Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis.

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

LSO:Ce Inorganic Scintillators Are Biocompatible With Neuronal and Circuit Function.

Optogenetics is widely used in neuroscience to control neural circuits. However, non-invasive methods for light delivery in brain are needed to avoid physical damage caused by current methods. One potential strategy could employ x-ray activation of radioluminescent particles (RPLs), enabling localized light generation within the brain. RPLs composed of inorganic scintillators can emit light at various wavelengths depending upon composition. Cerium doped lutetium oxyorthosilicate (LSO:Ce), an inorganic scintillator that emits blue light in response to x-ray or ultraviolet (UV) stimulation, could potentially be used to control neural circuits through activation of channelrhodopsin-2 (ChR2), a light-gated cation channel. Whether inorganic scintillators themselves negatively impact neuronal processes and synaptic function is unknown, and was investigated here using cellular, molecular, and electrophysiological approaches. As proof of principle, we applied UV stimulation to 4 µm LSO:Ce particles during whole-cell recording of CA1 pyramidal cells in acute hippocampal slices from mice that expressed ChR2 in glutamatergic neurons. We observed an increase in frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), indicating activation of ChR2 and excitation of neurons. Importantly, LSO:Ce particles did not affect survival of primary mouse cortical neurons, even after 24 h of exposure. In extracellular dendritic field potential recordings, no change in the strength of basal glutamatergic transmission was observed during exposure to LSO:Ce microparticles. However, the amplitude of the fiber volley was slightly reduced with high stimulation. Additionally, there was a slight decrease in the frequency of sEPSCs in whole-cell voltage-clamp recordings from CA1 pyramidal cells, with no change in current amplitudes. The amplitude and frequency of spontaneous inhibitory postsynaptic currents were unchanged. Finally, long term potentiation (LTP), a synaptic modification believed to underlie learning and memory and a robust measure of synaptic integrity, was successfully induced, although the magnitude was slightly reduced. Together, these results show LSO:Ce particles are biocompatible even though there are modest effects on baseline synaptic function and long-term synaptic plasticity. Importantly, we show that light emitted from LSO:Ce particles is able to activate ChR2 and modify synaptic function. Therefore, LSO:Ce inorganic scintillators are potentially viable for use as a new light delivery system for optogenetics.

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aß production.

The diversion of the membrane-bound ß-site amyloid precursor protein-(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aß) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aß production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aß production.

SnRK1 from Arabidopsis thaliana is an atypical AMPK.

SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic α subunit and non-catalytic ß and γ subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, ß(3) and ßγ, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic α subunit together with the unique ßγ subunit and one of three non-catalytic ß subunits: ß(1), ß(2) or the plant-specific ß(3) isoform. Thus, the AtSnRK1 heterotrimers contain the atypical ßγ subunit rather than a conventional γ subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the ß(1), ß(2) and ßγ subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

Molecular basis of impaired glycogen metabolism during ischemic stroke and hypoxia.

BACKGROUND: Ischemic stroke is the combinatorial effect of many pathological processes including the loss of energy supplies, excessive intracellular calcium accumulation, oxidative stress, and inflammatory responses. The brain's ability to maintain energy demand through this process involves metabolism of glycogen, which is critical for release of stored glucose. However, regulation of glycogen metabolism in ischemic stroke remains unknown. In the present study, we investigate the role and regulation of glycogen metabolizing enzymes and their effects on the fate of glycogen during ischemic stroke. RESULTS: Ischemic stroke was induced in rats by peri-vascular application of the vasoconstrictor endothelin-1 and forebrains were collected at 1, 3, 6 and 24 hours post-stroke. Glycogen levels and the expression and activity of enzymes involved in glycogen metabolism were analyzed. We found elevated glycogen levels in the ipsilateral hemispheres compared with contralateral hemispheres at 6 and 24 hours (25% and 39% increase respectively; P<0.05). Glycogen synthase activity and glycogen branching enzyme expression were found to be similar between the ipsilateral, contralateral, and sham control hemispheres. In contrast, the rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 58% lower activity (P<0.01) in the ipsilateral hemisphere (24 hours post-stroke), which corresponded with a 48% reduction in cAMP-dependent protein kinase A (PKA) activity (P<0.01). In addition, glycogen debranching enzyme expression 24 hours post-stroke was 77% (P<0.01) and 72% lower (P<0.01) at the protein and mRNA level, respectively. In cultured rat primary cerebellar astrocytes, hypoxia and inhibition of PKA activity significantly reduced glycogen phosphorylase activity and increased glycogen accumulation but did not alter glycogen synthase activity. Furthermore, elevated glycogen levels provided metabolic support to astrocytes during hypoxia. CONCLUSION: Our study has identified that glycogen breakdown is impaired during ischemic stroke, the molecular basis of which includes reduced glycogen debranching enzyme expression level together with reduced glycogen phosphorylase and PKA activity.

Defining the substrate specificity determinants recognized by the active site of C-terminal Src kinase-homologous kinase (CHK) and identification of ß-synuclein as a potential CHK physiological substrate.

C-Terminal Src kinase-homologous kinase (CHK) exerts its tumor suppressor function by phosphorylating the C-terminal regulatory tyrosine of the Src-family kinases (SFKs). The phosphorylation suppresses their activity and oncogenic action. In addition to phosphorylating SFKs, CHK also performs non-SFK-related functions by phosphorylating other cellular protein substrates. To define these non-SFK-related functions of CHK, we used the "kinase substrate tracking and elucidation" method to search for its potential physiological substrates in rat brain cytosol. Our search revealed ß-synuclein as a potential CHK substrate, and Y127 in ß-synuclein as the preferential phosphorylation site. Using peptides derived from ß-synuclein and positional scanning combinatorial peptide library screening, we defined the optimal substrate phosphorylation sequence recognized by the CHK active site to be E-x-[Φ/E/D]-Y-Φ-x-Φ, where Φ and x represent hydrophobic residues and any residue, respectively. Besides ß-synuclein, cellular proteins containing motifs resembling this sequence are potential CHK substrates. Intriguingly, the CHK-optimal substrate phosphorylation sequence bears little resemblance to the C-terminal tail sequence of SFKs, indicating that interactions between the CHK active site and the local determinants near the C-terminal regulatory tyrosine of SFKs play only a minor role in governing specific phosphorylation of SFKs by CHK. Our results imply that recognition of SFKs by CHK is mainly governed by interactions between motifs located distally from the active site of CHK and determinants spatially separate from the C-terminal regulatory tyrosine in SFKs. Thus, besides assisting in the identification of potential CHK physiological substrates, our findings shed new light on how CHK recognizes SFKs and other protein substrates.

A facile, click chemistry-based approach to assembling fluorescent chemosensors for protein tyrosine kinases.

A group of fluorophore-labeled peptide substrates of Src kinases have been synthesized with the aid of click chemistry. Some of the generated peptides exhibit an increase in fluorescence upon phosphorylation and are capable of detecting Src kinases with high sensitivity and specificity. Their availability permits real-time activity measurement of aberrantly activated oncogenic Src kinases in the crude lysate of chronic myelogenous leukemia cells. These new chemosensor peptides are highly useful tools that can be used for high-throughput screening to search for small molecule inhibitors of Src kinases as potential therapeutics for cancer treatment.