Faculty Bibliography

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.

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.

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

Encephalopathy of prematurity (EOP) is a complex form of cerebral injury that occurs in the setting of either primary or secondary hypoxia-ischemia (HI) in the premature infant. In this study we have investigated in the rat model of EOP whether neonatal HI of the brain may in vivo alter the expression of cystathionine b-synthase (CBS) and the components of the mammalian target of rapamycin (mTOR) signaling pathway. We have performed unilateral carotid ligation and HI (UCL/HI) in Long-Evans rats at P6 and found by western blot and immunohistochemical analyses an increased expression of CBS in the white matter as early as 24 hours (P7) post procedure. CBS remained elevated through P21, and to the lesser extent in early adulthood (P40). All tested mTOR downstream targets (p70S6K and phospho-p70S6K & S6 and phospho-S6) were also overexpressed at the same time points in the UCL/HI rats compared to healthy controls. Importantly, this overexpression of mTOR components was not observed in rats treated with the mTOR inhibitor everolimus. Behavioral assays-open field locomotion test and three-chambered social choice test-performed on young adult rats (P35-37) following UCL/HI at P6, indicated hyperactive behavior and impaired preference for social novelty. Everolimus prevented both of these hall-marks of autism and restored behavioral patterns otherwise observed in healthy controls. Gait analysis has shown motor deficits in the hind paws of UCL/HI rats that were also significantly reduced by everolimus, indicating neurological protection/recovery of treated animals. Our results suggest that neonatal HI brain injury may cause its long term sequelae via upregulation of CBS and mTOR signaling pathway, as recently observed by us in patients with EOP, and propose this signaling cascade as a possible new molecular therapeutic target for this still untreatable cause of later life motor and autism-like behavioral deficits

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.

The Heat Shock Response (HSR) is an evolutionarily conserved response to high temperatures and other stresses that controls adaptive proteostasis, and is primarily regulated by the factor, Heat shock transcription factor 1 (HSF1). In mammalian cells, HSF1 is converted from an inactive monomeric form to an active trimer in response to heat stress. A ribonucleoprotein complex comprising of eukaryotic translation elongation factor eEF1A1, and a long noncoding RNA HSR1 are the key components of HSF1 activation. Once activated, HSF1 is recruited to Heat Shock Protein (HSP) promoter regions, upregulating chaperone activity in the cell. Along with mediating initiation of HSR, eEF1A1 is also a vital component of protein synthesis machinery. Interestingly, another isoform of eEF1A, called eEF1A2, is expressed in some specialized terminally differentiated cells of skeletal muscle, heart, pancreatic islets and motor neurons, all of which are prone to protein aggregation. The two isoforms are 92% identical and are reciprocally regulated. To better understand the role of eEF1A1 and HSF1 proteins in humans, we use a CRISPR-Cas9 nickase system to knockout HSF1 and eEF1A1 in a human cell line. We showed that the hTERT-immortalized, normal diploid foreskin fibroblast cell line, BJ-5ta, produces both eEF1A isoforms. This will allow us to perform eEF1A1 knockout in these cells. We hypothesize that HSF1 knockout cell line will survive under normal conditions but express very low thermotolerance. Conjointly, we hypothesize that the elimination of eEF1A1 may be compensated by the upregulation of eEF1A2. If viable, the eEF1A1 knockout cell line will be used for screening mutants of eEF1A2 restoring activation of HSR. Both HSF1 and eEF1A1 knockout lines will also be used for future studies to improve upon the current model of the HSR pathway and potentially reveal therapeutic targets for diseases like ALS, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and amyloidosis

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.

The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.