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Darren Boehning

Publications and source records attributed to Darren Boehning.

11 recordsLinked to original sources
PubMed2006

Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.

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.

Apoptosis↗
PubMed2005

A peptide inhibitor of cytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways.

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.

Amino Acid Sequence↗
PubMed2004

Heme oxygenase-2 is activated by calcium-calmodulin.

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.

Animals↗
PubMed2004

Apoptosis and calcium: new roles for cytochrome c and inositol 1,4,5-trisphosphate.

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.

Animals↗
PubMed2004

Agonist-induced Ca2+ entry determined by inositol 1,4,5-trisphosphate recognition.

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.

Amino Acid Substitution↗
PubMed2004

Carbon monoxide mediates vasoactive intestinal polypeptide-associated nonadrenergic/noncholinergic neurotransmission.

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.

Anal Canal↗
PubMed2004

Inositol 1,4,5-trisphosphate receptors as signal integrators.

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.

Adenosine Triphosphate↗
PubMed2003

Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.

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.

Animals↗
PubMed2003

Carbon monoxide neurotransmission activated by CK2 phosphorylation of heme oxygenase-2.

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.

Animals↗
PubMed2003

Novel neural modulators.

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.

Amino Acids↗