Apoptosis is regulated by the VDAC1 N-terminal region and by VDAC oligomerization: release of cytochrome c,AIF and Smac/Diablo

Mitochondria, central to basic life functions due to their generation of cellular energy, also serve as the venue for cellular decisions leading to apoptosis. A key protein in mitochondria-mediated apoptosis is the voltage-dependent anion channel (VDAC), which also mediates the exchange of metabolites and energy between the cytosol and the mitochondria. In this study, the functions played by the N-terminal region of VDAC1 and by VDAC1 oligomerization in the release of cytochrome c, Smac/Diablo and apoptosis-inducing factor (AIF) and subsequent apoptosis were addressed. We demonstrate that cells undergoing apoptosis induced by STS or cisplatin and expressing N-terminally truncated VDAC1 do not release cytochrome c, Smac/Diablo or AIF. Ruthenium red (RuR), AzRu, DIDS and hexokinase-I (HK-I), all known to interact with VDAC, inhibited the release of cytochrome c, Smac/Diablo and AIF, while RuR-mediated inhibition was not observed in cells expressing RuR-insensitive E72Q-VDAC1. These findings suggest that VDAC1 is involved in the release of not only cytochrome c but also of Smac/Diablo and AIF. We also demonstrate that apoptosis induction is associated with VDAC oligomerization, as revealed by chemical cross-linking and monitoring in living cells using Bioluminescence Resonance Energy Transfer. Apoptosis induction by STS, H₂O₂ or selenite augmented the formation of VDAC oligomers several fold. The results show VDAC1 to be a component of the apoptosis machinery and offer new insight into the functions of VDAC1 oligomerization in apoptosis and of the VDAC1 N-terminal domain in the release of apoptogenic proteins as well as into regulation of VDAC by anti-apoptotic proteins, such as HK and Bcl2.     

Local unfolding in a destabilized, pathogenic variant of superoxide dismutase 1 observed with H/D exchange and mass spectrometry

Hydrogen exchange monitored by mass spectrometry has been used to study the structural behavior of the pathogenic A4V variant of superoxide dismutase 1 (SOD1) in the metal-free (apo) form. Mass spectrometric data revealed that in the disulfide-intact (S-S) form, the A4V variant is destabilized at residues 50-53, in the disulfide subloop of the dimer interface, but many other regions of the A4V protein exhibited hydrogen exchange properties identical to that of the wild type protein. Additionally, mass spectrometry revealed that A4V apoSOD1(S-S) undergoes slow localized unfolding in a large segment of the beta-barrel that included beta3, beta4, and loops II and III. In the disulfide-reduced form, A4V apoSOD1 exchanged like a "random coil" polypeptide at 20 degrees C and began to populate folded states at 4 degrees C. These local and global unfolding events could facilitate intermolecular protein-protein interactions that cause the aggregation or neurotoxicity of A4V SOD1.     

Discovery of a novel series of Notch-sparing gamma-secretase inhibitors

Using a cell-based assay, we have identified a new series of Notch-sparing γ-secretase inhibitors from HTS screening leads 2a and 2e. Lead optimization studies led to the discovery of analog 8e with improved γ-secretase inhibitory potency and Notch-sparing selectivity.     

Pancreatic LKB1 deletion leads to acinar polarity defects and cystic neoplasms

LKB1 is a key regulator of energy homeostasis through the activation of AMP-activated protein kinase (AMPK) and is functionally linked to vascular development, cell polarity, and tumor suppression. In humans, germ line LKB1 loss-of-function mutations cause Peutz-Jeghers syndrome (PJS), which is characterized by a predisposition to gastrointestinal neoplasms marked by a high risk of pancreatic cancer. To explore the developmental and physiological functions of Lkb1 in vivo, we examined the impact of conditional Lkb1 deletion in the pancreatic epithelium of the mouse. The Lkb1-deficient pancreas, although grossly normal at birth, demonstrates a defective acinar cell polarity, an abnormal cytoskeletal organization, a loss of tight junctions, and an inactivation of the AMPK/MARK/SAD family kinases. Rapid and progressive postnatal acinar cell degeneration and acinar-to-ductal metaplasia occur, culminating in marked pancreatic insufficiency and the development of pancreatic serous cystadenomas, a tumor type associated with PJS. Lkb1 deficiency also impacts the pancreas endocrine compartment, characterized by smaller and scattered islets and transient alterations in glucose control. These genetic studies provide in vivo evidence of a key role for LKB1 in the establishment of epithelial cell polarity that is vital for pancreatic acinar cell function and viability and for the suppression of neoplasia.     

Clusterin enhances AKT2-mediated motility of normal and cancer prostate cells through a PTEN and PHLPP1 circuit

Clusterin (CLU) is a chaperone-like protein with multiple functions. sCLU is frequently upregulated in prostate tumor cells after chemo- or radiotherapy and after surgical or pharmacological castration. Moreover, CLU has been documented to modulate the cellular homolog of murine thymoma virus akt8 oncogene (AKT) activity. Here, we investigated how CLU overexpression influences phosphatidylinositol 3′-kinase (PI3K)/AKT signaling in human normal and cancer epithelial prostate cells. Human prostate cells stably transfected with CLU were broadly profiled by reverse phase protein array (RPPA), with particular emphasis on the PI3K/AKT pathway. The effect of CLU overexpression on normal and cancer cell motility was also tested. Our results clearly indicate that CLU overexpression enhances phosphorylation of AKT restricted to isoform 2. Mechanistically, this can be explained by the finding that the phosphatase PH domain leucine-rich repeat-containing protein phosphatase 1 (PHLPP1), known to dephosphorylate AKT2 at S474, is markedly downregulated by CLU, whereas miR-190, a negative regulator of PHLPP1, is upregulated. Moreover, we found that phosphatase and tensin homolog (PTEN) was heavily phosphorylated at the inhibitory site S380, contributing to the hyperactivation of AKT signaling. By keeping AKT2 phosphorylation high, CLU dramatically enhances the migratory behavior of prostate epithelial cell lines with different migratory and invasive phenotypes, namely prostate normal epithelial 1A (PNT1A) and prostatic carcinoma 3 (PC3) cells. Altogether, our results unravel for the first time a circuit by which CLU can switch a low migration phenotype toward a high migration phenotype, through miR-190-dependent downmodulation of PHLPP1 expression and, in turn, stabilization of AKT2 phosphorylation.     

The benefits of clustering eggs: the role of egg predation and larval cannibalism in a predatory mite

Many arthropods produce clusters of eggs, but an unambiguous explanation for the evolution of egg clustering is still lacking. We test several hypotheses for the production of egg clusters by the predatory mite Iphiseius degenerans. This predator feeds on pollen, thrips larvae and nectar in flowers, but oviposits in clusters in tufts of leaf hairs (acarodomatia), where eggs run a lower risk of being killed by thrips, the prey of this predatory mite. The observed clustering is not caused by a shortage of oviposition sites; females preferably oviposit in a domatium containing eggs rather than in an empty domatium. To explain this preference, we first examined the effect of egg clusters on the risk of cannibalism. We found that eggs are invulnerable to cannibalism, whereas larvae emerging from single eggs or from clusters were equally vulnerable. Subsequently, we considered the killing of eggs resulting from counter-attacks by prey, i.e. the western flower thrips. We found no indication that a cluster of eggs protects eggs from predation by thrips. However, when eggs were clustered in a domatium rather than scattered over domatia, the proportion of eggs killed by thrips was lower. Hence, oviposition in clusters has no effect on its own and oviposition in domatia reduces predation risk by thrips, but oviposition in clusters in domatia leads to a synergistic effect on the survival of predator eggs. This synergism probably arises because eggs in clusters within tufts of leaf hairs are more difficult for thrips to reach. These experiments highlight a novel explanation of egg clustering, i.e. adaptation to counter-attacking prey. Moreover, they show that plant domatia protect predator eggs from predation.     

Assessment of new cationic porphyrin binding to plasma proteins by planar microarray and spectroscopic methods

Porphyrins have a unique aromatic structure determining particular photochemical properties that make them promising photosensitizers for anticancer therapy. Previously, we synthesized a set of artificial porphyrins by modifying side-chain functional groups and introducing different metals into the core structure. Here, we have performed a comparative study of the binding properties of 29 cationic porphyrins with plasma proteins by using microarray and spectroscopic approaches. The porphyrins were noncovalently immobilized onto hydrogel-covered glass slides and probed to bio-conjugated human and bovine serum albumins, as well as to human hemoglobin. The signal detection was carried out at the near-infrared fluorescence wavelength (800?nm) that enabled the effect of intrinsic visible wavelength fluorescence emitted by the porphyrins tested to be discarded. Competition assays on porphyrin microarrays indicated that long-chain fatty acids (FAs) (palmitic and stearic acids) decrease porphyrin binding to both serum albumin and hemoglobin. The binding affinity of different types of cationic porphyrins for plasma proteins was quantitatively assessed in the absence and presence of FAs by fluorescent and absorption spectroscopy. Molecular docking analysis confirmed results that new porphyrins and long-chain FAs compete for the common binding site FA1 in human serum albumin and meso-substituted functional groups in porphyrins play major role in the modulation of conformational rearrangements of the protein.     

Can the Neural Basis of Repression Be Studied in the MRI Scanner? New Insights from Two Free Association Paradigms

Background

The psychodynamic theory of repression suggests that experiences which are related to internal conflicts become unconscious. Previous attempts to investigate repression experimentally were based on voluntary, intentional suppression of stimulus material. Unconscious repression of conflict-related material is arguably due to different processes, but has never been studied with neuroimaging methods.

Methods

We used functional magnetic resonance imaging (fMRI) in addition with skin conductance recordings during two free association paradigms to identify the neural mechanisms underlying forgetting of freely associated words according to repression theory.

Results

In the first experiment, free association to subsequently forgotten words was accompanied by increases in skin conductance responses (SCRs) and reaction times (RTs), indicating autonomic arousal, and by activation of the anterior cingulate cortex. These findings are consistent with the hypothesis that these associations were repressed because they elicited internal conflicts. To test this idea more directly, we conducted a second experiment in which participants freely associated to conflict-related sentences. Indeed, these associations were more likely to be forgotten than associations to not conflict-related sentences and were accompanied by increases in SCRs and RTs. Furthermore, we observed enhanced activation of the anterior cingulate cortex and deactivation of hippocampus and parahippocampal cortex during association to conflict-related sentences.

Conclusions

These two experiments demonstrate that high autonomic arousal during free association predicts subsequent memory failure, accompanied by increased activation of conflict-related and deactivation of memory-related brain regions. These results are consistent with the hypothesis that during repression, explicit memory systems are down-regulated by the anterior cingulate cortex.     

Genome sequence of Leuconostoc carnosum KCTC 3525

We announce the draft genome sequence of the type strain Leuconostoc carnosum KCTC 3525 (3,234,408 bp with a G+C content of 40.9%), one of the most prevalent lactic acid bacteria present during the manufacturing process of vacuum-packaged meats, which consists of 2,407 large contigs (>500 bp in size). The genome sequence was obtained by a whole-genome shotgun strategy using Roche 454 GS (FLX Titanium) pyrosequencing, and all of the reads were assembled using Newbler Assembler 2.3.     

Genome sequence of Lactobacillus versmoldensis KCTC 3814

Lactobacillus versmoldensis KCTC 3814 was isolated from raw fermented poultry salami. The species was present in high numbers and frequently dominated the lactic acid bacteria (LAB) populations of the products. Here, we announce the draft genome sequence of Lactobacillus versmoldensis KCTC 3814, isolated from poultry salami, and describe major findings from its annotation.