sq-23377 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Endoplasmic reticulum (ER) stress causes neuronal dysfunction followed by cell death and is recognized as a feature of many neurodegenerative diseases. Using a phenotypic screen, we recently identified benzodiazepinone derivatives that reduce ER stress-mediated apoptosis in a rat neuronal progenitor cell line (CSM14.1). Herein we describe how structure-activity relationship (SAR) studies around these screening hits led to compounds that display robust cytoprotective activity against thapsigargin-induced ER stress in SH-SY5Y and H4 human neuronal cell lines. We demonstrate that the most potent of these derivatives, compound 4hh, inhibits the activation of p38 MAP kinase (p38) and c-Jun N-terminal kinase (JNK), protein kinases that are downstream signal effectors of the unfolded protein response (UPR). Compound 4hh specifically protects against thapsigargin-induced cell death and displays no protection against other insults known to induce cellular stress or activate p38. However, compound 4hh provides moderate inhibition of p38 activity stimulated by compounds that disrupt calcium homeostasis. Our data indicate that probe compound 4hh is a valuable small molecule tool that can be used to investigate the effects of ER stress on human neurons. This approach may provide the basis for the future development of therapeutics for the treatment of neurodegenerative diseases.

Topics: Animals; Benzodiazepinones; Calcium; Cell Death; Cell Line; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Enzyme Inhibitors; Homeostasis; Humans; Imidazoles; Ionomycin; Leupeptins; MAP Kinase Signaling System; Neurons; Oleanolic Acid; Rats; Structure-Activity Relationship; Thapsigargin

Studies of patients with congenital immunodeficiency due to mutation of the NF-kappaB essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-kappaB activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IkappaB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-kappaB activation. NK cell IkappaB degradation corresponded to increases in nuclear NF-kappaB as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-kappaB used. NF-kappaB function was evaluated in NK92 cells transduced with a kappaB GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-kappaB dependent proteins during the initiation of cytotoxicity.

Topics: Cell Line; Green Fluorescent Proteins; Humans; Ionomycin; K562 Cells; Killer Cells, Natural; Leupeptins; Natural Cytotoxicity Triggering Receptor 3; NF-kappa B; Receptors, Immunologic; Signal Transduction; Stilbenes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

Topics: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Humans; Ionomycin; Ionophores; Leupeptins; Mass Spectrometry; Molecular Sequence Data; Multienzyme Complexes; Mutation; Phosphorylation; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; ras Guanine Nucleotide Exchange Factors; ras Proteins; ras-GRF1; Son of Sevenless Proteins; Ubiquitins