Circadian Rhythms. Carbon Monoxide and Clocks

Science (New York, N.Y.). Dec, 2002  |  Pubmed ID: 12493901

Novel Neural Modulators

Annual Review of Neuroscience. 2003  |  Pubmed ID: 14527267

The discovery that nitric oxide (NO) is produced by neurons and regulates synaptic activity has challenged the definition of a neurotransmitter. NO is not stored in synaptic vesicles and does not act at conventional receptors on the surface of adjacent neurons. The toxic gases carbon monoxide (CO) and hydrogen sulfide (H2S) are also produced by neurons and modulate synaptic activity. D-serine synthesis and release by astrocytes as an endogenous ligand for the "glycine" site of N-methyl D-aspartate (NMDA) receptors defy the concept that a neurotransmitter must be synthesized by neurons. We review the properties of these "atypical" neural modulators.

Carbon Monoxide Neurotransmission Activated by CK2 Phosphorylation of Heme Oxygenase-2

Neuron. Sep, 2003  |  Pubmed ID: 14527438

Carbon monoxide (CO) is a putative gaseous neurotransmitter that lacks vesicular storage and must be synthesized rapidly following neuronal depolarization. We show that the biosynthetic enzyme for CO, heme oxygenase-2 (HO2), is activated during neuronal stimulation by phosphorylation by CK2 (formerly casein kinase 2). Phorbol ester treatment of hippocampal cultures results in the phosphorylation and activation of HO2 by CK2, implicating protein kinase C (PKC) in CK2 stimulation. Odorant treatment of olfactory receptor neurons augments HO2 phosphorylation and activity as well as cyclic guanosine monophosphate (cGMP) levels, with all of these effects selectively blocked by CK2 inhibitors. Likewise, CO-mediated nonadrenergic, noncholinergic (NANC) relaxation of the internal anal sphincter requires CK2 activity. Our findings provide a molecular mechanism for the rapid neuronal activation of CO biosynthesis, as required for a gaseous neurotransmitter.

Cytochrome C Binds to Inositol (1,4,5) Trisphosphate Receptors, Amplifying Calcium-dependent Apoptosis

Nature Cell Biology. Dec, 2003  |  Pubmed ID: 14608362

Mitochondrial cytochrome c release and inositol (1,4,5) trisphosphate receptor (InsP(3)R)-mediated calcium release from the endoplasmic reticulum mediate apoptosis in response to specific stimuli. Here we show that cytochrome c binds to the InsP(3)R during apoptosis. Addition of 1 nM cytochrome c blocks calcium-dependent inhibition of InsP(3)R function. Early in apoptosis, cytochrome c translocates to the endoplasmic reticulum where it selectively binds InsP(3)R, resulting in sustained, oscillatory cytosolic calcium increases. These calcium events are linked to the coordinate release of cytochrome c from all mitochondria. Our findings identify a feed-forward mechanism whereby early cytochrome c release increases InsP(3)R function, resulting in augmented cytochrome c release that amplifies the apoptotic signal.

Apoptosis and Calcium: New Roles for Cytochrome C and Inositol 1,4,5-trisphosphate

Cell Cycle (Georgetown, Tex.). Mar, 2004  |  Pubmed ID: 14726673

Mounting evidence suggests that calcium released from internal stores plays a critical role in the progression of apoptosis. The primary calcium release channel on endoplasmic reticulum membranes is the inositol 1,4,5-trisphosphate receptor (IP3R). Deletion of the gene for IP3R results in defects in apoptosis in response to multiple stimuli. Conversely, augmented IP3R levels are associated with increased cell death. A mechanistic basis for altered IP3R function during apoptosis was revealed with the discovery that cytochrome c binds to IP3R early in apoptosis. This interaction blocks the calcium-dependent inhibition of IP3R function, resulting in increased calcium release from internal stores. The resultant cytoplasmic and mitochondrial calcium overload culminates in cell-wide cytochrome c release and maximal caspase activation. These findings highlight the importance of intracellular calcium stores in apoptosis, and the multi-functional role of cytochrome c released from mitochondria in cell death.

Agonist-induced Ca2+ Entry Determined by Inositol 1,4,5-trisphosphate Recognition

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2004  |  Pubmed ID: 14983008

It has been considered that Ca2+ release is the causal trigger for Ca2+ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP3R), the lack of Ca2+ entry in response to receptor activation is attributed to the absence of Ca2+ release. We reveal in this article that IP3R recognition of IP3 determines agonist-induced Ca2+ entry (ACE), independent of its Ca2+ release activity. In DT40 IP3R(-/-) cells, endogenous ACE can be rescued with type 1 IP3R mutants (both a DeltaC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca2+ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP3R-dependent manner without Ca2+ store release. Conversely, ACE cannot be rescued with mutant IP3Rs lacking IP3 binding (both the Delta90-110 and R265Q IP3-binding site mutants). We conclude that an IP3-dependent conformational change in the IP3R, not endoplasmic reticulum Ca2+ pool release, triggers ACE.

Carbon Monoxide Mediates Vasoactive Intestinal Polypeptide-associated Nonadrenergic/noncholinergic Neurotransmission

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2004  |  Pubmed ID: 14983060

Carbon monoxide (CO) synthesized by heme oxygenase 2 (HO2) and nitric oxide (NO) produced by neuronal NO synthase (nNOS) mediate nonadrenergic/noncholinergic (NANC) intestinal relaxation. In many areas of the gastrointestinal tract, NO and CO function as coneurotransmitters. In the internal anal sphincter (IAS), NANC relaxation is mediated primarily by CO. Vasoactive intestinal polypeptide (VIP) has also been shown to participate in NANC relaxation throughout the intestine, including the IAS. By using a combination of pharmacology and genetic knockout of the biosynthetic enzymes for CO and NO, we show that the physiologic effects of exogenous and endogenous VIP in the IAS are mediated by HO2-synthesized CO.

Heme Oxygenase-2 is Activated by Calcium-calmodulin

The Journal of Biological Chemistry. Jul, 2004  |  Pubmed ID: 15175337

The heme oxygenase family of enzymes catalyzes the metabolism of heme to biliverdin, ferrous iron, and carbon monoxide (CO). At least two isoforms exist, heme oxygenase-1 (HO1) and heme oxygenase-2 (HO2), which are encoded by separate genes. HO2 is selectively enriched in neurons, and substantial evidence suggests that HO2-derived CO functions as a neurotransmitter/neuromodulator. However, a molecular mechanism for the rapid activation of HO2 during neuronal activity has not been described. Through a yeast two-hybrid screen we identified calmodulin as a potential regulator of HO2 activity. Calmodulin binds with nanomolar affinity to HO2 in a calcium-dependent manner via a canonical 1-10 motif, resulting in a 3-fold increase in catalytic activity. Mutations within this motif block calmodulin binding and calcium-dependent stimulation of enzyme activity in vitro and in intact cells. The calcium mobilizing agents ionomycin and glutamate stimulate endogenous HO2 activity in primary cortical cultures, establishing in vivo relevance. Calcium-calmodulin provides a mechanism for rapid and transient activation of HO2 during neuronal activity.

Inositol 1,4,5-trisphosphate Receptors As Signal Integrators

Annual Review of Biochemistry. 2004  |  Pubmed ID: 15189149

The inositol 1,4,5 trisphosphate (IP3) receptor (IP3R) is a Ca2+ release channel that responds to the second messenger IP3. Exquisite modulation of intracellular Ca2+ release via IP3Rs is achieved by the ability of IP3R to integrate signals from numerous small molecules and proteins including nucleotides, kinases, and phosphatases, as well as nonenzyme proteins. Because the ion conduction pore composes only approximately 5% of the IP3R, the great bulk of this large protein contains recognition sites for these substances. Through these regulatory mechanisms, IP3R modulates diverse cellular functions, which include, but are not limited to, contraction/excitation, secretion, gene expression, and cellular growth. We review the unique properties of the IP3R that facilitate cell-type and stimulus-dependent control of function, with special emphasis on protein-binding partners.

A Peptide Inhibitor of Cytochrome C/inositol 1,4,5-trisphosphate Receptor Binding Blocks Intrinsic and Extrinsic Cell Death Pathways

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2005  |  Pubmed ID: 15665074

Apoptotic stimuli augment intracellular calcium concentration through inositol 1,4,5-trisphosphate receptors (IP3R) on endoplasmic reticulum calcium stores. We previously discovered an apoptotic cascade wherein cytochrome c binds to IP3R early in apoptosis, resulting in dysregulated calcium release. Here we show that cytochrome c binding to IP3R depends on a cluster of glutamic acid residues within the C terminus of the channel. A cell permeant peptide derived from this sequence displaces cytochrome c from IP3R and abrogates cell death induced by staurosporine treatment of HeLa cells and Fas ligand stimulation of Jurkat cells. Small-molecule inhibitors of cytochrome c/IP3R interactions may prove useful in treating disorders associated with inappropriate intrinsic and extrinsic apoptotic signaling.

Requirement of Biphasic Calcium Release from the Endoplasmic Reticulum for Fas-mediated Apoptosis

The Journal of Cell Biology. Dec, 2006  |  Pubmed ID: 17130290

Fas receptor is a member of the tumor necrosis factor-alpha family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum-mediated calcium release in a mechanism dependent on phospholipase C-gamma1 (PLC-gamma1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-gamma1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-gamma1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.

Hepatitis C Virus Core Protein Increases Mitochondrial ROS Production by Stimulation of Ca2+ Uniporter Activity

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2007  |  Pubmed ID: 17392480

Many viruses have evolved mechanisms to alter mitochondrial function. The hepatitis C virus (HCV) produces a viral core protein that targets to mitochondria and increases Ca2+-dependent ROS production. The aim of this study was to determine whether core's effects are mediated by changes in mitochondrial Ca2+ uptake. Core expression caused enhanced mitochondrial Ca2+ uptake in response to ER Ca2+ release induced by thapsigargin or ATP. It also increased mitochondrial superoxide production and mitochondrial permeability transition (MPT). Incubating mouse liver mitochondria with an HCV core (100 ng/mg) in vitro increased Ca2+ entry rate by approximately 2-fold. Entry was entirely inhibited by the mitochondrial Ca2+ uniporter inhibitor, Ru-360, but not influenced by an Na+/Ca2+ exchanger inhibitor or ROS scavengers. These results indicate that core directly increases mitochondrial Ca2+ uptake via a primary effect on the uniporter. This enhanced the ability of mitochondria to sequester Ca2+ in response to ER Ca2+ release, and increased mitochondrial ROS production and MPT. Thus, the mitochondrial Ca2+ uniporter is a newly identified target for viral modification of cell function.

Gene Identification and Evidence for Expression of G Protein Alpha Subunits, Phospholipase C, and an Inositol 1,4,5-trisphosphate Receptor in Aplysia Californica Rhinophore

Genomics. Jul, 2007  |  Pubmed ID: 17498918

In the marine mollusk Aplysia californica, waterborne protein pheromones that are released during egg laying act in concert to stimulate mate attraction. However, molecular information concerning the cellular receptors and signaling mechanisms that may be involved in waterborne peptide and protein pheromonal communication is lacking. As a first step toward examining whether members of the G protein family and phosphoinositide signaling pathway are present in the primary peripheral chemosensory organs (i.e., rhinophores), we isolated five full-length cDNA clones from an A. californica central nervous system cDNA library. These clones encoded (1) the G protein alpha subunits of the Gq, Gi, and Go families, (2) a protein with homology to phospholipase C (PLC) isoforms, and (3) an inositol 1,4,5-trisphosphate receptor (IP3R). The expression of these genes was examined using laser capture microdissection/reverse transcription-polymerase chain reaction and in situ hybridization. All of them are expressed in the rhinophore sensory epithelium, suggesting that Galphaq, Galphai, Galphao, PLC-like protein, and IP3R may be involved in waterborne protein pheromone detection in Aplysia-possibly via a phosphoinositide signaling mechanism.

Effect of Insulin on the Inflammatory and Acute Phase Response After Burn Injury

Critical Care Medicine. Sep, 2007  |  Pubmed ID: 17713402

After a severe burn, the liver plays a pivotal role by modulating inflammatory processes, metabolic pathways, immune functions, and the acute phase response. Therefore, liver integrity and function are important for recovery. A thermal injury, however, causes hepatic damage by inducing hepatic edema, fatty infiltration, hepatocyte apoptosis, and metabolic derangements associated with insulin resistance and impaired insulin signaling. In preliminary studies, we found that these pathophysiological processes are related to hepatic inflammation, altered intracellular signaling, and mitochondrial dysfunction. We hypothesize that modulation of these processes with insulin could improve hepatic structure and function and, therefore, outcome of burned and critically ill patients. Insulin administration improves survival and decreases the rate of infections in severely burned and critically ill patients. Here, we show that insulin administration decreases the synthesis of proinflammatory cytokines and signal transcription factors and improves hepatic structure and function after a severe burn injury; insulin also restores hepatic homeostasis and improves hepatic dysfunction postburn via alterations in the signaling cascade.

Requirement of Inositol 1,4,5-trisphosphate Receptors for Tumor-mediated Lymphocyte Apoptosis

The Journal of Biological Chemistry. May, 2008  |  Pubmed ID: 18364356

Tumor cells strategically down-regulate Fas receptor expression to evade immune attack and up-regulate expression of Fas ligand to promote apoptosis of infiltrating T lymphocytes. Many pathways leading to apoptotic cell death require calcium release from inositol 1,4,5-trisphosphate receptors (IP3Rs). Here, we show that Fas-dependent killing of Jurkat T lymphoma cells by SW620 colon cancer cells requires calcium release from IP3R. General suppression of IP3R signaling significantly reduced SW620-mediated Jurkat cell apoptosis. Significantly, a specific inhibitor of apoptotic calcium release from IP3R strongly blocked lymphocyte apoptosis. Thus, selective pharmacological targeting of apoptotic calcium release from IP3R may enhance tumor cell immunogenicity.

Characterization of the Inflammatory Response During Acute and Post-acute Phases After Severe Burn

Shock (Augusta, Ga.). Nov, 2008  |  Pubmed ID: 18391855

Severe burn causes a pronounced hypermetabolic response characterized by catabolism and extensive protein wasting. We recently found that this hypermetabolic state is driven by a severe inflammatory response. We characterized in detail the kinetics of serum levels of a panel of cytokines in a rat model, which may serve as reference for the development of therapeutic interventions applicable to humans. Male Sprague-Dawley rats (n = 8) received a full-thickness burn of 60% total body surface area. Serum was harvested 1, 3, 6, 12, 24, 48, 96, and 168 h after burn. Eight serum cytokines commonly used to assess the inflammatory response in humans, such as IL-1beta, IL-6, IL-10, TNF, vascular endothelial growth factor, and monocyte chemotactic protein 1, and the rat-specific cytokines cytokine-induced neutrophil chemoattractant (CINC) 1, CINC-2, and CINC-3 were measured by enzyme-linked immunosorbent assay technique and were compared with controls (n = 4). Statistical analysis was conducted using the t test, with P < 0.05 considered as significantly different. Thermal injury resulted in significantly increased serum levels of IL-1beta, IL-6, IL-10, monocyte chemotactic protein 1, CINC-1, CINC-2, and CINC-3 when compared with the concentrations detected in nonburned rats (P < 0.05). Serum levels of TNF-alpha and vascular endothelial growth factor in burned rats were not found to be significantly different to controls. Burn causes a profound inflammatory response in rats. Specific cytokines known to increase in humans postburn such as IL-1 beta, IL-6, IL-10, MCP-1, and IL-8 (CINC-1, CINC-2, and CINC-3 in the rat) were also observed in our rat burn model, which now allows us to study new anti-inflammatory treatment options.

Role of Heme Oxygenase-2 in Pial Arteriolar Response to Acetylcholine in Mice with and Without Transfusion of Cell-free Hemoglobin Polymers

American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. Aug, 2008  |  Pubmed ID: 18495834

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.

Generation of Spinal Motor Neurons from Human Fetal Brain-derived Neural Stem Cells: Role of Basic Fibroblast Growth Factor

Journal of Neuroscience Research. Feb, 2009  |  Pubmed ID: 18803285

Neural stem cells (NSCs) have some specified properties but are generally uncommitted and so can change their fate after exposure to environmental cues. It is unclear to what extent this NSC plasticity can be modulated by extrinsic cues and what are the molecular mechanisms underlying neuronal fate determination. Basic fibroblast growth factor (bFGF) is a well-known mitogen for proliferating NSCs. However, its role in guiding stem cells for neuronal subtype specification is undefined. Here we report that in-vitro-expanded human fetal forebrain-derived NSCs can generate cholinergic neurons with spinal motor neuron properties when treated with bFGF within a specific time window. bFGF induces NSCs to express the motor neuron marker Hb9, which is blocked by specific FGF receptor inhibitors and bFGF neutralizing antibodies. This development of spinal motor neuron properties is independent of selective proliferation or survival and does not require high levels of MAPK activation. Thus our study indicates that bFGF can play an important role in modulating plasticity and neuronal fate of human NSCs and presumably has implications for exploring the full potential of brain NSCs for clinical applications, particularly in spinal motor neuron regeneration.

Severe Burn-induced Endoplasmic Reticulum Stress and Hepatic Damage in Mice

Molecular Medicine (Cambridge, Mass.). Sep-Oct, 2009  |  Pubmed ID: 19603103

Severe burn injury results in liver dysfunction and damage, with subsequent metabolic derangements contributing to patient morbidity and mortality. On a cellular level, significant postburn hepatocyte apoptosis occurs and likely contributes to liver dysfunction. However, the underlying mechanisms of hepatocyte apoptosis are poorly understood. The endoplasmic reticulum (ER) stress response/unfolded protein response (UPR) pathway can lead to hepatocyte apoptosis under conditions of liver dysfunction. Thus, we hypothesized that ER stress/UPR may mediate hepatic dysfunction in response to burn injury. We investigated the temporal activation of hepatic ER stress in mice after a severe burn injury. Mice received a scald burn over 35% of their body surface and were killed at 1, 7, 14, and 21 d postburn. We found that severe burn induces hepatocyte apoptosis as indicated by increased caspase-3 activity (P < 0.05). Serum albumin levels decreased postburn and remained lowered for up to 21 d, indicating that constitutive secretory protein synthesis was reduced. Significantly, upregulation of the ER stress markers glucose-related protein 78 (GRP78)/BIP, protein disulfide isomerase (PDI), p-protein kinase R-like endoplasmic reticulum kinase (p-PERK), and inositol-requiring enzyme 1alpha (IRE-1alpha) were found beginning 1 d postburn (P < 0.05) and persisted up to 21 d postburn (P < 0.05). Hepatic ER stress induced by burn injury was associated with compensatory upregulation of the calcium chaperone/storage proteins calnexin and calreticulin (P < 0.05), suggesting that ER calcium store depletion was the primary trigger for induction of the ER stress response. In summary, thermal injury in mice causes long-term adaptive and deleterious hepatic function alterations characterized by significant upregulation of the ER stress response.

Calcium and ER Stress Mediate Hepatic Apoptosis After Burn Injury

Journal of Cellular and Molecular Medicine. Aug, 2009  |  Pubmed ID: 20141609

A hallmark of the disease state following severe burn injury is decreased liver function, which results in gross metabolic derangements that compromise patient survival. The underlying mechanisms leading to hepatocyte dysfunction after burn are essentially unknown. The aim of the present study was to determine the underlying mechanisms leading to hepatocyte dysfunction and apoptosis after burn. Rats were randomized to either control (no burn) or burn (60% total body surface area burn) and sacrificed at various time-points. Liver was either perfused to isolate primary rat hepatocytes, which were used for in vitro calcium imaging, or liver was harvested and processed for immunohistology, transmission electron microscopy, mitochondrial isolation, mass spectroscopy or Western blotting to determine the hepatic response to burn injury in vivo. We found that thermal injury leads to severely depleted endoplasmic reticulum (ER) calcium stores and consequent elevated cytosolic calcium concentrations in primary hepatocytes in vitro. Burn-induced ER calcium depletion caused depressed hepatocyte responsiveness to signalling molecules that regulate hepatic homeostasis, such as vasopressin and the purinergic agonist ATP. In vivo, thermal injury resulted in activation of the ER stress response and major alterations in mitochondrial structure and function - effects which may be mediated by increased calcium release by inositol 1,4,5-trisphosphate receptors. Our results reveal that thermal injury leads to dramatic hepatic disturbances in calcium homeostasis and resultant ER stress leading to mitochondrial abnormalities contributing to hepatic dysfunction and apoptosis after burn injury.

T-cell Receptor Complex is Essential for Fas Signal Transduction

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2010  |  Pubmed ID: 20696918

The Fas receptor (also known as CD95 and APO-1) is a member of the tumor necrosis factor alpha-family of death receptors that mediate T-cell responses. Here, we show that Fas receptor signaling requires a functional T-cell receptor (TCR) complex. Fas receptor directly binds to and activates TCR components in a stimulus-dependent manner. Fas receptor stimulation does not activate canonical downstream TCR pathways, but instead the TCR complex is required specifically for Fas-mediated calcium release. Importantly, null mutations in Lck, ZAP70, and the TCR alpha- and beta-chains abrogate Fas signaling. Our results reveal a direct role for the TCR complex in mediating Fas-specific signaling events critical for T-cell homeostasis.

FAD Mutations in Amyloid Precursor Protein Do Not Directly Perturb Intracellular Calcium Homeostasis

PloS One. 2010  |  Pubmed ID: 20700539

Disturbances in intracellular calcium homeostasis are likely prominent and causative factors leading to neuronal cell death in Alzheimer's disease (AD). Familial AD (FAD) is early-onset and exhibits autosomal dominant inheritance. FAD-linked mutations have been found in the genes encoding the presenilins and amyloid precursor protein (APP). Several studies have shown that mutated presenilin proteins can directly affect calcium release from intracellular stores independently of Abeta production. Although less well established, there is also evidence that APP may directly modulate intracellular calcium homeostasis. Here, we directly examined whether overexpression of FAD-linked APP mutants alters intracellular calcium dynamics. In contrast to previous studies, we found that overexpression of mutant APP has no effects on basal cytosolic calcium, ER calcium store size or agonist-induced calcium release and subsequent entry. Thus, we conclude that mutated APP associated with FAD has no direct effect on intracellular calcium homeostasis independently of Abeta production.

Post-burn Hepatic Insulin Resistance is Associated with Endoplasmic Reticulum (ER) Stress

Shock (Augusta, Ga.). Mar, 2010  |  Pubmed ID: 22011639

Insulin resistance with its associated hyperglycemias represents one significant contributor to mortality in burned patients. A variety of cellular stress-signaling pathways are activated as a consequence of burn. A key player in the cellular stress response is the endoplasmic reticulum (ER). Here, we investigated a possible role for ER-stress pathways in the progression of insulin function dysregulation postburn. Rats received a 60% total body surface area thermal injury, and a laparotomy was performed at 24, 72, and 192 h postburn. Liver was harvested before and 1 min after insulin injection (1 IU/kg) into the portal vein, and expression patterns of various proteins known to be involved in insulin and ER-stress signaling were determined by Western blotting. mRNA expression of glucose-6-phosphatase and glucokinase were determined by reverse-transcriptase-polymerase chain reaction and fasting serum glucose and insulin levels by standard enzymatic and enzyme-linked immunosorbent assay techniques, respectively. Insulin resistance indicated by increased glucose and insulin levels occurred starting 24 h postburn. Burn injury resulted in activation of ER stress pathways, reflected by significantly increased accumulation of phospho-PKR-like ER-kinase and phosphorylated inositol requiring enzyme 1, leading to an elevation of phospho-c-Jun N-terminal kinase and serine phosphorylation of insulin receptor substrate (IRS) 1 postburn. Insulin administration caused a significant increase in tyrosine phosphorylation of IRS-1, leading to activation of the phosphatidylinositol 3 kinase/Akt pathway in normal liver. Postburn tyrosine phosphorylation of IRS-1 was significantly impaired, associated with an inactivation of signaling molecules acting downstream of IRS-1, leading to significantly elevated transcription of glucose-6-phosphatase and significantly decreased mRNA expression of glucokinase. Activation of ER-stress signaling cascades may explain metabolic abnormalities involving insulin action after burn.

Molecular Architecture of the Inositol 1,4,5-trisphosphate Receptor Pore

Current Topics in Membranes. 2010  |  Pubmed ID: 22353481

Insulin Protects Against Hepatic Damage Postburn

Molecular Medicine (Cambridge, Mass.). May-Jun, 2011  |  Pubmed ID: 21267509

Burn injury causes hepatic dysfunction associated with endoplasmic reticulum (ER) stress and induction of the unfolded protein response (UPR). ER stress/UPR leads to hepatic apoptosis and activation of the Jun-N-terminal kinase (JNK) signaling pathway, leading to vast metabolic alterations. Insulin has been shown to attenuate hepatic damage and to improve liver function. We therefore hypothesized that insulin administration exerts its effects by attenuating postburn hepatic ER stress and subsequent apoptosis. Male Sprague Dawley rats received a 60% total body surface area (TBSA) burn injury. Animals were randomized to receive saline (controls) or insulin (2.5 IU/kg q. 24 h) and euthanized at 24 and 48 h postburn. Burn injury induced dramatic changes in liver structure and function, including induction of the ER stress response, mitochondrial dysfunction, hepatocyte apoptosis, and up-regulation of inflammatory mediators. Insulin decreased hepatocyte caspase-3 activation and apoptosis significantly at 24 and 48 h postburn. Furthermore, insulin administration decreased ER stress significantly and reversed structural and functional changes in hepatocyte mitochondria. Finally, insulin attenuated the expression of inflammatory mediators IL-6, MCP-1, and CINC-1. Insulin alleviates burn-induced ER stress, hepatocyte apoptosis, mitochondrial abnormalities, and inflammation leading to improved hepatic structure and function significantly. These results support the use of insulin therapy after traumatic injury to improve patient outcomes.

Endoplasmic Reticulum Stress and Insulin Resistance Post-trauma: Similarities to Type 2 Diabetes

Journal of Cellular and Molecular Medicine. Aug, 2011  |  Pubmed ID: 21812914

Type 2 diabetes, a rapidly growing disease of modern etiology, has a profound impact on morbidity and mortality. Explosions in the understanding of the underlying cellular mechanisms which lead to type 2 diabetes have recently been elucidated. In particular, the central role of endoplasmic reticulum stress (ER stress) and the unfolding protein response (UPR) in insulin resistance in type 2 diabetes has recently been discovered. We hypothesize that ER stress and UPR are not only central for type 2 diabetes but also for stress-induced diabetes. We review here the evidence that post-burn insulin resistance and hyperglycemia have pathophysiologic mechanisms in common with type 2 diabetes. These recent discoveries not only highlight the importance of ER stress in the post-burn patient recovery, but furthermore enable new models to study fundamental and interventional aspects of type 2 diabetes.

Ubiquilin-1 is a Molecular Chaperone for the Amyloid Precursor Protein

The Journal of Biological Chemistry. Oct, 2011  |  Pubmed ID: 21852239

Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.

Propranolol Improves Impaired Hepatic Phosphatidylinositol 3-kinase/akt Signaling After Burn Injury

Molecular Medicine (Cambridge, Mass.). 2012  |  Pubmed ID: 22396018

Severe burn injury is associated with induction of the hepatic endoplasmic reticulum (ER) stress response. ER stress leads to activation of c-Jun N-terminal kinase (JNK), suppression of insulin receptor signaling via phosphorylation of insulin receptor substrate 1 and subsequent insulin resistance. Marked and sustained increases in catecholamines are prominent after a burn. Here, we show that administration of propranolol, a nonselective β1/2 adrenergic receptor antagonist, attenuates ER stress and JNK activation. Attenuation of ER stress by propranolol results in increased insulin sensitivity, as determined by activation of hepatic phosphatidylinositol 3-kinase and Akt. We conclude that catecholamine release is responsible for the ER stress response and impaired insulin receptor signaling after burn injury.

Structure-Function Of The Tumor Suppressor BRCA1

Computational and Structural Biotechnology Journal. Apr, 2012  |  Pubmed ID: 22737296

BRCA1, a multi-domain protein, is mutated in a large percentage of hereditary breast and ovarian cancers. BRCA1 is most often mutated in three domains or regions: the N-terminal RING domain, exons 11-13, and the BRCT domain. The BRCA1 RING domain is responsible for the E3 ubiquitin ligase activity of BRCA1 and mediates interactions between BRCA1 and other proteins. BRCA1 ubiquitinates several proteins with various functions. The BRCA1 BRCT domain binds to phosphoproteins with specific sequences recognized by both BRCA1 and ATM/ATR kinases. Structural studies of the RING and BRCT domains have revealed the molecular basis by which cancer causing mutations impact the functions of BRCA1. While no structural data is available for the amino acids encoded by exons 11-13, multiple binding sites and functional domains exist in this region. Many mutations in exons 11-13 have deleterious effects on the function of these domains. In this mini-review, we examine the structure-function relationships of the BRCA1 protein and the relevance to cancer progression.

Ubiquilin-1 Regulates Amyloid Precursor Protein Maturation and Degradation by Stimulating K63-linked Polyubiquitination of Lysine 688

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2012  |  Pubmed ID: 22847417

The pathogenesis of Alzheimer's disease (AD) is associated with proteolytic processing of the amyloid precursor protein (APP) to an amyloidogenic peptide termed Aβ. Although mutations in APP and the secretase enzymes that mediate its processing are known to result in familial forms of AD, the mechanisms underlying the more common sporadic forms of the disease are still unclear. Evidence suggests that the susceptibility of APP to amyloidogenic processing is related to its intracellular localization, and that secretase-independent degradation may prevent the formation of cytotoxic peptide fragments. Recently, single nucleotide polymorphisms in the UBQLN1 gene have been linked to late-onset AD, and its protein product, ubiquilin-1, may regulate the maturation of full-length APP. Here we show that ubiquilin-1 inhibits the maturation of APP by sequestering it in the early secretory pathway, primarily within the Golgi apparatus. This sequestration significantly delayed the proteolytic processing of APP by secretases and the proteasome. These effects were mediated by ubiquilin-1-stimulated K63-linked polyubiquitination of lysine 688 in the APP intracellular domain. Our results reveal the mechanistic basis by which ubiquilin-1 regulates APP maturation, with important consequences for the pathogenesis of late-onset AD.

Severe Injury is Associated with Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response

Annals of Surgery. Feb, 2012  |  Pubmed ID: 22241293

We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling.

Thermal Injury Activates EEF2k-Dependent EEF2 Pathway in Pediatric Patients

JPEN. Journal of Parenteral and Enteral Nutrition. Jan, 2012  |  Pubmed ID: 22269896

BACKGROUND: Burn induces a hypermetabolic state characterized by alterations in protein metabolism, which is associated with increased morbidity and mortality. Eukaryotic elongation factor 2 (eEF2) plays a crucial role in regulating protein synthesis in many diseases, but whether it participates in burn-induced hypermetabolism is unclear. The aim of this study was to determine the expression of eEF2 and the upstream eEF2-inactivating kinase, eEF2K, in severely burned pediatric patients. METHODS: Eight pediatric patients (> 40% total body surface area) and 3 nonburned pediatric volunteers were enrolled. Muscle and skin biopsies were collected at early (0-10 days postburn [dpb]), middle (11-49 dpb), and late (50-365 dpb) time points. Resting energy expenditure (REE), body composition, and muscle protein fractional synthesis rate (FSR) were measured. Proteins were extracted and analyzed by Western blotting. To further investigate the protein synthesis pathway, microarray data from muscle and skin were examined from 22 nonburned and 20 burned children. RESULTS: Burn patients exhibited a profound hypermetabolic response, as seen by a significant increase in REE (P < .05) and loss of lean body mass without altered muscle FSR, indicating a shift to catabolism after thermal injury. In muscle, the phosphorylation of eEF2K-dependent eEF2 was down regulated early and middle postburn. Similar changes in eEF2K and eEF2 levels occurred in skin at the early time point. Total amounts of eEF2 and eEF2K were not altered. Conclusion: Burn induces prolonged activation of eEF2K and eEF2. Alterations in these mediators may contribute to profound hypermetabolism in severely burned patients. (JPEN J Parenter Enteral Nutr. XXXX;xx:xx-xx).

Measurement of Hepatic Protein Fractional Synthetic Rate with Stable Isotope Labeling Technique in Thapsigargin Stressed HepG2 Cells

International Journal of Biological Sciences. 2012  |  Pubmed ID: 22298954

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97±0.02 and 0.99±0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68±0.03 and 0.60±0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.