Showing papers by "Peter G. Schultz published in 2010"

Adding New Chemistries to the Genetic Code

Abstract: The development of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs has led to the addition of approximately 70 unnatural amino acids (UAAs) to the genetic codes of Escherichia coli, yeast, and mammalian cells. These UAAs represent a wide range of structures and functions not found in the canonical 20 amino acids and thus provide new opportunities to generate proteins with enhanced or novel properties and probes of protein structure and function.

Aryl Hydrocarbon Receptor Antagonists Promote the Expansion of Human Hematopoietic Stem Cells

Abstract: Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

A facile system for encoding unnatural amino acids in mammalian cells

Abstract: This invention provides translation system components functional in both eubacterial and eukaryotic environments. The translation system components, such as aminoacyl-tRNA synthetases and tRNAs derived from Methanosarcina species are capable of charging unnatural amino acids, and can be shuttled from enterobacteria to mammalian cells.

High-Throughput Chemical Screen Identifies a Novel Potent Modulator of Cellular Circadian Rhythms and Reveals CKIα as a Clock Regulatory Kinase

Abstract: The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ∼120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.

Telomere-independent Rap1 is an IKK adaptor and regulates NF-kappaB-dependent gene expression.

Abstract: We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

Correction for Site-directed spin labeling of a genetically encoded unnatural amino acid

Abstract: The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.

Combination Therapy Targeting Both Tumor-Initiating and Differentiated Cell Populations in Prostate Carcinoma

Abstract: Purpose: The cancer stem cell hypothesis predicts that standard prostate cancer monotherapy eliminates bulk tumor cells but not a tumor-initiating cell population, eventually leading to relapse. Many studies have sought to determine the underlying differences between bulk tumor and cancer stem cells. Experimental Design: Our previous data suggest that the PTEN/PI3K/AKT pathway is critical for the in vitro maintenance of CD133 + /CD44 + prostate cancer progenitors and, consequently, that targeting PI3K signaling may be beneficial in treatment of prostate cancer. Results: Here, we show that inhibition of PI3K activity by the dual PI3K/mTOR inhibitor NVP-BEZ235 leads to a decrease in the population of CD133 + /CD44 + prostate cancer progenitor cells in vivo . Moreover, the combination of the PI3K/mTOR modulator NVP-BEZ235, which eliminates prostate cancer progenitor populations, and the chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors in a prostate cancer xenograft model than monotherapy. Conclusion: This combination treatment ultimately leads to the expansion of cancer progenitors with a PTEN E91D mutation, suggesting that the analysis of PTEN mutations could predict therapeutic response to the dual therapy. Clin Cancer Res; 16(23); 5692–702. ©2010 AACR.

A small molecule accelerates neuronal differentiation in the adult rat

Abstract: Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain. However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brain and resulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention directed at endogenous NPCs.

Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency

Abstract: Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFβ/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.

Site-specific labeling of proteins with NMR-active unnatural amino acids

Abstract: A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

Identification of RING finger protein 4 (RNF4) as a modulator of DNA demethylation through a functional genomics screen

Abstract: DNA methylation is an important epigenetic modification involved in transcriptional regulation, nuclear organization, development, aging, and disease. Although DNA methyltransferases have been characterized, the mechanisms for DNA demethylation remain poorly understood. Using a cell-based reporter assay, we performed a functional genomics screen to identify genes involved in DNA demethylation. Here we show that RNF4 (RING finger protein 4), a SUMO-dependent ubiquitin E3-ligase previously implicated in maintaining genome stability, plays a key role in active DNA demethylation. RNF4 reactivates methylation-silenced reporters and promotes global DNA demethylation. Rnf4 deficiency is embryonic lethal with higher levels of methylation in genomic DNA. Mechanistic studies show that RNF4 interacts with and requires the base excision repair enzymes TDG and APE1 for active demethylation. This activity appears to occur by enhancing the enzymatic activities that repair DNA G:T mismatches generated from methylcytosine deamination. Collectively, our study reveals a unique function for RNF4, which may serve as a direct link between epigenetic DNA demethylation and DNA repair in mammalian cells.

Genetic incorporation of unnatural amino acids into proteins in Mycobacterium tuberculosis.

Abstract: New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNATyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.

A Genetically Encoded ε-N-Methyl Lysine in Mammalian Cells

Abstract: The posttranslational methylation of lysine modulates the activity, stability, localization and biomolecular interactions of many eukaryotic proteins. For example, monomethylation of lysine 372 in the mammalian tumor suppressor p53 has been shown to affect protein stability and localization[1]. Protein methylation plays a particularly important role in gene expression due to its involvement in the histone code, in which specific modifications to histone proteins modulate the transcriptional status of specific genes. Methylation of distinct histone lysine residues has been correlated with both transcriptional activation and repression depending on the lysine modified.[2] To better understand the functional consequences of lysine methylation, methods are needed to generate proteins with defined methylation status both in vitro and in living cells.

A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression.

Abstract: During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 → R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2W56R can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3′-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP23′-UTR. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.

A New Strategy to Photoactivate Green Fluorescent Protein

Abstract: Photoactivatable fluorescent proteins have become an important addition to the set of molecular probes used to understand cellular function. Known as “molecular highlighters”, their fluorescence is switched on by irradiation, thereby enabling non-invasive tracking of protein trafficking and dynamics. They are also the basis for the imaging technique called photoactivated light microscopy (PALM) in which multiple emitted photons are observed from individual active fluorophores which are sequentially activated from a large pool of inactive proteins and then photobleached. By locating the center of the point spread function for these emitted photons, it is possible to determine the location of the active fluorophore at better resolution than the theoretical diffraction limit. Previous green fluorescent protein (GFP) mutants exhibiting photomodulatory behavior have been reported including Kaede and KikGR whose emission wavelength change irreversibly, Dronpa whose fluorescence can be reversibly activated with light, and photoactivatable GFP (paGFP), whose excitation wavelength can be irreversibly changed with light. For Kaede, KikGR, and Dronpa, the precise three-dimensional structure of the fluorophore must be preserved to maintain photoswitching behavior. In paGFP, the shift in excitation wavelength is mediated by a light dependent decarboxylation of Glu222. The loss of this carboxy group is believed to cause reorientation of an internal hydrogen bond network, which changes the protonation state of the fluorophore and leads to an irreversible shift in the excitation maximum from 397 nm to 475 nm. This mechanism of GFP photoactivation requires the preservation of active-site residues His203, His148, Ser205, and Glu222. Unfortunately, the sequence restrictions necessary to maintain photomodulatory behavior are not always compatible with the mutations necessary to produce other fluorescent protein variants including YFP and RFP. In addition, Kaede, KikGR, and paGFP exist in two forms with differing excitation and emission wavelengths, precluding the simultaneous use of these wavelengths for other purposes. To address these problems, we have developed a general strategy for photoactivating GFP based upon nonnatural amino acid mutagenesis with the photocaged tyrosine analogue o-nitrobenzyl-O-tyrosine (ONBY). Replacing the fluorophore tyrosine 66 with ONBY yields a GFP molecule that is non-fluorescent as observed for other onitrobenzyl appended fluorophores including fluoresceine, Texas Red, and quantum dots. An earlier nonnatural variant of GFP was produced using a similar strategy, however, the high pre-irradiation background fluorescence and low protein yield made this unsuitable for use as a molecular marker. Fast, time-resolved UV/Vis spectroscopy measurements indicate that the fluorescence quenching likely occurs through photo-induced electron transfer (PET). Irradiation at 365 nm is sufficient to remove the o-nitrobenzyl

Mesenchymal stem cell differentiation

Abstract: The present invention provides for methods and compositions for treating or preventing arthritis and joint injury.

Directed embryonic stem cell differentiation with small molecules.

Abstract: Embryonic stem cells (ESCs) are a promising cell source for regenerative medicine and transplantation therapy.ESCs are able to self-renew indefinitely in culture; however, the ability to differentiate ESCs into specific cell lineages is key to exploiting their therapeutic potential. Cell-based phenotypic and reporter-based screens have been used to identify small molecules that selectively promote ESC differentiation into a variety of cell lineages. Not only will such molecules facilitate the clinical applications of stem cells, the detailed study of their mechanism is providing new insights into the biology that regulates ESC self-renewal and differentiation. In this article we discuss key issues, challenges and opportunities in the application of this chemical approach to stem cell biology.

Differentiation of mesenchymal stem cells

Abstract: A polypeptide for use in a method to ameliorate or prevent arthritis or joint injuries in a mammal, wherein the method comprises administering to the mammal an effective amount of a polypeptide selected from: (a) a polypeptide consisting of SEQ ID NO: 1 or 25; (A) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1 or 25; (B) a polypeptide consisting of SEQ ID NOs: 2, 3, 4, 26, 27 or 28; (C) a polypeptide comprising an amino acid sequence of SEQ ID NOs: 2, 3, 4, 26, 27 or 28; (D) a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NOs: 5, 9, 13, 17 or 21; or (e) a polypeptide comprising an amino acid sequence of SEQ ID NOs: 5, 9, 13, 17 or 21 wherein said polypeptide has chondrogenic activity.

The Scripps Research Institute (La Jolla, CA)

Abstract: The invention provides proteins attached to solid supports, and methods of preparing such solid support-bound proteins are provided. The proteins are attached to solid supports by means of an unnatural amino acid incorporated into the protein, which unnatural amino acid includes a reactive group that can react with a second reactive group that is attached to a solid support.

Progress Toward an Antibody Glycosidase.

Abstract: The piperidine-based hapten 1 generates monoclonal antibodies that catalyze the hydrolysis of substrate 2 at pH5.5 (k_(cal) = 0.904 h^(−1), K_M = 324 μM). Hapten 1 is a competitive inhibitor of the antibody-catalyzed reaction. These results are significant for the development of antibodies for the sequence-specific hydrolysis of oligosaccharides.