Cannabidiol Impairs Brain Mitochondrial Metabolism and Neuronal Integrity.

Background: The mechanisms underlying the clinical effects of CBD remain poorly understood. Given the increasing evidence for CBD's effects on mitochondria, we sought to examine in more detail whether CBD impacts mitochondrial function and neuronal integrity. Methods: We utilized BE(2)-M17 neuroblastoma cells or acutely isolated brain mitochondria from rodents using a Seahorse extracellular flux analyzer and a fluorescent spectrofluorophotometer assay. Mitochondrial ion channel activity and hippocampal long-term potentiation were measured using standard cellular electrophysiological methods. Spatial learning/memory function was evaluated using the Morris water maze task. Plasma concentrations of CBD were assessed with liquid chromatography-mass spectrometry, and cellular viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction neuronal injury assay. Results: At low micromolar concentrations, CBD reduced mitochondrial respiration, the threshold for mitochondrial permeability transition, and calcium uptake, blocked a novel mitochondrial chloride channel, and reduced the viability of hippocampal cells. These effects were paralleled by in vitro and in vivo learning/memory deficits. We further found that these effects were independent of cannabinoid receptor 1 and mitochondrial G-protein-coupled receptor 55. Conclusion: Our results provide evidence for concentration- and dose-dependent toxicological effects of CBD, findings that may bear potential relevance to clinical populations.

Aberrant Mitochondrial Morphology and Function in the BTBR Mouse Model of Autism Is Improved by Two Weeks of Ketogenic Diet.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder that exhibits a common set of behavioral and cognitive impairments. Although the etiology of ASD remains unclear, mitochondrial dysfunction has recently emerged as a possible causative factor underlying ASD. The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that augments mitochondrial function, and has been shown to reduce autistic behaviors in both humans and in rodent models of ASD. The aim of the current study was to examine mitochondrial bioenergetics in the BTBR mouse model of ASD and to determine whether the KD improves mitochondrial function. We also investigated changes in mitochondrial morphology, which can directly influence mitochondrial function. We found that BTBR mice had altered mitochondrial function and exhibited smaller more fragmented mitochondria compared to C57BL/6J controls, and that supplementation with the KD improved both mitochondrial function and morphology. We also identified activating phosphorylation of two fission proteins, pDRP1S616 and pMFFS146, in BTBR mice, consistent with the increased mitochondrial fragmentation that we observed. Intriguingly, we found that the KD decreased pDRP1S616 levels in BTBR mice, likely contributing to the restoration of mitochondrial morphology. Overall, these data suggest that impaired mitochondrial bioenergetics and mitochondrial fragmentation may contribute to the etiology of ASD and that these alterations can be reversed with KD treatment.

Common Repository of FBS Proteins (cRFP) To Be Added to a Search Database for Mass Spectrometric Analysis of Cell Secretome.

We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretomes. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.

Enhanced glycolytic metabolism supports transmigration of brain-infiltrating macrophages in multiple sclerosis.

The migration of leukocytes into the CNS drives the neuropathology of multiple sclerosis (MS). This penetration likely utilizes energy resources that remain to be defined. Using the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined that macrophages within the perivascular cuff of post-capillary venules are highly glycolytic as manifested by strong expression of lactate dehydrogenase A (LDHA) that converts pyruvate to lactate. These macrophages expressed prominent levels of monocarboxylate transporter-4 (MCT-4) specialized in secreting lactate from glycolytic cells. The functional relevance of glycolysis was confirmed by siRNA-mediated knockdown of LDHA and MCT-4, which decreased lactate secretion and macrophage transmigration. MCT-4 was in turn regulated by EMMPRIN (CD147) as determined through co-expression/co-immunoprecipitation studies, and siRNA-mediated EMMPRIN silencing. The functional relevance of MCT-4/EMMPRIN interaction was affirmed by lower macrophage transmigration in culture using the MCT-4 inhibitor, α-cyano-4-hydroxy-cinnamic acid (CHCA), a cinnamon derivative. CHCA also reduced leukocyte infiltration and the clinical severity of EAE. Relevance to MS was corroborated by the strong expression of MCT-4, EMMPRIN and LDHA in perivascular macrophages in MS brains. These results detail the metabolism of macrophages for transmigration from perivascular cuffs into the CNS parenchyma and identifies CHCA and diet as potential modulators of neuro-inflammation in MS.

ER-stress mobilization of death-associated protein kinase-1-dependent xenophagy counteracts mitochondria stress-induced epithelial barrier dysfunction.

The gut microbiome contributes to inflammatory bowel disease (IBD), in which bacteria can be present within the epithelium. Epithelial barrier function is decreased in IBD, and dysfunctional epithelial mitochondria and endoplasmic reticulum (ER) stress have been individually associated with IBD. We therefore hypothesized that the combination of ER and mitochondrial stresses significantly disrupt epithelial barrier function. Here, we treated human colonic biopsies, epithelial colonoids, and epithelial cells with an uncoupler of oxidative phosphorylation, dinitrophenol (DNP), with or without the ER stressor tunicamycin and assessed epithelial barrier function by monitoring internalization and translocation of commensal bacteria. We also examined barrier function and colitis in mice exposed to dextran sodium sulfate (DSS) or DNP and co-treated with DAPK6, an inhibitor of death-associated protein kinase 1 (DAPK1). Contrary to our hypothesis, induction of ER stress (i.e. the unfolded protein response) protected against decreased barrier function caused by the disruption of mitochondrial function. ER stress did not prevent DNP-driven uptake of bacteria; rather, specific mobilization of the ATF6 arm of ER stress and recruitment of DAPK1 resulted in enhanced autophagic killing (xenophagy) of bacteria. Of note, epithelia with a Crohn's disease-susceptibility mutation in the autophagy gene ATG16L1 exhibited less xenophagy. Systemic delivery of the DAPK1 inhibitor DAPK6 increased bacterial translocation in DSS- or DNP-treated mice. We conclude that promoting ER stress-ATF6-DAPK1 signaling in transporting enterocytes counters the transcellular passage of bacteria evoked by dysfunctional mitochondria, thereby reducing the potential for metabolic stress to reactivate or perpetuate inflammation.

Uliginosibacterium sangjuense sp. nov., isolated from sediment of the Nakdong River.

A Gram-negative, aerobic, motile by flagella, and light yellow bacterium, designated SS1-76T, was isolated from sediment of the Nakdong River in Sangju-si, Republic of Korea. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate SS1-76T belongs to the genus Uliginosibacterium of the family Rhodocyclaceae, exhibiting high sequence similarity with the type strains of Uliginosibacterium gangwonense 5YN10-9T (96.0%) and Uliginosibacterium paludis KBP-13T (94.9%). Strain SS1-76T contains ubiquinone-8 as a respiratory quinone and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0, and C14:0 as major fatty acids. The cellular polar lipids are composed of phosphatidylethanolamine, phosphatidylglycerol, and unidentified aminophospholipids. The DNA G+C content was 65.3 mol%. Phenotypic, chemotaxonomic, and phylogenetic evidence clearly indicated that strain SS1-76T represents a novel species of the genus Uliginosibacterium, for which the name Uliginosibacterium sangjuense sp. nov. is proposed. The type strain is SS1-76T (= KCTC 52159T = JCM 31375T).

Vibrational Profiling of Brain Tumors and Cells.

This study reports vibration profiles of neuronal cells and tissues as well as brain tumor and neocortical specimens. A contact-free method and analysis protocol was designed to convert an atomic force microscope into an ultra-sensitive microphone with capacity to record and listen to live biological samples. A frequency of 3.4 Hz was observed for both cultured rat hippocampal neurons and tissues and vibration could be modulated pharmacologically. Malignant astrocytoma tissue samples obtained from operating room, transported in artificial cerebrospinal fluid, and tested within an hour, vibrated with a much different frequency profile and amplitude, compared to meningioma or lateral temporal cortex providing a quantifiable measurement to accurately distinguish the three tissues in real-time. Vibration signals were converted to audible sound waves by frequency modulation, thus demonstrating, acoustic patterns unique to meningioma, malignant astrocytoma and neocortex.

Carisbamate blockade of T-type voltage-gated calcium channels.

OBJECTIVES: Carisbamate (CRS) is a novel monocarbamate compound that possesses antiseizure and neuroprotective properties. However, the mechanisms underlying these actions remain unclear. Here, we tested both direct and indirect effects of CRS on several cellular systems that regulate intracellular calcium concentration [Ca2+ ]i . METHODS: We used a combination of cellular electrophysiologic techniques, as well as cell viability, Store Overload-Induced Calcium Release (SOICR), and mitochondrial functional assays to determine whether CRS might affect [Ca2+ ]i levels through actions on the endoplasmic reticulum (ER), mitochondria, and/or T-type voltage-gated Ca2+ channels. RESULTS: In CA3 pyramidal neurons, kainic acid induced significant elevations in [Ca2+ ]i and long-lasting neuronal hyperexcitability, both of which were reversed in a dose-dependent manner by CRS. Similarly, CRS suppressed spontaneous rhythmic epileptiform activity in hippocampal slices exposed to zero-Mg2+ or 4-aminopyridine. Treatment with CRS also protected murine hippocampal HT-22 cells against excitotoxic injury with glutamate, and this was accompanied by a reduction in [Ca2+ ]i . Neither kainic acid nor CRS alone altered the mitochondrial membrane potential (ΔΨ) in intact, acutely isolated mitochondria. In addition, CRS did not affect mitochondrial respiratory chain activity, Ca2+ -induced mitochondrial permeability transition, and Ca2+ release from the ER. However, CRS significantly decreased Ca2+ flux in human embryonic kidney tsA-201 cells transfected with Cav 3.1 (voltage-dependent T-type Ca2+ ) channels. SIGNIFICANCE: Our data indicate that the neuroprotective and antiseizure activity of CRS likely results in part from decreased [Ca2+ ]i accumulation through blockade of T-type Ca2+ channels.

Tissue Specific Impacts of a Ketogenic Diet on Mitochondrial Dynamics in the BTBRT+tf/j Mouse.

The ketogenic diet (KD) has been utilized as a dietary therapeutic for nearly a century. One experimental model particularly responsive to the KD is the BTBRT+tf/j (BTBR) mouse, which displays phenotypic characteristics of autism spectrum disorder (ASD) and insulin resistance. Recently, the study of impaired mitochondrial function has become a focal point of research investigating the pathophysiology of ASD. As highly dynamic organelles, mitochondria undergo constant fluctuations in morphology, biogenesis, and quality control in order to maintain cellular homeostasis. An important modifier of mitochondrial dynamics is energy availability. Therefore, the aim of this study was to examine the impact of a KD on mitochondrial dynamics in the liver and brain (prefrontal cortex) of the BTBR mouse model of ASD. Juvenile male C57Bl/6 (B6) and BTBR mice were age-matched to 5 weeks of age before being fed standard chow (CD, 13% kcal fat) or a KD (75% kcal fat) for 10-14 days. Analysis of brain tissue identified differences in mitochondrial gene expression but no correlation with protein levels. Unlike in the brain, KD led to decreased levels of mitochondrial proteins in the liver, despite increased gene expression. Consistent with decreased mitochondrial proteins, we also observed decreased mtDNA for all mice on the KD, demonstrating that the KD reduces the total amount of mitochondria in the liver. In order to explain the discrepancy between protein levels and gene expression, we investigated whether mitochondrial turnover via mitophagy was increased. To this end, we examined expression levels of the mitophagy regulator BNIP3 (BCL2/adenovirus E1B 19 kd-interacting protein 3). BNIP3 gene and protein expression were significantly elevated in liver of KD animals (p < 0.05), indicating the potential activation of mitophagy. Therefore, consumption of a KD exerts highly tissue-specific effects, ultimately increasing mitochondrial turnover in the liver, while gene and protein expression in the brain remaining tightly regulated.

Stoichiometric expression of mtHsp40 and mtHsp70 modulates mitochondrial morphology and cristae structure via Opa1L cleavage.

Deregulation of mitochondrial heat-shock protein 40 (mtHsp40) and dysfunction of mtHsp70 are associated with mitochondrial fragmentation, suggesting that mtHsp40 and mtHsp70 may play roles in modulating mitochondrial morphology. However, the mechanism of mitochondrial fragmentation induced by mtHsp40 deregulation and mtHsp70 dysfunction remains unclear. In addition, the functional link between mitochondrial morphology change upon deregulated mtHsp40/mtHsp70 and mitochondrial function has been unexplored. Our coimmunoprecipitation and protein aggregation analysis showed that both overexpression and depletion of mtHsp40 accumulated aggregated proteins in fragmented mitochondria. Moreover, mtHsp70 loss and expression of a mtHsp70 mutant lacking the client-binding domain caused mitochondrial fragmentation. Together the data suggest that the molecular ratio of mtHsp40 to mtHsp70 is important for their chaperone function and mitochondrial morphology. Whereas mitochondrial translocation of Drp1 was not altered, optic atrophy 1 (Opa1) short isoform accumulated in fragmented mitochondria, suggesting that mitochondrial fragmentation in this study results from aberration of mitochondrial inner membrane fusion. Finally, we found that fragmented mitochondria were defective in cristae development, OXPHOS, and ATP production. Taken together, our data suggest that impaired stoichiometry between mtHsp40 and mtHsp70 promotes Opa1L cleavage, leading to cristae opening, decreased OXPHOS, and triggering of mitochondrial fragmentation after reduction in their chaperone function.

Ketone bodies mediate antiseizure effects through mitochondrial permeability transition.

OBJECTIVE: Ketone bodies (KB) are products of fatty acid oxidation and serve as essential fuels during fasting or treatment with the high-fat antiseizure ketogenic diet (KD). Despite growing evidence that KB exert broad neuroprotective effects, their role in seizure control has not been firmly demonstrated. The major goal of this study was to demonstrate the direct antiseizure effects of KB and to identify an underlying target mechanism. METHODS: We studied the effects of both the KD and KB in spontaneously epileptic Kcna1-null mice using a combination of behavioral, planar multielectrode, and standard cellular electrophysiological techniques. Thresholds for mitochondrial permeability transition (mPT) were determined in acutely isolated brain mitochondria. RESULTS: KB alone were sufficient to: (1) exert antiseizure effects in Kcna1-null mice, (2) restore intrinsic impairment of hippocampal long-term potentiation and spatial learning-memory defects in Kcna1-null mutants, and (3) raise the threshold for calcium-induced mPT in acutely prepared mitochondria from hippocampi of Kcna1-null animals. Targeted deletion of the cyclophilin D subunit of the mPT complex abrogated the effects of KB on mPT, and in vivo pharmacological inhibition and activation of mPT were found to mirror and reverse, respectively, the antiseizure effects of the KD in Kcna1-null mice. INTERPRETATION: The present data reveal the first direct link between mPT and seizure control, and provide a potential mechanistic explanation for the KD. Given that mPT is increasingly being implicated in diverse neurological disorders, our results suggest that metabolism-based treatments and/or metabolic substrates might represent a worthy paradigm for therapeutic development.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

Decreased mTOR signaling pathway in human idiopathic autism and in rats exposed to valproic acid.

BACKGROUND: The molecular mechanisms underlying autistic behaviors remain to be elucidated. Mutations in genes linked to autism adversely affect molecules regulating dendritic spine formation, function and plasticity, and some increase the mammalian target of rapamycin, mTOR, a regulator of protein synthesis at spines. Here, we investigated whether the Akt/mTOR pathway is disrupted in idiopathic autism and in rats exposed to valproic acid, an animal model exhibiting autistic-like behavior. METHODS: Components of the mTOR pathway were assayed by Western blotting in postmortem fusiform gyrus samples from 11 subjects with idiopathic autism and 13 controls and in valproic acid versus saline-exposed rat neocortex. Additionally, protein levels of brain-derived neurotrophic factor receptor (TrkB) isoforms and the postsynaptic organizing molecule PSD-95 were measured in autistic versus control subjects. RESULTS: Full-length TrkB, PI3K, Akt, phosphorylated and total mTOR, p70S6 kinase, eIF4B and PSD-95 were reduced in autistic versus control fusiform gyrus. Similarly, phosphorylated and total Akt, mTOR and 4E-BP1 and phosphorylated S6 protein were decreased in valproic acid- versus saline-exposed rats. However, no changes in 4E-BP1 or eIF4E were found in autistic brains. CONCLUSIONS: In contrast to some monogenic disorders with high rates of autism, our data demonstrate down-regulation of the Akt/mTOR pathway, specifically via p70S6K/eIF4B, in idiopathic autism. These findings suggest that disruption of this pathway in either direction is widespread in autism and can have adverse consequences for synaptic function. The use of valproic acid, a histone deacetylase inhibitor, in rats successfully modeled these changes, implicating an epigenetic mechanism in these pathway disruptions.

The ketogenic diet modifies social and metabolic alterations identified in the prenatal valproic acid model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by abnormal social interactions, communication deficits and stereotyped or repetitive behaviors. Although the etiology of ASD remains elusive, converging lines of research indicate that mitochondrial dysfunction may play a substantive role in disease pathophysiology. Without an established causal link, the generation of therapeutic targets for ASD has been relatively unsuccessful and has focused solely on individual symptoms. The ketogenic diet (KD) is a high-fat low-carbohydrate diet that has previously been used for the treatment of intractable epilepsy and is known to enhance mitochondrial function. The purpose of this study was to determine if the KD could reverse the social deficits and mitochondrial dysfunction identified in the prenatal valproic acid (VPA) rodent model of ASD. Sprague-Dawley dams were administered VPA or saline on gestational day 12.5. The pups were treated with the KD or their standard diet (SD) for 10 days beginning on postnatal day 21 (PD21). On PD35 juvenile play behavior was tested with the play-fighting paradigm and rats were then sacrificed for mitochondrial bioenergetic analysis. The offspring exposed to VPA prenatally demonstrated a significant decrease in the number of play initiations/attacks and this was reversed with the KD. Prenatal VPA exposure also disrupted the pattern of play responses; VPA/SD animals used complete rotations more often than saline control animals. Treatment with the KD did not affect the number of complete rotations. In addition, while prenatal exposure to VPA altered mitochondrial respiration, the KD was able to restore aspects of bioenergetic dysfunction. As the KD was able to modify complex social behaviors and mitochondrial respiration, it may be a useful treatment option for ASD. Future studies will need to examine the effectiveness of the KD to reverse the two additional core deficits of ASD and to explore various treatment regimens to determine optimal treatment duration and formulation.

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor.

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFßR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFßR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFßR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFßR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Redox regulation of the tumor suppressor PTEN by glutathione.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Delta and gsh2Delta mutants. Expression of gamma-glutamylcysteine synthetase Gsh1 in the gsh1Delta mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.

Ribosomal protein S3, a new substrate of Akt, serves as a signal mediator between neuronal apoptosis and DNA repair.

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.

Differential profiling of breast cancer plasma proteome by isotope-coded affinity tagging method reveals biotinidase as a breast cancer biomarker.

BACKGROUND: Breast cancer is one of the leading causes of women's death worldwide. It is important to discover a reliable biomarker for the detection of breast cancer. Plasma is the most ideal source for cancer biomarker discovery since many cells cross-communicate through the secretion of soluble proteins into blood. METHODS: Plasma proteomes obtained from 6 breast cancer patients and 6 normal healthy women were analyzed by using the isotope-coded affinity tag (ICAT) labeling approach and tandem mass spectrometry. All the plasma samples used were depleted of highly abundant 6 plasma proteins by immune-affinity column chromatography before ICAT labeling. Several proteins showing differential abundance level were selected based on literature searches and their specificity to the commercially available antibodies, and then verified by immunoblot assays. RESULTS: A total of 155 proteins were identified and quantified by ICAT method. Among them, 33 proteins showed abundance changes by more than 1.5-fold between the plasmas of breast cancer patients and healthy women. We chose 5 proteins for the follow-up confirmation in the individual plasma samples using immunoblot assay. Four proteins, alpha1-acid glycoprotein 2, monocyte differentiation antigen CD14, biotinidase (BTD), and glutathione peroxidase 3, showed similar abundance ratio to ICAT result. Using a blind set of plasmas obtained from 21 breast cancer patients and 21 normal healthy controls, we confirmed that BTD was significantly down-regulated in breast cancer plasma (Wilcoxon rank-sum test, p = 0.002). BTD levels were lowered in all cancer grades (I-IV) except cancer grade zero. The area under the receiver operating characteristic curve of BTD was 0.78. Estrogen receptor status (p = 0.940) and progesterone receptor status (p = 0.440) were not associated with the plasma BTD levels. CONCLUSIONS: Our study suggests that BTD is a potential serological biomarker for the detection of breast cancer.

Mining of serum glycoproteins by an indirect approach using cell line secretome.

Glycosylation is the most important and abundant post-translational modification in serum proteome. Several specific types of glycan epitopes have been shown to be associated with various types of disease. Direct analysis of serum glycoproteins is challenging due to its wide dynamic range. Alternatively, glycoproteins can be discovered in the secretome of model cell lines and then confirmed in blood. However, there has been little experimental evidence showing cell line secretome as a tractable target for the study of serum glycoproteins. We used a hydrazine-based glycocapture method to selectively enrich glycoproteins from the secretome of the breast cancer cell line Hs578T. A total of 132 glycoproteins were identified by nanoLC-MS/MS analysis. Among the identified proteins, we selected 13 proteins that had one or more N-glycosylation motifs in the matched peptides, which were included in the Secreted Protein Database but not yet in the Plasma Proteome Database (PPD), and whose antibodies were commercially available. Nine out of the 13 selected proteins were detected from human blood plasma by western analysis. Furthermore, eight proteins were also detected from the plasma by targeted LC-MS/MS, which had never been previously identified by data-dependent LC-MS/MS. Our results provide novel proteins that should be enrolled in PPD and suggest that analysis of cell line secretome with subfractionation is an efficient strategy for discovering disease-relevant serum proteins.