Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

scholarly journals Expression of Hepatitis C Virus E2 Ectodomain in E. coli and Its Application in the Detection of Anti-E2 Antibodies in Human Sera

2004 ◽  
Vol 36 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Jing Liu ◽  
Xin-Xin Zhang ◽  
Shen-Ying Zhang ◽  
Min Lu ◽  
Yu-Ying Kong ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Western Blot ◽  
Mammalian Cells ◽  
Rabbit Antiserum ◽  
Hcv Rna ◽  
Target Cells ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Human Sera

Abstract The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385–730) with a four-amino-acid mutation (aa 568–571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2-specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glycoproteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previously reported C-terminally truncated E2 fragment (aa 385–565). Association between anti-E2 antibodies in patient sera and HCV RNA status was also demonstrated using this E. coli-derived protein. E2N730(m) might serve as an inexpensive alternative to mammalian cell-expressed E2 proteins in clinical and research applications.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Targeting xanthine oxidase by natural products as a therapeutic approach for mental disorders

2020 ◽  
Vol 26 ◽  
Author(s):  
Miquel Martorell ◽  
Xavier Lucas ◽  
Pedro Alarcón-Zapata ◽  
Xavier Capó ◽  
Maria Magdalena Quetglas-Llabrés ◽  
...  
Keyword(s):  
Mental Disorders ◽  
Small Molecules ◽  
Xanthine Oxidase ◽  
Cell Damage ◽  
Bipolar Affective Disorder ◽  
Oxidative Reaction ◽  
Mental Diseases ◽  
Potential Therapeutic Application ◽  
Increased Activity ◽  
Dietary Flavonoids

: Mental disorders comprise diverse human pathologies including depression, bipolar affective disorder, schizophrenia, and dementia that affect millions of people around the world. The causes of mental disorders are unclear but growing evidence suggests that oxidative stress and the purine/adenosine system play a key role in their development and progression. Xanthine oxidase (XO) is a flavoprotein enzyme essential for the catalysis of the oxidative hydroxylation of purines -hypoxanthine and xanthine- to generate uric acid. As a consequence of the oxidative reaction of XO, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are produced and, further, contribute to the pathogenesis of mental disorders. Altered XO activity has been associated with free radical-mediated neurotoxicity inducing cell damage and inflammation. Diverse studies reported a direct association between an increased activity of XO and diverse mental diseases including depression or schizophrenia. Small-molecule inhibitors, such as the well-known allopurinol, and dietary flavonoids, can modulate the XO activity and subsequent ROS production. In the present work, we review the available literature on XO inhibition by small molecules and their potential therapeutic application in mental disorders. In addition, we discuss the chemistry and molecular mechanism of XO inhibitors, as well as the use of structure-based and computational methods to design specific inhibitors with the capability of modulating XO activity.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Nucleoside Analogues Are Potent Inducers of Pol V-mediated Mutagenesis

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 843
Author(s):  
Balagra Kasim Sumabe ◽  
Synnøve Brandt Ræder ◽  
Lisa Marie Røst ◽  
Animesh Sharma ◽  
Eric S. Donkor ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutation Frequency ◽  
Nucleoside Analogues ◽  
Cancer Therapies ◽  
E Coli ◽  
Replicative Stress ◽  
Pol V ◽  
Dna And Rna ◽  
Sos Induction ◽  
Anti Cancer

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli, using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Cell specificity in metabolic activation of aflatoxin B1 and benzo(a)pyrene to mutagens for mammalian cells

Nature ◽  
1978 ◽  
Vol 276 (5685) ◽  
pp. 277-280 ◽  
Author(s):  
ROBERT LANGENBACH ◽  
HEATHER J. FREED ◽  
DINA RAVEH ◽  
ELIEZER HUBERMAN
Keyword(s):  
Aflatoxin B1 ◽  
Mammalian Cells ◽  
Metabolic Activation ◽  
Cell Specificity

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

scholarly journals Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Carrie L. Shaffer ◽  
James A. D. Good ◽  
Santosh Kumar ◽  
K. Syam Krishnan ◽  
Jennifer A. Gaddy ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Small Molecules ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Effector Protein ◽  
Secretion Systems ◽  
Type Iv ◽  
H Pylori

ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori , KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori , we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

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