h-89 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Cyclooxygenases (COXs) catalyze the committed step in prostaglandin (PG) biosynthesis. COX-1 is constitutively expressed and stable, whereas COX-2 is inducible and short lived. COX-2 is degraded via endoplasmic reticulum (ER)-associated degradation (ERAD) following post-translational glycosylation of Asn-594. COX-1 and COX-2 are found in abundance on the luminal surfaces of the ER and inner membrane of the nuclear envelope. Using confocal immunocytofluorescence, we detected both COX-2 and microsomal PGE synthase-1 (mPGES-1) but not COX-1 in the Golgi apparatus. Inhibition of trafficking between the ER and Golgi retarded COX-2 ERAD. COX-2 has a C-terminal STEL sequence, which is an inefficient ER retention signal. Substituting this sequence with KDEL, a robust ER retention signal, concentrated COX-2 in the ER where it was stable and slowly glycosylated on Asn-594. Native COX-2 and a recombinant COX-2 having a Golgi targeting signal but not native COX-1 exhibited efficient catalytic coupling to mPGES-1. We conclude that N-glycosylation of Asn-594 of COX-2 occurs in the ER, leading to anterograde movement of COX-2 to the Golgi where the Asn-594-linked glycan is trimmed prior to retrograde COX-2 transport to the ER for ERAD. Having an inefficient ER retention signal leads to sluggish Golgi to ER transit of COX-2. This permits significant Golgi residence time during which COX-2 can function catalytically. Cytosolic phospholipase A2α, which mobilizes arachidonic acid for PG synthesis, preferentially translocates to the Golgi in response to physiologic Ca(2+) mobilization. We propose that cytosolic phospholipase A2α, COX-2, and mPGES-1 in the Golgi comprise a dedicated system for COX-2-dependent PGE2 biosynthesis.

Topics: Amino Acid Sequence; Animals; Asparagine; Cyclooxygenase 2; Cysteine Proteinase Inhibitors; Dinoprostone; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Fibroblasts; Glycosylation; Golgi Apparatus; Group IV Phospholipases A2; HEK293 Cells; Humans; Immunoblotting; Intramolecular Oxidoreductases; Isoquinolines; Leupeptins; Mice; Microscopy, Confocal; Mutation; NIH 3T3 Cells; Prostaglandin-E Synthases; Protein Kinase Inhibitors; Protein Transport; Sulfonamides

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gαs plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.

Topics: Apoptosis; Blotting, Western; Cell Line, Tumor; Cyclic AMP; Dinoprostone; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; GTP-Binding Protein alpha Subunits, Gs; Humans; Isoquinolines; Leupeptins; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Phosphorylation; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-raf; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sirtuins; Sulfonamides; Ubiquitin

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

Topics: Animals; Biopterins; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Cysteine Proteinase Inhibitors; Dopamine; Gene Expression Regulation; Isoquinolines; Leupeptins; Mesencephalon; Neurons; PC12 Cells; Phosphorylation; Primary Cell Culture; Proteasome Endopeptidase Complex; Proteolysis; Rats; Receptors, Dopamine D2; Serine; Signal Transduction; Sulfonamides; Tyrosine 3-Monooxygenase; Ubiquitin; Ubiquitination

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

Topics: Acute Disease; Adamantane; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cinnamates; Cyclic AMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Histones; Humans; Isoquinolines; Leukemia, Myeloid; Leupeptins; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Protein Binding; Signal Transduction; Sulfonamides; Surface Plasmon Resonance

The proteasome is a multicatalytic cellular complex present in human sperm that plays a significant role during several steps of mammalian fertilization. Here, we present evidence that the proteasome is involved in human sperm capacitation. Aliquots of highly motile sperm were incubated with proteasome inhibitors MG132 or epoxomicin. The percentage of capacitated sperm, the chymotrypsin-like activity of the proteasome, cAMP content, and the pattern of protein phosphorylation were assayed by using the chlortetracycline hydrochloride assay, a fluorogenic substrate, the cAMP enzyme immunoassay kit, and Western blot analysis, respectively. Our results indicate that treatment of sperm with proteasome inhibitors blocks the capacitation process, does not alter cAMP concentration, and changes the pattern of protein phosphorylation. To elucidate how proteasome activity is regulated during capacitation, sperm were incubated with: 1) tyrosine kinase (TK) inhibitors (genistein or herbimycin A); 2) protein kinase (PK) A inhibitors or activators (H89 and Rp-cAMPS, and 8-Br-cAMP, respectively); or 3) PKC inhibitors (tamoxifen or staurosporin) at different capacitation times. The chymotrypsin-like activity and degree of phosphorylation of the proteasome were then assayed. The results indicate that sperm treatment with TK and PKA inhibitors significantly decreases the chymotrypsin-like activity of the proteasome during capacitation. Immunoprecipitation and Western blot results suggest that the proteasome is phosphorylated during capacitation in a TK- and PKA-dependent pathway. In conclusion, we suggest that the sperm proteasome participates in the capacitation process, and that its activity is modulated by PKs.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Benzoquinones; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Enzyme Inhibitors; Genistein; Humans; In Vitro Techniques; Isoquinolines; Lactams, Macrocyclic; Leupeptins; Male; Oligopeptides; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein-Tyrosine Kinases; Rifabutin; Sperm Capacitation; Spermatozoa; Sulfonamides; Thionucleotides

Chronic exposure of pancreatic islets to elevated plasma lipids (lipotoxicity) can lead to beta-cell dysfunction, with overtime becoming irreversible. We examined, by confocal microscopy and biochemistry, whether the expression of islet inducible nitric oxide synthase (iNOS) and the concomitant inhibition of glucose-stimulated insulin release seen after lipid infusion in rats was modulated by the islet neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)27. Lipid infusion for 8 days induced a strong expression of islet iNOS, which was mainly confined to beta-cells and was still evident after incubating islets at 8.3 mmol/l glucose. This was accompanied by a high iNOS-derived NO generation, a decreased insulin release, and increased cyclic GMP accumulation. No iNOS expression was found in control islets. Addition of PACAP27 to incubated islets from lipid-infused rats resulted in loss of iNOS protein expression, increased cyclic AMP, decreased cyclic GMP, and suppression of the activities of neuronal constitutive (nc)NOS and iNOS and increased glucose-stimulated insulin response. These effects were reversed by the PKA inhibitor H-89. The suppression of islet iNOS expression induced by PACAP27 was not affected by the proteasome inhibitor MG-132, which by itself induced the loss of iNOS protein, making a direct proteasomal involvement less likely. Our results suggest that PACAP27 through its cyclic AMP- and PKA-stimulating capacity strongly suppresses not only ncNOS but, importantly, also the lipid-induced stimulation of iNOS expression, possibly by a nonproteasomal mechanism. Thus PACAP27 restores the impairment of glucose-stimulated insulin release and additionally might induce cytoprotection against deleterious actions of iNOS-derived NO in beta-cells.

Topics: Animals; Cyclic AMP; Cyclic GMP; Drug Interactions; Fat Emulsions, Intravenous; Glucose; In Vitro Techniques; Insulin; Insulin Secretion; Islets of Langerhans; Isoquinolines; Leupeptins; Male; Microscopy, Confocal; Neurotransmitter Agents; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Parenteral Nutrition, Total; Pituitary Adenylate Cyclase-Activating Polypeptide; Protease Inhibitors; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sulfonamides

When isolated mouse fat pads were incubated with insulin or sodium orthovanadate (vanadate) for up to 4h, the intracellular leptin content was increased by insulin, while it was decreased by vanadate. Bupranolol, a beta3-adrenergic receptor antagonist, prevented both effects of vanadate, i.e., the decrease in intracellular leptin and increase in cellular cAMP content, while BRL 37344, a beta3-adrenergic receptor antagonist mimicked the action of vanadate. H-89 prevented the vanadate-induced decrease in intracellular leptin, suggesting the involvement of a cAMP-dependent protein kinase (PKA). No detectable difference in the incorporation of [3H]leucine into leptin was observed between incubations of the fat pads with and without vanadate, suggesting that the action of vanadate is independent of decreasing synthesis. Similar concentrations of MG-132, a membrane-permeable proteasome inhibitor, prevented the vanadate-induced decrease in both intracellular leptin content and leptin secretion, suggesting the involvement of the proteasome in the vanadate action. The proteasome fraction separated from the vanadate-treated fat pads increased the degradation of exogenous [125I]leptin in the presence of an ATP-regenerating system together with an ubiqutination system. The endopeptidase activity against Cbz-Leu-Leu-Glu-beta-naphthylamine also was increased by the proteasome fraction. MG-132 prevented both increased effects. The 8-Br-cAMP-treated proteasome fraction increased the degradation of the exogenous leptin. H-89 prevented the effect of 8-Br-cAMP. These results indicate that vanadate decreases the intracellular leptin content by increased degradation via a cAMP/PKA-dependent process involving proteasome activation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphatases; Adipose Tissue; Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Epididymis; Insulin; Isoquinolines; Leptin; Leupeptins; Male; Mice; Mice, Inbred Strains; Multienzyme Complexes; Proteasome Endopeptidase Complex; Rabbits; Sulfonamides; Ubiquitin; Vanadates

In patients with human granulocytic ehrlichiosis (HGE), the HGE agent has been seen only in the peripheral blood granulocytes, which have a life span too short for ehrlichial proliferation. To determine if the HGE agent delays the apoptosis of human peripheral blood neutrophils for its advantage, peripheral blood granulocytes consisting mostly of neutrophils were incubated with freshly freed host cell-free HGE agent in vitro. The HGE agent induced a significant delay in morphological apoptosis and the cytoplasmic appearance of histone-associated DNA fragments in the granulocytes. This antiapoptotic effect was dose dependent. Although much weaker than the HGE agent freshly freed from the host cells, noninfectious purified HGE agent stored frozen and thawed also had antiapoptotic effect, which was lost with proteinase K treatment but not with periodate treatment. Treatment of neutrophils with a transglutaminase inhibitor, monodansylcadaverine, blocked the antiapoptotic effect of the HGE agent. Addition of oxytetracycline, however, did not prevent or reverse the antiapoptotic effect of the HGE agent. These results suggest that binding of a protein component(s) of the HGE agent to neutrophils and subsequent cross-linking and/or internalization of the receptor and ehrlichiae are required for antiapoptotic signaling, but ehrlichial protein synthesis and/or proliferation is not required. MG-132, a proteasome inhibitor, and cycloheximide accelerated the apoptosis of neutrophils and overrode the antiapoptotic effect of the HGE agent. Studies with specific inhibitors suggest that protein kinase A, NF-kappaB, and interleukin 1beta are not involved in the antiapoptotic mechanism of the HGE agent.

Topics: Apoptosis; Cadaverine; DNA Fragmentation; Ehrlichia; Genistein; HL-60 Cells; Humans; Interleukin-1; Isoquinolines; Leupeptins; Neutrophils; NF-kappa B; Oxytetracycline; Sulfonamides

Depolarization and subsequent calcium entry exert essential neuroprotective effects but the ultimate effector by which calcium blocks apoptosis is not known. Here we show that inhibition of calcium entry into cerebellar neurons by switching from high to low extracellular K+ concentrations (30-5 mM) induces apoptosis, that correlates with a rapid accumulation of cyclin D1 (CD1), an early marker of the G1/S transition of the cell cycle. These effects on apoptosis and cyclin D1 are mimicked either by blocking calcium entry into neurons (LaCl3, 100 microM or nifedipine, 10(-6) M) or by inhibiting the calcium/calmodulin pathway (calmidazolium, 10(-7) M). The increased CD1 protein levels do not result from a transcriptional upregulation of the CD1 gene by the Ca2+/calmodulin pathway but rather reflect an accumulation due to the lack of degradation by the proteasome-dependent pathway. Specific proteasome antagonists: carbobenzoxyl-leucinyl-leucinyl-norvalinal-H (MG-115), carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG-132) and clastolactacystin beta-lactone, induce neuronal apoptosis by themselves. Finally, this pathway is functional only at neuroprotective concentrations of K+ (30 mM), suggesting that calcium/CamK signalling pathway may regulate neuronal death by regulating the proteasome-mediated degradation activity of rapidly turning-over proteins (constitutively expressed genes or pre-existing pools of mRNA).

Topics: Animals; Apoptosis; Calcium; Calmodulin; Cerebellum; Cyclic AMP-Dependent Protein Kinases; Cyclin D1; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Primers; Enzyme Inhibitors; Gene Expression; Imidazoles; In Situ Nick-End Labeling; Isoquinolines; Leupeptins; Membrane Potentials; Mice; Mice, Inbred Strains; Multienzyme Complexes; Neurons; Potassium Chloride; Protease Inhibitors; Proteasome Endopeptidase Complex; RNA, Messenger; Signal Transduction; Sulfonamides; Ubiquitins