Cullin-4B E3 ubiquitin ligase mediates Apaf-1 ubiquitination to regulate caspase-9 activity.

Apoptotic protease-activating factor 1 (Apaf-1) is a component of apoptosome, which regulates caspase-9 activity. In addition to apoptosis, Apaf-1 plays critical roles in the intra-S-phase checkpoint; therefore, impaired expression of Apaf-1 has been demonstrated in chemotherapy-resistant malignant melanoma and nuclear translocation of Apaf-1 has represented a favorable prognosis of patients with non-small cell lung cancer. In contrast, increased levels of Apaf-1 protein are observed in the brain in Huntington's disease. The regulation of Apaf-1 protein is not yet fully understood. In this study, we show that etoposide triggers the interaction of Apaf-1 with Cullin-4B, resulting in enhanced Apaf-1 ubiquitination. Ubiquitinated Apaf-1, which was degraded in healthy cells, binds p62 and forms aggregates in the cytosol. This complex of ubiquitinated Apaf-1 and p62 induces caspase-9 activation following MG132 treatment of HEK293T cells that stably express bcl-xl. These results show that ubiquitinated Apaf-1 may activate caspase-9 under conditions of proteasome impairment.

CED-4 is an mRNA-binding protein that delivers ced-3 mRNA to ribosomes.

Cell death abnormal (ced)-3 and ced-4 genes regulate apoptosis to maintain tissue homeostasis in Caenorhabditis elegans. Apoptosome formation and CED-4 translocation drive CED-3 activation. However, the precise role of CED-4 translocation is not yet fully understood. In this study, using a combination of immunoprecipitation and reverse transcription-polymerase chain reaction methods in cells and a glutathione-S-transferase pull down assay in a cell-free system, we show that CED-4 binds ced-3 mRNA. In the presence of ced-3 mRNA, CED-4 protein is enriched in the microsomal fraction and interacts with ribosomal protein L10a in mammalian cells, increasing the levels of CED-3. These results suggest that CED-4 forms a complex with ced-3 mRNA and delivers it to ribosomes for translation.

Inhibition of Small Maf Function in Pancreatic ß-Cells Improves Glucose Tolerance Through the Enhancement of Insulin Gene Transcription and Insulin Secretion.

The large-Maf transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) has been found to be crucial for insulin transcription and synthesis and for pancreatic ß-cell function and maturation. However, insights about the effects of small Maf factors on ß-cells are limited. Our goal was to elucidate the function of small-Maf factors on ß-cells using an animal model of endogenous small-Maf dysfunction. Transgenic (Tg) mice with ß-cell-specific expression of dominant-negative MafK (DN-MafK) experiments, which can suppress the function of all endogenous small-Mafs, were fed a high-fat diet, and their in vivo phenotypes were evaluated. Phenotypic analysis, glucose tolerance tests, morphologic examination of ß-cells, and islet experiments were performed. DN-MafK-expressed MIN6 cells were also used for in vitro analysis. The results showed that DN-MafK expression inhibited endogenous small-Maf binding to insulin promoter while increasing MafA binding. DN-MafK Tg mice under high-fat diet conditions showed improved glucose metabolism compared with control mice via incremental insulin secretion, without causing changes in insulin sensitivity or MafA expression. Moreover, up-regulation of insulin and glucokinase gene expression was observed both in vivo and in vitro under DN-MafK expression. We concluded that endogenous small-Maf factors negatively regulates ß-cell function by competing for MafA binding, and thus, the inhibition of small-Maf activity can improve ß-cell function.

Polyglutamine expansion disturbs the endoplasmic reticulum formation, leading to caspase-7 activation through Bax.

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington's disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.

Olfaxin as a novel Prune2 isoform predominantly expressed in olfactory system.

Prune homolog 2 (Drosophila) (PRUNE2) encodes a BCH motif-containing protein that shares homology with the Cayman ataxia-related protein Caytaxin. Caytaxin is a substrate of caspase-3 and is specifically expressed at the presynapse of vesicular-type glutamate transporter (VGLUT)-positive neurons, where it plays a role in glutamate neurotransmission primarily in the cerebellum and hippocampus. Here, we showed that a novel Prune2 isoform contains a BCH motif and localizes predominantly to the synaptic cytosol, similar to Caytaxin. However, the isoform is expressed predominantly in the olfactory bulb and layer Ia of the piriform cortex, where Caytaxin is scarcely expressed. The isoform expression is upregulated during development, similar to that in the presynaptic-localizing proteins Synapsin I and Bassoon. Prune2 and its previously identified isoforms have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein, and an antagonist of cellular transformation. In addition, a novel isoform may develop new roles for Prune2 at the synapse in olfactory systems.

Endoplasmic reticulum stress enhances γ-secretase activity.

The endoplasmic reticulum (ER) copes with unfolded proteins in the lumen (ER stress) by activating three distinct intracellular signaling pathways of unfolded protein response (UPR). ER stress contributes to the pathogenesis of obesity and diabetes, which are risk factors for Alzheimer's disease (AD) that accelerate the pathogenesis of AD. However, whether ER stress is involved in the development of AD remains unclear. In this study, we demonstrate that ER stress induces presenilin-1 expression through activating transcription factor 4 (ATF4), resulting in increased amyloid-ß (Aß) secretion by γ-secretase activity, which is suppressed by quercetin by modifying UPR signaling. This result suggests that ER stress may be stimulated in obesity and type 2 diabetes, thereby enhancing γ-secretase activity that is the underlying molecular mechanism affecting the pathogenesis of AD.

The expression and localization of Prune2 mRNA in the central nervous system.

A family of Bcl-2/adenovirus E1B 19kDa-interacting proteins (BNIPs) plays critical roles in several cellular processes such as cellular transformation, apoptosis, neuronal differentiation, and synaptic function, which are mediated by the BNIP2 and Cdc42GAP homology (BCH) domain. Prune homolog 2 (Drosophila) (PRUNE2) and its isoforms -C9orf65, BCH motif-containing molecule at the carboxyl terminal region 1 (BMCC1), and BNIP2 Extra Long (BNIPXL) - have been shown to be a susceptibility gene for Alzheimer's disease, a biomarker for leiomyosarcomas, a proapoptotic protein in neuronal cells, and an antagonist of cellular transformation, respectively. However, precise localization of PRUNE2 in the brain remains unclear. Here, we identified the distribution of Prune2 mRNA in the adult mouse brain. Prune2 mRNA is predominantly expressed in the neurons of the cranial nerve motor nuclei and the motor neurons of the spinal cord. The expression in the dorsal root ganglia (DRG) is consistent with the previously described reports. In addition, we observed the expression in another sensory neuron in the mesencephalic trigeminal nucleus. These results suggest that Prune2 may be functional in these restricted brain regions.

Cayman ataxia-related protein is a presynapse-specific caspase-3 substrate.

Caspase plays an important role in apoptosis and physiological processes such as synaptic plasticity. However, the caspase substrate at the synapse is still unknown. Here we used an in vitro cleavage assay with a small-pool human brain cDNA library. We identified the presynaptic protein Caytaxin as a substrate of caspase-3 and caspase-7. Deficiency in Caytaxin causes Cayman ataxia, a disorder characterized by cerebellar dysfunction and mental retardation. Caytaxin cleavage in cerebellar granule neurons is dependent on caspase-3 activation. The cleavage site is upstream of the cellular retinal and the TRIO guanine exchange factor domain, producing a C-terminal fragment that may play an alternative role in inhibiting MEK2 signaling. Thus, we concluded that Caytaxin is a novel substrate of caspase-3 at the presynapse.

Autophagy impairment stimulates PS1 expression and gamma-secretase activity.

Gamma-secretase plays an important role in the development of Alzheimer disease (AD). Gamma-secretase activity is enriched in autophagic vacuoles and it augments amyloid-beta (Abeta) synthesis. Autophagy-lysosomal dysfunction has been implicated in AD, but whether gamma-secretase activity is affected by autophagy remains unclear. Here we report that gamma-secretase activity is enhanced in basal autophagy-disturbed cells through the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) kinase, general control nonderepressible 2 (GCN2). Presenilin-1 (PS1) expression was increased even in the presence of nutrients in autophagy-related 5 knockdown (Atg5KD) human embryonic kidney (HE K293) cells expressing a short hairpin RNA as well as in chloroquine-treated HE K293 cells. However, PS1 expression induction was prevented in GCN2KD and ATF4KD cells. Furthermore, Atg5KD cells showed an increase in Abeta production and Notch1 cleavage. These were reduced by an autophagy inducer, resveratrol. Thus, we conclude that the autophagy-lysosomal system regulates gamma-secretase activity through GCN2.

An alternative spliced mouse presenilin-2 mRNA encodes a novel gamma-secretase inhibitor.

The gamma-secretase, composed of presenilin-1 (PS1) or presenilin-2 (PS2), nicastrin (NCT), anterior pharynx-defective phenotype 1 (APH-1), and PEN-2, is critical for the development of Alzheimer's disease (AD). PSs are autoproteolytically cleaved, producing an N-terminal fragment (NTF) and a hydrophilic loop domain-containing C-terminal fragment. However, the role of the loop domain in the gamma-secretase complex assembly remains unknown. Here, we report a novel PS2 isoform generated by alternative splicing, named PS2beta, which is composed of an NTF with a hydrophilic loop domain. PS2beta disturbed the interaction between NCT and APH-1, resulting in the inhibition of amyloid-beta production. We concluded that PS2beta may inhibit gamma-secretase activity by affecting the gamma-secretase complex assembly.