MT7 via four complementary techniques: native‐PAGE electrophoresis analysed by both Western blotting and autoradiography on solubilized membrane preparations of CHO‐M1 cells (Chinese‐hamster ovary cells expressing muscarinic M₁ receptors); FRET (fluorescence resonance energy transfer) experiments on cells expressing differently tagged M₁ receptors using either an acceptor photobleaching approach or a novel fluorescence emission anisotropy technique; and, finally, by BRET (bioluminescence resonance energy transfer) assays. Our results reveal that MT7 seems to protect the M₁ receptor from the dissociating effect of the detergent and induces an increase in the FRET and BRET signals, highlighting its ability to affect the dimeric form of the receptor. Conclusions. Our results suggest that MT7 binds to a dimeric form of hM₁ receptor, favouring the stability of this receptor state at the cellular level, probably by inducing some conformational rearrangements of the pre‐existing muscarinic receptor homodimers.     

Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes

Autophagy defines the lifespan of eukaryotic organisms by ensuring cellular survival through regulated bulk clearance of proteins, organelles and membranes. Pathophysiological consequences of improper autophagy give rise to a variety of age-related human diseases such as cancer and neurodegeneration. Rational therapeutic implementation of autophagy modulation remains problematic, as fundamental molecular details such as the generation of autophagosomes, unique double-membrane vesicles formed to permit the process of autophagy, are insufficiently understood. Here, freeze-fracture replica immunolabelling reveals WD-repeat protein interacting with phosphoinositides 1 and 2 (WIPI-1 and WIPI-2) as membrane components of autophagosomes and the plasma membrane (PM). In addition, WIPI-1 is also present in membranes of the endoplasmic reticulum (ER) and WIPI-2 was further detected in membranes close to the Golgi cisternae. Our results identify WIPI-1 and WIPI-2 as novel protein components of autophagosomes, and of membrane sites from which autophagosomes might originate (ER, PM, Golgi area). Hence therapeutic modulation of autophagy could involve approaches that functionally target human WIPI proteins.     

E2F-1- and E2Ftr-mediated apoptosis: the role of DREAM and HRK

E2F-1-deleted mutant, 'truncated E2F' (E2Ftr, E2F-1[1-375]), lacking the carboxy-terminal transactivation domain, was shown to be more potent at inducing cancer cell apoptosis than wild-type E2F-1 (wtE2F-1; full-length E2F-1). Mechanisms by which wtE2F-1 and E2Ftr induce apoptosis, however, are not fully elucidated. Our study demonstrates molecular effects of pro-apoptotic BH3-only Bcl-2 family member Harakiri (Hrk) in wtE2F-1- and E2Ftr-induced melanoma cell apoptosis. We found that Hrk mRNA and Harakiri (HRK) protein expression was highly up-regulated in melanoma cells in response to wtE2F-1 and E2Ftr overexpression. HRK up-regulation did not require the E2F-1 transactivation domain. In addition, Hrk gene up-regulation and HRK protein expression did not require p53 in cancer cells. Hrk knockdown by Hrk siRNA was associated with significantly reduced wtE2F-1- and E2Ftr-induced apoptosis. We also found that an upstream factor, 'downstream regulatory element antagonist modulator' (DREAM), may be involved in HRK-mediated apoptosis in response to wtE2F-1 and E2Ftr overexpression. DREAM expression levels increased following wtE2F-1 and E2Ftr overexpression. Western blotting detected increased DREAM primarily in dimeric form. The homodimerization of DREAM resulting from wtE2F-1 and E2Ftr overexpression may contribute to the decreased binding activity of DREAM to the 3'-untranslated region of the Hrk gene as shown by electromobility shift assay. Results showed wtE2F-1- and E2Ftr-induced apoptosis is partially mediated by HRK. HRK function is regulated in response to DREAM. Our findings contribute to understanding the mechanisms that regulate wtE2F-1- and E2Ftr-induced apoptosis and provide insights into the further evaluation of how E2Ftr-induced apoptosis may be used for therapeutic gain.     

A comparative molecular force spectroscopy study of homophilic JAM-A interactions and JAM-A interactions with reovirus attachment protein sigma1

JAM-A belongs to a family of immunoglobulin-like proteins called junctional adhesion molecules (JAMs) that localize at epithelial and endothelial intercellular tight junctions. JAM-A is also expressed on dendritic cells, neutrophils, and platelets. Homophilic JAM-A interactions play an important role in regulating paracellular permeability and leukocyte transmigration across epithelial monolayers and endothelial cell junctions, respectively. In addition, JAM-A is a receptor for the reovirus attachment protein, sigma1. In this study, we used single molecular force spectroscopy to compare the kinetics of JAM-A interactions with itself and sigma1. A chimeric murine JAM-A/Fc fusion protein and the purified sigma1 head domain were used to probe murine L929 cells, which express JAM-A and are susceptible to reovirus infection. The bond half-life (t(1/2)) of homophilic JAM-A interactions was found to be shorter (k(off)(o) = 0.688 +/- 0.349 s(-1)) than that of sigma1/JAM-A interactions (k(off)(o) = 0.067 +/- 0.041 s(-1)). These results are in accordance with the physiological functions of JAM-A and sigma1. A short bond lifetime imparts a highly dynamic nature to homophilic JAM-A interactions for regulating tight junction permeability while stable interactions between sigma1 and JAM-A likely anchor the virus to the cell surface and facilitate viral entry.     

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.     

Atomic force microscopy study of the antigen‐antibody binding force on patient cancer cells based on ROR1 fluorescence recognition

Knowledge of drug–target interaction is critical to our understanding of drug action and can help design better drugs. Due to the lack of adequate single‐molecule techniques, the information of individual interactions between ligand‐receptors is scarce until the advent of atomic force microscopy (AFM) that can be used to directly measure the individual ligand‐receptor forces under near‐physiological conditions by linking ligands onto the surface of the AFM tip and then obtaining force curves on cells. Most of the current AFM single‐molecule force spectroscopy experiments were performed on cells grown in vitro (cell lines) that are quite different from the human cells in vivo. From the view of clinical practice, investigating the drug–target interactions directly on the patient cancer cells will bring more valuable knowledge that may potentially serve as an important parameter in personalized treatment. Here, we demonstrate the capability of AFM to measure the binding force between target (CD20) and drug (rituximab, an anti‐CD20 monoclonal antibody targeted drug) directly on lymphoma patient cancer cells under the assistance of ROR1 fluorescence recognition. ROR1 is a receptor expressed on some B‐cell lymphomas but not on normal cells. First, B‐cell lymphoma Raji cells (a cell line) were used for ROR1 fluorescence labeling and subsequent measurement of CD20‐rituximab binding force. The results showed that Raji cells expressed ROR1, and the labeling of ROR1 did not influence the measurement of CD20‐rituximab binding force. Then the established experimental procedures were performed on the pathological samples prepared from the bone marrow of a follicular lymphoma patient. Cancer cells were recognized by ROR1 fluorescence. Under the guidance of fluorescence, with the use of a rituximab‐conjugated tip, the cellular topography was visualized by using AFM imaging and the CD20‐Rituximab binding force was measured by single‐molecule force spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.