GRIM-19 and p16INK4a Synergistically Regulate Cell Cycle Progression and E2F1-responsive Gene Expression

GRIM-19 (Gene associated with Retinoid-IFN-induced Mortality-19) was originally isolated as a growth suppressor in a genome-wide knockdown screen with antisense libraries. Like classical tumor suppressors, mutations, and/or loss of GRIM-19 expression occur in primary human tumors; and it is inactivated by viral gene products. Our search for potential GRIM-19-binding proteins, using mass spectrometry, that permit its antitumor actions led to the inhibitor of cyclin-dependent kinase 4, CDKN2A. The GRIM-19/CDKN2A synergistically suppressed cell cycle progression via inhibiting E2F1-driven gene expression. The N terminus of GRIM-19 and the fourth ankyrin repeat of CDKN2A are crucial for their interaction. The biological relevance of these interactions is underscored by observations that GRIM-19 promotes the inhibitory effect of CDKN2A on CDK4; and mutations from primary tumors disrupt its ability to interact with GRIM-19 and suppress E2F1-driven gene expression.     

24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.     

Multiorgan autoimmunity in a Turner syndrome patient with partial monosomy 2q and trisomy 10p

Turner syndrome is a condition caused by numeric and structural abnormalities of the X chromosome, and is characterized by a series of clinical features, the most common being short stature and gonadal dysgenesis. An increased frequency of autoimmune diseases as well as an elevated incidence of autoantibodies has been observed in Turner patients.     

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

Molecular and structural analysis of C4-specific PEPC isoform from Pennisetum glaucum plays a role in stress adaptation

As an activator of adenylate cyclase, the neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) impacts levels of cyclic AMP, a key second messenger available in brain cells. PACAP is involved in certain adult behaviors. To elucidate PACAP interactions, a compendium of microarrays representing mRNA expression in the adult mouse whole brain was pooled from the Phenogen database for analysis. A regulatory network was computed based on mutual information between gene pairs using gene expression data across the compendium. Clusters among genes directly linked to PACAP, and probable interactions between corresponding proteins were computed. Database “experts” affirmed some of the inferred relationships. The findings suggest ADCY7 is probably the adenylate cyclase isoform most relevant to PACAP's action. They also support intervening roles for kinases including GSK3B, PI 3-kinase, SGK3 and AMPK. Other high-confidence interactions are hypothesized for future testing. This new information has implications for certain behavioral and other disorders.     

Protonation and hydrogen bonding of Ca2+ site residues in the E2P phosphoenzyme intermediate of sarcoplasmic reticulum Ca2+-ATPase studied by a combination of infrared spectroscopy and electrostatic calculations

Protonation of the Ca²⁺ ligands of the SR Ca²⁺-ATPase (SERCA1a) was studied by a combination of rapid scan FTIR spectroscopy and electrostatic calculations. With FTIR spectroscopy, we investigated the pH dependence of CO bands of the Ca²⁺-free phosphoenzyme (E2P) and obtained direct experimental evidence for the protonation of carboxyl groups upon Ca²⁺ release. At least three of the infrared signals from protonated carboxyl groups of E2P are pH dependent with pK_a values near 8.3: a band at 1758 cm⁻¹ characteristic of nonhydrogen-bonded carbonyl groups, a shoulder at 1720 cm⁻¹, and part of a band at 1710 cm⁻¹, both characteristic of hydrogen-bonded carbonyl groups. The bands are thus assigned to H⁺ binding residues, some of which are involved in H⁺ countertransport. At pH 9, bands at 1743 and 1710 cm⁻¹ remain which we do not attribute to Ca²⁺/H⁺ exchange. We also obtained evidence for a pH-dependent conformational change in β-sheet or turn structures of the ATPase. With MCCE on the E2P analog E2(), we assigned infrared bands to specific residues and analyzed whether or not the carbonyl groups of the acidic Ca²⁺ ligands are hydrogen bonded. The carbonyl groups of Glu⁷⁷¹, Asp⁸⁰⁰, and Glu⁹⁰⁸ were found to be hydrogen bonded and will thus contribute to the lower wave number bands. The carbonyl group of some side-chain conformations of Asp⁸⁰⁰ is left without a hydrogen-bonding partner; they will therefore contribute to the higher wave number band.