Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer.

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin-proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

Chronic glucolipotoxic conditions in pancreatic islets impair insulin secretion due to dysregulated calcium dynamics, glucose responsiveness and mitochondrial activity.

BACKGROUND: In the progression towards diabetes, glucolipotoxicity is one of the main causes of pancreatic beta cell pathology. The aim of this study was to examine the in vitro effects of chronic glucolipotoxic conditions on cellular responses in pancreatic islets, including glucose and fat metabolism, Calcium mobilization, insulin secretion and insulin content. RESULTS: Exposure of islets to chronic glucolipotoxic conditions decreased glucose stimulated insulin secretion in vitro. Reduced protein levels of Glut2/slc2a2, and decreased glucokinase and pyruvate carboxylase mRNA levels indicated a significant lowering in glucose sensing. Concomitantly, both fatty acid uptake and triglyceride accumulation increased significantly while fatty acid oxidation decreased. This general suppression in glucose metabolism correlated well with a decrease in mitochondrial number and activity, reduction in cellular ATP content and dampening of the TCA cycle. Further, we also observed a decrease in IP3 levels and lower Calcium mobilization in response to glucose. Importantly, chronic glucolipotoxic conditions in vitro decreased insulin gene expression, insulin content, insulin granule docking (to the plasma membrane) and insulin secretion. CONCLUSIONS: Our results present an integrated view of the effects of chronic glucolipotoxic conditions on known and novel signaling events, in vitro, that results in reduced glucose responsiveness and insulin secretion.

MLN4924, a NEDD8-activating enzyme inhibitor, is active in diffuse large B-cell lymphoma models: rationale for treatment of NF-{kappa}B-dependent lymphoma.

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIkappaBalpha, decrease in nuclear p65 content, reduction of nuclear factor-kappaB (NF-kappaB) transcriptional activity, and G(1) arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-kappaB pathway inhibition. Treatment of germinal-center B cell-like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-kappaB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-kappaB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-kappaB-dependent lymphomas.

FMRP phosphorylation reveals an immediate-early signaling pathway triggered by group I mGluR and mediated by PP2A.

Fragile X syndrome is a common form of inherited mental retardation and is caused by loss of fragile X mental retardation protein (FMRP), a selective RNA-binding protein that influences the translation of target messages. Here, we identify protein phosphatase 2A (PP2A) as an FMRP phosphatase and report rapid FMRP dephosphorylation after immediate group I metabotropic glutamate receptor (mGluR) stimulation (<1 min) in neurons caused by enhanced PP2A enzymatic activity. In contrast, extended mGluR activation (1-5 min) resulted in mammalian target of rapamycin (mTOR)-mediated PP2A suppression and FMRP rephosphorylation. These activity-dependent changes in FMRP phosphorylation were also observed in dendrites and showed a temporal correlation with the translational profile of select FMRP target transcripts. Collectively, these data reveal an immediate-early signaling pathway linking group I mGluR activity to rapid FMRP phosphorylation dynamics mediated by mTOR and PP2A.

Nicotinic receptor expression following nicotine exposure via maternal milk.

Studies have shown nicotine is excreted into maternal milk, so that suckling offspring would be a target of the drug during the pre-weaning period. Since nicotine exposure leads to an upregulation of neuronal nicotinic receptors, this study examines the hypothesis that nicotine delivered via maternal milk is capable of altering neuronal nicotinic receptor regulation in the drug-exposed rat pups. The present study showed that postnatal nicotine exposure via maternal milk was sufficient to induce an upregulation in brain nicotinic receptors similar to that seen in adults that smoke. Such exposure may result in altered neuronal development and synaptic activity and structure, potentially leading to long-term behavioral, learning, and memory deficits.

Novel DNA damage checkpoints mediating cell death induced by the NEDD8-activating enzyme inhibitor MLN4924.

MLN4924 is an investigational small-molecule inhibitor of the NEDD8-activating enzyme (NAE) in phase I clinical trials. NAE inhibition prevents the ubiquitination and proteasomal degradation of substrates for cullin-RING ubiquitin E3 ligases that support cancer pathophysiology, but the genetic determinants conferring sensitivity to NAE inhibition are unknown. To address this gap in knowledge, we conducted a genome-wide siRNA screen to identify genes and pathways that affect the lethality of MLN4924 in melanoma cells. Of the 154 genes identified, approximately one-half interfered with components of the cell cycle, apoptotic machinery, ubiquitin system, and DNA damage response pathways. In particular, genes involved in DNA replication, p53, BRCA1/BRCA2, transcription-coupled repair, and base excision repair seemed to be important for MLN4924 lethality. In contrast, genes within the G(2)-M checkpoint affected sensitivity to MLN4924 in colon cancer cells. Cell-cycle analysis in melanoma cells by flow cytometry following RNAi-mediated silencing showed that MLN4924 prevented the transition of cells from S-G(2) phase after induction of rereplication stress. Our analysis suggested an important role for the p21-dependent intra-S-phase checkpoint and extensive rereplication, whereas the ATR-dependent intra-S-phase checkpoint seemed to play a less dominant role. Unexpectedly, induction of the p21-dependent intra-S-phase checkpoint seemed to be independent of both Cdt1 stabilization and ATR signaling. Collectively, these data enhance our understanding of the mechanisms by which inhibition of NEDD8-dependent ubiquitination causes cell death, informing clinical development of MLN4924.

Distribution, enrichment and principal component analysis for possible sources of naturally occurring and anthropogenic radionuclides in the agricultural soil of Punjab state, India.

Enrichment factor (EF) of elements including geo-accumulation indices for soil quality and principal component analysis (PCA) were used to identify the contributions of the origin of sources in the studied area. Results of (40)K, (137)Cs, (238)U and (232)Th including their decay series isotopes in the agricultural soil of Mansa and Bathinda districts in the state of Punjab were presented and discussed. The measured mean radioactivity concentrations for (238)U, (232)Th and (40)K in the agricultural soil of the studied area differed from nationwide average crustal abundances by 51, 17 and 43 %, respectively. The sequence of the EFs of radionuclides in soil from the greatest to the least was found to be (238)U > (40)K > (226)Ra > (137)Cs > (232)Th > (228)Ra. Even though the enrichment of naturally occurring radionuclides was found to be higher, they remained to be in I(geo) class of '0', indicating that the soil is uncontaminated with respect to these radionuclides. Among non-metals, N showed the highest EF and belonged to I(geo) class of '2', indicating that soil is moderately contaminated due to intrusion of fertiliser. The resulting data set of elemental contents in soil was also interpreted by PCA, which facilitates identification of the different groups of correlated elements. The levels of the (40)K, (238)U and (232)Th radionuclides showed a significant positive correlation with each other, suggesting a similar origin of their geochemical sources and identical behaviour during transport in the soil system.

Treatment-emergent mutations in NAEß confer resistance to the NEDD8-activating enzyme inhibitor MLN4924.

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEß as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEß mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEß mutations.

Inhibition of NEDD8-activating enzyme induces rereplication and apoptosis in human tumor cells consistent with deregulating CDT1 turnover.

Loss of NEDD8-activating enzyme (NAE) function by siRNA knockdown or inhibition by the small molecule NAE inhibitor MLN4924 leads to increased steady-state levels of direct Cullin-RING ligase (CRL) substrates by preventing their ubiquitination and proteasome-dependent degradation. Many of these CRL substrates are involved in cell cycle progression, including a critical DNA replication licensing factor CDT1. Cell cycle analysis of asynchronous and synchronous cultures after NAE inhibition revealed effects on cell cycle distribution and activation of DNA break repair signaling pathways similar to that reported for CDT1 overexpression. The siRNA knockdown of cullins critical for the turnover of CDT1 recapitulated the aberrant rereplication phenotype while CDT1 knockdown was suppressing. Although NAE inhibition leads to deregulation of many CRL substrates, these data demonstrate that CDT1 accumulation mediates the DNA rereplication phenotype resulting from loss of NAE function. DNA rereplication is an unrecoverable cellular insult and the small molecule inhibitor MLN4924, currently in phase I trials, represents an unprecedented opportunity to explore this mechanism of cytotoxicity for the treatment of cancer.

NEDD8-targeting drug MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering checkpoint activation, apoptosis, and senescence in cancer cells.

MLN4924 is a first-in-class experimental cancer drug that inhibits the NEDD8-activating enzyme, thereby inhibiting cullin-RING E3 ubiquitin ligases and stabilizing many cullin substrates. The mechanism by which MLN4924 inhibits cancer cell proliferation has not been defined, although it is accompanied by DNA rereplication and attendant DNA damage. Here we show that stabilization of the DNA replication factor Cdt1, a substrate of cullins 1 and 4, is critical for MLN4924 to trigger DNA rereplication and inhibit cell proliferation. Even only 1 hour of exposure to MLN4924, which was sufficient to elevate Cdt1 for 4-5 hours, was found to be sufficient to induce DNA rereplication and to activate apoptosis and senescence pathways. Cells in S phase were most susceptible, suggesting that MLN4924 will be most toxic on highly proliferating cancers. Although MLN4924-induced cell senescence seems to be dependent on induction of p53 and its downstream effector p21(Waf1), we found that p53(-/-) and p21(-/-) cells were even more susceptible than wild-type cells to MLN4924. Our results suggested that apoptosis, not senescence, might be more important for the antiproliferative effect of MLN4924. Furthermore, our findings show that transient exposure to this new investigational drug should be useful for controlling p53-negative cancer cells, which often pose significant clinical challenge.

Selective separation of iron from uranium in quantitative determination of traces of uranium by alpha spectrometry in soil/sediment sample.

During this work, controlled redox potential methodology was adopted for the complete separation of traces of uranium from the host matrix of mixed hydroxide of Iron. Precipitates of Fe(+2) and Fe(+3) along with other transuranic elements were obtained from acid leached solution of soil by raising the pH to 9 with 14N ammonia solution. The concentration of the uranium observed in the soil samples was 200-600 ppb, whereas in sediment samples, the concentration range was 61-400 ppb.

S6K1 phosphorylates and regulates fragile X mental retardation protein (FMRP) with the neuronal protein synthesis-dependent mammalian target of rapamycin (mTOR) signaling cascade.

Fragile X syndrome is a common form of cognitive deficit caused by the functional absence of fragile X mental retardation protein (FMRP), a dendritic RNA-binding protein that represses translation of specific messages. Although FMRP is phosphorylated in a group I metabotropic glutamate receptor (mGluR) activity-dependent manner following brief protein phosphatase 2A (PP2A)-mediated dephosphorylation, the kinase regulating FMRP function in neuronal protein synthesis is unclear. Here we identify ribosomal protein S6 kinase (S6K1) as a major FMRP kinase in the mouse hippocampus, finding that activity-dependent phosphorylation of FMRP by S6K1 requires signaling inputs from mammalian target of rapamycin (mTOR), ERK1/2, and PP2A. Further, the loss of hippocampal S6K1 and the subsequent absence of phospho-FMRP mimic FMRP loss in the increased expression of SAPAP3, a synapse-associated FMRP target mRNA. Together these data reveal a S6K1-PP2A signaling module regulating FMRP function and place FMRP phosphorylation in the mGluR-triggered signaling cascade required for protein-synthesis-dependent synaptic plasticity.

Fragile X mental retardation protein deficiency leads to excessive mGluR5-dependent internalization of AMPA receptors.

Fragile X syndrome (FXS), a common inherited form of mental retardation, is caused by the functional absence of the fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates the translation of specific mRNAs at synapses. Altered synaptic plasticity has been described in a mouse FXS model. However, the mechanism by which the loss of FMRP alters synaptic function, and subsequently causes the mental impairment, is unknown. Here, in cultured hippocampal neurons, we used siRNAs against Fmr1 to demonstrate that a reduction of FMRP in dendrites leads to an increase in internalization of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) subunit, GluR1, in dendrites. This abnormal AMPAR trafficking was caused by spontaneous action potential-driven network activity without synaptic stimulation by an exogenous agonist and was rescued by 2-methyl-6-phenylethynyl-pyridine (MPEP), an mGluR5-specific inverse agonist. Because AMPAR internalization depends on local protein synthesis after mGluR5 stimulation, FMRP, a negative regulator of translation, may be viewed as a counterbalancing signal, wherein the absence of FMRP leads to an apparent excess of mGluR5 signaling in dendrites. Because AMPAR trafficking is a driving process for synaptic plasticity underlying learning and memory, our data suggest that hypersensitive AMPAR internalization in response to excess mGluR signaling may represent a principal cellular defect in FXS, which may be corrected by using mGluR antagonists.

Coupled in vitro import of U snRNPs and SMN, the spinal muscular atrophy protein.

Cytoplasmic assembly of Sm-class small nuclear ribonucleoproteins (snRNPs) is a central process in eukaryotic gene expression. A large macromolecular complex containing the survival of motor neurons (SMN) protein is required for proper snRNP assembly in vivo. Defects in SMN function lead to a human neuromuscular disorder, spinal muscular atrophy (SMA). SMN protein localizes to both nuclear and cytoplasmic compartments, and a reduction in nuclear levels of SMN is correlated with the disease. The mechanism of SMN nuclear import, however, is unknown. Using digitonin-permeabilized cells, we show that SMN import depends on the presence of Sm snRNPs. Conversely, import of labeled U1 snRNPs was SMN complex dependent. Thus, import of SMN and U snRNPs are coupled in vitro. Furthermore, we identify nuclear import defects in SMA patient-derived SMN mutants, uncovering a potential mechanism for SMN dysfunction.

SMN, the spinal muscular atrophy protein, forms a pre-import snRNP complex with snurportin1 and importin beta.

The survival of motor neuron (SMN) protein is mutated in patients with spinal muscular atrophy (SMA). SMN is part of a multiprotein complex required for biogenesis of the Sm class of small nuclear ribonucleoproteins (snRNPs). Following assembly of the Sm core domain, snRNPs are transported to the nucleus via importin beta. Sm snRNPs contain a nuclear localization signal (NLS) consisting of a 2,2,7-trimethylguanosine (TMG) cap and the Sm core. Snurportin1 (SPN) is the adaptor protein that recognizes both the TMG cap and importin beta. Here, we report that a mutant SPN construct lacking the importin beta binding domain (IBB), but containing an intact TMG cap-binding domain, localizes primarily to the nucleus, whereas full-length SPN localizes to the cytoplasm. The nuclear localization of the mutant SPN was not a result of passive diffusion through the nuclear pores. Importantly, we found that SPN interacts with SMN, Gemin3, Sm snRNPs and importin beta. In the presence of ribonucleases, the interactions with SMN and Sm proteins were abolished, indicating that snRNAs mediate this interplay. Cell fractionation studies showed that SPN binds preferentially to cytoplasmic SMN complexes. Notably, we found that SMN directly interacts with importin beta in a GST-pulldown assay, suggesting that the SMN complex might represent the Sm core NLS receptor predicted by previous studies. Therefore, we conclude that, following Sm protein assembly, the SMN complex persists until the final stages of cytoplasmic snRNP maturation and may provide somatic cell RNPs with an alternative NLS.

Interaction between the small-nuclear-RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron.

The biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) requires the cytoplasmic assembly of the Sm-core complex, followed by the hypermethylation of the small nuclear RNA (snRNA) 5' cap. Both the Sm-core complex and the snRNA trimethylguanosine cap are required for the efficient nuclear import of snRNPs. Here, we show that trimethylguanosine synthase 1 (TGS1), the human homologue of the yeast snRNA cap hypermethylase, interacts directly with the survival of motor neuron (SMN) protein. Both proteins are similarly distributed, localizing in the cytoplasm and in nuclear Cajal bodies. The interaction between TGS1 and SMN is disrupted by a mutation in SMN that mimics the predominant isoform of the protein that is expressed in patients with the neurodegenerative disease, spinal muscular atrophy. These data indicate that, in addition to its function in cytoplasmic Sm-core assembly, the SMN protein also functions in the recruitment of the snRNA cap hypermethylase.