Natalya Smirnova - Academia.edu (original) (raw)

Papers by Natalya Smirnova

Journal of Bacteriology, 1997

Environmental stress activates sigma B, the general stress response sigma factor of Bacillus subt... more Environmental stress activates sigma B, the general stress response sigma factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of sigma B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a sigma B-dependent promoter, stress activation of sigma B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived sigma B activation. The data suggest that RsbX is not a direct participant in the sigma B stress induction process but rather serves as a device to limit the magnitude of the stress response.

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jun 8, 2016

A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related facto... more A promising approach to neurotherapeutics involves activating the nuclear-factor-E2-related factor 2 (Nrf2)/antioxidant response element signaling, which regulates expression of antioxidant, anti-inflammatory, and cytoprotective genes. Tecfidera, a putative Nrf2 activator, is an oral formulation of dimethylfumarate (DMF) used to treat multiple sclerosis. We compared the effects of DMF and its bioactive metabolite monomethylfumarate (MMF) on Nrf2 signaling and their ability to block 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced experimental Parkinson's disease (PD). We show that in vitro DMF and MMF activate the Nrf2 pathway via S-alkylation of the Nrf2 inhibitor Keap1 and by causing nuclear exit of the Nrf2 repressor Bach1. Nrf2 activation by DMF but not MMF was associated with depletion of glutathione, decreased cell viability, and inhibition of mitochondrial oxygen consumption and glycolysis rates in a dose-dependent manner, whereas MMF increased these activities...

Environmental stress activates B , the general stress response factor of Bacillus subtilis, by a ... more Environmental stress activates B , the general stress response factor of Bacillus subtilis, by a pathway that is negatively controlled by the RsbX protein. To determine whether stress activation of B occurs by a direct effect of stress on RsbX, we constructed B. subtilis strains which synthesized various amounts of RsbX or lacked RsbX entirely and subjected these strains to ethanol stress. Based on the induction of a B-dependent promoter, stress activation of B can occur in the absence of RsbX. Higher levels of RsbX failed to detectably influence stress induction, but reduced levels of RsbX resulted in greater and longer-lived B activation. The data suggest that RsbX is not a direct participant in the B stress induction process but rather serves as a device to limit the magnitude of the stress response.

Journal of Molecular Medicine, 2007

The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functio... more The brain demands oxygen and glucose to fulfill its roles as the master regulator of body functions as diverse as bladder control and creative thinking. Chemical and electrical transmission in the nervous system is rapidly disrupted in stroke as a result of hypoxia and hypoglycemia. Despite being highly evolved in its architecture, the human brain appears to utilize phylogenetically conserved homeostatic strategies to combat hypoxia and ischemia. Specifically, several converging lines of inquiry have demonstrated that the transcription factor hypoxia-inducible factor-1 (HIF1-1) mediates the activation of a large cassette of genes involved in adaptation to hypoxia in surviving neurons after stroke. Accordingly, pharmacological or molecular approaches that engage hypoxic adaptation at the point of one of its sensors (e.g., inhibition of HIF prolyl 4 hydroxylases) leads to profound sparing of brain tissue and enhanced recovery of function. In this review, we discuss the potential mechanisms that could subserve protective and restorative effects of augmenting hypoxic adaptation in the brain. The strategy appears to involve HIF-dependent and HIF-independent pathways and more than 70 genes and proteins activated transcriptionally and post-transcriptionally that can act at cellular, local, and system levels to compensate for oxygen insufficiency. The breadth and depth of this homeostatic program offers a hopeful alternative to the current pessimism towards stroke therapeutics. Keywords Brain. Stroke. Hypoxia. HIF. HIF prolyl hydroxylase. Therapeutics Stroke is defined as injury to the brain accruing from a vascular etiology. Strikingly, it has emerged as the third leading cause of death and the leading cause of disability in the USA. Accordingly, the estimated financial costs of stroke are more than 50 billion dollars a year in the USA alone. These financial costs do not begin to tell the story of the personal suffering that amasses from the silent epidemic of stroke disability-over 5 million Americans face the challenges of handicaps from stroke each day. The recognition of stroke as a leading age-associated public health issue has led the government and the pharmaceutical industry to expend enormous resources on developing

Journal of Industrial Microbiology and Biotechnology, 2001

Ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in ... more Ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in straight-chain fatty acid (SCFA) biosynthesis in Escherichia coli. Overproduction of the corresponding KASIII gene, or the Brassica napus KASIII gene has previously been observed to lead to an increase in the amount of shorter-chain fatty acids produced by E. coli. In this study it is shown that overexpression of the KASIII gene, which initiates branched-chain fatty acid (BCFA) in Streptomyces glaucescens, does not lead to a change in the fatty acid profiles of E. coli. E. coli produces trace levels of BCFAs when grown in the presence of isobutyric acid, but the amounts of these are not significantly altered by expression of the S. glaucescens KASIII gene. In contrast, the amounts of BCFAs produced from isobutyryl CoA in vitro by E. coli cell-free extracts can be increased at least four-fold by the presence of the S. glaucescens KASIII. These observations suggest that in vivo production of isopalmitate by E. coli expressing the S. glaucescens KASIII is limited by availability of the appropriate BCFA biosynthetic primers.

Journal of Bacteriology, 2001

The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiate... more The Streptomyces glaucescens β-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) initiates straight- and branched-chain fatty acid biosynthesis by catalyzing the decarboxylative condensation of malonyl-ACP with different acyl-coenzyme A (CoA) primers. This KASIII has one cysteine residue, which is critical for forming an acyl-enzyme intermediate in the first step of the process. Three mutants (Cys122Ala, Cys122Ser, Cys122Gln) were created by site-directed mutagenesis. Plasmid-based expression of these mutants in S. glaucescens resulted in strains which generated 75 (Cys122Ala) to 500% (Cys122Gln) more straight-chain fatty acids (SCFA) than the corresponding wild-type strain. In contrast, plasmid-based expression of wild-type KASIII had no effect on fatty acid profiles. These observations are attributed to an uncoupling of the condensation and decarboxylation activities in these mutants (malonyl-ACP is thus converted to acetyl-ACP, a SCFA precursor). Incorporation experiments...

Biochemical and Biophysical Research Communications, 1999

The general stress regulon of Bacillus subtilis is controlled by the transcription factor B. Envi... more The general stress regulon of Bacillus subtilis is controlled by the transcription factor B. Environmental stress activates B via a phosphatase/kinase cascade that triggers B 's release from an anti sigma factor complex. To determine if the members of the phosphatase/kinase cascade are sufficient to detect environmental stress and activate B , we expressed B and its regulators in E. coli. In E. coli, as in B. subtilis, the intact collection of regulators silenced B , while allowing B to be active if the cascade's most upstream negative regulator was deleted. The regulators could not, however, activate B in response to ethanol treatment or heat shock. In other experiments, the GroEL and DnaK chaperones, known to be important in controlling stress factors in E. coli, were found to be unimportant for B activity in B. subtilis. The findings argue that stress induction of B requires novel factors that are B. subtilis specific.

Annals of the New York Academy of Sciences, 2008

A major challenge for neurological therapeutics is the development of small molecule drugs that c... more A major challenge for neurological therapeutics is the development of small molecule drugs that can activate a panoply of downstream pathways without toxicity. Over the past decade our group has shown that a family of enzymes that regulate posttranscriptional and transcriptional adaptive responses to hypoxia are viable targets for neuronal protection and repair. The family is a group of iron, oxygen, and 2‐oxoglutarate‐dependent dioxygenases, known as the HIF prolyl 4‐hydroxylases (HIF PHDs). We have previously shown that pluripotent protection offered by iron chelators is mediated, in part, via the ability of these agents to inhibit the HIF PHDs. Our group and others have implicated the transcriptional activator HIF‐1 in some of the salutary effects of iron chelation‐induced PHD inhibition. While some iron chelators are currently employed in humans for conditions such as hemochromatosis, the diverse utilization of iron in physiological processes in the brain makes the development o...

Annals of Neurology, 2012

Targeting newly identified damage pathways in the ischemic brain can help to circumvent the curre... more Targeting newly identified damage pathways in the ischemic brain can help to circumvent the currently severe limitations of acute stroke therapy. Here we show that the activity of 12/15lipoxygenase was increased in the ischemic mouse brain, and 12/15-lipoxygenase co-localized with a marker for oxidized lipids MDA2. This co-localization was also detected in the brain of two human stroke patients, where it also coincided with increased apoptosis-inducing factor, AIF. A novel inhibitor of 12/15-lipoxygenase, LOXBlock-1 protected neuronal HT22 cells against oxidative stress. In a mouse model of transient focal ischemia, the inhibitor reduced infarct sizes both 24 hours and 14 days post stroke, with improved behavioral parameters. Even when treatment was delayed until at least four hours after onset of ischemia, LOXBlock-1 was protective. Furthermore, it reduced tPA-associated hemorrhage in a clot model of ischemia/ reperfusion. This study establishes inhibition of 12/15-lipoxygenase as a viable strategy for first line stroke treatment.

Drug Discovery and Development - Present and Future, 2011

Although these reporters are obviously of great help in research, they provide an integrated resp... more Although these reporters are obviously of great help in research, they provide an integrated response and are affected by many steps such as transcription factor stabilization, phosphorylation, translocation to nucleus, interaction with transcription cofactors, etc. As a result, the response of these reporters is not immediate: it is delayed by many hours, the magnitude of activation effect is very modest, and there is always uncertainty in the actual site of their effect along the path leading to activation of a particular promoter. The important general pathway effectively regulating the cellular response to hypoxia, oxidation and inflammation is specific regulation of protein stability of the corresponding transcription factors or their modulators by ubiquitination and proteasomal degradation. Protein degradation by the proteasome is one of the major regulatory mechanisms in the cell. The proteasome mediates the degradation of most short-lived proteins that control cell cycle, transcription, DNA repair, apoptosis and other cellular processes. Under normal conditions, the stress response transcription factors are expressed constitutively, but on the protein level, these transcriptional factors are unstable: they undergo ubiquitination either directly, or upon specific covalent modification(s) of the targeted amino acids residues (like hypoxia inducible factor, HIF). Considering the protein stability of transcription factors as the most important and limiting step in the stress response we have recently developed a different approach to high throughput screening (HTS) of stabilizers of transcription factors. The approach is based on stable expression of a fusion between luciferase and a transcription factor minimum domain recognized by ubiquitination machinery (Fig.1). Minimum domain is the portion of a transciption factor that is necessary and sufficient for recognition and ubiquitination steps to occur. The overexpressed luciferase-labeled surrogate of a transcription factor undergoes the same recognition and transformation steps as an endogenous one. The time-course of reporter signal changes can be easily followed: the luminescent readings are extremely sensitive and provide monitoring just minutes after drug administration. This new approach to HTS was developed in this laboratory and successfully used to discover novel activators of HIF1 and Nrf2 as exemplified below. 2. Development, validation and application of novel reporters for the purposes of HTS 2.1 Neh2-luciferase reporter construction and performance (Smirnova et al 2011) The key transcription factor that orchestrates antioxidant response is Nrf2 (Moi et al 1994, Motoyashi & Yamamoto 2004, Kaspar et al 2009). Compounds that activate Nrf2 make the cell more resistant to subsequent xenobiotic attack or oxidative stress. This has major implications for human health: (1) Nrf2 activators can be considered as medications for cancer prevention; (2) while an increased level of Nrf2 makes cancerous cells more resistant to chemotherapy; and (3) Nrf2 activators can both prevent and treat neurodegenerative diseases. There is a consensus that oxidative stress either derived from gene mutations or environmental toxins is a mediator of neurodegenerative diseases and thus, Nrf2 has been justified as a pharmacological target for neuroprotective therapies in Parkinson's, Huntington's and other neurodegenerative diseases. 2.1.1 Nrf2-Keap1 interaction mode Nrf2 is composed of Neh1-Neh6 domains, among which Neh2 (1-98 aa) is the putative negative regulatory domain that interacts with Keap1, while Neh4 and Neh5 are www.intechopen.com Novel Approach to High Throughput Screening for Activators of Transcription Factors 297 transactivation domains, and Neh1 is the binding domain for ARE. The functional domains of Keap1 are the Broad complex, Tramtrack and Bric-a-Brac, the intervening region, the double glycine repeats domain, and the C-terminal region (Zhang et al 2006). Two motifs in the Neh2 domain, i.e. ETGE and DLG (Tong et al 2006 a, 2006b), are recognized by the Keap1 homodimer in a hinge-latch mode (see Fig.2 and 3). Keap1 mediates polyubiquitination of the lysines positioned within the central α-helix of the Neh2 domain under homeostatic conditions. Under oxidative/electrophilic stress, most reactive cysteines within Keap1 (Cys 151, Cys273, Cys288) are modified and Keap1 undergoes conformational changes which lead to Nrf2 stabilization (see Fig.3) (Cullinan et al 2004).