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1.
Methods Mol Biol ; 2676: 117-129, 2023.
Article in English | MEDLINE | ID: mdl-37277628

ABSTRACT

Phage display facilitates the evolution of peptides and proteins for affinity selection against targets, but it is mostly limited to the chemical diversity provided by the naturally encoded amino acids. The combination of phage display with genetic code expansion allows the incorporation of noncanonical amino acids (ncAAs) into proteins expressed on the phage. In this method, we describe incorporation of one or two ncAAs in a single-chain fragment variable (scFv) antibody in response to amber or quadruplet codon. We take advantage of the pyrrolysyl-tRNA synthetase/tRNA pair to incorporate a lysine derivative and an orthogonal tyrosyl-tRNA synthetase/tRNA pair to incorporate a phenylalanine derivative. The encoding of novel chemical functionalities and building blocks in proteins displayed on phage provides the foundation for further phage display applications in fields such as imaging, protein targeting, and the production of new materials.


Subject(s)
Amino Acyl-tRNA Synthetases , Bacteriophages , Amino Acids/chemistry , Lysine/metabolism , Codon , RNA, Transfer/genetics , Bacteriophages/genetics , Bacteriophages/metabolism , Amino Acyl-tRNA Synthetases/metabolism
2.
J Am Chem Soc ; 143(12): 4600-4606, 2021 03 31.
Article in English | MEDLINE | ID: mdl-33750116

ABSTRACT

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.


Subject(s)
Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Cell Line, Tumor , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Signal Transduction/drug effects
3.
Nat Methods ; 18(3): 240-241, 2021 03.
Article in English | MEDLINE | ID: mdl-33637971
4.
Neuron ; 103(3): 412-422.e4, 2019 08 07.
Article in English | MEDLINE | ID: mdl-31221560

ABSTRACT

Selective synaptic and axonal degeneration are critical aspects of both brain development and neurodegenerative disease. Inhibition of caspase signaling in neurons is a potential therapeutic strategy for neurodegenerative disease, but no neuron-specific modulators of caspase signaling have been described. Using a mass spectrometry approach, we discovered that RUFY3, a neuronally enriched protein, is essential for caspase-mediated degeneration of TRKA+ sensory axons in vitro and in vivo. Deletion of Rufy3 protects axons from degeneration, even in the presence of activated CASP3 that is competent to cleave endogenous substrates. Dephosphorylation of RUFY3 at residue S34 appears required for axon degeneration, providing a potential mechanism for neurons to locally control caspase-driven degeneration. Neuronally enriched RUFY3 thus provides an entry point for understanding non-apoptotic functions of CASP3 and a potential target to modulate caspase signaling specifically in neurons for neurodegenerative disease.


Subject(s)
Axons/pathology , Nerve Degeneration/pathology , Nerve Tissue Proteins/physiology , Animals , Axons/enzymology , Caspase 3/physiology , Cells, Cultured , Cytoskeletal Proteins , Enzyme Activation , Ganglia, Spinal/cytology , Ganglia, Spinal/embryology , Mice , Mice, Knockout , Nerve Degeneration/enzymology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/deficiency , Phosphorylation , Protein Processing, Post-Translational , Receptor, trkA/physiology , Sensory Receptor Cells/physiology , Structure-Activity Relationship
5.
Cell Chem Biol ; 24(10): 1250-1258.e4, 2017 Oct 19.
Article in English | MEDLINE | ID: mdl-28919041

ABSTRACT

To dissect the cellular roles of individual kinases, it is useful to design tools for their selective activation. We describe the engineering of a split-cAbl kinase (sKin-Abl) that is rapidly activated in cells with rapamycin and allows temporal, dose, and compartmentalization control. Our design strategy involves an empirical screen in mammalian cells and identification of split site in the N lobe. This split site leads to complete loss of activity, which can be restored upon small-molecule-induced dimerization in cells. Remarkably, the split site is transportable to the related Src Tyr kinase and the distantly related Ser/Thr kinase, AKT, suggesting broader applications to kinases. To quantify the fold induction of phosphotyrosine (pTyr) modification, we employed quantitative proteomics, NeuCode SILAC. We identified a number of known Abl substrates, including autophosphorylation sites and novel pTyr targets, 432 pTyr sites in total. We believe that this split-kinase technology will be useful for direct activation of protein kinases in cells.


Subject(s)
Protein Engineering , Proto-Oncogene Proteins c-abl/metabolism , Enzyme Activation/drug effects , HEK293 Cells , Humans , Phosphorylation , Phosphotyrosine/metabolism , Proto-Oncogene Proteins c-abl/genetics , Sirolimus/pharmacology , src-Family Kinases/genetics
6.
Nat Chem Biol ; 12(9): 680-5, 2016 09.
Article in English | MEDLINE | ID: mdl-27376690

ABSTRACT

Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.


Subject(s)
Ferrous Compounds/analysis , Ferrous Compounds/metabolism , Fluorescent Dyes/analysis , Fluorescent Dyes/chemistry , Fluorescent Antibody Technique , Fluorescent Dyes/chemical synthesis , Humans , Molecular Structure , Puromycin/chemistry , Puromycin/pharmacology , Spiro Compounds/analysis , Spiro Compounds/chemical synthesis , Spiro Compounds/chemistry
7.
Proc Natl Acad Sci U S A ; 112(27): 8344-9, 2015 Jul 07.
Article in English | MEDLINE | ID: mdl-26106156

ABSTRACT

Cellular demolition during apoptosis is completed by executioner caspases, that selectively cleave more than 1,500 proteins but whose individual roles are challenging to assess. Here, we used an optimized site-specific and inducible protease to examine the role of a classic apoptotic node, the caspase-activated DNase (CAD). CAD is activated when caspases cleave its endogenous inhibitor ICAD, resulting in the characteristic DNA laddering of apoptosis. We describe a posttranscriptional gene replacement (PTGR) approach where endogenous biallelic ICAD is knocked down and simultaneously replaced with an engineered allele that is susceptible to inducible cleavage by tobacco etch virus protease. Remarkably, selective activation of CAD alone does not induce cell death, although hallmarks of DNA damage are detected in human cancer cell lines. Our data strongly support that the highly cooperative action of CAD and inhibition of DNA repair systems are critical for the DNA laddering phenotype in apoptosis. Furthermore, the PTGR approach provides a general means for replacing wild-type protein function with a precisely engineered mutant at the transcriptional level that should be useful for cell engineering studies.


Subject(s)
Apoptosis , Caspases/metabolism , Deoxyribonucleases/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Cycle , Cell Survival , Deoxyribonucleases/genetics , Flow Cytometry , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Mutation , Proteolysis , Reverse Transcriptase Polymerase Chain Reaction , Transfection
8.
Methods Enzymol ; 544: 179-213, 2014.
Article in English | MEDLINE | ID: mdl-24974291

ABSTRACT

Caspases, aspartate-specific cysteine proteases, have fate-determining roles in many cellular processes including apoptosis, differentiation, neuronal remodeling, and inflammation (for review, see Yuan & Kroemer, 2010). There are a dozen caspases in humans alone, yet their individual contributions toward these phenotypes are not well understood. Thus, there has been considerable interest in activating individual caspases or using their activity to drive these processes in cells and animals. We envision that such experimental control of caspase activity can not only afford novel insights into fundamental biological problems but may also enable new models for disease and suggest possible routes to therapeutic intervention. In particular, localized, genetic, and small-molecule-controlled caspase activation has the potential to target the desired cell type in a tissue. Suppression of caspase activation is one of the hallmarks of cancer and thus there has been significant enthusiasm for generating selective small-molecule activators that could bypass upstream mutational events that prevent apoptosis. Here, we provide a practical guide that investigators have devised, using genetics or small molecules, to activate specific caspases in cells or animals. Additionally, we show genetically controlled activation of an executioner caspase to target the function of a defined group of neurons in the adult mammalian brain.


Subject(s)
Caspases/genetics , Caspases/metabolism , Enzyme Activation/drug effects , Small Molecule Libraries/pharmacology , Animals , Apoptosis/drug effects , Caspases/analysis , Cell Engineering/methods , Cloning, Molecular/methods , Humans , Models, Molecular , Neurons/cytology , Neurons/enzymology , Neurons/metabolism , Protein Multimerization/drug effects , Small Molecule Libraries/chemistry
9.
Brain Res ; 1033(2): 117-27, 2005 Feb 08.
Article in English | MEDLINE | ID: mdl-15694915

ABSTRACT

The axons of sacral parasympathetic preganglionic neurons (PGNs) originate on a primary dendrite between 10 and 110 mum from the soma. Therefore, it was hypothesized that the location of the axon origin would impact the relative efficacy of ipsilateral and contralateral synaptic inputs. The morphology of two PGNs was reconstructed, and the transfer impedance was used to quantify the influence of synaptic inputs on the transmembrane potential at the axon initial segment. The ratio of ipsilateral transfer impedance to contralateral transfer impedance (termed the relative gain) was increased by 14-29% for axons originating from the dendrite vs. axons originating from the soma. The addition of 50 synchronized "gating" synapses on the proximal dendrites increased the relative gain by 17-38% when the axon originated from the dendrite, but only by 11-15% when the axon originated from the soma. The efficacy of synaptic inputs and the ability of proximal gating synapses to regulate synaptic efficacy were strongly influenced by the site of origin of the axon. The position of axon origin is an effective structural mechanism to regulate the relative efficacy of synaptic inputs arriving at different locations on the dendritic tree.


Subject(s)
Action Potentials/physiology , Dendrites/physiology , Synapses/physiology , Animals , Axons/physiology , Cats , Cell Size
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