Protein acetylation in prokaryotes increases stress resistance

Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes.     

Protein intake and energy balance

Maintaining energy balance in the context of body-weight regulation requires a multifactorial approach. Recent findings suggest that an elevated protein intake plays a key role herein, through (i) increased satiety related to increased diet-induced thermogenesis, (ii) its effect on thermogenesis, (iii) body composition, and (iv) decreased energy-efficiency, all of which are related to protein metabolism. Supported by these mechanisms, relatively larger weight loss and subsquent stronger body-weight maintenance have been observed. Elevated thermogenesis and GLP-1 appear to play a role in high protein induced satiety. Moreover, a negative fat-balance and positive protein-balance is shown in the short-term, whereby fat-oxidation is increased. Furthermore, a high protein diet shows a reduced energy efficiency related to the body-composition of the body-weight regained, i.e. favor of fat free mass. Since protein intake is studied under various energy balances, absolute and relative protein intake needs to be discriminated. In absolute grams, a normal protein diet becomes a relatively high protein diet in negative energy balance and at weight maintenance. Therefore 'high protein negative energy balance diets' aim to keep the grams of proteins ingested at the same level as consumed at energy balance, despite lower energy intakes.     

Methoxychlor‐induced alteration in the levels of HSP70 and clusterin is accompanied with oxidative stress in adult rat testis

Methoxychlor, an organochlorine pesticide, has been reported to induce abnormalities in male reproductive tract. However, the insight into the mechanisms of gonadal toxicity induced by methoxychlor is not well known. We investigated whether treatment with methoxychlor would alter the levels of stress proteins, heat shock proteins (HSP), and clusterin (CLU), and oxidative stress‐related parameters in the testis of adult male rats. Animals were exposed to a single dose of methoxychlor (50 mg/kg body weight) orally and were terminated at various time points (0, 3, 6, 12, 24, and 72 h) using anesthetic ether. The levels of HSP70, CLU, and the activities of superoxide dismutase (SOD), catalase, and lipid peroxidation levels were evaluated in a 10% testis homogenate. A sequential reduction in the activities of catalase and SOD with concomitant increase in the levels of thiobarbituric acid reactive substance (TBARS) was observed. These changes elicited by methoxychlor were very significant between 6–12 h of posttreatment. Immunoblot analysis of HSP revealed the expression of HSP72, an inducible form of HSP, at certain time points (3–24 h) following exposure to methoxychlor. Similarly, the levels of secretory CLU (sCLU) were also found to be elevated between 3–24 h of treatment. The present data demonstrate methoxychlor‐elicited increase in the levels of inducible HSP72 and sCLU, which could be a part of protective mechanism mounted to reduce cellular oxidative damage. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:29–35, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20262     

Parkinsonism-associated Protein DJ-1/Park7 Is a Major Protein Deglycase That Repairs Methylglyoxal- and Glyoxal-glycated Cysteine,Arginine, and Lysine Residues

Glycation is an inevitable nonenzymatic covalent reaction between proteins and endogenous reducing sugars or dicarbonyls (methylglyoxal, glyoxal) that results in protein inactivation. DJ-1 was reported to be a multifunctional oxidative stress response protein with poorly defined function. Here, we show that human DJ-1 is a protein deglycase that repairs methylglyoxal- and glyoxal-glycated amino acids and proteins by acting on early glycation intermediates and releases repaired proteins and lactate or glycolate, respectively. DJ-1 deglycates cysteines, arginines, and lysines (the three major glycated amino acids) of serum albumin, glyceraldehyde-3-phosphate dehydrogenase, aldolase, and aspartate aminotransferase and thus reactivates these proteins. DJ-1 prevented protein glycation in an Escherichia coli mutant deficient in the DJ-1 homolog YajL and restored cell viability in glucose-containing media. These results suggest that DJ-1-associated Parkinsonism results from excessive protein glycation and establishes DJ-1 as a major anti-glycation and anti-aging protein.     

Protein Phosphatases and Signaling Cascades in Higher Plants

Protein kinases and phosphatases play a central role in cellular signaling. Although protein kinases have been widely studied in higher plants, protein phosphatases have been largely neglected until recently. The article focuses on the most recent studies that have placed protein phosphatases in the context of several signal cascades in higher plants. These pathways include stomatal regulation and abscisic acid signal transduction, pathogen and stress responses, and developmental control. Studies clearly have demonstrated that protein phosphatases function not only to counterbalance the protein kinases but also take a leading role in many signaling processes.     

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

cAMP-regulated Protein Lysine Acetylases in Mycobacteria

Cyclic AMP synthesized by Mycobacterium tuberculosis has been shown to play a role in pathogenesis. However, the high levels of intracellular cAMP found in both pathogenic and non-pathogenic mycobacteria suggest that additional and important biological processes are regulated by cAMP in these organisms. We describe here the biochemical characterization of novel cAMP-binding proteins in M. smegmatis and M. tuberculosis (MSMEG_5458 and Rv0998, respectively) that contain a cyclic nucleotide binding domain fused to a domain that shows similarity to the GNAT family of acetyltransferases. We detect protein lysine acetylation in mycobacteria and identify a universal stress protein (USP) as a substrate of MSMEG_5458. Acetylation of a lysine residue in USP is regulated by cAMP, and using a strain deleted for MSMEG_5458, we show that USP is indeed an in vivo substrate for MSMEG_5458. The Rv0998 protein shows a strict cAMP-dependent acetylation of USP, despite a lower affinity for cAMP than MSMEG_5458. Thus, this report not only represents the first demonstration of protein lysine acetylation in mycobacteria but also describes a unique functional interplay between a cyclic nucleotide binding domain and a protein acetyltransferase.     

Unfolded Protein Response Signaling and Metabolic Diseases

The endoplasmic reticulum (ER) is a central organelle for protein biosynthesis, folding, and traffic. Perturbations in ER homeostasis create a condition termed ER stress and lead to activation of the complex signaling cascade called the unfolded protein response (UPR). Recent studies have documented that the UPR coordinates multiple signaling pathways and controls various physiologies in cells and the whole organism. Furthermore, unresolved ER stress has been implicated in a variety of metabolic disorders, such as obesity and type 2 diabetes. Therefore, intervening in ER stress and modulating signaling components of the UPR would provide promising therapeutics for the treatment of human metabolic diseases.     

Co-ruminating increases stress hormone levels in women

Same-sex friendships are an important source of social support and typically contribute to positive adjustment. However, there can be adjustment trade-offs if the friends co-ruminate (i.e., talk excessively about problems) in that co-rumination is related to having close friendships but also to increased internalizing symptoms. The current study utilized an experimental manipulation that elicited co-rumination in young women and thus mirrored an everyday response to stress. Observed co-rumination was associated with a significant increase in the stress hormone, cortisol (after controlling for self-reported co-rumination and for cortisol levels assessed before the discussion of problems). These findings suggest that co-rumination can amplify, rather than mitigate, the hormonal stress response to personal life stressors.     

Glial fibrillary acidic protein is greatly modified by oxidative stress in aceruloplasminemia brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Proteasomes Associated with the Blm10 Activator Protein Antagonize Mitochondrial Fission through Degradation of the Fission Protein Dnm1

The conserved Blm10/PA200 activators bind to the proteasome core particle gate and facilitate turnover of peptides and unfolded proteins in vitro. We report here that Blm10 is required for the maintenance of functional mitochondria. BLM10 expression is induced 25-fold upon a switch from fermentation to oxidative metabolism. In the absence of BLM10, Saccharomyces cerevisiae cells exhibit a temperature-sensitive growth defect under oxidative growth conditions and produce colonies with dysfunctional mitochondria at high frequency. Loss of BLM10 leads to reduced respiratory capacity, increased mitochondrial oxidative damage, and reduced viability in the presence of oxidative stress or death stimuli. In the absence of BLM10, increased fragmentation of the mitochondrial network under oxidative stress is observed indicative of elevated activity of the mitochondrial fission machinery. The degradation of Dnm1, the main factor mediating mitochondrial fission, is impaired in the absence of BLM10 in vitro and in vivo. These data suggest that the mitochondrial functional and morphological changes observed are related to elevated Dnm1 levels. This hypothesis is supported by the finding that cells that constitutively overexpress DNM1 display the same mitochondrial defects as blm10Δ cells. The data are consistent with a model in which Blm10 proteasome-mediated turnover of Dnm1 is required for the maintenance of mitochondrial function and provides cytoprotection under conditions that induce increased mitochondrial damage and programmed cell death.     

Effects of Polyhydroxyl Alcohols on Protein Synthesis in Brain Slices

Stressors such as tissue slicing, toxic chemicals, and heat shock applied to cultured cells, organ tissues, or whole animals in vivo induce the synthesis of a 71,000-kilodalton stress protein (SP71) that is not normally present in most organ tissues. In the present experiment, an attempt was made to inhibit selectively the synthesis of SP71 in rat brain tissue slices. Of several manipulations to the brain slice incubation medium that were examined, only addition of very high concentrations of certain polyhydroxyl alcohols, i.e., 1.0 M glycerol, selectively inhibited SP71 synthesis. Glycerol also selectively inhibited SP71 synthesis in heat-shocked cerebral microvascular cells in culture but failed to inhibit SP71 synthesis in anesthetized rats in vivo in response to heat shock. The effects of glycerol on SP71 synthesis are discussed in relationship to current hypotheses concerning the function of SP71.     

Diurnal urinary 6-sulfatoxymelatonin levels among healthy Danish nurses during work and leisure time

The present study aims to examine the influence of evening and night shift work, compared to day shift work, on melatonin secretion in nurses in a field setting. Effects were examined during a workday and during a day off. Both fixed schedules and mixed or rotating schedules were studied. In total, 170 nurses were studied: 89 nurses worked fixed schedules, 27 nurses worked the day shift, 12 nurses worked the evening shift, 50 nurses worked the night shift, and 82 nurses worked mixed schedules, with data collected during a day (n=17), evening (n=14), or night shift (n=50). All spot urine samples were collected during 24 h from the participants on a work day and on a day off and were analyzed for 6-sulphatoxymelatonin. On the day of urine sampling, participants filled in the Karolinska Sleep Diary. Additional information was collected through a telephone interview. Data were analyzed using a mixed procedure with autoregressive covariance structure. The present study showed that shift work affected the concentrations of 6-sulphatoxymelatonin in the short term by lower excretion in urine from nurses working the night compared to day shift on a workday and on a day off as well. No significant differences were observed between a workday and a day off when doing day and evening shifts, irrespective of mixed and fixed schedules. Sleep length was reduced workdays (from 6.1-6.8 h) among all nurses, compared to days off (from 7.8-8.7 h).     

Dual Sites of Protein Initiation Control the Localization and Myristoylation of Methionine Sulfoxide Reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system, which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. In mammals, one gene encodes two forms of the reductase, one targeted to the cytosol and the other to mitochondria. The cytosolic form displays faster mobility than the mitochondrial form, suggesting a lower molecular weight for the former. The apparent size difference and targeting to two cellular compartments had been proposed to result from differential splicing of mRNA. We now show that differential targeting is effected by use of two initiation sites, one of which includes a mitochondrial targeting sequence, whereas the other does not. We also demonstrate that the mass of the cytosolic form is not less than that of the mitochondrial form; the faster mobility of cytosolic form is due to its myristoylation. Lipidation of methionine sulfoxide reductase A occurs in the mouse, in transfected tissue culture cells, and even in a cell-free protein synthesis system. The physiologic role of myristoylation of MsrA remains to be elucidated.     

血浆蛋白C、蛋白S抗凝体系的研究进展

蛋白C是一种维生素K依赖糖蛋白,它为血浆丝氨酸蛋白酶酶原;凝血酶能将蛋白C转化为活化蛋白C（APC）,在一种叫蛋白S（PS）的APC协同因子存在时,APC发挥抗凝活性,蛋白C和蛋白S缺陷或缺乏可引起动、静脉血栓形成。     

Altered Patterns of Protein Phosphorylation and Synthesis Caused by Methyl Mercury in Cerebellar Granule Cell Culture

In the preceding report we demonstrated a dose-dependent increase in 32P-phosphoprotein labeling after 24-h exposure of cultured cerebellar granule neurons to methyl mercury (MeHg), a response that was not observed in glial cultures. In the present study we have examined 32P-labeled phosphoproteins by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. At concentrations of 0.5 and 1 microM, which were not extensively cytotoxic, MeHg enhanced phosphorylation of numerous acidic proteins, particularly a cluster of proteins with Mr approximately 28,000 and pI approximately 5.7-5.9 (pp 28/5.7-5.9) and a protein with Mr approximately 58,000 and pI approximately 5.6. The pp28 cluster displayed considerable two-dimensional pattern variability from one experiment to the next, suggesting susceptibility to subtle structural modifications. Time course studies revealed that increased 32P phospholabeling of pp28/5.7-5.9 was detectable after 12-h exposure to 3 microM MeHg and reached values of 300-500% of control by 24 h. These studies also showed that among the 21 proteins analyzed by two-dimensional densitometry, 32P phospholabeling of four proteins increased by 20-50% and of two proteins decreased by 20-50% after 24-h treatment. However, exposure to 10 microM MeHg produced stimulation of pp28/5.7-5.9 32P phospholabeling within 2 h. Under these conditions a relatively high stimulation (sevenfold) of pp28/5.7 phospholabeling occurred, while pp28/5.9 32P phospholabeling was only moderately (5-20%) enhanced. 35S and 32P double-label analysis of cells treated with 0, 0.5, and 1 microM MeHg indicated specific stimulation of 32P phospholabeling of these proteins without increased polypeptide synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleolar Stress Induces Ubiquitination-independent Proteasomal Degradation of PICT1 Protein

The nucleolar protein PICT1 regulates tumor suppressor p53 by tethering ribosomal protein L11 within the nucleolus to repress the binding of L11 to the E3 ligase MDM2. PICT1 depletion results in the release of L11 to the nucleoplasm to inhibit MDM2, leading to p53 activation. Here, we demonstrate that nucleolar stress induces proteasome-mediated degradation of PICT1 in a ubiquitin-independent manner. Treatment of H1299 cells with nucleolar stress inducers, such as actinomycin D, 5-fluorouridine, or doxorubicin, induced the degradation of PICT1 protein. The proteasome inhibitors MG132, lactacystin, and epoxomicin blocked PICT1 degradation, whereas the inhibition of E1 ubiquitin-activating enzyme by a specific inhibitor and genetic inactivation fail to repress PICT1 degradation. In addition, the 20 S proteasome was able to degrade purified PICT1 protein in vitro. We also found a PICT1 mutant showing nucleoplasmic localization did not undergo nucleolar stress-induced degradation, although the same mutant underwent in vitro degradation by the 20 S proteasome, suggesting that nucleolar localization is indispensable for the stress-induced PICT1 degradation. These results suggest that PICT1 employs atypical proteasome-mediated degradation machinery to sense nucleolar stress within the nucleolus.