Biomedical subjects

Peter G Schultz

Publications and source records attributed to Peter G Schultz.

At least 19 recordsLinked to original sources

PubMed2025

A chemical epigenetic tool to probe site-specific DNA-binding protein complexes.

Site-specific DNA binding by proteins is critical for regulating transcriptional activity and cell fate decision. However, identifying proteins bound to specific genomic regions (e.g., promoter or enhancer regions) remains challenging. To address this, we developed a chemical epigenetic tool, named Site-specific noncanonical amino acid-mediated capture of protein (SCOPE), incorporating a photo-crosslinking amino acid into a nuclease-deficient dCas9 mutant. Human pluripotent stem cells (hPSCs) carrying SCOPE enable the capture of proteins bound to, in theory, any genomic location, facilitating the study of the cell context-dependent DNA-protein interactions. Using SCOPE, we identified the OCT4/SOX2/CARHSP1 complex binding to the NANOG promoter to maintain pluripotency in hPSCs. During ectoderm differentiation, ZIC2 acts as a competitive inhibitor, binding the same promoter region to downregulate NANOG expression and promote differentiation. Additionally, SCOPE identified that ZNF8 binds to the distal regulatory region of OCT4 to maintain naïve pluripotency. In summary, SCOPE provides a robust system for uncovering cell context-dependent, site-specific genome regulators, offering valuable insights into gene regulation networks driving cell fate transitions.

Humans↗

PubMed2025

An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

Bacteriophage T7↗

PubMed2024

An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 β-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.

Journal Article↗

PubMed2003

A rugged energy landscape mechanism for trapping of transmembrane receptors during endocytosis.

Efficient clathrin-mediated endocytosis of transmembrane receptors requires that clathrin-coated pits retain the receptors long enough to allow vesicle formation and internalization. In many cases, however, the receptors can exhibit mean lifetimes in coated pits much shorter than the lifetime of the pit at the plasma membrane. A rugged energy landscape for binding, which produces a broad distribution of residence times, ensures a significant probability of times much greater than the mean and would allow efficient trapping of proteins in these cases. We used fluorescence correlation spectroscopy and total internal reflection microscopy to measure the kinetics of movement of a C5a receptor-yellow fluorescent protein fusion in living cells. These experiments demonstrate that clusters of trapped receptors exhibit fluctuations in fluorescence intensity that vary in time scale over 2 orders of magnitude. Most of the variation in intensity is likely due to the motion of the receptors in the plane of the plasma membrane, although it is not possible to rule out a small contribution from motion orthogonal to the plane of the membrane. The broad time scale distribution of the intensity fluctuations is consistent with a rugged energy landscape mechanism for trapping of the receptors. This mechanism, which allows efficient trapping to coexist with rapid exchange, may also be relevant to other biological processes involving binding in heterogeneous chemical environments.

Antigens, CD↗

PubMed2003

A method for the generation of glycoprotein mimetics.

A general method for preparing glycoprotein mimetics with defined glycan structure using the Z domain protein as an example is reported. An unnatural amino acid containing the keto group was site-specifically incorporated into a target protein, Z domain, in response to the amber nonsense codon with high translational fidelity and efficiency. An aminooxy saccharide derivative was then selectively coupled to this genetically encoded keto group. The resulting saccharide core was elaborated to a glycan complex with glycosyltransferases. Alternatively, aminooxy analogues of more complicated glycans were prepared and directly attached to the keto group. Homogeneous glycoprotein mimetics thus prepared should prove useful for the study of carbohydrate effects on glycoprotein structure and function. This method may also lead to the production of glycoprotein therapeutics from Escherichia coli.

Acetylglucosamine↗

PubMed2003

Generation of a bacterium with a 21 amino acid genetic code.

We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.

Amino Acyl-tRNA Synthetases↗

PubMed2003

Structural evidence for substrate strain in antibody catalysis.

The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-A resolution. The antibody-bound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration. The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved. A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.

Antibodies↗

PubMed2003

Small-molecule switches for zinc finger transcription factors.

We have developed a general approach to generate selective small-molecule switches for zinc finger transcription factors based on the notion of structural complementation. A small molecule, 2-(4'-quinoline)benzimidazole (7), was identified by screening of a heterocycle library, which functionally rescues a structurally compromised transcription factor C7-F116A/H125G-VP16. Compound 7 induces the activity of the mutant zinc finger in a luciferase-based transactivation assay by about 18-fold at 100 muM concentration. Surface plasmon resonance measurements revealed that compound 7 enhances the affinity between the mutant C7 and its cognate DNA sequence by 16-fold while a structurally analogous compound, 2-(2'-naphthyl)benzimidazole (8), showed no affinity enhancement, indicating there is a specific interaction between compound 7 and the mutant zinc finger. Taken together, these results suggest that the partial affinity recovery of the mutant transcription factor may be the mechanism for the functional rescue of the zinc finger transcription factor by compound 7.

Amino Acid Sequence↗

PubMed2003

Unnatural amino acid mutagenesis of green fluorescent protein.

Unnatural amino acid mutagenesis has been used to selectively substitute tyrosine 66 of green fluorescent protein (GFP) with five novel amino acids: p-amino-L-phenylalanine, p-methoxy-L-phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, and L-3-(2-naphthyl)alanine. The absorbance and emission maxima of the resulting mutant GFPs span the range from 375 to 435 nm and 428 to 498 nm, respectively. The spectral properties of the mutant GFPs, including the absorbance and fluorescence maxima and quantum yields, correlate with the structural and electronic properties of the substituents on the amino acids.

Amino Acid Substitution↗

PubMed2003

Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.

Amino Acid Sequence↗

PubMed2003

Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes.

We have previously shown that alpha/beta interferon (IFN-alpha/beta) and IFN-gamma inhibit hepatitis B virus (HBV) replication noncytopathically in the livers of HBV transgenic mice and in hepatocyte cell lines derived from these mice. The present study was designed to identify transcriptionally controlled hepatocellular genes that are tightly associated with the inhibition of HBV replication and that might, therefore, mediate the antiviral effect of these cytokines. Twenty-nine genes were identified, many of which have known or potential antiviral activity. Notably, multiple components of the immunoproteasome and ubiquitin-like proteins were strongly induced by both IFN-alpha/beta and IFN-gamma, as were a number of GTP-binding proteins, including GTPases with known antiviral activity, chemokines, signaling molecules, and miscellaneous genes associated with antigen processing, DNA-binding, or cochaperone activity and several expressed sequence tags. The results suggest that one or more members of this relatively small subset of genes may mediate the antiviral effect of IFN-alpha/beta and IFN-gamma against HBV. We have already exploited this information by demonstrating that the antiviral activity of IFN-alpha/beta and IFN-gamma is proteasome dependent.

Animals↗

PubMed2002

Addition of the keto functional group to the genetic code of Escherichia coli.

Although the keto group is the most versatile of the functional groups in organic chemistry, it is absent in the genetically encoded amino acids. To overcome this natural limitation on protein biosynthesis, we have evolved an orthogonal tRNA-synthetase pair that makes possible the efficient incorporation of a keto amino acid, p-acetyl-l-phenylalanine, into proteins in E. coli with high translational fidelity in response to the amber nonsense codon. To demonstrate the utility of this keto amino acid, we have used it to modify a protein selectively with a small molecule fluorophore and biotin derivative. This additional genetically encoded amino acid should greatly expand our ability to manipulate protein structure and function both in vitro and in living cells.

Amino Acid Substitution↗

PubMed2002

A potent and highly selective sulfotransferase inhibitor.

In the present work, we have used a newly developed, fluorescence-based assay to screen a library of >30 000 compounds as potential beta-arylsulfotransferase-IV inhibitors. A total of 11 inhibitors were discovered. Most of the compounds discovered showed low micromolar inhibition, but one of the compounds showed potent inhibition (Ki = 96 nM). The most potent of these inhibitors was tested against a variety of other purine binding enzymes and showed remarkable specificity.

Adenosine Diphosphate↗

PubMed2002

Enzymatic phosphorylation of unnatural nucleosides.

In an effort to expand the genetic alphabet, a number of unnatural, predominantly hydrophobic, nucleoside analogues have been developed which pair selectively in duplex DNA and during enzymatic synthesis. Significant progress has been made toward the efficient in vitro replication of DNA containing these base pairs. However, the in vivo expansion of the genetic alphabet will require that the unnatural nucleoside triphosphates be available within the cell at sufficient concentrations for DNA replication. We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes. The first step of this pathway is catalyzed by the D. melanogaster nucleoside kinase, which catalyzes the phosphorylation of nucleosides to the corresponding monophosphates. We demonstrate that each unnatural nucleoside is phosphorylated with a rate that should be sufficient for the in vivo replication of DNA.

Animals↗

PubMed2002

A small molecule with osteogenesis-inducing activity in multipotent mesenchymal progenitor cells.

Purmorphamine, which is a 2,6,9-trisubstituted purine compound, was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development.

Alkaline Phosphatase↗

PubMed2002

The chemistry of the antibody molecule.

Immunochemistry has traditionally focused on understanding the molecular basis of antibody binding affinity and specificity. In the last 20 years a major shift has occurred in the field. We are now asking how we can exploit the remarkable properties of the antibody molecule and the immune response in chemistry-from testing basic theories of enzymatic catalysis to the synthesis of new catalysts. Moreover, the concept of molecular diversity, which underlies these experiments, is now being applied to a large number of other problems-from libraries of biopolymers to libraries of solid-state materials. Importantly, theses studies are providing fundamental molecular insights into many biological, chemical, and physical phenomena, as well as changing the way we approach the synthesis of new molecular function.

Antibodies↗

PubMed2002

A novel silver(i)-mediated DNA base pair.

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base pairs dA:dT and dC:dG. UV-melting experiments revealed that the dSPy:dSPy self-pair can replace natural base pairs at multiple sites and still form stable DNA duplexes.

Base Pairing↗

PubMed2002

Genomic analysis of the host response to hepatitis C virus infection.

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-alpha response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-gamma-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

Animals↗