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Upregulation of cystathione beta-synthase and p70S6K/S6 in neonatal hypoxic ischemic brain injury
Lechpammer, Mirna; Tran, Yen P; Wintermark, Pia; Martinez-Cerdeno, Veronica; Krishnan, Viswanathan V; Ahmed, Waseem; Berman, Robert F; Jensen, Frances E; Nudler, Evgeny; Zagzag, David
Encephalopathy of prematurity (EOP) is a complex form of cerebral injury that occurs in the setting of hypoxia-ischemia (HI) in premature infants. Using a rat model of EOP, we investigated whether neonatal HI of the brain may alter the expression of cystathionine beta-synthase (CBS) and the components of the mammalian target of rapamycin (mTOR) signaling. We performed unilateral carotid ligation and induced HI (UCL/HI) in Long-Evans rats at P6 and found increased CBS expression in white matter (i.e., corpus callosum, cingulum bundle and external capsule) as early as 24 hours (P7) post-procedure. CBS remained elevated through P21, and, to a lesser extent, at P40. The mTOR downstream target 70 kDa ribosomal protein S6 kinase (p70S6K and phospho-p70S6K) and 40S ribosomal protein S6 (S6 and phospho-S6) were also overexpressed at the same time points in the UCL/HI rats compared to healthy controls. Overexpression of mTOR components was not observed in rats treated with the mTOR inhibitor everolimus. Behavioral assays performed on young rats (postnatal day 35-37) following UCL/HI at P6 indicated impaired preference for social novelty, a behavior relevant to autism spectrum disorder, and hyperactivity. Everolimus restored behavioral patterns to those observed in healthy controls. A gait analysis has shown that motor deficits in the hind paws of UCL/HI rats were also significantly reduced by everolimus. Our results suggest that neonatal HI brain injury may inflict long-term damage by upregulation of CBS and mTOR signaling. We propose this cascade as a possible new molecular target for E
PMID: 27465493
ISSN: 1750-3639
CID: 2191582
Glycogen controls Caenorhabditis elegans lifespan and resistance to oxidative stress
Gusarov, Ivan; Pani, Bibhusita; Gautier, Laurent; Smolentseva, Olga; Eremina, Svetlana; Shamovsky, Ilya; Katkova-Zhukotskaya, Olga; Mironov, Alexander; Nudler, Evgeny
A high-sugar diet has been associated with reduced lifespan in organisms ranging from worms to mammals. However, the mechanisms underlying the harmful effects of glucose are poorly understood. Here we establish a causative relationship between endogenous glucose storage in the form of glycogen, resistance to oxidative stress and organismal aging in Caenorhabditis elegans. We find that glycogen accumulated on high dietary glucose limits C. elegans longevity. Glucose released from glycogen and used for NADPH/glutathione reduction renders nematodes and human hepatocytes more resistant against oxidative stress. Exposure to low levels of oxidants or genetic inhibition of glycogen synthase depletes glycogen stores and extends the lifespan of animals fed a high glucose diet in an AMPK-dependent manner. Moreover, glycogen interferes with low insulin signalling and accelerates aging of long-lived daf-2 worms fed a high glucose diet. Considering its extensive evolutionary conservation, our results suggest that glycogen metabolism might also have a role in mammalian aging.
PMCID:5481799
PMID: 28627510
ISSN: 2041-1723
CID: 2603802
Mechanism of H2S-mediated protection against oxidative stress in Escherichia coli
Mironov, Alexander; Seregina, Tatyana; Nagornykh, Maxim; Luhachack, Lyly G; Korolkova, Natalya; Lopes, Liubov Errais; Kotova, Vera; Zavilgelsky, Gennady; Shakulov, Rustem; Shatalin, Konstantin; Nudler, Evgeny
Endogenous hydrogen sulfide (H2S) renders bacteria highly resistant to oxidative stress, but its mechanism remains poorly understood. Here, we report that 3-mercaptopyruvate sulfurtransferase (3MST) is the major source of endogenous H2S in Escherichia coli Cellular resistance to H2O2 strongly depends on the activity of mstA, a gene that encodes 3MST. Deletion of the ferric uptake regulator (Fur) renders mstA cells hypersensitive to H2O2 Conversely, induction of chromosomal mstA from a strong pLtetO-1 promoter (P tet -mstA) renders fur cells fully resistant to H2O2 Furthermore, the endogenous level of H2S is reduced in fur or sodA sodB cells but restored after the addition of an iron chelator dipyridyl. Using a highly sensitive reporter of the global response to DNA damage (SOS) and the TUNEL assay, we show that 3MST-derived H2S protects chromosomal DNA from oxidative damage. We also show that the induction of the CysB regulon in response to oxidative stress depends on 3MST, whereas the CysB-regulated l-cystine transporter, TcyP, plays the principle role in the 3MST-mediated generation of H2S. These findings led us to propose a model to explain the interplay between l-cysteine metabolism, H2S production, and oxidative stress, in which 3MST protects E. coli against oxidative stress via l-cysteine utilization and H2S-mediated sequestration of free iron necessary for the genotoxic Fenton reaction.
PMCID:5468659
PMID: 28533366
ISSN: 1091-6490
CID: 2574702
Alterations of Behavior, Motor Functions and Brain Morphology in Young Rats Following Exposure to Common Organic Solvents in Infancy [Meeting Abstract]
Lechpammer, Mirna; Thongphanh, Krista; Gonzales, Hilary; Chan, Joanne; Martinez-Cerdeno, Veronica; Krishnan, Viswanathan; Nudler, Evgeny; Zagzag, David; Berman, Robert
ISI:000404906900128
ISSN: 1554-6578
CID: 2645112
S-nitrosylation of peroxiredoxin 1 contributes to viability of lung epithelial cells during Bacillus anthracis infection
Chung, Myung-Chul; Alem, Farhang; Hamer, Sarah G; Narayanan, Aarthi; Shatalin, Konstantin; Bailey, Charles; Nudler, Evgeny; Hakami, Ramin M
BACKGROUND: Using Bacillus anthracis as a model gram-positive bacterium, we investigated the effects of host protein S-nitrosylation during bacterial infection. B. anthracis possess a bacterial nitric oxide synthase (bNOS) that is important for its virulence and survival. However, the role of S-nitrosylation of host cell proteins during B. anthracis infection has not been determined. METHODS: Nitrosoproteomic analysis of human small airway epithelial cells (HSAECs) infected with toxigenic B. anthracis Sterne was performed, identifying peroxiredoxin 1 (Prx1) as one predominant target. Peroxidase activity of Prx during infection was measured using 2-Cys-Peroxiredoxin activity assay. Chaperone activity of S-nitrosylated Prx1 was measured by insulin aggregation assay, and analysis of formation of multimeric species using Native PAGE. Griess assay and DAF-2DA fluorescence assay were used to measure NO production. Cell viability was measured using the Alamar Blue assay and the ATPlite assay (Perkin Elmer). RESULTS: S-nitrosylation of Prx1 in Sterne-infected HSAECs leads to a decrease in its peroxidase activity while enhancing its chaperone function. Treatment with bNOS inhibitor, or infection with bNOS deletion strain, reduces S-nitrosylation of Prx1 and decreases host cell survival. Consistent with this, siRNA knockdown of Prx1 lowers bNOS-dependent protection of HSAEC viability. CONCLUSIONS: Anthrax infection results in S-nitrosylation of multiple host proteins, including Prx1. The nitrosylation-dependent decrease in peroxidase activity of Prx1 and increase in its chaperone activity is one factor contributing to enhancing infected cell viability. GENERAL SIGNIFICANCE: These results provide a new venue of mechanistic investigation for inhalational anthrax that could lead to novel and potentially effective countermeasures.
PMID: 27612662
ISSN: 0006-3002
CID: 2238822
A Magic Spot in Genome Maintenance
Rasouly, Aviram; Pani, Bibhusita; Nudler, Evgeny
Nucleotide excision repair (NER) is the key DNA repair system that eliminates the majority of DNA helix-distorting lesions. RNA polymerase (RNAP) expedites the recognition of DNA damage by NER components via transcription-coupled DNA repair (TCR). In bacteria, a modified nucleotide ppGpp ('magic spot') is a pleiotropic second messenger that mediates the response to nutrient deficiencies by altering the initiation properties of RNAP. In this review, we discuss newly elucidated roles of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in transcription elongation that couple this alarmone to DNA damage repair and maintenance.
PMCID:5182163
PMID: 27931778
ISSN: 0168-9525
CID: 2354392
Strategies and Methods of Transcription-Coupled Repair Studies In Vitro and In Vivo
Epshtein, Vitaly; Kamarthapu, Venu; Nudler, Evgeny
Transcription-coupled repair (TCR) serves an important role in preserving genome integrity and maintaining fidelity of replication. Coupling transcription to DNA repair requires a coordinated action of several factors, including transcribing RNA polymerase and various transcription modulators and repair proteins. To study TCR in molecular detail, it is important to employ defined protein complexes in vitro and defined genetic backgrounds in vivo. In this chapter, we present methods to interrogate various aspects of TCR at different stages of repair. We describe promoter-initiated and nucleic acid scaffold-initiated transcription as valid approaches to recapitulate various stages of TCR, and discuss their strengths and weaknesses. We also outline an approach to study TCR in its cellular context using Escherichia coli as a model system.
PMID: 28645373
ISSN: 1557-7988
CID: 2604572
CYSTATHIONE beta-SYNTHASE EXPRESSION IN ASTROCYTOMAS INCREASES WITH HISTOPATHOLOGICAL GRADE [Meeting Abstract]
Lechpammer, Mirna; Thongphanh, Krista; Shahlaie, Kiarash; Azeem, Syed; Bishop, John; Nudler, Evgeny; Zagzag, David
ISI:000398604102188
ISSN: 1523-5866
CID: 2545122
sRNA-Mediated Control of Transcription Termination in E. coli
Sedlyarova, Nadezda; Shamovsky, Ilya; Bharati, Binod K; Epshtein, Vitaly; Chen, Jiandong; Gottesman, Susan; Schroeder, Renee; Nudler, Evgeny
Bacterial small RNAs (sRNAs) have been implicated in various aspects of post-transcriptional gene regulation. Here, we demonstrate that sRNAs also act at the level of transcription termination. We use the rpoS gene, which encodes a general stress sigma factor sigma(S), as a model system, and show that sRNAs DsrA, ArcZ, and RprA bind the rpoS 5'UTR to suppress premature Rho-dependent transcription termination, both in vitro and in vivo. sRNA-mediated antitermination markedly stimulates transcription of rpoS during the transition to the stationary phase of growth, thereby facilitating a rapid adjustment of bacteria to global metabolic changes. Next generation RNA sequencing and bioinformatic analysis indicate that Rho functions as a global "attenuator" of transcription, acting at the 5'UTR of hundreds of bacterial genes, and that its suppression by sRNAs is a widespread mode of bacterial gene regulation.
PMCID:5040353
PMID: 27662085
ISSN: 1097-4172
CID: 2255022
Rates and mechanisms of bacterial mutagenesis from maximum-depth sequencing
Jee, Justin; Rasouly, Aviram; Shamovsky, Ilya; Akivis, Yonatan; R Steinman, Susan; Mishra, Bud; Nudler, Evgeny
In 1943, Luria and Delbruck used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 104-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcription-replication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.
PMCID:4940094
PMID: 27338792
ISSN: 1476-4687
CID: 2159262