Structural basis of TMPRSS2 zymogen activation and recognition by the HKU1 seasonal coronavirus.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

Oncolytic attenuated measles virus encoding NY-ESO-1 induces HLA I and II presentation of this tumor antigen by melanoma and dendritic cells.

Antitumor virotherapy stimulates the antitumor immune response during tumor cell lysis induced by oncolytic viruses (OVs). OV can be modified to express additional transgenes that enhance their therapeutic potential. In this study, we armed the spontaneously oncolytic Schwarz strain of measles viruses (MVs) with the gene encoding the cancer/testis antigen NY-ESO-1 to obtain MVny. We compared MV and MVny oncolytic activity and ability to induce NY-ESO-1 expression in six human melanoma cell lines. After MVny infection, we measured the capacity of melanoma cells to present NY-ESO-1 peptides to CD4 + and CD8 + T cell clones specific for this antigen. We assessed the ability of MVny to induce NY-ESO-1 expression and presentation in monocyte-derived dendritic cells (DCs). Our results show that MVny and MV oncolytic activity are similar with a faster cell lysis induced by MVny. We also observed that melanoma cell lines and DC expressed the NY-ESO-1 protein after MVny infection. In addition, MVny-infected melanoma cells and DCs were able to stimulate NY-ESO-1-specific CD4 + and CD8 + T cells. Finally, MVny was able to induce DC maturation. Altogether, these results show that MVny could be an interesting candidate to stimulate NY-ESO-1-specific T cells in melanoma patients with NY-ESO-1-expressing tumor cells.

Endoplasmic Reticulum Calcium Pumps and Tumor Cell Differentiation.

Endoplasmic reticulum (ER) calcium homeostasis plays an essential role in cellular calcium signaling, intra-ER protein chaperoning and maturation, as well as in the interaction of the ER with other organelles. Calcium is accumulated in the ER by sarco/endoplasmic reticulum calcium ATPases (SERCA enzymes) that generate by active, ATP-dependent transport, a several thousand-fold calcium ion concentration gradient between the cytosol (low nanomolar) and the ER lumen (high micromolar). SERCA enzymes are coded by three genes that by alternative splicing give rise to several isoforms, which can display isoform-specific calcium transport characteristics. SERCA expression levels and isoenzyme composition vary according to cell type, and this constitutes a mechanism whereby ER calcium homeostasis is adapted to the signaling and metabolic needs of the cell, depending on its phenotype, its state of activation and differentiation. As reviewed here, in several normal epithelial cell types including bronchial, mammary, gastric, colonic and choroid plexus epithelium, as well as in mature cells of hematopoietic origin such as pumps are simultaneously expressed, whereas in corresponding tumors and leukemias SERCA3 expression is selectively down-regulated. SERCA3 expression is restored during the pharmacologically induced differentiation of various cancer and leukemia cell types. SERCA3 is a useful marker for the study of cell differentiation, and the loss of SERCA3 expression constitutes a previously unrecognized example of the remodeling of calcium homeostasis in tumors.

Polycystins and intercellular mechanotransduction: A precise dosage of polycystin 2 is necessary for alpha-actinin reinforcement of junctions upon mechanical stimulation.

Polycystins 1 and 2, which are mutated in Autosomal Polycystic Kidney Disease, are involved in mechanotransduction through various mechanisms. In renal cells, polycystins not only have an important mechanotransductive role in primary cilia but are also present in intercellular contacts but their role there remains unclear. Here, we address the hypothesis that polycystins are involved in mechanotransduction via intercellular junctions, which would be expected to have consequences on tissue organization. We focused on the role of polycystin 2, which could be involved in mechanical organization at junctions either by its channel activity or by the direct recruitment of cytoskeleton components such as the F-actin cross-linker α-actinin. After mechanical stimulation of intercellular junctions in MDCK renal epithelial cells, α-actinin is rapidly recruited but this is inhibited upon overexpression of PC2 or the D509V mutant that lacks channel activity, and is also decreased upon PC2 silencing. This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive α-actinin recruitment at junctions. At the multicellular level, a change in PC2 expression was associated with changes in velocity in confluent epithelia and during wound healing together with a loss of orientation. This study suggests that the mechanosensitive regulation of cytoskeleton by polycystins in intercellular contacts may be important in the context of ADPKD.

Loss of endoplasmic reticulum calcium pump expression in choroid plexus tumours.

AIMS: Sarco/Endoplasmic Reticulum Calcium ATPase-type calcium pumps (SERCA enzymes) control cell activation by sequestering calcium ions from the cytosol into the endoplasmic reticulum. Although endoplasmic reticulum calcium signalling plays an important role in the regulation of choroid plexus epithelial function, SERCA expression in the choroid plexus has not been investigated so far. METHODS: In this work we investigated the expression of the SERCA3-type calcium pump in choroid plexus epithelial cells grown in vitro, and in normal and hyperplastic choroid plexus tissue, in choroid plexus papillomas displaying various degrees of atypia, and in choroid plexus carcinoma by immunohistochemistry in situ. RESULTS: Whereas normal choroid plexus epithelial cells express SERCA3 abundantly, SERCA3 expression is strongly decreased in papillomas, and is absent in choroid plexus carcinoma, while expression in hyperplastic epithelium is high, similarly to normal epithelium. SERCA3 expression was detected also in normal primary choroid plexus epithelial cells grown in vitro, and expression was markedly enhanced by short-chain fatty acid-type cell differentiation inducing agents, including valproate. CONCLUSION: These observations show that SERCA3 is a new phenotypic marker of normal choroid plexus epithelial differentiation, and that SERCA3 constitutes an early tumour marker 'by loss of expression' in the choroid plexus that may be useful to distinguish hyperplastic processes from papillomas. Endoplasmic reticulum calcium homeostasis becomes anomalous, due to loss of SERCA3 expression, already in benign neoplastic lesions of the choroid plexus epithelium.

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells.

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.

Mutations in calmodulin-binding domains of TRPV4/6 channels confer invasive properties to colon adenocarcinoma cells.

Transient receptor potential (TRP) channels form a family of polymodal cation channels gated by thermal, mechanical, or chemical stimuli, with many of them involved in the control of proliferation, apoptosis, or cell cycle. From an evolutionary point of view, TRP family is characterized by high conservation of duplicated genes originating from whole-genome duplication at the onset of vertebrates. The conservation of such "ohnolog" genes is theoretically linked to an increased probability of generating phenotypes deleterious for the organism upon gene mutation. We aimed to test experimentally the hypothesis that TRP mutations, in particular gain-of-function, could be involved in the generation of deleterious phenotypes involved in cancer, such as gain of invasiveness. Indeed, a number of TRP channels have been linked to cancer progression, and exhibit changes in expression levels in various types of cancers. However, TRP mutations in cancer have been poorly documented. We focused on 2 TRPV family members, TRPV4 and TRPV6, and studied the effect of putative gain-of-function mutations on invasiveness properties. TRPV channels have a C-terminal calmodulin-binding domain (CaMBD) that has important functions for regulating protein function, through different mechanisms depending on the channel (channel inactivation/potentiation, cytoskeleton regulation). We studied the effect of mutations mimicking constitutive phosphorylation in TRPV4 and TRPV6 CaMBDs: TRPV4 S823D, S824D and T813D, TRPV6 S691D, S692D and T702. We found that most of these mutants induced a strong gain of invasiveness of colon adenocarcinoma SW480 cells, both for TRPV4 and TRPV6. While increased invasion with TRPV6 S692D and T702D mutants was correlated to increased mutant channel activity, it was not the case for TRPV4 mutants, suggesting different mechanisms with the same global effect of gain in deleterious phenotype. This highlights the potential importance to search for TRP mutations involved in cancer.

Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus latent membrane protein-1.

BACKGROUND: Calcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated. RESULTS: Two Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles. CONCLUSION: The data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.

Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation.

BACKGROUND: Endoplasmic reticulum (ER) calcium storage and release play important roles in B lymphocyte maturation, survival, antigen-dependent cell activation and immunoglobulin synthesis. Calcium is accumulated in the endoplasmic reticulum (ER) by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes). Because lymphocyte function is critically dependent on SERCA activity, it is important to understand qualitative and quantitative changes of SERCA protein expression that occur during B lymphoid differentiation and leukemogenesis. METHODS: In this work we investigated the modulation of SERCA expression during the pharmacologically induced differentiation of leukemic precursor B lymphoblast cell lines that carry the E2A-PBX1 fusion oncoprotein. Changes of SERCA levels during differentiation were determined and compared to those of established early B lymphoid differentiation markers. SERCA expression of the cells was compared to that of mature B cell lines as well, and the effect of the direct inhibition of SERCA-dependent calcium transport on the differentiation process was investigated. RESULTS: We show that E2A-PBX1+ leukemia cells simultaneously express SERCA2 and SERCA3-type calcium pumps; however, their SERCA3 expression is markedly inferior to that of mature B cells. Activation of protein kinase C enzymes by phorbol ester leads to phenotypic differentiation of the cells, and this is accompanied by the induction of SERCA3 expression. Direct pharmacological inhibition of SERCA-dependent calcium transport during phorbol ester treatment interferes with the differentiation process. CONCLUSION: These data show that the calcium pump composition of the ER is concurrent with increased SERCA3 expression during the differentiation of precursor B acute lymphoblastic leukemia cells, that a cross-talk exists between SERCA function and the control of differentiation, and that SERCA3 may constitute an interesting new marker for the study of early B cell phenotype.

Synergistic Effect of H2O2 and NO2 in Cell Death Induced by Cold Atmospheric He Plasma.

Cold atmospheric pressure plasmas (CAPPs) have emerged over the last decade as a new promising therapy to fight cancer. CAPPs' antitumor activity is primarily due to the delivery of reactive oxygen and nitrogen species (RONS), but the precise determination of the constituents linked to this anticancer process remains to be done. In the present study, using a micro-plasma jet produced in helium (He), we demonstrate that the concentration of H2O2, NO2(-) and NO3(-) can fully account for the majority of RONS produced in plasma-activated buffer. The role of these species on the viability of normal and tumour cell lines was investigated. Although the degree of sensitivity to H2O2 is cell-type dependent, we show that H2O2 alone cannot account for the toxicity of He plasma. Indeed, NO2(-), but not NO3(-), acts in synergy with H2O2 to enhance cell death in normal and tumour cell lines to a level similar to that observed after plasma treatment. Our findings suggest that the efficiency of plasma treatment strongly depends on the combination of H2O2 and NO2(-) in determined concentrations. We also show that the interaction of the He plasma jet with the ambient air is required to generate NO2(-) and NO3(-) in solution.

Modulation of endoplasmic reticulum calcium pump expression during lung cancer cell differentiation.

Cellular calcium signaling plays important roles in several signal transduction pathways that control proliferation, differentiation and apoptosis. In epithelial cells calcium signaling is initiated mainly by calcium release from endoplasmic-reticulum-associated intracellular calcium pools. Because calcium is accumulated in the endoplasmic reticulum by sarco/endoplasmic reticulum calcium ATPases (SERCA), these enzymes play a critical role in the control of calcium-dependent cell activation, growth and survival. We investigated the modulation of SERCA expression and function in human lung adenocarcinoma cells. In addition to the ubiquitous SERCA2 enzyme, the SERCA3 isoform was also expressed at variable levels. SERCA3 expression was selectively enhanced during cell differentiation in lung cancer cells, and marked SERCA3 expression was found in fully differentiated normal bronchial epithelium. As studied by using a recombinant fluorescent calcium probe, induction of the expression of SERCA3, a lower calcium affinity pump, was associated with decreased intracellular calcium storage, whereas the amplitude of capacitative calcium influx remained unchanged. Our observations indicate that the calcium homeostasis of the endoplasmic reticulum in lung adenocarcinoma cells presents a functional defect due to decreased SERCA3 expression that is corrected during pharmacologically induced differentiation. The data presented in this work show, for the first time, that endoplasmic reticulum calcium storage is anomalous in lung cancer cells, and suggest that SERCA3 may serve as a useful new phenotypic marker for the study of lung epithelial differentiation.

Endoplasmic reticulum calcium pumps and cancer cell differentiation.

The endoplasmic reticulum (ER) is a major intracellular calcium storage pool and a multifunctional organelle that accomplishes several calcium-dependent functions involved in many homeostatic and signaling mechanisms. Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA)-type calcium pumps. SERCA activity can determine ER calcium content available for intra-ER functions and for calcium release into the cytosol, and can shape the spatiotemporal characteristics of calcium signals. SERCA function therefore constitutes an important nodal point in the regulation of cellular calcium homeostasis and signaling, and can exert important effects on cell growth, differentiation and survival. In several cell types such as cells of hematopoietic origin, mammary, gastric and colonic epithelium, SERCA2 and SERCA3-type calcium pumps are simultaneously expressed, and SERCA3 expression levels undergo significant changes during cell differentiation, activation or immortalization. In addition, SERCA3 expression is decreased or lost in several tumor types when compared to the corresponding normal tissue. These observations indicate that ER calcium homeostasis is remodeled during cell differentiation, and may present defects due to decreased SERCA3 expression in tumors. Modulation of the state of differentiation of the ER reflected by SERCA3 expression constitutes an interesting new aspect of cell differentiation and tumor biology.

Endoplasmic reticulum calcium pumps and cancer.

Endoplasmic reticulum calcium homeostasis is involved in a multitude of signaling, as well as "house-keeping" functions that control cell growth, differentiation or apoptosis in every human/eukaryotic cell. Calcium is actively accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium transport ATPases (SERCA enzymes). SERCA-dependent calcium transport is the only calcium uptake mechanism in this organelle, and therefore the regulation of SERCA function by the cell constitutes a key mechanism to adjust calcium homeostasis in the endoplasmic reticulum depending on the cell type and its state of differentiation. The direct pharmacological modulation of SERCA activity affects cell differentiation and survival. SERCA expression levels can undergo significant changes during cell differentiation or tumorigenesis, leading to modified endoplasmic reticulum calcium storage. In several cell types such as cells of hematopoietic origin or various epithelial cells, two SERCA genes (SERCA2 and SERCA3) are simultaneously expressed. Expression levels of SERCA3, a lower calcium affinity calcium pump are highly variable. In several cell systems SERCA3 expression is selectively induced during differentiation, whereas during tumorigenesis and blastic transformation SERCA3 expression is decreased. These observations point at the existence of a cross-talk, via the regulation of SERCA3 levels, between endoplasmic reticulum calcium homeostasis and the control of cell differentiation, and show that endoplasmic reticulum calcium homeostasis itself can undergo remodeling during differentiation. The investigation of the anomalies of endoplasmic reticulum differentiation in tumor and leukemia cells may be useful for a better understanding of the contribution of calcium signaling to the establishment of malignant phenotypes.