C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in β-cells.

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

Cation Transport Coupled to ATP Hydrolysis By the (Na, K)-ATPase : an integrated, animated model

An Adobe (R) animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na+ and K+ translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P-2c-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also known as an E-1/E-2-ATPase as it undergoes conformational changes between the E-1 and E-2 forms during the pumping cycle, altering the affinity and accessibility of the transmembrane ion-binding sites. The animation is based on Horisberger's scheme that incorporates the most recent significant findings to have improved our understanding of the (Na, K)-ATPase structure function relationship. The movements of the various domains within the (Na, K)-ATPase alpha-subunit illustrate the conformational changes that occur during Na+ and K+ translocation across the membrane and emphasize involvement of the actuator, nucleotide, and phosphorylation domains, that is, the "core engine" of the pump, with respect to ATP binding, cation transport, and ADP and P-i release.

Molecular characterization of the processing of arenavirus envelope glycoprotein precursors by subtilisin kexin isozyme-1/site-1 protease.

A crucial step in the life cycle of arenaviruses is the biosynthesis of the mature fusion-active viral envelope glycoprotein (GP) that is essential for virus-host cell attachment and entry. The maturation of the arenavirus GP precursor (GPC) critically depends on proteolytic processing by the cellular proprotein convertase (PC) subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P). Here we undertook a molecular characterization of the SKI-1/S1P processing of the GPCs of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and the pathogenic Lassa virus (LASV). Previous studies showed that the GPC of LASV undergoes processing in the endoplasmic reticulum (ER)/cis-Golgi compartment, whereas the LCMV GPC is cleaved in a late Golgi compartment. Herein we confirm these findings and provide evidence that the SKI-1/S1P recognition site RRLL, present in the SKI-1/S1P prodomain and LASV GPC, but not in the LCMV GPC, is crucial for the processing of the LASV GPC in the ER/cis-Golgi compartment. Our structure-function analysis revealed that the cleavage of arenavirus GPCs, but not cellular substrates, critically depends on the autoprocessing of SKI-1/S1P, suggesting differences in the processing of cellular and viral substrates. Deletion mutagenesis showed that the transmembrane and intracellular domains of SKI-1/S1P are dispensable for arenavirus GPC processing. The expression of a soluble form of the protease in SKI-I/S1P-deficient cells resulted in the efficient processing of arenavirus GPCs and rescued productive virus infection. However, exogenous soluble SKI-1/S1P was unable to process LCMV and LASV GPCs displayed at the surface of SKI-I/S1P-deficient cells, indicating that GPC processing occurs in an intracellular compartment. In sum, our study reveals important differences in the SKI-1/S1P processing of viral and cellular substrates.

Regulation of the transforming growth factor beta-responsive transcription factor CTF-1 by calcineurin and calcium/calmodulin-dependent protein kinase IV.

Transforming growth factor beta (TGF-beta) is a pluripotent peptide hormone that regulates various cellular activities, including growth, differentiation, and extracellular matrix protein gene expression. We previously showed that TGF-beta induces the transcriptional activation domain (TAD) of CTF-1, the prototypic member of the CTF/NF-I family of transcription factors. This induction correlates with the proposed role of CTF/NF-I binding sites in collagen gene induction by TGF-beta. However, the mechanisms of TGF-beta signal transduction remain poorly understood. Here, we analyzed the role of free calcium signaling in the induction of CTF-1 transcriptional activity by TGF-beta. We found that TGF-beta stimulates calcium influx and mediates an increase of the cytoplasmic calcium concentration in NIH3T3 cells. TGF-beta induction of CTF-1 is inhibited in cells pretreated with thapsigargin, which depletes the endoplasmic reticulum calcium stores, thus further arguing for the potential relevance of calcium mobilization in TGF-beta action. Consistent with this possibility, expression of a constitutively active form of the calcium/calmodulin-dependent phosphatase calcineurin or of the calcium/calmodulin-dependent kinase IV (DeltaCaMKIV) specifically induces the CTF-1 TAD and the endogenous mouse CTF/NF-I proteins. Both calcineurin- and DeltaCaMKIV-mediated induction require the previously identified TGF-beta-responsive domain of CTF-1. The immunosuppressants cyclosporin A and FK506 abolish calcineurin-mediated induction of CTF-1 activity. However, TGF-beta still induces the CTF-1 TAD in cells treated with these compounds or in cells overexpressing both calcineurin and DeltaCaMKIV, suggesting that other calcium-sensitive enzymes might mediate TGF-beta action. These results identify CTF/NF-I as a novel calcium signaling pathway-responsive transcription factor and further suggest multiple molecular mechanisms for the induction of CTF/NF-I transcriptional activity by growth factors.

Effects of verapamil and ryanodine on activity of the embryonic chick heart during anoxia and reoxygenation.

Perturbations of the trans-sarcolemmal and sarcoplasmic Ca2+ transport contribute to the abnormal myocardial activity provoked by anoxia and reoxygenation. Whether Ca2+ pools of the extracellular compartment and sarcoplasmic reticulum (SR) are involved to the same extent in the dysfunction of the anoxic-reoxygenated immature heart has not been investigated. Spontaneously contracting hearts isolated from 4-day-old chick embryos were submitted to repeated anoxia (1 min) followed by reoxygenation (5 min). Heart rate, atrioventricular propagation velocity, ventricular shortening, velocities of contraction and relaxation, and incidence of arrhythmias were studied, recorded continuously. Addition of verapamil (10 nM), which blocks selectively sarcolemmal L-type Ca2+ channels, was expected to protect against excessive entry of extracellular Ca2+, whereas addition of ryanodine (10 nM), which opens the SR Ca2+ release channel, was expected to increase cytosolic Ca2+ concentration. Verapamil (a) had no dromotropic effect by contrast to adult heart, (b) attenuated ventricular contracture induced by repeated anoxia, (c) shortened cardioplegia induced by reoxygenation, and (d) had remarkable antiarrhythmic properties during reoxygenation specially. On the other hand, ryanodine potentiated markedly arrhythmias both during anoxia and at reoxygenation. Thus despite its immaturity, the SR seems to be functional early in the developing chick heart and involved in the reversible dysfunction induced by anoxia-reoxygenation. Moreover, Ca2+ entry through L-type channels appears to worsen arrhythmias especially during reoxygenation. These findings show that the Ca2+-handling systems involved in irregular activity in immature heart, such as the embryonic chick heart, may differ from those in the adult.

Interplay of oxidative, nitrosative/nitrative stress, inflammation, cell death and autophagy in diabetic cardiomyopathy.

Diabetes is a recognized risk factor for cardiovascular diseases and heart failure. Diabetic cardiovascular dysfunction also underscores the development of diabetic retinopathy, nephropathy and neuropathy. Despite the broad availability of antidiabetic therapy, glycemic control still remains a major challenge in the management of diabetic patients. Hyperglycemia triggers formation of advanced glycosylation end products (AGEs), activates protein kinase C, enhances polyol pathway, glucose autoxidation, which coupled with elevated levels of free fatty acids, and leptin have been implicated in increased generation of superoxide anion by mitochondria, NADPH oxidases and xanthine oxidoreductase in diabetic vasculature and myocardium. Superoxide anion interacts with nitric oxide forming the potent toxin peroxynitrite via diffusion limited reaction, which in concert with other oxidants triggers activation of stress kinases, endoplasmic reticulum stress, mitochondrial and poly(ADP-ribose) polymerase 1-dependent cell death, dysregulates autophagy/mitophagy, inactivates key proteins involved in myocardial calcium handling/contractility and antioxidant defense, activates matrix metalloproteinases and redox-dependent pro-inflammatory transcription factors (e.g. nuclear factor kappaB) promoting inflammation, AGEs formation, eventually culminating in myocardial dysfunction, remodeling and heart failure. Understanding the complex interplay of oxidative/nitrosative stress with pro-inflammatory, metabolic and cell death pathways is critical to devise novel targeted therapies for diabetic cardiomyopathy, which will be overviewed in this brief synopsis. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

GLUT8 subcellular localization and absence of translocation to the plasma membrane in PC12 cells and hippocampal neurons.

GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.

How do plants feel the heat?

In plants, the heat stress response (HSR) is highly conserved and involves multiple pathways, regulatory networks and cellular compartments. At least four putative sensors have recently been proposed to trigger the HSR. They include a plasma membrane channel that initiates an inward calcium flux, a histone sensor in the nucleus, and two unfolded protein sensors in the endoplasmic reticulum and the cytosol. Each of these putative sensors is thought to activate a similar set of HSR genes leading to enhanced thermotolerance, but the relationship between the different pathways and their hierarchical order is unclear. In this review, we explore the possible involvement of different thermosensors in the plant response to warming and heat stress.

Clic4, a novel protein that sensitizes ß-cells to apoptosis.

Introduction: La préservation et/ou l'expansion de la masse des cellules ß pourraient constituer des approches prometteuses dans le traitement du diabète. L'une des stratégies clés serait de réduire l'apoptose des cellules ß. Le chloride intracellular channel protein 4 (Clic4) est une protéine exprimée de manière ubiquitaire et supposée agir dans de nombreux processus cellulaires tels que le contrôle du cycle cellulaire, la différenciation cellulaire et l'apoptose. Ici, nous avons étudié le rôle de Clic4 dans l'apoptose des cellules ß pancréatiques en utilisant des cellules ßTC-tet et des îlots de Langerhans issus de souris knockout pour Clic4 (ßClic4KO). Résultats: L'expression de l'ARNm et de la protéine Clic4 était augmentée par un traitement aux cytokines dans les cellules ßTC-tet et encore plus fortement dans des îlots isolés de souris. De plus, la sous-expression de Clic4 dans les cellules ßTC-tet diminuait leur sensibilité à l'apoptose induite par les cytokines. La sous-expression de Clic4 dans les cellules ßTC-tet n'affectait pas l'expression des ARNm de Bcl-2 et Bad, mais augmentait leur expression protéique ainsi que la forme phosphorylée de Bad. Les mêmes résultats ont été obtenus sur des îlots isolés de souris contrôles et ßClic4KO. De plus, les îlots issus de souris ßClic4KO présentaient une augmentation de l'expression de la protéine Bcl-xL. Dans le but de déterminer si Clic4 augmentait l'expression de Bcl-2 et Bad via une interaction protéique directe, nous avons immunoprécipité Clic4 à partir de cellules ßTC-tet à l'aide d'anticorps dirigés contre la partie C- ou N-terminale de la protéine, puis nous avons soumis les immunoprécipités à une analyse de spectrométrie de masse. Aucune co- immunoprécipitation avec Bcl-2 ou d'autres protéines de la famille Bcl-2 n'a été détectée. Cependant, de manière intéressante, Clic4 était co-purifié avec plusieurs protéines du protéasome suggérant un rôle de Clic4 dans la dégradation des protéines. Par conséquent, nous avons étudié la demi-vie de Bcl-2 et Bad, et avons observé que la sous-expression de Clic4 dans les cellules ßTC-tet augmentait la demi-vie de ces protéines. De plus, l'expression de l'ARNm et de la protéine Clic4 était également augmentée lors d'un stress du réticulum endoplasmique induit par la thapsigargine dans les cellules ßTC-tet. La sous-expression de Clic4 dans les cellules ßTC-tet ou chez les KO diminuait la sensibilité des cellules ß à l'apoptose induite par la thapsigargine ou l'acide palmitique, respectivement. Conclusion: Ces résultats suggèrent que Clic4 sensibilise les cellules ß à l'apoptose induite par les cytokines ou l'acide palmitique/thapsigargine (stress du réticulum endoplasmique). De plus, la sous-expression de Clic4 améliore la survie des cellules ß en diminuant la dégradation de Bcl-2 et Bcl-xL, et en augmentant le niveau total de Bad phosphorylé, peut-être suite à une interaction de Clic4 avec le protéasome.

Morbillivirus glycoprotein expression induces ER stress, alters Ca2+ homeostasis and results in the release of vasostatin.

Although the pathology of Morbillivirus in the central nervous system (CNS) is well described, the molecular basis of neurodegenerative events still remains poorly understood. As a model to explore Morbillivirus-mediated CNS dysfunctions, we used canine distemper virus (CDV) that we inoculated into two different cell systems: a monkey cell line (Vero) and rat primary hippocampal neurons. Importantly, the recombinant CDV used in these studies not only efficiently infects both cell types but recapitulates the uncommon, non-cytolytic cell-to-cell spread mediated by virulent CDVs in brain of dogs. Here, we demonstrated that both CDV surface glycoproteins (F and H) markedly accumulated in the endoplasmic reticulum (ER). This accumulation triggered an ER stress, characterized by increased expression of the ER resident chaperon calnexin and the proapoptotic transcription factor CHOP/GADD 153. The expression of calreticulin (CRT), another ER resident chaperon critically involved in the response to misfolded proteins and in Ca(2+) homeostasis, was also upregulated. Transient expression of recombinant CDV F and H surface glycoproteins in Vero cells and primary hippocampal neurons further confirmed a correlation between their accumulation in the ER, CRT upregulation, ER stress and disruption of ER Ca(2+) homeostasis. Furthermore, CDV infection induced CRT fragmentation with re-localisation of a CRT amino-terminal fragment, also known as vasostatin, on the surface of infected and neighbouring non-infected cells. Altogether, these results suggest that ER stress, CRT fragmentation and re-localization on the cell surface may contribute to cytotoxic effects and ensuing cell dysfunctions triggered by Morbillivirus, a mechanism that might potentially be relevant for other neurotropic viruses.

Calreticulin levels determine onset of early muscle denervation by fast motoneurons of ALS model mice.

Although the precise signaling mechanisms underlying the vulnerability of some sub-populations of motoneurons in ALS remain unclear, critical factors such as metallo-proteinase 9 expression, neuronal activity and endoplasmic reticulum stress have been shown to be involved. In the context of SOD1(G93A) ALS mouse model, we previously showed that a two-fold decrease in calreticulin (CRT) is occurring in the vulnerable fast motoneurons. Here, we asked to which extent the decrease in CRT levels was causative to muscle denervation and/or motoneuron degeneration. Toward this goal, a hemizygous deletion of the crt gene in SOD1(G93A) mice was generated since the complete ablation of crt is embryonic lethal. We observed that SOD1(G93A);crt(+/-) mice display increased and earlier muscle weakness and muscle denervation compared to SOD1(G93A) mice. While CRT reduction in motoneurons leads to a strong upregulation of two factors important in motoneuron dysfunction, ER stress and mTOR activation, it does not aggravate motoneuron death. Our results underline a prevalent role for CRT levels in the early phase of muscle denervation and support CRT regulation as a potential therapeutic approach.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Lack of GDAP1 induces neuronal calcium and mitochondrial defects in a knockout mouse model of charcot-marie-tooth neuropathy.

Mutations in GDAP1, which encodes protein located in the mitochondrial outer membrane, cause axonal recessive (AR-CMT2), axonal dominant (CMT2K) and demyelinating recessive (CMT4A) forms of Charcot-Marie-Tooth (CMT) neuropathy. Loss of function recessive mutations in GDAP1 are associated with decreased mitochondrial fission activity, while dominant mutations result in impairment of mitochondrial fusion with increased production of reactive oxygen species and susceptibility to apoptotic stimuli. GDAP1 silencing in vitro reduces Ca2+ inflow through store-operated Ca2+ entry (SOCE) upon mobilization of endoplasmic reticulum (ER) Ca2+, likely in association with an abnormal distribution of the mitochondrial network. To investigate the functional consequences of lack of GDAP1 in vivo, we generated a Gdap1 knockout mouse. The affected animals presented abnormal motor behavior starting at the age of 3 months. Electrophysiological and biochemical studies confirmed the axonal nature of the neuropathy whereas histopathological studies over time showed progressive loss of motor neurons (MNs) in the anterior horn of the spinal cord and defects in neuromuscular junctions. Analyses of cultured embryonic MNs and adult dorsal root ganglia neurons from affected animals demonstrated large and defective mitochondria, changes in the ER cisternae, reduced acetylation of cytoskeletal α-tubulin and increased autophagy vesicles. Importantly, MNs showed reduced cytosolic calcium and SOCE response. The development and characterization of the GDAP1 neuropathy mice model thus revealed that some of the pathophysiological changes present in axonal recessive form of the GDAP1-related CMT might be the consequence of changes in the mitochondrial network biology and mitochondria-endoplasmic reticulum interaction leading to abnormalities in calcium homeostasis.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

β-Adrenergic modulation of skeletal muscle contraction: key role of excitation-contraction coupling.

Our aim is to describe the acute effects of catecholamines/β-adrenergic agonists on contraction of non-fatigued skeletal muscle in animals and humans, and explain the mechanisms involved. Adrenaline/β-agonists (0.1-30 μm) generally augment peak force across animal species (positive inotropic effect) and abbreviate relaxation of slow-twitch muscles (positive lusitropic effect). A peak force reduction also occurs in slow-twitch muscles in some conditions. β2 -Adrenoceptor stimulation activates distinct cyclic AMP-dependent protein kinases to phosphorylate multiple target proteins. β-Agonists modulate sarcolemmal processes (increased resting membrane potential and action potential amplitude) via enhanced Na(+) -K(+) pump and Na(+) -K(+) -2Cl(-) cotransporter function, but this does not increase force. Myofibrillar Ca(2+) sensitivity and maximum Ca(2+) -activated force are unchanged. All force potentiation involves amplified myoplasmic Ca(2+) transients consequent to increased Ca(2+) release from sarcoplasmic reticulum (SR). This unequivocally requires phosphorylation of SR Ca(2+) release channels/ryanodine receptors (RyR1) which sensitize the Ca(2+) -induced Ca(2+) release mechanism. Enhanced trans-sarcolemmal Ca(2+) influx through phosphorylated voltage-activated Ca(2+) channels contributes to force potentiation in diaphragm and amphibian muscle, but not mammalian limb muscle. Phosphorylation of phospholamban increases SR Ca(2+) pump activity in slow-twitch fibres but does not augment force; this process accelerates relaxation and may depress force. Greater Ca(2+) loading of SR may assist force potentiation in fast-twitch muscle. Some human studies show no significant force potentiation which appears to be related to the β-agonist concentration used. Indeed high-dose β-agonists (∼0.1 μm) enhance SR Ca(2+) -release rates, maximum voluntary contraction strength and peak Wingate power in trained humans. The combined findings can explain how adrenaline/β-agonists influence muscle performance during exercise/stress in humans.