Girish Rachakonda

Covalent modification at Cys151 dissociates the electrophile sensor Keap1 from the ubiquitin ligase CUL3.

The regulation of cellular stress responses to electrophiles and oxidants is mediated by the transcription factor NF-E2-related factor 2 (Nrf2), which, in turn, is regulated by CUL-E3 (CUL3) ligase-mediated ubiquitylation. The Kelch-like ECH-associated protein 1 (Keap1) serves as an adapter between CUL3 and Nrf2. We used the model electrophile N-iodoacetyl- N-biotinylhexylenediamine (IAB) to define the relationship among the adduction of Keap1 cysteine residues, structure, and function. Exposure of Keap1 to IAB in vitro was accompanied by progressive loss of protein secondary structure, as monitored by CD spectroscopy and a loss of the ability to associate with recombinant CUL3. Dissociation of Keap1 from CUL3 in vitro was dependent upon C151 in Keap1. A quantitative mass spectrometry-based kinetic analysis of adduction in HEK293 cells expressing FLAG-Keap1 revealed that Cys151 was one of the most reactive residues in vivo and that it was required for IAB-mediated dissociation of the Keap1-CUL3 interaction. These results demonstrate that Cys151 adduction confers a critical alkylation sensor function upon Keap1, making Keap1 unique among BTB CUL3 adapter proteins.

Cysteine-based Regulation of the CUL3 Adaptor Protein Keap1

Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.

Activating Transcription Factor 3 Is a Novel Repressor of the Nuclear Factor Erythroid-Derived 2–Related Factor 2 (Nrf2)–Regulated Stress Pathway

The transcription factor nuclear factor erythroid-derived 2-related factor 2 (Nrf2) regulates induction of an extensive cellular stress response network when complexed with the cAMP-responsive element binding protein (CBP) at antioxidant response elements (ARE) located in the promoter region of target genes. Activating transcription factor 3 (ATF3) can repress Nrf2-mediated signaling in a manner that is not well understood. Here, we show that ATF3-mediated suppression is a consequence of direct ATF3-Nrf2 protein-protein interactions that result in displacement of CBP from the ARE. This work establishes ATF3 as a novel repressor of the Nrf2-directed stress response pathway.

Increased cell migration and plasticity in Nrf2-deficient cancer cell lines

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequences of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or the expression of independent short hairpin RNA (shRNA)/siRNA sequences enhanced cellular levels of reactive oxygen species, Smad-dependent tumor cell motility and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-cadherin transcriptional repressor Slug and suppression of the cell–cell adhesion protein E-cadherin. Ectopic expression of the wildtype but not dominant-negative Nrf2 suppressed the activity of a synthetic transforming growth factor-β1-responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using transforming growth factor-β/Smad signaling.

NRF2 deficiency reduces life span of mice administered thoracic irradiation

Subsets of cancer survivors who have been subjected to thoracic irradiation face the prospect of developing pulmonary injury. Radiation-induced pulmonary fibrosis is an insidious injury that presents 6 to 24 months after irradiation and continues to progress over a period of years. TGF-β and reactive oxygen species contribute significantly to the pathogenesis of this injury. The transcription factor NRF2 controls antioxidant gene expression and therefore regulates the cellular oxidant burden. This work demonstrates an additional paradigm for NRF2: suppression of TGF-β-mediated signaling, assessed by measuring expression of a surrogate TGF-β1 target gene (PAI-1) in lung fibroblasts. Thoracic irradiation of Nfe2l2(-/-) mice resulted in rapid expression of PAI-1 and FSP-1 compared to irradiated wild-type mice. Examination of lung tissue 16 weeks after thoracic irradiation of Nfe2l2(-/-) mice revealed the presence of distended alveoli and decreased numbers of alveoli compared to wild-type mice. Suppression of NRF2 expression shortened life span in mice administered 16 Gy to the thorax. Nfe2l2(+/-) and Nfe2l2(-/-) mice exhibited a mean life span of 176 days compared to wild-type mice, which lived an average of 212 days. These novel results identify NRF2 as a susceptibility factor for the development of late tissue injury.

Genetic diversity of noroviruses and sapoviruses in children with acute sporadic gastroenteritis in New Delhi, India

BACKGROUND Human caliciviruses (HuCVs) cause gastroenteritis throughout the world. Limited information is available on molecular epidemiology of caliciviruses from developing countries including India. OBJECTIVES Standardization and evaluation of a two-step multiplex RT-PCR assay for HuCVs and characterization of strains. STUDY DESIGN Two hundred and twenty-six stool samples were collected from children with acute gastroenteritis (AGE) over a one and half year to study the prevalence and diversity of HuCVs in children with AGE in New Delhi, India. A multiplex two-step RT-PCR using 3 sets of external and 4 sets of internal primers from the RdRp gene was standardized for detection of NoVs and SaVs. Molecular characterization of some HuCV strains was done by sequencing followed by phylogenetic analysis. RESULTS Fifty-nine HuCVs strains were detected in 54 (24%) of the samples; 5 samples had mixed infections. Of these 59 HuCVs, 36 (61%) were norovirus (34 were GGII; 2 were GGI) and 23 (39%) were sapovirus (22 were GGI; 1 was GGII). Phylogenetic analysis of partial RdRp gene of 12 HuCV strains identified three genotypes (GGI/4, GGII/3 and a newly identified GIIb/Hilversum cluster) in NoVs and one genotype (GGI/1) in SaVs. CONCLUSION This is one of the few reports from India on detection and characterization of HuCVs by multiplex RT-PCR assay. This assay can be a useful tool for epidemiological studies of HuCV infections.

The Novel Chemical Entity YTR107 Inhibits Recruitment of Nucleophosmin to Sites of DNA Damage, Suppressing Repair of DNA Double-Strand Breaks and Enhancing Radiosensitization

Purpose: Radiation therapy continues to be an important therapeutic strategy for providing definitive local/regional control of human cancer. However, oncogenes that harbor driver mutations and/or amplifications can compromise therapeutic efficacy. Thus, there is a need for novel approaches that enhance the DNA damage produced by ionizing radiation. Experimental Design: A forward chemical genetic approach coupled with cell-based phenotypic screening of several tumor cell lines was used to identify a novel chemical entity (NCE) that functioned as a radiation sensitizer. Proteomics, comet assays, confocal microscopy, and immunoblotting were used to identify the biological target. Results: The screening process identified a 5-((N-benzyl-1H-indol-3-yl)-methylene)pyrimidine-2,4,6(1H,3H,5H)trione as an NCE that radiosensitized cancer cells expressing amplified and/or mutated RAS, ErbB, PIK3CA, and/or BRAF oncogenes. Affinity-based solid-phase resin capture followed by liquid chromatography/tandem mass spectrometry identified the chaperone nucleophosmin (NPM) as the NCE target. SiRNA suppression of NPM abrogated radiosensitization by the NCE. Confocal microscopy showed that the NCE inhibited NPM shuttling to radiation-induced DNA damage repair foci, and the analysis of comet assays indicated a diminished rate of DNA double-strand break repair. Conclusion: These data support the hypothesis that inhibition of DNA repair due to inhibition of NPM shuttling increases the efficacy of DNA-damaging therapeutic strategies. Clin Cancer Res; 17(20); 6490–9. ©2011 AACR.

Indolyl-quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation-mediated control of tumor vasculature

There is a need for novel strategies that target tumor vasculature, specifically those that synergize with cytotoxic therapy, in order to overcome resistance that can develop with current therapeutics. A chemistry‐driven drug discovery screen was employed to identify novel compounds that inhibit endothelial cell tubule formation. Cell‐based phenotypic screening revealed that noncytotoxic concentrations of (Z)‐(±)‐2– (1‐benzenesulfonylindol‐3–ylmethylene)‐1‐azabicyclo[2. 2.2]octan‐3‐ol (analog I) and (Z)‐(±)‐2‐(l‐benzylindol‐3‐ylmethylene)‐1‐azabicyclo[2.2.2]octan‐3‐ol (analog II) inhibited endothelial cell migration and the ability to form capillary‐like structures in Matrigel by ≥70%. The ability to undergo neoangiogenesis, as measured in a window‐chamber model, was also inhibited by 70%. Screening of biochemical pathways revealed that analog II inhibited the enzyme ENOX1 (EC50 = 10 µM). Retroviral‐mediated shRNA suppression of endothelial ENOX1 expression inhibited cell migration and tubule formation, recapitulating the effects observed with the small‐molecule analogs. Genetic or chemical suppression of ENOX1 significantly increased radiation‐mediated Caspase3‐activated apoptosis, coincident with suppression of p70S6K1 phosphorylation. Administration of analog II prior to fractionated X‐irradiation significantly diminished the number and density of tumor microvessels, as well as delayed syngeneic and xenograft tumor growth compared to results obtained with radiation alone. Analysis of necropsies suggests that the analog was well tolerated. These results suggest that targeting ENOX1 activity represents a novel therapeutic strategy for enhancing the radiation response of tumors.—Geng, L., Rachakonda, G., Morre, D. J., Morre, D. M., Crooks, P. A., Sonar, V. N., Roti Roti, J. L., Rogers, B. E., Greco, S., Ye, F., Salleng, K. J., Sasi, S., Freeman, M. L., Sekhar, K. R. Indolyl‐quinuclidinols inhibit ENOX activity and endothelial cell morphogenesis while enhancing radiation‐mediated control of tumor vasculature. FASEB J. 23, 2986–2995 (2009). www.fasebj.org

The novel antiangiogenic VJ115 inhibits the NADH oxidase ENOX1 and cytoskeleton-remodeling proteins

SummaryTargeting tumor vasculature represents a rational strategy for controlling cancer. (Z)-(+/−)-2-(1-benzylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol (denoted VJ115) is a novel chemical entity that inhibits the enzyme ENOX1, a NADH oxidase. Genetic and small molecule inhibition of ENOX1 inhibits endothelial cell tubule formation and tumor-mediated neo-angiogenesis. Inhibition of ENOX1 radiosensitizes tumor vasculature, a consequence of enhanced apoptosis. However, the molecular mechanisms underlying these observations are not well understood. Herein, we mechanistically link ENOX1-mediated regulation of cellular NADH concentrations with proteomics profiling of endothelial cell protein expression following exposure to VJ115. Pathway Studios network analysis of potential effector molecules identified by the proteomics profiling indicated that a VJ115 exposure capable of altering intracellular NADH concentrations impacted proteins involved in cytoskeletal reorganization. The analysis was validated using RT-PCR and immunoblotting of selected proteins. RNAi knockdown of ENOX1 was shown to suppress expression of stathmin and lamin A/C, proteins identified by the proteomics analysis to be suppressed upon VJ115 exposure. These data support the hypothesis that VJ115 inhibition of ENOX1 can impact expression of proteins involved in cytoskeletal reorganization and support a hypothesis in which ENOX1 activity links elevated cellular NADH concentrations with cytoskeletal reorganization and angiogenesis.