Piceatannol-3'-O-ß-D-glucopyranoside attenuates colistin-induced neurotoxicity by suppressing oxidative stress via the NRF2/HO-1 pathway.

BACKGROUND: Multidrug-resistant Gram-negative bacteria are the most pressing problem in treating infectious diseases. As one of the primary drugs for multidrug-resistant Gram-negative bacteria, the neurotoxicity of colistin has become a significant challenge in clinical practice. PURPOSE: This study aimed to investigate the potential effect of piceatannol-3'-O-ß-D glucopyranoside (PG) on colistin-induced neurotoxicity and the underlying mechanism. METHODS: In vitro, nerve cell damage models were established by exposing N2a cells to 400 µM colistin for 24 h. The effects of PG on cell viability, apoptosis level, and oxidative stress level were analyzed. A western blot experiment was performed to determine the NRF2 pathway, apoptosis, and autophagy-related proteins. Mitochondrial morphology and mitochondrial membrane potential were detected after staining using laser confocal microscopy. In vivo, nerve injury mouse model was established by intracerebroventricular colistin administration. Morphological changes in brain tissues were observed using HE and Nissl staining. RESULTS: PG significantly reduced colistin-induced neuronal apoptosis levels. The apoptosis-related protein expressions were suppressed after PG intervention. Mechanistically, PG increased the levels of antioxidant factors and decreased the levels of oxidative factors, which might be related to the activation of the NRF2 pathway. In addition, PG treatment reversed the deviations in mitochondrial morphology and membrane potential. PG suppressed autophagy levels in N2a cells, possibly because PG inhibited colistin-induced apoptosis, thus reducing the level of spontaneous protective autophagy in cells. Nrf2 knockdown N2a cell models were applied to confirm that the activation of the NRF2 pathway played a vital role in PG alleviating the nerve damage caused by colistin. CONCLUSION: PG is a potential treatment option for colistin-induced neurotoxicity. It mitigated colistin-induced oxidative stress-associated injury and mitochondrial damage by activating the NRF2/HO-1 pathway, thus reducing nerve cell apoptosis.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span.

Caloric restriction (CR) is known to extend life span across species; however, the molecular mechanisms are not well understood. We investigate the mechanism by which glucose restriction (GR) extends yeast replicative life span, by combining ribosome profiling and RNA-seq with microfluidic-based single-cell analysis. We discovered a cross-talk between glucose sensing and the regulation of intracellular methionine: GR down-regulated the transcription and translation of methionine biosynthetic enzymes and transporters, leading to a decreased intracellular methionine concentration; external supplementation of methionine cancels the life span extension by GR. Furthermore, genetic perturbations that decrease methionine synthesis/uptake extend life span. These observations suggest that intracellular methionine mediates the life span effects of various nutrient and genetic perturbations, and that the glucose-methionine cross-talk is a general mechanism for coordinating the nutrient status and the translation/growth of a cell. Our work also implicates proteasome as a downstream effector of the life span extension by GR.

See & Catch method for studying protein complexes in yeast cells: a technique unifying fluorescence microscopy and mass spectrometry.

We have developed a method for studying proteins and protein complexes in yeast cells based on unification of fluorescence microscopy and mass spectrometry techniques. To apply the method, termed by us as "See & Catch," we first produced a variety of DNA plasmids used as PCR templates for genomic tagging of proteins with a modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. Among those modular tags, we found several combinations which were optimal for determining protein subcellular localization and for purifying the tagged proteins and protein complexes for detailed analysis by mass spectrometry. Combining fluorescence microscopy and mass spectrometry into a single method provides a unique possibility to obtain a unified view of the processes regulating dynamic properties of the proteins and protein complexes in living cells.

An N-terminal splice variant of human Stat5a that interacts with different transcription factors is the dominant form expressed in invasive ductal carcinoma.

We have identified a new variant of human Stat5a, found at higher ratios to full-length Stat5a in invasive ductal carcinoma versus contiguous normal tissue. The variant, missing exon 5, inhibits p21 and Bax production and increases cell number. After prolactin stimulation, only full-length Stat5a interacts with the vitamin D and retinoid X receptors, whereas only Δ5 Stat5a interacts with activating protein 1-2 and specificity protein 1. Prolactin also oppositely regulates interaction of the two Stat5a forms with ß-catenin. We propose that a change in splicing leading to upregulation of this new isoform is a pathogenic aspect of invasive ductal carcinoma.

Cricket paralysis virus antagonizes Argonaute 2 to modulate antiviral defense in Drosophila.

Insect viruses have evolved strategies to control the host RNAi antiviral defense mechanism. In nature, Drosophila melanogaster C virus (DCV) infection causes low mortality and persistent infection, whereas the closely related cricket paralysis virus (CrPV) causes a lethal infection. We show that these viruses use different strategies to modulate the host RNAi defense machinery. The DCV RNAi suppressor (DCV-1A) binds to long double-stranded RNA and prevents processing by Dicer2. In contrast, the CrPV suppressor (CrPV-1A) interacts with the endonuclease Argonaute 2 (Ago2) and inhibits its activity without affecting the microRNA (miRNA)-Ago1-mediated silencing. We examined the link between viral RNAi suppressors and the outcome of infection using recombinant Sindbis viruses encoding either CrPV-1A or DCV-1A. Flies infected with Sindbis virus expressing CrPV-1A showed a marked increase in virus production, spread and mortality. In contrast, Sindbis pathogenesis was only modestly increased by expression of DCV- 1A. We conclude that RNAi suppressors function as virulence factors in insects and can target the Drosophila RNAi pathway at different points.

GABAergic interneuron dysfunction impairs hippocampal neurogenesis in adult apolipoprotein E4 knockin mice.

Apolipoprotein (apo) E, a polymorphic protein with three isoforms (apoE2, apoE3, and apoE4), is essential for lipid homeostasis. Carriers of apoE4 are at higher risk for developing Alzheimer's disease. We have investigated adult neurogenesis in mice with knockout (KO) for apoE or with knockin (KI) alleles for human apoE3 or apoE4, and we report that neurogenesis is reduced in both apoE-KO and apoE4-KI mice. In apoE-KO mice, increased BMP signaling promoted glial differentiation at the expense of neurogenesis. In contrast, in apoE4-KI mice, presynaptic GABAergic input-mediated maturation of newborn neurons was diminished. Tau phosphorylation, an Alzheimer's disease characteristic, and levels of neurotoxic apoE fragments were both elevated in apoE4-KI hippocampal neurons concomitant with decreased GABAergic interneuron survival. Potentiating GABAergic signaling restored neuronal maturation and neurogenesis in apoE4-KI mice to normal levels. These findings suggest that GABAergic signaling can be targeted to mitigate the deleterious effects of apoE4 on neurogenesis.

Unifying fluorescence microscopy and mass spectrometry for studying protein complexes in cells.

We have developed and applied a method unifying fluorescence microscopy and mass spectrometry for studying spatial and temporal properties of proteins and protein complexes in yeast cells. To combine the techniques, first we produced a variety of DNA constructs that can be used for genomic tagging of proteins with modular fluorescent and affinity tags. The modular tag consists of one of the multiple versions of monomeric fluorescent proteins fused to a variety of small affinity epitopes. After this step we tested the constructs by tagging two yeast proteins, Pil1 and Lsp1, the core components of eisosomes, the large protein complexes involved in endocytosis in Saccharomyces cerevisiae, with a variety of fluorescent and affinity probes. Among the modular tags produced we found several combinations that were optimal for determining subcellular localization and for purifying the tagged proteins and protein complexes for the detailed analysis by mass spectrometry. And finally, we applied the designed method for finding the new protein components of eisosomes and for gaining new insights into molecular mechanisms regulating eisosome assembly and disassembly by reversible phosphorylation and dephosphorylation. Our results indicate that this approach combining fluorescence microscopy and mass spectrometry into a single method provides a unique perspective into molecular mechanisms regulating composition and dynamic properties of the protein complexes in living cells.

A role for protein phosphorylation in cytochrome P450 3A4 ubiquitin-dependent proteasomal degradation.

Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr(264) and Ser(420). We now document that liver cytosolic kinases additionally target Ser(478) as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Delta strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser(478), Thr(264), and Ser(420) residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation.

Prolactin blocks nuclear translocation of VDR by regulating its interaction with BRCA1 in osteosarcoma cells.

Based on their content of prolactin receptors, osteosarcoma cells were predicted to be responsive to prolactin (PRL), but whether PRL would be beneficial or contribute to pathogenesis was unclear. 1,25(OH)(2) vitamin D(3) [1alpha,25(OH)(2)D(3)] has antiproliferative effects on osteosarcoma cells, and a complex interregulatory situation exists between PRL and 1alpha,25(OH)(2)D(3). Using osteosarcoma cells, Western blot, real time RT-PCR, and promoter-luciferase assays, we have examined the interaction between PRL and 1alpha,25(OH)(2)D(3) and demonstrated that physiological concentrations of PRL block increased osteocalcin and vitamin D receptor (VDR) expression in response to 1alpha,25(OH)(2)D(3.) This blockade was shown to be the result of lack of nuclear accumulation of the VDR in response to 1alpha,25(OH)(2)D(3). Although inhibition of proteasomic degradation with MG132 had no effect on the VDR itself in a 30-min time frame, it relieved the blockade by PRL. Analysis of ubiquitinated proteins brought down by immunoprecipitation with anti-VDR showed PRL regulation of a 250-kDa protein-VDR complex. P250 was identified as the breast cancer tumor suppressor gene product, BRCA1, by Western blot of the VDR immunoprecipitate and confirmed by immunoprecipitation with anti-BRCA1 and blotting for the VDR in the absence and presence of PRL. Knockdown of BRCA1 inhibited nuclear translocation of the VDR and the ability of 1alpha,25(OH)(2)D(3) to induce the VDR. This, to our knowledge, is the first demonstration of a role for BRCA1 in nuclear accumulation of a steroid hormone and the first demonstration that PRL has the potential to affect the cell cycle through effects on BRCA1.

Modular mass spectrometric tool for analysis of composition and phosphorylation of protein complexes.

The combination of high accuracy, sensitivity and speed of single and multiple-stage mass spectrometric analyses enables the collection of comprehensive sets of data containing detailed information about complex biological samples. To achieve these properties, we combined two high-performance matrix-assisted laser desorption ionization mass analyzers in one modular mass spectrometric tool, and applied this tool for dissecting the composition and post-translational modifications of protein complexes. As an example of this approach, we here present studies of the Saccharomyces cerevisiae anaphase-promoting complexes (APC) and elucidation of phosphorylation sites on its components. In general, the modular concept we describe could be useful for assembling mass spectrometers operating with both matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources into powerful mass spectrometric tools for the comprehensive analysis of complex biological samples.

Opposite effects of unmodified prolactin and a molecular mimic of phosphorylated prolactin on morphology and the expression of prostate specific genes in the normal rat prostate.

BACKGROUND: In the current study, we have investigated the individual roles of unmodified, wild-type prolactin (WT PRL) and a molecular mimic of phosphorylated prolactin (S179D PRL) in the normal rat prostate. METHODS: In the first animal experiment, recombinant WT PRL and S179D PRL were delivered to adult male rats at a rate of 14 microg/kg per day for 3 weeks. In the second animal experiment, two subcutaneous (200 microg/kg) injections of long-acting forms of the two PRLs were given to adult male rats on day 1 and day 22 for a total of 5.5 weeks of treatment. RESULTS: The different forms of PRL had opposite effects on the normal rat prostate, independently of androgens. WT PRL promoted morphologic changes in prostate epithelium consistent with preparation for cell proliferation, whereas S179D PRL produced morphologic evidence of a more differentiated epithelium. Northern blot analysis of expression of the two major prostate specific proteins, prostatein and probasin, showed that WT PRL decreased, whereas S179D PRL increased, the expression of the mRNAs for these two proteins. At the same time, S179D PRL reduced both testosterone and dihydrotestosterone levels. CONCLUSION: We conclude that PRL is an important modulator of normal rat prostate biology and that different forms of PRL have specific functions. The molecular mimic of phosphorylated PRL, S179D PRL, is the most important in terms of epithelial cell differentiation.