PubMed

Recent Publications

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Sci Rep. 2016 Nov 29;6:37942

Authors: Soloveychik M, Xu M, Zaslaver O, Lee K, Narula A, Jiang R, Rosebrock AP, Caudy AA, Meneghini MD

Abstract
Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2's impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation.

PMID: 27897198 [PubMed - in process]



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Brief Report: Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early-vs. late-stage OA synovial fluid.

Brief Report: Stage-specific differences in secretory profile of mesenchymal stromal cells (MSCs) subjected to early-vs. late-stage OA synovial fluid.

Osteoarthritis Cartilage. 2016 Nov 25;:

Authors: Gómez-Aristizábal A, Sharma A, Bakooshli MA, Kapoor M, Gilbert PM, Viswanathan S, Gandhi R

Abstract
OBJECTIVE: Although, mesenchymal stromal cells (MSCs) are being clinically investigated for their use in osteoarthritis (OA), it is unclear whether their postulated therapeutic properties are equally effective in the early- and late-stages of OA. In this study we investigated MSC cytokine secretion post-exposure to synovial fluid (SF), obtained from early-vs. late-stage knee OA patients to justify a potential patient stratification strategy to maximize MSC-mediated treatment effects.
METHOD: Subjects were recruited and categorized into early- [Kellgren-Lawrence (KL) grade I/II, n = 12] and late-stage (KL-III/IV, n = 12) knee OA groups. SF samples were obtained, and their proteome was tested using multiplex assays, after three-days culture, with and without MSCs. SFs cultured without MSCs were used as a baseline to identify MSC-secreted factors into SFs cultured with MSCs. Linear mixed-effect models and non-parametric tests were used to identify alterations in the MSC secretome during exposure to OA SF (three-days). MSCs cultured for three-days in 0.5% fetal bovine serum-supplemented medium were used to compare SF results with culture medium.
RESULTS: Following exposure to OA SF, the MSC secretome contained proteins that are involved in tissue repair, angiogenesis, chemotaxis, matrix remodeling and the clotting process. However, CXCL8 (chemokine (C-X-C motif) ligand-8; chemoattractant), interleukin-6 and CCL2 (chemokine (C-C motif) ligand 2) were elevated in the MSC-secretome in response to early-vs. late-stage OA SF.
CONCLUSION: Early-vs. late-stage OA SF samples elicit a differential MSC secretome response, arguing for stratification of OA patients to maximize MSC-mediated therapeutic effects.

PMID: 27894935 [PubMed - as supplied by publisher]



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A Highly Diverse and Functional Naïve Ubiquitin Variant Library for Generation of Intracellular Affinity Reagents.

A Highly Diverse and Functional Naïve Ubiquitin Variant Library for Generation of Intracellular Affinity Reagents.

J Mol Biol. 2016 Nov 22;:

Authors: Leung I, Jarvik N, Sidhu S

Abstract
We report the design, construction and validation of a highly diverse phage-displayed naïve ubiquitin variant (Ubv) library. We first conducted a mutation tolerance scan of 27 residues and confirmed that 24 of these could be substituted by chemically diverse amino acids without compromising the display of Ubvs on phage. Subsequently, we constructed a library containing 6.8×10(10) unique members in which these 24 positions were diversified with a degenerate codon that encodes for six amino acids that are prevalent in protein interaction sites. To ensure optimal structural stability of the Ubvs, the library was constructed in a two-step process whereby 12 positions were randomized first, and following selection for displayed Ubvs, the resulting pool was further diversified at the other 12 positions. The resulting library was validated by conducting binding selections against a panel of 40 diverse protein antigens and was found to be as functional as a highly validated synthetic antibody library, yielding binders against 30 of the antigens. Detailed characterization of an Ubv that bound to the cell-surface receptor Her3 revealed tight binding in the single-digit nanomolar range. Moreover, Ubvs that bound to two distinct sites on the intracellular adapter Grb2 could be combined to generate a potent inhibitor that functioned in cells. These results validate ubiquitin as a robust scaffold for the construction of naïve libraries that can be used to generate Ubvs that target signaling networks both outside and inside cells.

PMID: 27887869 [PubMed - as supplied by publisher]



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Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Protein-phosphotyrosine proteome profiling by superbinder-SH2 domain affinity purification mass spectrometry, sSH2-AP-MS.

Proteomics. 2016 Nov 23;:

Authors: Tong J, Cao B, Martyn GD, Krieger JR, Taylor P, Yates B, Sidhu SS, Li SS, Mao X, Moran MF

Abstract
Recently, "superbinder" SH2 domain variants with three amino acid substitutions (sSH2) were reported to have 100-fold or greater affinity for protein-phosphotyrosine (pY) than natural SH2 domains. Here we report a protocol in which His-tagged Src sSH2 efficiently captures pY-peptides from protease-digested HeLa cell total protein extracts. Affinity purification of pY-peptides by this method shows little bias for pY-proximal amino acid sequences, comparable to that achieved by using antibodies to pY, but with equal or higher yield. Superbinder-SH2 affinity purification mass spectrometry (sSH2-AP-MS) therefore provides an efficient and economical approach for unbiased pY-directed phospho-proteome profiling without the use of antibodies. This article is protected by copyright. All rights reserved.

PMID: 27880036 [PubMed - as supplied by publisher]



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Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

Related Articles

Large-Scale Interaction Profiling of Protein Domains Through Proteomic Peptide-Phage Display Using Custom Peptidomes.

Methods Mol Biol. 2017;1518:213-226

Authors: Seo MH, Nim S, Jeon J, Kim PM

Abstract
Protein-protein interactions are essential to cellular functions and signaling pathways. We recently combined bioinformatics and custom oligonucleotide arrays to construct custom-made peptide-phage libraries for screening peptide-protein interactions, an approach we call proteomic peptide-phage display (ProP-PD). In this chapter, we describe protocols for phage display for the identification of natural peptide binders for a given protein. We finally describe deep sequencing for the analysis of the proteomic peptide-phage display.

PMID: 27873209 [PubMed - in process]



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Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

Nat Med. 2016 Nov 21;:

Authors: Steinhart Z, Pavlovic Z, Chandrashekhar M, Hart T, Wang X, Zhang X, Robitaille M, Brown KR, Jaksani S, Overmeer R, Boj SF, Adams J, Pan J, Clevers H, Sidhu S, Moffat J, Angers S

Abstract
Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome. Our results uncover an underappreciated level of context-dependent specificity at the Wnt receptor level. We further derived a panel of recombinant antibodies that reports the expression of nine FZD proteins and confirms that FZD5 functional specificity cannot be explained by protein expression patterns. Additionally, antibodies that specifically bind FZD5 and FZD8 robustly inhibited the growth of RNF43-mutant PDAC cells grown in vitro and as xenografts in vivo, providing orthogonal support for the functional specificity observed genetically. Proliferation of a patient-derived PDAC cell line harboring an RNF43 variant was also selectively inhibited by the FZD5 antibodies, further demonstrating their use as a potential targeted therapy. Tumor organoid cultures from colorectal carcinoma patients that carried RNF43 mutations were also sensitive to the FZD5 antibodies, highlighting the potential generalizability of these findings beyond PDAC. Our results show that CRIPSR-based genetic screens can be leveraged to identify and validate cell surface targets for antibody development and therapy.

PMID: 27869803 [PubMed - as supplied by publisher]



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Engineering cell signaling modulators from native protein-protein interactions.

Engineering cell signaling modulators from native protein-protein interactions.

Curr Opin Struct Biol. 2016 Nov 17;45:25-35

Authors: Zhang W, Ben-David M, Sidhu SS

Abstract
Recent studies on genome sequencing and genetic screens with RNAi and CRISPR technology have revolutionized our understanding of aberrant signaling networks in human diseases. A strategy combining both genetic and protein-based technologies should be at the heart of modern drug-development efforts, particularly in the era of precision medicine. Thus, there is an urgent need for efficient platforms to develop probes that can modulate protein function in cells to validate drug targets and to develop therapeutic leads. Advanced protein engineering has enabled the rapid production of monoclonal antibodies and small protein scaffold affinity reagents for diverse protein targets. Here, we review the most recent progress on engineering natural protein-protein interactions for modulation of cell signaling.

PMID: 27866084 [PubMed - as supplied by publisher]



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Controlling DNA-nanoparticle serum interactions.

Related Articles

Controlling DNA-nanoparticle serum interactions.

Proc Natl Acad Sci U S A. 2016 Nov 16;:

Authors: Zagorovsky K, Chou LY, Chan WC

Abstract
Understanding the interaction of molecularly assembled nanoparticles with physiological fluids is critical to their use for in vivo delivery of drugs and contrast agents. Here, we systematically investigated the factors and mechanisms that govern the degradation of DNA on the nanoparticle surface in serum. We discovered that a higher DNA density, shorter oligonucleotides, and thicker PEG layer increased protection of DNA against serum degradation. Oligonucleotides on the surface of nanoparticles were highly resistant to DNase I endonucleases, and degradation was carried out exclusively by protein-mediated exonuclease cleavage and full-strand desorption. These results enabled the programming of the degradation rates of the DNA-assembled nanoparticle system from 0.1 to 0.7 h(-1) and the engineering of superstructures that can release two different preloaded dye molecules with distinct kinetics and half-lives ranging from 3.3 to 9.8 h. This study provides a general framework for investigating the serum stability of DNA-containing nanostructures. The results advance our understanding of engineering principles for designing nanoparticle assemblies with controlled in vivo behavior and present a strategy for storage and multistage release of drugs and contrast agents that can facilitate the diagnosis and treatment of cancer and other diseases.

PMID: 27856755 [PubMed - as supplied by publisher]



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Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes.

Transcription Factors Encoded on Core and Accessory Chromosomes of Fusarium oxysporum Induce Expression of Effector Genes.

PLoS Genet. 2016 Nov;12(11):e1006401

Authors: van der Does HC, Fokkens L, Yang A, Schmidt SM, Langereis L, Lukasiewicz JM, Hughes TR, Rep M

Abstract
Proteins secreted by pathogens during host colonization largely determine the outcome of pathogen-host interactions and are commonly called 'effectors'. In fungal plant pathogens, coordinated transcriptional up-regulation of effector genes is a key feature of pathogenesis and effectors are often encoded in genomic regions with distinct repeat content, histone code and rate of evolution. In the tomato pathogen Fusarium oxysporum f. sp. lycopersici (Fol), effector genes reside on one of four accessory chromosomes, known as the 'pathogenicity' chromosome, which can be exchanged between strains through horizontal transfer. The three other accessory chromosomes in the Fol reference strain may also be important for virulence towards tomato. Expression of effector genes in Fol is highly up-regulated upon infection and requires Sge1, a transcription factor encoded on the core genome. Interestingly, the pathogenicity chromosome itself contains 13 predicted transcription factor genes and for all except one, there is a homolog on the core genome. We determined DNA binding specificity for nine transcription factors using oligonucleotide arrays. The binding sites for homologous transcription factors were highly similar, suggesting that extensive neofunctionalization of DNA binding specificity has not occurred. Several DNA binding sites are enriched on accessory chromosomes, and expression of FTF1, its core homolog FTF2 and SGE1 from a constitutive promoter can induce expression of effector genes. The DNA binding sites of only these three transcription factors are enriched among genes up-regulated during infection. We further show that Ftf1, Ftf2 and Sge1 can activate transcription from their binding sites in yeast. RNAseq analysis revealed that in strains with constitutive expression of FTF1, FTF2 or SGE1, expression of a similar set of plant-responsive genes on the pathogenicity chromosome is induced, including most effector genes. We conclude that the Fol pathogenicity chromosome may be partially transcriptionally autonomous, but there are also extensive transcriptional connections between core and accessory chromosomes.

PMID: 27855160 [PubMed - in process]



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Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Kin17 facilitates multiple double-strand break repair pathways that govern B cell class switching.

Sci Rep. 2016 Nov 17;6:37215

Authors: Le MX, Haddad D, Ling AK, Li C, So CC, Chopra A, Hu R, Angulo JF, Moffat J, Martin A

Abstract
Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

PMID: 27853268 [PubMed - in process]



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