Binding Protein Calreticulin Research Articles

Novel tetrasubstituted 5-Arylamino pyrazoles able to interfere with angiogenesis and Ca2+ mobilization

In the last years, 5-pyrazolyl ureas and 5-aminopyrazoles have been investigated for their antiangiogenetic properties and their potential interaction with the ubiquitous Ca2+ binding protein Calreticulin. Based on the structure of the active compounds I and GeGe-3, novel 5-arylamino pyrazoles 2 and 3 were synthesized through a stepwise procedure. In MTT assays, all the new derivatives proved to be non-cytotoxic against eight different tumor cell lines, normal fibroblasts, and endothelial cells. Furthermore, selected derivatives showed relevant antiangiogenetic properties, resulting more effective than reference molecules I and GeGe-3 in inhibiting HUVEC endothelial tube formation. 5-Arylamino pyrazoles 2a and 2d were identified as the most interesting compounds and significantly prevented tube formation of tumor secretome-stimulated HUVEC. Furthermore, the two compounds inhibited HUVEC migration in wound healing assay and altered cell invasion capability. Additionally, 2a and 2d strongly affected Ca2+ mobilization and cytoskeletal organization of HUVEC cells, being as active as the reference compound GeGe-3. Differently from previous studies, molecular docking simulations suggested a poor affinity of 2a towards Calreticulin, one of the interacting partners of the lead compound GeGe-3. Collectively, this new amino-pyrazole library further extends the structure-activity relationships of the previously prepared derivatives and confirmed the biological attractiveness of this chemical scaffold as antiangiogenetic agents.

Anticancer Effects of the Novel Pyrazolyl-Urea GeGe-3.

In a screen of over 200 novel pyrazole compounds, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a potential anticancer compound. GeGe-3 displays potent anti-angiogenic properties through the presumptive targeting of the protein kinase DMPK1 and the Ca2+-binding protein calreticulin. We further explored the anticancer potential of GeGe-3 on a range of established cancer cell lines, including PC3 (prostate adenocarcinoma), SKMEL-28 (cutaneous melanoma), SKOV-3 (ovarian adenocarcinoma), Hep-G2 (hepatocellular carcinoma), MDA-MB231, SKBR3, MCF7 (breast adenocarcinoma), A549 (lung carcinoma), and HeLa (cervix epithelioid carcinoma). At concentrations in the range of 10 μM, GeGe-3 significantly restricted cell proliferation and metabolism. GeGe-3 also reduced PC3 cell migration in a standard wound closure and trans-well assay. Together, these results confirm the anticancer potential of GeGe-3 and underline the need for more detailed pre-clinical investigations into its molecular targets and mechanisms of action.

Characterization of antigenic proteins of the Taenia solium postoncospheral form

Neurocysticercosis is the leading cause for acquired epilepsy worldwide, and it is caused by the larval stage of the parasite Taenia solium. Several proteins of this stage have been characterized and studied to understand the parasite-host interaction, however, the proteins from the early cysticercus stages (the postoncospheral form) have not yet been characterized. The study of the postoncospheral form proteins is important to understand the host-parasite relationship in the early stages of infection. The aim of this work was to identify postoncospheral form antigenic proteins using sera from neurocysticercosis patients. T. solium activated oncospheres were cultured in HCT-8 cells to obtain the postoncospheral form. Soluble total and excretory/secretory proteins were obtained from the postoncospheral form and were incubated with both pool sera and individual serum of neurocysticercosis positive human patients. Immunoblotting showed target antigenic proteins with apparent molecular weights of 23 kDa and 46–48 kDa. The 46–48 kDa antigen bands present in soluble total and excretory/secretory postoncospheral form proteins were analyzed by LC-MS/MS; proteins identified were: nuclear elongation factor 1 alpha, enolase, unnamed protein product/antigen diagnostic GP50, calcium binding protein calreticulin precursor and annexin. The postoncospheral form expresses proteins related to interaction with the host, some of these proteins are predicted to be exosomal proteins. In conclusion, postoncospheral proteins are consistent targets of the humoral immune response in human and may serve as targets for diagnosis and vaccines.

The ER-localized Ca2+-binding protein calreticulin couples ER stress to autophagy by associating with microtubule-associated protein 1A/1B light chain 3

Autophagy is of key importance for eliminating aggregated proteins during the maintenance of cellular proteostasis in response to endoplasmic reticulum (ER) stress. However, the upstream signaling that mediates autophagy activation in response to ER stress is incompletely understood. In this study, in vivo and in vitro approaches were utilized that include gain- and loss-of-function assays and mouse livers and human cell lines with tunicamycin-induced pharmacological ER stress. We report that calreticulin, a quality control chaperone that binds to misfolded glycoproteins for refolding in the ER, is induced under ER stress. Calreticulin overexpression stimulated the formation of autophagosomes and increased autophagic flux. Interestingly, calreticulin was sufficient for attenuating ER stress in tunicamycin- or thapsigargin-treated HeLa cells, whereas lentivirus-mediated shRNA calreticulin knockdown exacerbated ER stress. Mechanistically, we noted that calreticulin induces autophagy by interacting with microtubule-associated protein 1A/1B-light chain 3 (LC3). Confocal microscopy revealed that the colocalization of calreticulin and LC3 at the autophagosome was enhanced under ER stress conditions. Importantly, a conserved LC3-interacting region was necessary for calreticulin-mediated stimulation of autophagy and for reducing ER stress. These findings indicate a calreticulin-based mechanism that couples ER stress to autophagy activation, which, in turn, attenuates cellular stress, likely by alleviating the formation of aberrantly folded proteins. Pharmacological or genetic approaches that activate calreticulin-autophagy signaling may have potential for managing ER stress and related cellular disorders.

Usefulness of gel filtration fraction as potential biomarker for neurocysticercosis in serum: towards a new diagnostic tool.

There is an increasing interest in improving neurocysticercosis (NCC) diagnosis through the search of new and alternative antigenic sources, as those obtained from heterologous antigens. The aim of this study was to obtain potential biomarkers for NCC diagnosis after gel filtration chromatography [gel filtration fraction (GFF)] from the total saline extract (SE) from Taenia saginata metacestodes, followed by protein identification and application in immunodiagnostic. SE and GFF proteic profiles were characterized in gel electrophoresis, and diagnostic performance was verified by testing 160 serum samples through enzyme-linked immunosorbent assay and immunoblotting. Sensitivity (Se), specificity (Sp) and other diagnostic parameters were calculated. Polypeptides of interest in the diagnosis of human NCC present at GFF were analysed by mass spectrometry (MS) and B-cell epitopes were predicted. GFF had the best diagnostic parameters: Se 93·3%; Sp 93%; AUC 0·990; LR+ = 13·42 and LR- = 0·07, and proved to be useful reacting with serum samples in immunoblotting. Proteic profile ranged from 64 to 68 kDa and enolase and calcium binding protein calreticulin precursor were identified after MS. The enolase and calcium-binding protein calreticulin precursor showed 18 and 10 predicted B-cell epitopes, respectively. In conclusion we identified important markers in the GFF with high efficiency to diagnose NCC.

A(a)LS: Ammonia-induced amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a dreadful, devastating and incurable motor neuron disease. Aetiologically, it is a multigenic, multifactorial and multiorgan disease. Despite intense research, ALS pathology remains unexplained. Following extensive literature review, this paper posits a new integrative explanation. This framework proposes that ammonia neurotoxicity is a main player in ALS pathogenesis. According to this explanation, a combination of impaired ammonia removal— mainly because of impaired hepatic urea cycle dysfunction—and increased ammoniagenesis— mainly because of impaired glycolytic metabolism in fast twitch skeletal muscle—causes chronic hyperammonia in ALS. In the absence of neuroprotective calcium binding proteins (calbindin, calreticulin and parvalbumin), elevated ammonia—a neurotoxin—damages motor neurons. Ammonia-induced motor neuron damage occurs through multiple mechanisms such as macroautophagy-endolysosomal impairment, endoplasmic reticulum (ER) stress, CDK5 activation, oxidative/nitrosative stress, neuronal hyperexcitability and neuroinflammation. Furthermore, the regional pattern of calcium binding proteins’ loss, owing to either ER stress and/or impaired oxidative metabolism, determines clinical variability of ALS. Most importantly, this new framework can be generalised to explain other neurodegenerative disorders such as Huntington’s disease and Parkinsonism.

A(a)LS: Ammonia-induced amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a dreadful, devastating and incurable motor neuron disease. Aetiologically, it is a multigenic, multifactorial and multiorgan disease. Despite intense research, ALS pathology remains unexplained. Following extensive literature review, this paper posits a new integrative explanation. This framework proposes that ammonia neurotoxicity is a main player in ALS pathogenesis. According to this explanation, a combination of impaired ammonia removal- mainly because of impaired hepatic urea cycle dysfunction-and increased ammoniagenesis- mainly because of impaired glycolytic metabolism in fast twitch skeletal muscle-causes chronic hyperammonia in ALS. In the absence of neuroprotective calcium binding proteins (calbindin, calreticulin and parvalbumin), elevated ammonia-a neurotoxin-damages motor neurons. Ammonia-induced motor neuron damage occurs through multiple mechanisms such as macroautophagy-endolysosomal impairment, endoplasmic reticulum (ER) stress, CDK5 activation, oxidative/nitrosative stress, neuronal hyperexcitability and neuroinflammation. Furthermore, the regional pattern of calcium binding proteins' loss, owing to either ER stress and/or impaired oxidative metabolism, determines clinical variability of ALS. Most importantly, this new framework can be generalised to explain other neurodegenerative disorders such as Huntington's disease and Parkinsonism.

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

BackgroundLegumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca2+ concentration, which is a fundamental prerequisite for any Ca2+-based signalling system, is accomplished by complex mechanisms including Ca2+ binding proteins acting as Ca2+ buffers. In this work we investigated the occurrence of Ca2+ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.ResultsA soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca2+-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca2+-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca2+ binding ability of the M. loti protein was demonstrated by 45Ca2+-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca2+ adducts.ConclusionsThe present data indicate that ferredoxin II is a major Ca2+ binding protein in M. loti that may participate in Ca2+ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca2+ regulatory systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

Monitoring residual disease in the ph-negative myeloproliferative neoplasms post-allogeneic stem cell transplantation: more mutations and more methodologies.

The Membrane-Proximal KXGFFKR Motif of α-Integrin Mediates Chemoresistance

Cell adhesion-mediated drug resistance contributes to minimal residual disease and relapse in hematological malignancies. Here, we show that adhesion of Jurkat T-acute lymphoblastic leukemia cells to substrates engaging α4β1-integrin or α5β1-integrin promotes chemoresistance to doxorubicin-induced apoptosis. Reconstituted expression of α4δ, a truncated α4-integrin with KXGFFKR as the cytoplasmic motif, in α4-deficient cells promoted chemoresistance to doxorubicin in a manner independent of α4-mediated adhesion. The adhesion-independent chemoresistance did not require β1-integrin as the heterodimeric pair, since expression of Tacδ, a monomeric nonintegrin transmembrane protein fused to the juxtamembrane KXGFFKR, was sufficient to reproduce the phenomenon. The requirement for integrin-mediated adhesion in stimulation of Akt phosphorylation and activation was bypassed for cells expressing α4δ and Tacδ. Cells expressing α4δ and Tacδ exhibited a high influx of extracellular Ca(2+), and inhibition of Ca(2+) channels with verapamil attenuated the adhesion-independent chemoresistance. Tacδ cells also exhibited greater rates of drug efflux. α4δ and Tacδ interacted with the Ca(2+)-binding protein calreticulin, in a manner dependent on the KXGFFKR motif. Adhesion-mediated engagement of α4-integrins promoted an increased calreticulin-α4 association and greater influx of extracellular Ca(2+) than in nonadherent cells. The α-integrin KXGFFKR motif is involved in adhesion-mediated control of chemoresistance in T cells.

The Conserved Kxgffkr Motif of α-Integrin Cytoplasmic Tail Mediates Chemoresistance to Doxorubicin in T-Cell Leukemia.

Abstract 2467Integrins mediate bi-directional signaling between the extracellular ligand binding sites and the cytoplasmic tails, linking the cytoskeleton and intracellular processes to the cellular microenvironment. Interactions between the integrin cytoplasmic tail with accessory proteins can alter the integrins’ ligand binding affinity, modulate cytoskeletal remodeling necessary for adhesion and motility and promote cell survival signaling. Prior studies have implicated involvement of α4 integrins in cell adhesion mediated drug resistance (CAM-DR) in cell models for multiple myeloma and AML. In the present study, we used a genetic reconstitution model to examine the requirement of α4-extracellular ligand interaction in CAM-DR and to delineate the α4 tail sequences mediating drug resistance. Methods:JB4 cells, a Jurkat T-cell derivative lacking α4 expression, was reconstituted with wildtype α4 (JB4-α4) and a cytoplasmic tail truncated mutant (JB4-α4Δ). Cells were plated on CS1, Fn9.11 or BSA to specifically engage respectively, α4β1, α5β1, or no integrins. Adhered or non-adhered cells were untreated or treated with doxorubicin, and % apoptosis determined by flow cytometry using Annexin V binding. Results:The percentage of apoptotic Jurkat and JB4-α4 cells plated on CS1 or Fn9.11 and subjected to doxorubicin were half of those plated on BSA, indicating that both α4β1 and α5β1 integrin ligation confer enhanced chemoresistance. In contrast, JB4 cells lacking α4β1 expression exhibited CAM-DR when plated on Fn9.11, but not on CS1 or BSA, indicating α4β1-ligation to its substrate is necessary to support α4-mediated CAM-DR. Unexpectedly, JB4-α4Δ cells exhibited chemoresistance to doxorubicin when plated on all 3 substrates suggesting α4Δ expression resulted in an adhesion independent chemoresistant phenotype. α4Δ is truncated at the C-terminal tail following the highly conserved KxGFFKR sequence which is required for α-β heterodimer formation. This deletion is also known to disrupt α4Δβ1 binding with its extracellular ligand; a phenomenon which we confirmed using adhesion assays. The adhesion independent chemoresistance exhibited by JB4-α4Δ could be attributed to a gain in β1 expression (as α4Δβ1). To eliminate formation of the heterodimer, we created JB4-Tac and JB4-TacΔ cells, where TacΔ is a fusion of KxGFFKR to the extracellular and transmembrane epitope of the monomeric Tac. Treatment of JB4-TacΔ, but not JB4-Tac cells with doxorubicin in suspension recapitulated the low levels of apoptosis exhibited by JB4-α4Δ cells, indicating that the membrane proximal KxGFFKR sequence is sufficient to promote an adhesion independent form of chemoresistance.The effects are mediated in part via stimulation of the PI3K/Akt/Bad cell survival pathway. Adhesion of α4β1-expressing cells to CS1, or of α5β1 to Fn9.11, stimulated an increase in phospho-Akt and phospho-Bad. Conversely, JB4-α4Δ and JB4-TacΔ cells exhibit constitutively high levels of phospho-Akt in an adhesion independent manner. We also show that the membrane proximal KxGFFKR associates with the Ca2+ binding protein calreticulin (CRT) that is known to regulate L-type Ca2+ channels. Finally, co-treatment of cells with doxorubicin and Verapamil, an L-type Ca2+ channel inhibitor, enhanced the chemosensitivity of JB4-TacΔ cells. Thus, the highly conserved membrane proximal KxGFFKR motif of α-integrins mediates chemoresistance via its interaction with a Ca2+ binding protein involved in regulation of an L-type calcium channel. With the increasing number of integrin-ligand interactions shown to promote CAM-DR, we speculate that interactions involving the KxGFFKR motif of α-integrins may be the common denominator for effecting cell adhesion mediated survival signaling and drug resistance. Disclosures:No relevant conflicts of interest to declare.

Anthracycline Treatment Induces Extracellular Calreticulin in Acute Myeloid Leukemia

Abstract 1376The myeloid key transcription factor CEBPA (CCAAT-enhancer binding protein alpha) is blocked by the mRNA binding protein calreticulin (CRT) thereby affecting myeloid differentiation. We previously reported that the unfolded protein response (UPR) is activated in 24.5% of AML patients, involving the up-regulation of endoplasmatic reticulum (ER) stressors, such as CRT. In addition, anthracycline treatment was shown to trigger the UPR and to specifically induce CRT cell membrane surface expression in some solid tumor types. This ecto-CRT is specifically targeting dendritic cells and initiates immunogenic cell death of cancer cells by acting as an “eat-me” signal. However, UPR induced translocation of CRT in leukemogenesis has hardly been investigated so far.This study aimed at analyzing anthracycline induced CRT exposure and extracellular release of CRT in leukemic cells as well as at elucidating the candidate pathways involved such as the UPR transducers PERK, ATF6 and IRE1.In leukemic cell lines, we observed that anthracycline treatment was inducing CRT expression on the cell membrane surface within 2 hours. This effect was caused by intracellular translocation, but not de novo synthesis. Exposure of CRT was decreased by Brefeldin A, which blocks the vesicle transport from the ER to the Golgi apparatus within 2 hours of treatment, suggesting that exocytosis is critical for CRT delocalization. By specific inhibition of the PERK pathway of the UPR using Salubrinal, we observed reduced CRT expression on the cell membrane. In contrast, blocking the ATF6 or the IRE1 pathway did not affect CRT exposure.Mean CRT levels on the cell membrane of blasts from 53 AML patients at diagnosis were significantly (21-fold; p<0.0005) increased compared to CRT levels of myeloid cells from healthy volunteers (n=75). Using an ELISA assay investigating serum from 104 AML patients at diagnosis and 86 healthy volunteers, we found that extracellular CRT was significantly (2.2-fold; p<0.0001) increased in AML patients compared to healthy controls. We observed a more favorable outcome in AML patients with increased CRT expression on the cell membrane, as well as in patients with higher levels of extracellular CRT in the serum. Finally, we identified a strong correlation between CRT serum levels and CRT expression on the cell surface of leukemic cells. Ongoing studies aim at elucidating the mechanisms of CRT secretion from the cell membrane to the serum.In conclusion, CRT translocation to the cell membrane and increasing levels of secreted CRT in the serum represent early events of anthracycline treatment in leukemic cells, and both are associated with a more favorable outcome in AML patients. Disclosures:No relevant conflicts of interest to declare.

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca(2+)-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca(2+) binding. A significant conformational change was observed for the Ca(2+)-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca(2+) binding. Based on the crystal structure of Ca(2+)-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca(2+) binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.

Serpins Save Cells

Biologically active proteases are held in check in part by a family of peptidase inhibitors known as serpins. Most serpins are secreted, but some are intracellular proteins implicated in regulating lysosomal proteases. Luke et al. found that in the worm Caenorhabditis elegans, loss of the intracellular serpin SRP-6 caused animals to become highly sensitive to hypo-osmotic stress and to die as a result of necrotic cell death. This effect appeared to require inhibition of cysteine peptidases because survival of the knockout worms was improved in mutant animals engineered to express wild-type SRP-6 but not in animals that expressed a mutant serpin that lacked inhibitory activity. Calcium mobilization appeared to be required for cell death because SRP-6 knockout animals lacking the ryanodine receptor, the inositol-1,4,5-trisphosphate receptor, or the Ca2+-binding protein calreticulin showed suppression of cell death. Lysosome-like gut granules also appeared to be required, because death was suppressed in animals lacking a guanosine triphosphatase required for formation of these acidic granules. Animals lacking SRP-6 were also more susceptible than wild-type animals to heat shock, hypoxia, or hyperoxia. The authors argue that the common effects of serpins on these stimuli may indicate that there is a central necrotic death mechanism that can be regulated by serpins. If so, serpins could act as an antidote to cells undergoing, for example, hypoxic stress during heart attacks. Such a prosurvival function of intracellular serpins might also explain why increased expression of some serpin family members portends a poor prognosis in various human cancers. — LBR

Serpins Save Cells

Biologically active proteases are held in check in part by a family of peptidase inhibitors known as serpins. Most serpins are secreted proteins, but some are intracellular proteins implicated in regulating lysosomal proteases. Knockout mice have failed to reveal the biological roles of these inhibitors, though--possibly because mice have some 28 members of the intracellular variety. Therefore, Luke et al . turned to the worm Caenorhabditis elegans , which has about a third as many intracellular serpins. Loss of one of these, SRP-6, caused animals to become highly sensitive to hypo-osmotic stress and to die as a result of necrotic cell death. This effect appeared to require inhibition of cysteine peptidases because survival of the knockout worms was improved in mutant animals engineered to express wild-type SRP-6 but not in animals that expressed a mutant serpin that lacked inhibitory activity. Calcium mobilization appeared to be required for cell death as SRP-6 knockout animals lacking the ryanodine receptor, the inositol-1,4,5-trisphosphate receptor, or the Ca 2+ -binding protein calreticulin showed suppression of cell death. Lysosome-like gut granules also appeared to be required, because death was suppressed in animals lacking the Rab-like guanosine triphosphatase Glo-1, which is required for formation of these acidic granules. Animals lacking SRP-6 were also more susceptible than wild-type animals to heat shock, hypoxia, or hyperoxia. The authors argue that the common effects of serpins on these stimuli, which cause distinct biochemical alterations to initiate cell death, indicate that there may be a central necrotic death mechanism that can be regulated by serpins. If so, serpins could act as an antidote to cells undergoing damage by such stresses, which occur, for example, during heart attacks. Such a prosurvival function of intracellular serpins may help explain why increased expression of some serpin family members is associated with a poor prognosis in various human cancers. C. J. Luke, S. C. Pak, Y. S. Askew, T. L. Naviglia, D. J. Askew, S. M. Nobar, A. C. Vetica, O. S. Long, S. C. Watkins, D. B. Stolz, R. J. Barstead, G. L. Moulder, D. Brömme, G. A. Silvermann, An intracellular serpin regulates necrosis by inhibiting the induction and sequelae of lysosomal injury. Cell , 130 , 1108-1119 (2007). [PubMed]

Chain-reaction Ca2+ signaling in the heart

Mutations in Ca(2+) -handling proteins in the heart have been linked to exercise-induced sudden cardiac death. The best characterized of these have been mutations in the cardiac Ca(2+) release channel known as the ryanodine receptor type 2 (RyR2). RyR2 mutations cause "leaky" channels, resulting in diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR) that can trigger fatal cardiac arrhythmias during stress. In this issue of the JCI, Song et al. show that mutations in the SR Ca(2+)-binding protein calsequestrin 2 (CASQ2) in mice result not only in reduced CASQ2 expression but also in a surprising, compensatory elevation in expression of both the Ca(2+)-binding protein calreticulin and RyR2, culminating in premature Ca(2+) release from cardiac myocytes and stress-induced arrhythmia (see the related article beginning on page 1814). In the context of these findings and other recent reports studying CASQ2 mutations, we discuss how CASQ2 influences the properties of Ca(2+)-dependent regulation of RyR2 and how this contributes to cardiac arrhythmogenesis.

Interaction of HTLV-1 Tax protein with calreticulin: Implications for Tax nuclear export and secretion

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 transcriptional transactivator protein Tax plays an integral role in virus replication and disease progression. Traditionally, Tax is described as a nuclear protein where it performs its primary role as a transcriptional transactivator. However, recent studies have clearly shown that Tax can also be localized to the cytoplasm where it has been shown to interact with a number of host transcription factors most notably NF-κB, constitutive expression of which is directly related to the T cell transforming properties of Tax in ATL patients. The presence of a functional nuclear export signal (NES) within Tax and the secretion of full-length Tax have also been demonstrated previously. Additionally, release of Tax from HTLV-1-infected cells and the presence of cell-free Tax was demonstrated in the CSF of HAM/TSP patients suggesting that the progression to HAM/TSP might be mediated by the ability of Tax to function as an extracellular cytokine. Therefore, in both ATL and HAM/TSP Tax nuclear export and nucleocytoplasmic shuttling may play a critical role, the mechanism of which remains unknown. In this study, we have demonstrated that the calcium binding protein calreticulin interacts with Tax by co-immunoprecipitation. This interaction was found to localize to a region at or near the nuclear membrane. In addition, differential expression of calreticulin was demonstrated in various cell types that correlated with their ability to retain cytoplasmic Tax, particularly in astrocytes. Finally, a comparison of a number of HTLV-1-infected T cell lines to non-infected T cells revealed higher expression of calreticulin in infected cells implicating a direct role for this protein in HTLV-1 infection.

Key Role of the Postsynaptic Density Scaffold Proteins Shank and Homer in the Functional Architecture of Ca2+Homeostasis at Dendritic Spines in Hippocampal Neurons

A key aspect of postsynaptic function, also important for plasticity, is the segregation within dendritic spines of Ca2+ rises attributable to release from intracellular stores. Previous studies have shown that overexpression in hippocampal neurons of two postsynaptic density (PSD) scaffold proteins, Shank1B and Homer1b, induces spine maturation, including translocation of the intracellular Ca2+ channel inositol trisphosphate receptor (IP3R). The structural and functional significance of these processes remained undefined. Here, we show that in its relocation, IP3R is accompanied by other endoplasmic reticulum (ER) proteins: the Ca2+ pump sarcoendoplasmic reticulum calcium ATPase, the lumenal Ca2+-binding protein calreticulin, the ER lumen-addressed green fluorescent protein, and, to a lesser extent, the membrane chaperone calbindin. The specificity of these translocations was demonstrated by their inhibition by both a Shank1 fragment and the dominant-negative Homer1a. Activation in Shank1B-transfected neurons of the metabotropic glutamatergic receptors 1/5 (mGluRs1/5), which induce IP3 generation with ensuing Ca2+ release from the stores, triggered considerable increases in Ca2+-dependent responses: activation of the big K+ channel, which was revealed by patch clamping, and extracellular signal-regulated protein kinase (ERK) phosphorylation. The interaction of Shank1B and Homer1b appears as the molecular mechanism linking mGluRs1/5, strategically located in the spines, to IP3R with the integration of entire ER cisternas in the PSD and with consequences on both local Ca2+ homeostasis and overall neuronal signaling.

Overexpression of the Ca2+‐binding protein calreticulin in the endoplasmic reticulum improves growth of tobacco cell suspensions (Nicotiana tabacum) in high‐Ca2+ medium

Calreticulin (CRT) is a eukaryotic, highly conserved, Ca2+‐binding protein predominantly located in the endoplasmic reticulum (ER) lumen. In addition to being involved in the regulation of cellular Ca2+, calreticulin is a key quality control element during protein folding in the ER lumen. Tobacco (Nicotiana tabacum L.) suspension cells overexpressing a maize CRT (CRT1a) were used here to examine the properties of CRT in growing plant cells with respect to stress exposure. The endogenous CRT gene was induced rapidly after subculturing of the cells to new medium. In accordance, the CRT protein levels increased, peaking at days 3–4. At day 5, when the CRT transcript levels had levelled off, a further increase in endogenous CRT expression was obtained when the cells were treated with excess Ca2+ or the N‐linked glycosylation inhibitor tunicamycin. Whereas the response to Ca2+ occurred within 30 min, the induction by tunicamycin took several hours to be established. Transforming tobacco cells with maize CRT1a, under a constitutive mannopine synthase promoter, resulted in a stable level of expressed CRT1a during the growth cycle compared with endogenous CRT. The CRTs showed differences in attached glycans, but both contained the high mannose‐rich‐type glycans characteristic of ER proteins. In agreement with an ER location, both tobacco CRT and the transgene product CRT1a codistributed with the ER marker NADH cytochrome c reductase after density gradient centrifugation of microsomal fractions from tobacco cells. Increased production of CRT, as was obtained in the transgenic tobacco cell lines, made cells more tolerant than wild‐type cells to high Ca2+ during growth. These data suggest that overexpression of CRT1a in plant cells results in a more efficient calcium buffering capacity in the ER.

Tubular aggregates are from whole sarcoplasmic reticulum origin: alterations in calcium binding protein expression in mouse skeletal muscle during aging

Tubular aggregates are observed in various muscle disorders and appear as densely packed tubules believed to arise from sarcoplasmic reticulum of striated muscle. They are found both in human skeletal muscle, especially from patients suffering from ‘tubular aggregate myopathy’ and in fast twitch skeletal muscle of the male inbred mouse during aging. In this work, we studied tubular aggregates present in inbred male mouse skeletal muscle using electron microscopy as well as histochemistry and Western blotting with the main markers of the sarcoplasmic reticulum. We show that mouse tubular aggregates include the proteins SERCA 1, sarcalumenin (longitudinal sarcoplasmic reticulum), calsequestrin (terminal cisternae) and RyR1 (junctional sarcoplasmic reticulum). We demonstrate also that 95 and 51 kDa triadin isoforms are present in mouse skeletal muscle and are both components of tubular aggregates. These results support the hypothesis that tubular aggregates form a tubular arrangement of a complete sarcoplasmic reticulum containing the junctional, cisternae and longitudinal components of sarcoplasmic reticulum implicated in calcium homeostasis. During mouse skeletal muscle aging, however, densitometry of Western blots reveals a persistent decrease in the expression of the calcium binding protein calreticulin as well as a continuous increase in calsequestrin-like protein expression which both appear unrelated to the tubular aggregate formation.