Publications by authors named "Lewis M Brown"

32 Publications

Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence.

Nat Commun 2021 07 29;12(1):4613. Epub 2021 Jul 29.

Department of Biological Sciences, Columbia University, New York, NY, USA.

R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes. PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.
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http://dx.doi.org/10.1038/s41467-021-24796-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322103PMC
July 2021

Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome.

J Proteome Res 2021 08 22;20(8):4001-4009. Epub 2021 Jul 22.

Department of Otolaryngology-Head and Neck Surgery, Columbia University Vagelos College of Physicians and Surgeons, New York, New York 10032, United States.

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone ( = 6), IP dexamethasone ( = 8), or untreated for control ( = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 μL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.
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http://dx.doi.org/10.1021/acs.jproteome.1c00322DOI Listing
August 2021

Formation of Biomolecular Condensates in Bacteria by Tuning Protein Electrostatics.

ACS Cent Sci 2020 Dec 12;6(12):2301-2310. Epub 2020 Nov 12.

Department of Chemical Engineering, Columbia University, New York, New York 10027, United States.

While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates-organelles that lack a membrane-provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid-liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in . Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.
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http://dx.doi.org/10.1021/acscentsci.0c01146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760465PMC
December 2020

Novel 3D-printed hollow microneedles facilitate safe, reliable, and informative sampling of perilymph from guinea pigs.

Hear Res 2021 02 2;400:108141. Epub 2020 Dec 2.

Department of Otolaryngology - Head and Neck Surgery, Columbia University Vagelos College of Physicians and Surgeons, 180 Fort Washington Avenue, Harkness Pavilion, 8th Floor, New York, NY 10032, United States; Department of Mechanical Engineering, Columbia University, New York, NY, United States. Electronic address:

Background: Inner ear diagnostics is limited by the inability to atraumatically obtain samples of inner ear fluid. The round window membrane (RWM) is an attractive portal for accessing perilymph samples as it has been shown to heal within one week after the introduction of microperforations. A 1 µL volume of perilymph is adequate for proteome analysis, yet the total volume of perilymph within the scala tympani of the guinea pig is limited to less than 5 µL. This study investigates the safety and reliability of a novel hollow microneedle device to aspirate perilymph samples adequate for proteomic analysis.

Methods: The guinea pig RWM was accessed via a postauricular surgical approach. 3D-printed hollow microneedles with an outer diameter of 100 µm and an inner diameter of 35 µm were used to perforate the RWM and aspirate 1 µL of perilymph. Two perilymph samples were analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics as part of a preliminary study. Hearing was assessed before and after aspiration using compound action potential (CAP) and distortion product otoacoustic emissions (DPOAE). RWMs were harvested 72 h after aspiration and evaluated for healing using confocal microscopy.

Results: There was no permanent damage to hearing at 72 h after perforation as assessed by CAP (n = 7) and DPOAE (n = 8), and all perforations healed completely within 72 h (n = 8). In the two samples of perilymph analyzed, 620 proteins were detected, including the inner ear protein cochlin, widely recognized as a perilymph marker.

Conclusion: Hollow microneedles can facilitate aspiration of perilymph across the RWM at a quality and volume adequate for proteomic analysis without causing permanent anatomic or physiologic dysfunction. Microneedles can mediate safe and effective intracochlear sampling and show great promise for inner ear diagnostics.
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http://dx.doi.org/10.1016/j.heares.2020.108141DOI Listing
February 2021

Time Course of Changes in Sorafenib-Treated Hepatocellular Carcinoma Cells Suggests Involvement of Phospho-Regulated Signaling in Ferroptosis Induction.

Proteomics 2020 05 18;20(10):e2000006. Epub 2020 May 18.

Department of Biological Sciences, Columbia University, New York, NY, 10027, USA.

Ferroptosis is a form of regulated, non-apoptotic cell death characterized by excessive lipid peroxidation that can be triggered by inhibition of the cystine-glutamate antiporter, system X . Sorafenib, an FDA-approved multi-kinase inhibitor drug that is used for treatment of hepatocellular carcinoma (HCC), has been shown to induce ferroptosis. Protein phosphorylation changes upon sorafenib treatment have been previously reported in patient studies and in cell culture. However, early phosphorylation changes during induction of ferroptosis are not reported. This work highlights these changes through a time course from 7 to 60 min of sorafenib treatment in human (SKHep1) HCC cells. A total of 6170 unique phosphosites from 2381 phosphoproteins are quantified, and phosphorylation changes occur after as little as 30 min of sorafenib treatment. By 60 min, notable changes included phosphosites significantly changing on p53 (P04637), CAD protein (P27708), and proteins important for iron homeostasis, such as heavy chain ferritin (FTH1; P02794), heme oxygenase 1 (HMOX1; P09601), and PCBP1 (Q15365). Additional sites on proteins in key regulatory pathways are identified, including sites in ferroptosis-related proteins, indicating the likely involvement of phospho-regulated signaling during ferroptosis induction.
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http://dx.doi.org/10.1002/pmic.202000006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305944PMC
May 2020

Application of Ion Mobility Mass Spectrometry in Lipidomics.

Adv Exp Med Biol 2019 ;1140:317-326

Department of Biological Sciences, Quantitative Proteomics and Metabolomics Center, Columbia University, New York, NY, USA.

Lipids play significant roles in biological system, and the study of lipid metabolisms may provide a new insight into the diagnosis and pathophysiology of diseases. Recent developments in high-resolution mass spectrometry techniques combined with high-performance chromatographic methods provide deep insight into lipid analysis. Addition of ion mobility mass spectrometry orthogonal to LC-MS analysis workflow enhances separation of complex lipids, improve isomers resolution, and intensify confidence in lipid identification and characterization. In this chapter, we describe the principle of travelling wave ion mobility mass spectrometry (TWIMS) and its applications in untargeted LC-MS analysis for characterizing the structural diversity and complexity of lipid species in biological samples.
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http://dx.doi.org/10.1007/978-3-030-15950-4_18DOI Listing
September 2019

Imidazole Ketone Erastin Induces Ferroptosis and Slows Tumor Growth in a Mouse Lymphoma Model.

Cell Chem Biol 2019 05 21;26(5):623-633.e9. Epub 2019 Feb 21.

Department of Chemistry, Columbia University, New York, NY 10027, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Electronic address:

Ferroptosis is a form of regulated cell death that can be induced by inhibition of the cystine-glutamate antiporter, system x. Among the existing system x inhibitors, imidazole ketone erastin (IKE) is a potent, metabolically stable inhibitor of system x and inducer of ferroptosis potentially suitable for in vivo applications. We investigated the pharmacokinetic and pharmacodynamic features of IKE in a diffuse large B cell lymphoma (DLBCL) xenograft model and demonstrated that IKE exerted an antitumor effect by inhibiting system x, leading to glutathione depletion, lipid peroxidation, and the induction of ferroptosis biomarkers both in vitro and in vivo. Using untargeted lipidomics and qPCR, we identified distinct features of lipid metabolism in IKE-induced ferroptosis. In addition, biodegradable polyethylene glycol-poly(lactic-co-glycolic acid) nanoparticles were employed to aid in IKE delivery and exhibited reduced toxicity compared with free IKE in a DLBCL xenograft model.
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http://dx.doi.org/10.1016/j.chembiol.2019.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525071PMC
May 2019

Dual IFN-γ/hypoxia priming enhances immunosuppression of mesenchymal stromal cells through regulatory proteins and metabolic mechanisms.

J Immunol Regen Med 2018 Mar 25;1:45-56. Epub 2018 Apr 25.

Department of Biomedical Engineering, Columbia University, New York, NY, USA.

The immunosuppressive capacity of human mesenchymal stromal cells (MSCs) renders them promising candidates for treating diverse immune disorders. However, after hundreds of clinical trials, there are still no MSC therapies approved in the United States. MSCs require specific cues to adopt their immunosuppressive phenotype, and yet most clinical trials use cells expanded in basic culture medium and growth conditions. We propose that priming MSCs prior to administration will improve their therapeutic efficacy. Interferon-gamma (IFN-γ) priming are cues common to situations of immune escape that have individually shown promise as MSC priming cues but have not been systematically compared. Using mixed lymphocyte reactions, we show that priming MSCs with either cue alone improves T-cell inhibition. However, combining the two cues results in additive effects and markedly enhances the immunosuppressive phenotype of MSCs. We demonstrate that IFN-γ induces expression of numerous immunosuppressive proteins (IDO, PD-L1, HLA-E, HLA-G), whereas hypoxia switches MSCs to glycolysis, causing rapid glucose consumption and production of T-cell inhibitory lactate levels. Dual IFN-γ/hypoxia primed MSCs display both attributes and have even higher induction of immunosuppressive proteins over IFN-γ priming alone (IDO and HLA-G), which may reflect another benefit of metabolic reconfiguration.
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http://dx.doi.org/10.1016/j.regen.2018.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197483PMC
March 2018

HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

Aging Cell 2018 08 5;17(4):e12710. Epub 2018 May 5.

Edward S. Harkness Eye Institute, New York-Presbyterian Hospital, New York, NY, USA.

High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.
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http://dx.doi.org/10.1111/acel.12710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052470PMC
August 2018

The influence of hypoxia and IFN-γ on the proteome and metabolome of therapeutic mesenchymal stem cells.

Biomaterials 2018 06 15;167:226-234. Epub 2018 Mar 15.

Department of Biomedical Engineering, Columbia University, New York, NY, USA; Department of Medicine, Columbia University, New York, NY, USA. Electronic address:

Over the past 15 years, mesenchymal stem cells (MSCs) have been assessed for their capacity to suppress inflammation and promote tissue repair. Regardless of whether the cells are primed (exposed to instructive cues) before administration, their phenotype will respond to environmental signals present in the pathophysiological setting being treated. Since hypoxia and inflammation coexist in the settings of acute injury and chronic disease we sought to explore how the proteome and metabolome of MSCs changes when cells were exposed to 48 h of 1% oxygen, interferon gamma (IFN-γ), or both cues together. We specifically focused on changes in cell metabolism, immune modulation, extracellular matrix secretion and modification, and survival capacity. IFN-γ promoted expression of anti-pathogenic proteins and induced MSCs to limit inflammation and fibrosis while promoting their own survival. Hypoxia instead led to cell adaptation to low oxygen, including upregulation of proteins involved in anaerobic metabolism, autophagy, angiogenesis, and cell migration. While dual priming resulted in additive effects, we also found many instances of synergy. These data lend insight to how MSCs may behave after administration to a patient and suggest how priming cells beforehand could improve their therapeutic capacity.
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http://dx.doi.org/10.1016/j.biomaterials.2018.03.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894357PMC
June 2018

Design of Small Molecules That Compete with Nucleotide Binding to an Engineered Oncogenic KRAS Allele.

Biochemistry 2018 02 6;57(8):1380-1389. Epub 2018 Feb 6.

Quantitative Proteomics and Metabolomics Center, Columbia University , New York, New York 10027, United States.

RAS mutations are found in 30% of all human cancers, with KRAS the most frequently mutated among the three RAS isoforms (KRAS, NRAS, and HRAS). However, directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has been difficult because of the high affinity of KRAS for GDP and GTP. We designed an engineered allele of KRAS and a covalent inhibitor that competes for GTP and GDP. This ligand-receptor combination demonstrates that the high affinity of GTP and GDP for RAS proteins can be overcome with a covalent inhibitor and a suitably engineered binding site. The covalent inhibitor irreversibly modifies the protein at the engineered nucleotide-binding site and is able to compete with GDP and GTP. This provides a new tool for studying KRAS function and suggests strategies for targeting the nucleotide-binding site of oncogenic RAS proteins.
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http://dx.doi.org/10.1021/acs.biochem.7b01113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960803PMC
February 2018

Left-Ventricular Assist Device Impact on Aortic Valve Mechanics, Proteomics and Ultrastructure.

Ann Thorac Surg 2018 Feb 7;105(2):572-580. Epub 2017 Dec 7.

Division of Cardiac, Thoracic and Vascular Surgery, Columbia University Medical Center, New York, New York.

Background: Aortic regurgitation is a prevalent, detrimental complication of left ventricular assist devices (LVADs). The altered hemodynamics of LVADs results in aortic valves (AVs) having distinct mechanical stimulation. Our hypothesis was that the altered AV hemodynamics modulates the valve cells and matrix, resulting in changes in valvular mechanical properties that then can lead to regurgitation.

Methods: AVs were collected from 16 LVAD and 6 non-LVAD patients at time of heart transplant. Standard demographic and preoperative data were collected and comparisons between the two groups were calculated using standard statistical methods. Samples were analyzed using biaxial mechanical tensile testing, mass spectrometry-based proteomics, and transmission electron microscopy to assess ultrastructure.

Results: The maximum circumferential leaflet strain in LVAD patients was less than in non-LVAD patients (0.35 ± 0.10MPa versus 0.52 ± 0.18 MPa, p = 0.03) with a trend of reduced radial strain (p = 0.06) and a tendency for the radial strain to decrease with increasing LVAD duration (p = 0.063). Numerous proteins associated with actin and myosin, immune signaling and oxidative stress, and transforming growth factor β were increased in LVAD patients. Ultrastructural analysis showed a trend of increased fiber diameter in LVAD patients (46.2 ± 7.2 nm versus 45.1 ± 6.9 nm, p = 0.10), but no difference in fiber density.

Conclusions: AVs in LVAD patients showed decreased compliance and increased expression of numerous proteins related to valve activation and injury compared to non-LVAD patients. Further knowledge of AV changes leading to regurgitation in LVAD patients and the pathways by which they occur may provide an opportunity for interventions to prevent and/or reverse this detrimental complication.
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http://dx.doi.org/10.1016/j.athoracsur.2017.08.030DOI Listing
February 2018

Nuclear Proximity of Mtr4 to RNA Exosome Restricts DNA Mutational Asymmetry.

Cell 2017 04;169(3):523-537.e15

Department of Microbiology and Immunology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and senataxin) with the noncoding RNA processing function of RNA exosome determine the strand-specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development.
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http://dx.doi.org/10.1016/j.cell.2017.03.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515252PMC
April 2017

Multivalent Small-Molecule Pan-RAS Inhibitors.

Cell 2017 02;168(5):878-889.e29

Department of Chemistry, Columbia University, New York, NY 10027, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA. Electronic address:

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.
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http://dx.doi.org/10.1016/j.cell.2017.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362268PMC
February 2017

Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

Nat Chem Biol 2016 07 9;12(7):497-503. Epub 2016 May 9.

Department of Biological Sciences, Columbia University, New York, New York, USA.

Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.
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http://dx.doi.org/10.1038/nchembio.2079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920070PMC
July 2016

Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective.

Proc Natl Acad Sci U S A 2015 Apr 6;112(17):E2245-52. Epub 2015 Apr 6.

Department of Biological Sciences, Columbia University, New York, NY 10027; Department of Chemistry, Columbia University, New York, NY 10027; Howard Hughes Medical Institute and Department of Systems Biology, Columbia University, New York, NY 10027

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI's role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease.
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http://dx.doi.org/10.1073/pnas.1500439112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4418888PMC
April 2015

Quantitative shotgun proteomics with data-independent acquisition and traveling wave ion mobility spectrometry: a versatile tool in the life sciences.

Authors:
Lewis M Brown

Adv Exp Med Biol 2014 ;806:79-91

Quantitative Proteomics Center, Department of Biological Sciences, Columbia University, New York, NY, 10027, USA,

Data-independent acquisition (DIA) implemented in a method called MS(E) can be performed in a massively parallel, time-based schedule rather than by sampling masses sequentially in shotgun proteomics. In MS(E) alternating low and high energy spectra are collected across the full mass range. This approach has been very successful and stimulated the development of variants modeled after the MS(E) protocol, but over narrower mass ranges. The massively parallel MS(E) and other DIA methodologies have enabled effective label-free quantitation methods that have been applied to a wide variety of samples including affinity pulldowns and studies of cells, tissues, and clinical samples. Another complementary technology matches accurate mass and retention times of precursor ions across multiple chromatographic runs. This further enhances the impact of MS(E) in counteracting the stochastic nature of mass spectrometry as applied in proteomics. Otherwise significant amounts of data in typical large-scale protein profiling experiments are missing. A variety of software packages perform this function similar in concept to matching of accurate mass tags. Another enhancement of this method involves a variation of MS(E) coupled with traveling wave ion mobility spectrometry to provide separations of peptides based on cross-sectional area and shape in addition to mass/charge (m/z) ratio. Such a two-dimensional separation in the gas phase considerably increases protein coverage as well as typically a doubling of the number of proteins detected. These developments along with advances in ultrahigh pressure liquid chromatography have resulted in the evolution of a robust and versatile platform for label-free protein profiling.
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http://dx.doi.org/10.1007/978-3-319-06068-2_4DOI Listing
October 2014

Growth factor priming differentially modulates components of the extracellular matrix proteome in chondrocytes and synovium-derived stem cells.

PLoS One 2014 7;9(2):e88053. Epub 2014 Feb 7.

Department of Biomedical Engineering, Columbia University, New York, New York, United States of America.

To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB) in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC)-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes) and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs). However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0088053PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917883PMC
May 2015

Validation of genome-wide association study (GWAS)-identified disease risk alleles with patient-specific stem cell lines.

Hum Mol Genet 2014 Jul 4;23(13):3445-55. Epub 2014 Feb 4.

Barbara and Donald Jonas Laboratory of Stem Cells and Regenerative Medicine, Department of Ophthalmology, Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA, Edward S. Harkness Eye Institute, Columbia University, New York, NY 10032, USA, New York-Presbyterian Hospital, Columbia University Medical Center, New York, NY 10032, USA

While the past decade has seen great progress in mapping loci for common diseases, studying how these risk alleles lead to pathology remains a challenge. Age-related macular degeneration (AMD) affects 9 million older Americans, and is characterized by the loss of the retinal pigment epithelium (RPE). Although the closely linked genome-wide association studies ARMS2/HTRA1 genes, located at the chromosome 10q26 locus, are strongly associated with the risk of AMD, their downstream targets are unknown. Low population frequencies of risk alleles in tissue banks make it impractical to study their function in cells derived from autopsied tissue. Moreover, autopsy eyes from end-stage AMD patients, where age-related RPE atrophy and fibrosis are already present, cannot be used to determine how abnormal ARMS2/HTRA1 expression can initiate RPE pathology. Instead, induced pluripotent stem (iPS) cell-derived RPE from patients provides us with earlier stage AMD patient-specific cells and allows us to analyze the underlying mechanisms at this critical time point. An unbiased proteome screen of A2E-aged patient-specific iPS-derived RPE cell lines identified superoxide dismutase 2 (SOD2)-mediated antioxidative defense in the genetic allele's susceptibility of AMD. The AMD-associated risk haplotype (T-in/del-A) impairs the ability of the RPE to defend against aging-related oxidative stress. SOD2 defense is impaired in RPE homozygous for the risk haplotype (T-in/del-A; T-in/del-A), while the effect was less pronounced in RPE homozygous for the protective haplotype (G-Wt-G; G-Wt-G). ARMS2/HTRA1 risk alleles decrease SOD2 defense, making RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis.
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http://dx.doi.org/10.1093/hmg/ddu053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049304PMC
July 2014

Regulation of ferroptotic cancer cell death by GPX4.

Cell 2014 Jan;156(1-2):317-331

Department of Biological Sciences, Columbia University, 1208 Northwest Corner Building, 12 Floor, 550 West 120 Street, MC 4846, New York, NY 10027, USA.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
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http://dx.doi.org/10.1016/j.cell.2013.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076414PMC
January 2014

Interactions of the TnaC nascent peptide with rRNA in the exit tunnel enable the ribosome to respond to free tryptophan.

Nucleic Acids Res 2014 Jan 16;42(2):1245-56. Epub 2013 Oct 16.

Department of Biology, Texas A&M University, College Station, TX 77843, USA, Department of Biological Sciences, University of Alabama in Huntsville, Huntsville, AL 35899, USA, Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA, Quantitative Proteomics Center, Department of Biological Sciences, Columbia University, New York, NY 10027, USA and Department of Biology, Stanford University, Stanford, CA 94305, USA.

A transcriptional attenuation mechanism regulates expression of the bacterial tnaCAB operon. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp). In this study we demonstrate, using genetic and biochemical analyses, that in Escherichia coli, TnaC residue I19 and 23S rRNA nucleotide A2058 are essential for the ribosome's ability to sense free L-Trp. We show that the mutational change A2058U in 23S rRNA reduces the concentration dependence of L-Trp-mediated tna operon induction, whereas the TnaC I19L change suppresses this phenotype, restoring the sensitivity of the translating A2058U mutant ribosome to free L-Trp. These findings suggest that interactions between TnaC residue I19 and 23S rRNA nucleotide A2058 contribute to the creation of a regulatory L-Trp binding site within the translating ribosome.
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http://dx.doi.org/10.1093/nar/gkt923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3902921PMC
January 2014

Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome.

J Virol Methods 2014 Jan 12;195:9-17. Epub 2013 Oct 12.

Cabrini College, Department of Science, 610 King of Prussia Road, Radnor, PA 19087, United States.

In this work, a method was developed to study the structural proteome of mycobacteriophage Marvin, a recent isolate from soil with 107 predicted coding sequences. This prototype method was applied for semi-quantitative analysis of the composition of this mycobacteriophage virion using ion mobility spectrometry and data-independent acquisition (MS(E)-IMS). MS(E)-IMS was compared to a more conventional proteomics technique employing mass spectrometry with a data-dependent acquisition strategy. MS(E)-IMS provided broad coverage of the virion proteome and high sequence coverage for individual proteins. This shotgun method does not depend on the limited sensitivity of visualization of protein bands by staining reagents inherent in gel-based methods. The method is comprehensive, provides high sequence coverage and is proposed as a particularly efficient method for the study of bacteriophage proteomes.
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http://dx.doi.org/10.1016/j.jviromet.2013.10.007DOI Listing
January 2014

dMyc expression in the fat body affects DILP2 release and increases the expression of the fat desaturase Desat1 resulting in organismal growth.

Dev Biol 2013 Jul 19;379(1):64-75. Epub 2013 Apr 19.

Department of Biology, City College of the City University of New York, New York, NY 10031, USA.

Drosophila dMyc (dMyc) is known for its role in cell-autonomous regulation of growth. Here we address its role in the fat body (FB), a metabolic tissue that functions as a sensor of circulating nutrients to control the release of Drosophila Insulin-like peptides (Dilps) from the brain influencing growth and development. Our results show that expression of dMyc in the FB affects development and animal size. Expression of dMyc, but not of CycD/cdk4 or Rheb, in the FB diminishes the ability to retain Drosophila Insulin-like peptide-2 (DILP2) in the brain during starvation, suggesting that expression of dMyc mimics the signal that remotely controls the release of Dilps into the hemolymph. dMyc also affects glucose metabolism and increases the transcription of Glucose-transporter-1 mRNA, and of Hexokinase and Pyruvate-Kinase mRNAs, key regulators of glycolysis. These animals are able to counteract the increased levels of circulating trehalose induced by a high sugar diet leading to the conclusion that dMyc activity in the FB promotes glucose disposal. dMyc expression induces cell autonomous accumulation of triglycerides, which correlates with increased levels of Fatty Acid Synthase and Acetyl CoA Carboxylase mRNAs, enzymes responsible for lipid synthesis. We also found the expression of Stearoyl-CoA desaturase, Desat1 mRNA significantly higher in FB overexpressing dMyc. Desat1 is an enzyme that is necessary for monosaturation and production of fatty acids, and its reduction affects dMyc's ability to induce fat storage and resistance to animal survival. In conclusion, here we present novel evidences for dMyc function in the Drosophila FB in controlling systemic growth. We discovered that dMyc expression triggers cell autonomous mechanisms that control glucose and lipid metabolism to favor the storage of nutrients (lipids and sugars). In addition, the regulation of Desat1 controls the synthesis of triglycerides in FB and this may affect the humoral signal that controls DILP2 release in the brain.
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http://dx.doi.org/10.1016/j.ydbio.2013.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712331PMC
July 2013

Neisseria infection of rhesus macaques as a model to study colonization, transmission, persistence, and horizontal gene transfer.

Proc Natl Acad Sci U S A 2013 Feb 4;110(8):3059-64. Epub 2013 Feb 4.

BIO5 Institute and Department of Immunobiology, University of Arizona, Tucson, AZ 85721, USA.

The strict tropism of many pathogens for man hampers the development of animal models that recapitulate important microbe-host interactions. We developed a rhesus macaque model for studying Neisseria-host interactions using Neisseria species indigenous to the animal. We report that Neisseria are common inhabitants of the rhesus macaque. Neisseria isolated from the rhesus macaque recolonize animals after laboratory passage, persist in the animals for at least 72 d, and are transmitted between animals. Neisseria are naturally competent and acquire genetic markers from each other in vivo, in the absence of selection, within 44 d after colonization. Neisseria macacae encodes orthologs of known or presumed virulence factors of human-adapted Neisseria, as well as current or candidate vaccine antigens. We conclude that the rhesus macaque model will allow studies of the molecular mechanisms of Neisseria colonization, transmission, persistence, and horizontal gene transfer. The model can potentially be developed further for preclinical testing of vaccine candidates.
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http://dx.doi.org/10.1073/pnas.1217420110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581930PMC
February 2013

N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae.

PLoS One 2011 22;6(6):e21373. Epub 2011 Jun 22.

Department of Immunobiology and the BIO5 Institute, University of Arizona, Tucson, Arizona, United States of America.

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021373PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120873PMC
November 2011

Label-free protein profiling of adipose-derived human stem cells under hyperosmotic treatment.

J Proteome Res 2011 Jul 14;10(7):3050-9. Epub 2011 Jun 14.

Department of Biomedical Engineering, Columbia University, New York, New York, USA.

Our previous work suggested that treatment of cells with hyperosmotic media during 2D passaging primes cells for cartilage tissue engineering applications. Here, we used label-free proteomic profiling to evaluate the effects of control and hyperosmotic treatment environments on the phenotype of multipotent adipose-derived stem cells (ASCs) cultivated with a chondrogenic growth factor cocktail. Spectra were recorded in a data-independent fashion at alternate low (precursor) and high (product) fragmentation voltages (MS(E)). This method was supplemented with data mining of accurate mass and retention time matches in precursor ion spectra across the experiment. The results indicated a complex cellular response to osmotic treatment, with a number of proteins differentially expressed between control and treated cell groups. The roles of some of these proteins have been documented in the literature as characteristic of the physiological states studied, especially aldose reductase (osmotic stress). This protein acted as a positive control in this work, providing independent corroborative validation. Other proteins, including 5'-nucleotidase and transgelin, have been previously linked to cell differentiation state. This study demonstrates that label-free profiling can serve as a useful tool in characterizing cellular responses to chondrogenic treatment regimes, recommending its use in optimization of cell priming protocols for cartilage tissue engineering.
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http://dx.doi.org/10.1021/pr200030vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153440PMC
July 2011

The C terminus of p53 binds the N-terminal domain of MDM2.

Nat Struct Mol Biol 2010 Aug 18;17(8):982-9. Epub 2010 Jul 18.

Department of Biological Sciences, Columbia University, New York, New York, USA.

The p53 tumor suppressor interacts with its negative regulator Mdm2 via the former's N-terminal region and core domain, yet the extreme p53 C-terminal region contains lysine residues ubiquitinated by Mdm2 and can bear post-translational modifications that inhibit Mdm2-p53 association. We show that the Mdm2-p53 interaction is decreased upon deletion, mutation or acetylation of the p53 C terminus. Mdm2 decreases the association of full-length but not C-terminally deleted p53 with a DNA target sequence in vitro and in cells. Further, using multiple approaches, we show that a peptide from the p53 C terminus directly binds the Mdm2 N terminus in vitro. We also show that p300-acetylated p53 inefficiently binds Mdm2 in vitro, and Nutlin-3 treatment induces C-terminal modification(s) of p53 in cells, explaining the low efficiency of Nutlin-3 in dissociating p53-MDM2 in vitro.
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http://dx.doi.org/10.1038/nsmb.1872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922928PMC
August 2010

Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli.

Science 2008 May;320(5878):935-8

Department of Biochemistry, New York University School of Medicine, New York, NY 10016, USA.

Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes.
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http://dx.doi.org/10.1126/science.1152763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059013PMC
May 2008

A metabonomic and proteomic analysis of changes in IMCD3 cells chronically adapted to hypertonicity.

Nephron Physiol 2008 5;109(1):p1-10. Epub 2008 May 5.

Department of Anesthesiology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA.

Background: The genomic response to adaptation of IMCD3 cells to hypertonicity results in both upregulation and downregulation of a variety of genes.

Method: The present study was undertaken to assess the metabonomic and proteomic response of IMCD3 cells that have been chronically adapted to hypertonicity (600 and 900 mosm/kg H(2)O) as compared to cells under isotonic conditions.

Results: Adaptation of IMCD3 cells to hypertonic conditions resulted in a change of a wide range of organic osmolytes, including sorbitol (+8,291%), betaine (+1,099%), myo-inositol (+669%), taurine (+113%) and glycerophosphorylcholine (+61%). Evaluation of the polyol pathway for sorbitol production revealed a reduction in sorbitol dehydrogenase and an increase in aldose reductase mRNA in adapted cells. Proteome analysis revealed increased expression of six glycolytic proteins, including malic enzyme and pyruvate carboxylase, indicating the activation of the pyruvate shunt and changes in glucose metabolism. This study showed that the observed reduction in cell replication could possibly reflect a redirection of cellular energy from cell growth and replication to maintenance of intracellular ion levels in chronically adapted cells.

Conclusion: The combined metabonomic and proteomic analysis was shown to be a very helpful tool for the analysis of the effects caused by chronic adaptation to hypertonicity. It made it possible to better evaluate the importance of certain changes that occur in the process of adaptation.
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http://dx.doi.org/10.1159/000129074DOI Listing
August 2008

Expression of the calcium-binding protein S100A4 is markedly up-regulated by osmotic stress and is involved in the renal osmoadaptive response.

J Biol Chem 2007 Mar 2;282(9):6644-52. Epub 2007 Jan 2.

Division of Renal Diseases and Hypertension, School of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.

Proteomic analysis of Inner Medullary Collecting Duct (IMCD3) cells adapted to increasing levels of tonicity (300, 600, and 900 mosmol/kg H(2)O) by two-dimensional difference gel electrophoresis and mass spectrometry revealed several proteins as yet unknown to be up-regulated in response to hypertonic stress. Of these proteins, one of the most robustly up-regulated (22-fold) was S100A4. The identity of the protein was verified by high pressure liquid chromatography-mass spectrometry. Western blot analysis confirmed increased expression with increased tonicity, both acute and chronic. S100A4 protein expression was further confirmed by immunocytochemical analysis. Cells grown in isotonic conditions showed complete absence of immunostaining, whereas chronically adapted IMCD3 cells had uniform cytoplasmic localization. The protein is also regulated in vivo as in mouse kidney tissues S100A4 expression was many -fold greater in the papilla as compared with the cortex and increased further in the papilla upon 36 h of thirsting. Increased expression of S100A4 was also observed in the medulla and papilla, but not the cortex of a human kidney. Data from Affymetrix gene chip analysis and quantitative PCR also revealed increased S100A4 message in IMCD3 cells adapted to hypertonicity. The initial expression of message increased at 8-10 h following exposure to acute sublethal hypertonic stress (550 mosmol/kg H(2)O). Protein and message half-life in IMCD3 cells were 85.5 and 6.8 h, respectively. Increasing medium tonicity with NaCl, sucrose, mannitol, and choline chloride stimulated S100A4 expression, whereas urea did not. Silencing of S100A4 expression using a stable siRNA vector (pSM2; Open Biosystems) resulted in a 48-h delay in adaptation of IMCD3 cells under sublethal osmotic stress, suggesting S100A4 is involved in the osmoadaptive response. In summary, we describe the heretofore unrecognized up-regulation of a small calcium-binding protein, both in vitro and in vivo, whose absence profoundly delays osmoadaptation and slows cellular growth under hypertonic conditions.
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http://dx.doi.org/10.1074/jbc.M609432200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2547994PMC
March 2007
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