Biomedical subjects

Jon B Klein

Publications and source records attributed to Jon B Klein.

At least 19 recordsLinked to original sources

PubMed2007

Oxidative stress in the aging murine olfactory bulb: redox proteomics and cellular localization.

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

Aging↗

PubMed2006

Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: relevance to Alzheimer's disease.

Aging and age-related disorders such as Alzheimer's disease (AD) are usually accompanied by oxidative stress as one of the main mechanisms contributing to neurodegeneration and cognitive decline. Aging canines develop cognitive dysfunction and neuropathology similar to those seen in humans, and the use of antioxidants results in reductions in oxidative damage and in improvement in cognitive function in this canine model of human aging. In the present study, the effect of a long-term treatment with an antioxidant-fortified diet and a program of behavioral enrichment on oxidative damage was studied in aged canines. To identify the neurobiological mechanisms underlying these treatment effects, the parietal cortex from 23 beagle dogs (8.1-12.4 years) were treated for 2.8 years in one of four treatment groups: i.e., control food-control behavioral enrichment (CC); control food-behavioral enrichment (CE); antioxidant food-control behavioral enrichment (CA); enriched environment-antioxidant-fortified food (EA). We analyzed the levels of the oxidative stress biomarkers, i.e., protein carbonyls, 3-nitrotyrosine (3-NT), and the lipid peroxidation product, 4-hydroxynonenal (HNE), and observed a decrease in their levels on all treatments when compared to control, with the most significant effects found in the combined treatment, EA. Since EA treatment was most effective, we also carried out a comparative proteomics study to identify specific brain proteins that were differentially expressed and used a parallel redox proteomics approach to identify specific brain proteins that were less oxidized following EA. The specific protein carbonyl levels of glutamate dehydrogenase [NAD (P)], glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-enolase, neurofilament triplet L protein, glutathione-S-transferase (GST) and fascin actin bundling protein were significantly reduced in brain of EA-treated dogs compared to control. We also observed significant increases in expression of Cu/Zn superoxide dismutase, fructose-bisphosphate aldolase C, creatine kinase, glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The increased expression of these proteins and in particular Cu/Zn SOD correlated with improved cognitive function. In addition, there was a significant increase in the enzymatic activities of glutathione-S-transferase (GST) and total superoxide dismutase (SOD), and significant increase in the protein levels of heme oxygenase (HO-1) in EA treated dogs compared to control. These findings suggest that the combined treatment reduces the levels of oxidative damage and improves the antioxidant reserve systems in the aging canine brain, and may contribute to improvements in learning and memory. These observations provide insights into a possible neurobiological mechanism underlying the effects of the combined treatment. These results support the combination treatments as a possible therapeutic approach that could be translated to the aging human population who are at risk for age-related neurodegenerative disorders, including Alzheimer's disease.

Aldehydes↗

PubMed2006

Redox proteomic identification of oxidized cardiac proteins in adriamycin-treated mice.

Adriamycin (ADR) is a potent anticancer drug, but its use is limited by a dose-dependent cardiotoxicity. Oxidative stress is regarded as the mediating mechanism of ADR cardiotoxicity. However, cardiac proteins that are oxidatively modified have not been well characterized. We took a redox proteomics approach to identify increasingly oxidized murine cardiac proteins after a single injection of ADR (ip, 20 mg/kg body wt). The specific carbonyl levels of three proteins were significantly increased, and these proteins were identified as triose phosphate isomerase (TPI), beta-enolase, and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). TPI and enolase are key enzymes in the glycolytic pathway, and ETF-QO serves as the transporter for electrons derived from a variety of oxidative processes to the mitochondria respiratory chain. Cardiac enolase activity in ADR-treated mice was reduced by 25%, whereas the cardiac TPI activity remained unchanged. Oxidation of purified enolase or TPI via Fenton chemistry led to a 17 or 23% loss of activity, respectively, confirming that a loss of activity was the consequence of oxidation. The observation that these cardiac enzymes involved in energy production are more oxidized resulting from ADR treatment indicates that the bioenergetic pathway is an important target in ADR-initiated oxidative stress.

Animals↗

PubMed2006

Protective effect of D609 against amyloid-beta1-42-induced oxidative modification of neuronal proteins: redox proteomics study.

Oxidative stress has been implicated in the pathophysiology of a number of diseases, including neurodegenerative disorders such as Alzheimer's disease (AD), a neurodegenerative disorder associated with cognitive decline and enhanced oxidative stress. Amyloid-beta peptide(1-42) (Abeta(1-42)), one of the main component of senile plaques, can induce in vitro and in vivo oxidative damage to neuronal cells through its ability to produce free radicals. The aim of this study was to investigate the protective effect of the xanthate D609 on Abeta(1-42)-induced protein oxidation by using a redox proteomics approach. D609 was recently found to be a free radical scavenger and antioxidant. In the present study, rat primary neuronal cells were pretreated with 50 microM of D609, followed by incubation with 10 microM Abeta(1-42) for 24 hr. In the cells treated with Abeta(1-42) alone, four proteins that were significantly oxidized were identified: glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, malate dehydrogenase, and 14-3-3 zeta. Pretreatment of neuronal cultures with D609 prior to Abeta(1-42) protected all the identified oxidized proteins in the present study against Abeta(1-42)-mediated protein oxidation. Therefore, D609 may ameliorate the Abeta(1-42)-induced oxidative modification. We discuss the implications of these Abeta(1-42)-mediated oxidatively modified proteins for AD pathology and for potential therapeutic intervention in this dementing disorder.

14-3-3 Proteins↗

PubMed2006

Urinary protein expression patterns in children with sleep-disordered breathing: preliminary findings.

BACKGROUND: Obstructive sleep apnea (OSA) is the most common form of sleep-disordered breathing, with almost 15 million Americans affected and many more at risk. Current diagnostic approach to OSA requires polysomnography, which is laborious, onerous, and time-consuming. There is ample evidence that inflammatory responses to the perturbations associated with OSA trigger a variety of genes and signaling cascades that ultimately lead to end-organ injury and changes in kidney function and protein expression. The aim of this study was therefore to analyze proteins in human urine and assess whether differential expression of particular proteins is associated with the presence of OSA. METHODS: Eleven OSA and 11 control children between the ages of three and 14 (males=17; females=5) underwent overnight sleep studies followed by a first-morning urine sample. Proteomic analysis of urine samples yielded a unique map of proteins, of which, five spots were selected for further analysis due to their significant intensity differences between OSA and control groups. RESULTS: Mass spectrometry followed by peptide mass fingerprinting conclusively identified four of the five spots as gelsolin, perlecan (a heparan sulfate proteoglycan), albumin, and immunoglobulin (P<0.05 and protein scores>67). CONCLUSIONS: Overall, increased expression of gelsolin and perlecan in the urinary proteome of children with OSA suggests that the episodic hypoxia associated with OSA may lead to changes in protein permeability or alternatively to increased catabolism of these proteins and their excretion in urine.

Body Mass Index↗

PubMed2006

Redox proteomics identification of oxidatively modified hippocampal proteins in mild cognitive impairment: insights into the development of Alzheimer's disease.

Mild cognitive impairment (MCI) is generally referred to the transitional zone between normal cognitive function and early dementia or clinically probable Alzheimer's disease (AD). Oxidative stress plays a significant role in AD and is increased in the superior/middle temporal gyri of MCI subjects. Because AD involves hippocampal-resident memory dysfunction, we determined protein oxidation and identified the oxidized proteins in the hippocampi of MCI subjects. We found that protein oxidation is significantly increased in the hippocampi of MCI subjects when compared to age- and sex-matched controls. By using redox proteomics, we determined the oxidatively modified proteins in MCI hippocampus to be alpha-enolase (ENO1), glutamine synthetase (GLUL), pyruvate kinase M2 (PKM2) and peptidyl-prolyl cis/trans isomerase 1 (PIN1). The interacteome of these proteins revealed that these proteins functionally interact with SRC, hypoxia-inducible factor 1, plasminogen (PLG), MYC, tissue plasminogen activator (PLAT) and BCL2L1. Moreover, the interacteome indicates the functional involvement of energy metabolism, synaptic plasticity and mitogenesis/proliferation. Therefore, oxidative inactivation of ENO1, GLUL and PIN1 may alter these cellular processes and lead to the development of AD from MCI. We conclude that protein oxidation plays a significant role in the development of AD from MCI and that the oxidative inactivation of ENO1, GLUL, PKM2 and PIN1 is involved in the progression of AD from MCI. The current study provides a framework for future studies on the development of AD from MCI relevant to oxidative stress.

Aged↗

PubMed2006

A proteomic screen identified stress-induced chaperone proteins as targets of Akt phosphorylation in mesangial cells.

The serine-threonine kinase Akt regulates mesangial cell apoptosis, proliferation, and hypertrophy. To define Akt signaling pathways in mesangial cells, we performed a functional proteomic screen for rat mesangial cell proteins phosphorylated by Akt. A group of chaperone proteins, heat shock protein (Hsp) 70, Hsp90alpha, Hsp90beta, Glucose-regulated protein (Grp) Grp78, Grp94, and protein disulfide isomerase (PDI) were identified as potential Akt substrates by two techniques: (a) in vitro phosphorylation of mesangial cell lysate by recombinant active Akt followed by protein separation by SDS-PAGE or 2-DE and phosphoprotein identification by peptide mass fingerprinting using MALDI-MS, or (b) immunoblot analysis of proteins from PDGF-stimulated mesangial cells using an anti-Akt phospho-motif antibody. In vitro kinase reactions using recombinant proteins confirmed that Akt phosphorylates Hsp70, Hsp90alpha and beta, Grp94, and PDI. Immunoprecipitation of Akt from mesangial cell lysate coprecipitated Grp78 and Hsp70. PDGF stimulation of mesangial cells caused an acidic shift in the isoelectric point of Hsp70, Hsp90, and PDI that was dependent on PI-3K activity for Hsp70 and Hsp90. The data suggest that Akt-mediated phosphorylation of stress-induced chaperones represents a mechanism for regulation of chaperone function during mesangial cell responses to physiologic and pathologic stimuli.

Animals↗

PubMed2006

AGRO100 inhibits activation of nuclear factor-kappaB (NF-kappaB) by forming a complex with NF-kappaB essential modulator (NEMO) and nucleolin.

AGRO100, also known as AS1411, is an experimental anticancer drug that recently entered human clinical trials. It is a member of a novel class of antiproliferative agents known as G-rich oligonucleotides (GRO), which are non-antisense, guanosine-rich phosphodiester oligodeoxynucleotides that form stable G-quadruplex structures. The biological activity of GROs results from their binding to specific cellular proteins as aptamers. One important target protein of GROs has been previously identified as nucleolin, a multifunctional protein expressed at high levels by cancer cells. Here, we report that AGRO100 also associates with nuclear factor-kappaB (NF-kappaB) essential modulator (NEMO), which is a regulatory subunit of the inhibitor of kappaB (IkappaB) kinase (IKK) complex, and also called IKKgamma. In the classic NF-kappaB pathway, the IKK complex is required for phosphorylation of IkappaBalpha and subsequent activation of the transcription factor NF-kappaB. We found that treatment of cancer cells with AGRO100 inhibits IKK activity and reduces phosphorylation of IkappaBalpha in response to tumor necrosis factor-alpha stimulation. Using a reporter gene assay, we showed that AGRO100 blocks both tumor necrosis factor-alpha-induced and constitutive NF-kappaB activity in human cancer cell lines derived from cervical, prostate, breast, and lung carcinomas. In addition, we showed that, in AGRO100-treated cancer cells, NEMO is coprecipitated by nucleolin, indicating that both proteins are present in the same complex. Our studies suggest that abrogation of NF-kappaB activity may contribute to the anticancer effects of AGRO100 and that nucleolin may play a previously unknown role in regulating the NF-kappaB pathway.

Antineoplastic Agents↗

PubMed2006

Proteomic analysis of human neutrophils.

Proteomics is the study of the set of proteins, or proteome, expressed by a cell under specific conditions. Proteomics methodology consists of protein extraction, protein separation, and protein identification. Currently, two-dimensional gel electrophoresis (2DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry are the most widespread methods for proteomic studies. The recent introduction of precast immobilized pH gradient gel strips, precast gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and well-designed electrophoresis equipment has made 2DE a highly reproducible and relatively simple method for protein separation. Inherent limitations of the procedure, however, require approaches in sample preparation that may be cell- or tissue-dependent. This chapter describes a methodology for proteomic analysis of human neutrophils and discusses its applications.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Redox proteomics identification of oxidatively modified brain proteins in inherited Alzheimer's disease: an initial assessment.

OBJECTIVE: To identify oxidatively modified proteins in brains of persons with inherited Alzheimer's disease. METHODS: Redox proteomics was used to identify oxidatively modified brain proteins in persons with mutations in the genes for presenilin-1 (PS-1). RESULTS: An initial redox proteomics assessment of oxidatively modified proteins from brains of individuals with PS-1 mutations was performed. These PS1 mutations, Q222H and M233T, are completely penetrant causing early-onset familial AD as previously reported in these Australian families. We show that oxidative modifications of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), gamma-enolase, actin, and dimethylarginine dimethylaminohydrolase 1 (DMDMAH-1) are present in the brain of familial AD subjects. CONCLUSIONS: These initial results suggest that oxidatively modified proteins are important common features in both familial and sporadic AD.

Actins↗

PubMed2005

Identification of nitrated proteins in Alzheimer's disease brain using a redox proteomics approach.

Nitric oxide (NO) has been implicated in the pathophysiology of a number of neurodegenerative diseases including Alzheimer's disease (AD). In the present study, using a proteomics approach, we identified enolase, glyceraldehyde-3-phosphate dehydrogenase, ATP synthase alpha chain, carbonic anhydrase-II, and voltage-dependent anion channel-protein as the targets of nitration in AD hippocampus, a region that shows a extensive deposition of amyloid beta-peptide, compared with the age-matched control brains. Immunoprecipitation and Western blotting techniques were used to validate the correct identification of these proteins. Our results are discussed in context of the role of oxidative stress as one of the important mechanisms of neurodegeneration in AD.

ATP Synthetase Complexes↗

PubMed2005

Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD.

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles, senile plaques and loss of synapses. There is accumulating evidence that oxidative stress plays an important role in AD pathophysiology. Previous redox proteomics studies from our laboratory on AD inferior parietal lobule led to the identification of oxidatively modified proteins that were consistent with biochemical or pathological alterations in AD. The present study was focused on the identification of specific targets of protein oxidation in AD and control hippocampus and cerebellum using a redox proteomics approach. In AD hippocampus, peptidyl prolyl cis-trans isomerase, phosphoglycerate mutase 1, ubiquitin carboxyl terminal hydrolase 1, dihydropyrimidinase related protein-2 (DRP-2), carbonic anhydrase II, triose phosphate isomerase, alpha-enolase, and gamma-SNAP were identified as significantly oxidized protein with reduced enzyme activities relative to control hippocampus. In addition, no significant excessively oxidized protein spots were identified in cerebellum compared to control, consistent with the lack of pathology in this brain region in AD. The identification of oxidatively modified proteins in AD hippocampus was verified by immunochemical means. The identification of common oxidized proteins in different brain regions of AD brain suggests a potential role for these oxidized proteins and thereby oxidative stress in the pathogenesis of Alzheimer's disease.

Aged↗

PubMed2005

Effects of ACE inhibition on proximal tubule sodium transport.

Angiotensin-converting enzyme (ACE) inhibitors such as captopril, which block ANG II formation, are commonly used for treatment of hypertension. There is substantial evidence that the proximal tubule (PT) is a primary target site for captopril but the molecular mechanisms for its action in PT are not well defined. The aim of this study was to determine the physiological and molecular changes in PT provoked by acute captopril treatment in the absence of changes in blood pressure or glomerular filtration rate (GFR). Captopril (infused at 12 microg/min for 20 min) did not change blood pressure or GFR but induced an immediate (<10 min) increase in PT flow measured with a nonobstructive optical method (to 117 +/- 14% of baseline) along with a rapid diuresis from 2.1 +/- 0.6 mg/min (baseline) to 3.7 +/- 0.9 mg/min (captopril). Captopril also provoked a significant retraction of PT Na(+)/H(+) exchanger isoform 3 (NHE3), NHE regulatory factor (NHERF)-1, myosin-VI, and Na(+)-P(i) cotransporter type 2 (NaPi2), but not ACE, out of apical microvillus-enriched membranes. Proteomic analysis with MALDI-TOF MS revealed an additional eight abundant membrane-associated proteins that redistributed out of the microvillus-enriched membrane during captopril treatment: megalin, myosin II-A, clathrin, aminopeptidase N, DPPIV, ezrin, moesin, and vacuolar H(+)-ATPase subunit beta(2). In summary, captopril can rapidly depress PT reabsorption in the absence of a change in GFR or BP and provokes the redistribution of a set of transporters and transporter-associated proteins that likely participate in the decrease in PT reabsorption and may also contribute to the blood pressure-lowering effect of ACE inhibitors.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2005

Modulation of cytochrome C oxidase-va is possibly involved in metallothionein protection from doxorubicin cardiotoxicity.

Previous studies using a cardiac-specific metallothionein (MT)-overexpressing transgenic (MT-TG) mouse model have demonstrated that MT protects from doxorubicin (DOX)-induced oxidative heart injury. The molecular mechanisms that underlie this cardioprotection, however, have yet to be defined. In the present study, we tested the hypothesis that MT overexpression activates cytoprotective mechanisms, leading to cardiac protection from DOX toxicity. MT-TG mice and nontransgenic wild-type (WT) controls were treated i.p. with DOX at a single dose of 20 mg/kg and sacrificed on the third day after the treatment. An expression proteomic analysis involving two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to identify MT-induced changes in cytoprotection-related proteins. We identified 18 proteins that were modified by DOX treatment in the heart. These proteins included those involved in cellular antioxidant defense, enzymes of the mitochondrial electron transport chain, enzymes involved in beta-oxidation of fatty acids and glycolysis, and proteins involved in regulation of cardiac muscle contraction. However, the most dominant modification by MT is the cytochrome c oxidase subunit Va (CCO-Va). In response to DOX treatment, a specific isoform of CCO-Va was enhanced in the MT-TG but not in the WT mouse hearts. Because CCO-Va is a critical component in the mitochondrial electron transport chain, the results suggest that the cardioprotective effect of MT may be related to an increased expression or a differential modification of CCO-Va.

Amino Acid Sequence↗

PubMed2005

Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Abeta(1-42): implications for Alzheimer's disease.

Protein oxidation has been shown to lead to loss of protein function, increased protein aggregation, decreased protein turnover, decreased membrane fluidity, altered cellular redox poteintial, loss of Ca2+ homeostaisis, and cell death. There is increasing evidence that protein oxidation is involved in the pathogenesis of Alzheimer's disease and amyloid beta-peptide (1-42) has been implicated as a mediator of oxidative stress in AD. However, the specific implications of the oxidation induced by Abeta(1-42) on the neurodegeneration evident in AD are unknown. In this study, we used proteomic techniques to identify specific targets of oxidation in transgenic Caenorhabditis elegans (C. elegans) expressing human Abeta(1-42). We identified 16 oxidized proteins involved in energy metabolism, proteasome function, and scavenging of oxidants that are more oxidized compared to control lines. These results are discussed with reference to Alzheimer's disease.

Amyloid beta-Peptides↗

PubMed2005

Proteomic identification of less oxidized brain proteins in aged senescence-accelerated mice following administration of antisense oligonucleotide directed at the Abeta region of amyloid precursor protein.

Amyloid beta-peptide (Abeta) is the major constituent of senile plaques, a pathological hallmark of Alzheimer's disease (AD) brain. It is generally accepted that Abeta plays a central role in the pathophysiology of AD. Abeta is released from cells under entirely normal cellular conditions during the internalization and endosomal processing of amyloid precursor protein (APP). However, accumulation of Abeta can induce neurotoxicity. Our previous reports showed that decreasing the production of Abeta by giving an intracerebroventricular injection of a 42-mer phosphorothiolated antisense oligonucleotide (AO) directed at the Abeta region of the APP gene reduces lipid peroxidation and protein oxidation and improves cognitive deficits in aged senescence-accelerated mice prone 8 (SAMP8) mice. In order to investigate how Abeta level reduction improves learning and memory performance of SAMP8 mice through reduction of oxidative stress in brains, we used proteomics to identify the proteins that are less oxidized in 12-month-old SAMP8 mice brains treated with AO against the Abeta region of APP (12 mA) compared to that of the age-control SAMP8 mice. We found that the specific protein carbonyl levels of aldoase 3 (Aldo3), coronin 1a (Coro1a) and peroxiredoxin 2 (Prdx2) are significantly decreased in the brains of 12 mA SAMP8 mice compared to the age-controlled SAMP8 treated with random AO (12 mR). We also found that the expression level of alpha-ATP synthase (Atp5a1) was significantly decreased, whereas the expression of profilin 2 (Pro-2) was significantly increased in brains from 12 mA SAMP8 mice. Our results suggest that decreasing Abeta levels in aged brain in aged accelerated mice may contribute to the mechanism of restoring the learning and memory improvement in aged SAMP8 mice and may provide insight into the role of Abeta in the memory and cognitive deficits in AD.

Acetaldehyde↗

PubMed2005

Akt-mediated valosin-containing protein 97 phosphorylation regulates its association with ubiquitinated proteins.

Hypoxia is a common environmental stress that influences signaling pathways and cell function. Previous studies from our laboratory have identified significant differences in cellular responses to sustained or intermittent hypoxia with the latter proving more cytotoxic. We hypothesized that differences in susceptibility of neurons to intermittent (IH) and sustained hypoxia (SH) are mediated by altered Akt signaling. SH, but not IH, induced a significant increase in Akt activation in rat CA1 hippocampal region extracts compared with room air controls. Akt immunoprecipitations followed by proteomic analysis identified valosin-containing protein (VCP) as an Akt-binding protein. In addition, VCP expression and association with Akt was enhanced during SH, and this association was decreased upon phosphoinositide 3-kinase/Akt pathway blockade with LY294002. Active recombinant Akt phosphorylated recombinant VCP in vitro. Site-directed mutagenesis studies identified Ser352, Ser746, and Ser748 as Akt phosphorylation sites on VCP. In addition, rat CA1 hippocampal tissue exposed to SH exhibited an acidic pI shift of VCP. Protein phosphatase 2A treatment inhibited this acidic shift consistent with SH-induced phosphorylation of VCP in vivo. PC-12 cells transfected with active Akt, but not dominant negative Akt or vector, induced VCP expression and an acidic shift in VCP pI, which was inhibited by protein phosphatase 2A treatment. Furthermore, VCP association with ubiquitinated proteins was demonstrated in vector-transfected PC-12 cell lysates, whereas active Akt-transfected cells demonstrated a marked decrease in association of VCP with ubiquitinated proteins. We concluded that Akt phosphorylates VCP in vitro and in vivo, and VCP phosphorylation releases it from ubiquitinated substrate protein(s) possibly allowing ubiquitinated protein(s) to be degraded by the proteosome.

Adenosine Triphosphatases↗

PubMed2005

Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis.

Age-related impairment of functionality of the central nervous system (CNS) is associated with increased susceptibility to develop many neurodegenerative diseases. Increased oxidative stress in the CNS of aged animals is manifested by increased protein oxidation, which is believed to contribute to the age-related learning and memory deficits. Glutamate dysregulation, mitochondrial dysfunction and impaired protein synthesis are observed in aged brains, along with increased protein oxidation. Interestingly, all of these age-related cellular alterations can be improved by caloric restriction (CR), which can also improve the plasticity and recovery of the CNS. Although the beneficial effects of CR on brains are well established, the mechanism(s) of its action remains unclear. In order to gain insight into the mechanism of CR in the brain, we located the brain regions that are benefited the most from reduced oxidative stress by CR. Along with other brain regions, striatum (ST) showed significantly decreased bulk protein carbonyl levels and hippocampus (HP) showed decreased bulk protein 3-nitrotyrosine (3-NT) levels in CR aged rats when compared to those of age matched controls. To determine which proteins were oxidatively modified in these brain regions, we used parallel proteomics approach to identify the proteins that are altered in oxidation and expression. The specific carbonyl levels of pyruvate kinase M2 (PKM2), alpha-enolase (ENO1), inositol monophosphatase (INSP1), and F1-ATPase Chain B (ATP-F1B) were significantly decreased in ST of aged CR rats. In contrast, the expression levels of phosphoglycerate kinase 1 (PKG1), inosine monophosphate cyclohydrolase (IMPCH) and F1-ATPase Chain A (ATP-F1A) were significantly increased in the ST of CR rats. In the hippocampus of CR rats, the specific 3-NT levels of malate dehydrogenase (MDH), phosphoglycerate kinase 1 (PKG1) and 14-3-3 zeta protein were significantly decreased and expression levels of DLP1 splice variant 1 (DLP1), mitochondrial aconitase (ACO2), dihydrolipoamide dehydrogenase (DLDH), neuroprotective peptide H3 (NPH3), and eukaryotic translation initiation factor 5A (eIF-5A) are increased. Moreover, an unnamed protein product (UNP1) with similar sequence to initiation factor 2 (IF-2) was decreased in the HP of CR rats. Our data support the hypothesis that CR induces a mild metabolic stress response by increasing the production of neurotrophic proteins, therefore, priming neurons against apoptosis. Moreover, our study shows that the improvement of glutamate dysregulation, mitochondrial dysfunction and protein synthesis by CR is, at least partially, due to the CR-mediated alteration of the oxidation or the expression of PKM2, ENO1, INSP1, ATP-F1B, PKG1, IMPCH, ATP-F1A MDH, PKG1 and 14-3-3 zeta protein, DLP1, ACO2, DLDH, NPH3, eIF-5A and UNP1. This study provides valuable insights into the mechanisms of the beneficial factors on brain aging by CR.

Aging↗