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1.
Nat Methods ; 21(3): 406-410, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38253843

ABSTRACT

The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.


Subject(s)
Eukaryota , Luminescence , Animals , Mammals
2.
Nat Biomed Eng ; 8(2): 193-200, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37996615

ABSTRACT

Owing to the immunogenicity of adeno-associated viruses (AAVs), gene therapies using AAVs face considerable obstacles. Here, by leveraging ex vivo T-cell assays, the prediction of epitope binding to major histocompatibility complex class-II alleles, sequence-conservation analysis in AAV phylogeny and site-directed mutagenesis, we show that the replacement of amino acid residues in a promiscuous and most immunodominant T-cell epitope in the AAV9 capsid with AAV5 sequences abrogates the immune responses of peripheral blood mononuclear cells to the chimaeric vector while preserving its functions, potency, cellular specificity, transduction efficacy and biodistribution. This rational approach to the immunosilencing of capsid epitopes promiscuously binding to T cells may be applied to other AAV vectors and epitope regions.


Subject(s)
Capsid , Dependovirus , Capsid/chemistry , Capsid/metabolism , Dependovirus/genetics , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/analysis , Epitopes, T-Lymphocyte/metabolism , Leukocytes, Mononuclear , Tissue Distribution , Capsid Proteins/genetics , Capsid Proteins/chemistry , Capsid Proteins/metabolism
3.
Org Biomol Chem ; 17(46): 9906-9912, 2019 11 27.
Article in English | MEDLINE | ID: mdl-31720665

ABSTRACT

Collagen is an abundant component of the extracellular matrix and connective tissues. Some collagen-mimetic peptides (CMPs) that do not form homotrimers can anneal to damaged tissue. Here, through a computational screen, we identify (flpHypGly)7 as an optimal monomeric CMP for heterotrimer formation. We find that (flpHypGly)7 forms stable triple helices with (ProProGly)7 but not with itself. The nonnatural amino acid HflpOH, which is (2S,4S)-4-fluoroproline, is not toxic to human fibroblasts or keratinocytes. Conjugation of (flpHypGly)7 to a fluorescent dye enables the facile detection of burned collagenous tissue with high specificity. The ubiquity of collagen and the prevalence of injuries and diseases that disrupt endogenous collagen suggests widespread utility for this approach.


Subject(s)
Burns/diagnosis , Collagen/chemistry , Peptides/chemistry , Humans , Models, Molecular
4.
J Biol Chem ; 294(41): 15095-15103, 2019 10 11.
Article in English | MEDLINE | ID: mdl-31431502

ABSTRACT

The angiogenin (ANG) gene is mutated frequently in individuals with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Delivering human ANG to mice that display ALS-like symptoms extends their lifespan and improves motor function. ANG is a secretory vertebrate RNase that enters neuronal cells and cleaves a subset of tRNAs, leading to the inhibition of translation initiation and the assembly of stress granules. Here, using murine neuronal and astrocytic cell lines, we find that ANG triggers the activation of the Nrf2 (nuclear factor erythroid 2-related factor 2) pathway, which provides a critical cellular defense against oxidative stress. This activation, which occurred in astrocytes but not in neurons, promoted the survival of proximal neurons that had oxidative injury. These findings extend the role of ANG as a neuroprotective agent and underscore its potential utility in ALS management.


Subject(s)
Antioxidant Response Elements/drug effects , Astrocytes/drug effects , Astrocytes/metabolism , NF-E2-Related Factor 2/metabolism , Neurons/drug effects , Oxidative Stress/drug effects , Ribonuclease, Pancreatic/pharmacology , Animals , Astrocytes/cytology , Cell Line , Mice , Neurons/cytology , Neurons/metabolism , Neuroprotective Agents/pharmacology , Signal Transduction/drug effects
5.
Mol Cancer Ther ; 17(12): 2622-2632, 2018 12.
Article in English | MEDLINE | ID: mdl-30282811

ABSTRACT

Pancreatic-type ribonucleases (ptRNases) are prevalent secretory enzymes that catalyze the cleavage of RNA. Ribonuclease inhibitor (RI) is a cytosolic protein that has femtomolar affinity for ptRNases, affording protection from the toxic catalytic activity of ptRNases, which can invade human cells. A human ptRNase variant that is resistant to inhibition by RI is a cytotoxin that is undergoing a clinical trial as a cancer chemotherapeutic agent. We find that the ptRNase and protein kinases in the ERK pathway exhibit strongly synergistic toxicity toward lung cancer cells (including a KRASG12C variant) and melanoma cells (including BRAFV600E variants). The synergism arises from inhibiting the phosphorylation of RI and thereby diminishing its affinity for the ptRNase. These findings link seemingly unrelated cellular processes, and suggest that the use of a kinase inhibitor to unleash a cytotoxic enzyme could lead to beneficial manifestations in the clinic.


Subject(s)
MAP Kinase Signaling System , Mutation/genetics , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/toxicity , Ribonucleases/antagonists & inhibitors , Ribonucleases/genetics , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , HEK293 Cells , Humans , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinase Kinases/metabolism , Models, Biological , Phosphorylation/drug effects , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Substrate Specificity/drug effects
6.
RNA ; 24(8): 1018-1027, 2018 08.
Article in English | MEDLINE | ID: mdl-29748193

ABSTRACT

Angiogenin (ANG) is a secretory ribonuclease that promotes the proliferation of endothelial cells, leading to angiogenesis. This function relies on its ribonucleolytic activity, which is low for simple RNA substrates. Upon entry into the cytosol, ANG is sequestered by the ribonuclease inhibitor protein (RNH1). We find that ANG is a potent cytotoxin for RNH1-knockout HeLa cells, belying its inefficiency as a nonspecific catalyst. The toxicity does, however, rely on the ribonucleolytic activity of ANG and a cytosolic localization, which lead to the accumulation of particular tRNA fragments (tRFs), such as tRF-5 Gly-GCC. These up-regulated tRFs are highly cytotoxic at physiological concentrations. Although ANG is well-known for its promotion of cell growth, our results reveal that ANG can also cause cell death.


Subject(s)
Carrier Proteins/metabolism , Cell Death/physiology , Cytotoxins/metabolism , RNA, Transfer/genetics , Ribonuclease, Pancreatic/metabolism , CRISPR-Cas Systems , Carrier Proteins/genetics , Cell Death/genetics , Cell Line, Tumor , Cell Proliferation , Cytotoxins/genetics , Gene Knockout Techniques , HeLa Cells , Humans , MicroRNAs/genetics , Oxidative Stress , Protein Binding/genetics , Ribonuclease, Pancreatic/genetics
7.
Anal Biochem ; 555: 67-72, 2018 08 15.
Article in English | MEDLINE | ID: mdl-29733811

ABSTRACT

Neddylation is a posttranslational modification that regulates protein stability, activity, and subcellular localization. Here, we describe a new tool for exploring the neddylation cycle of cullin1 (Cul1) directly in a cellular context. This assay utilizes the NanoLuc® Binary Technology (NanoBiT) to monitor the covalent neddylation status of Cul1. A stable clonal cell line derived from HEK293 was developed that expressed a C-terminus LgBiT tagged-Cul1 and N-terminus SmBiT tagged-Nedd8. Using this cell line, we screened inhibitors that are known to disrupt Nedd8 biology and demonstrated that both inhibitors of Nedd8-activating enzyme (NAE) and Constitutive photomorphogenesis 9 signalosome (CSN) complex produce concentration and time dependent signal decreases and increases, respectively. The kinetics of both responses could be monitored in real time and demonstrated that modulation of the Nedd8 pathway occurs rapidly. Further characterization of the cellular components of this cell line was performed in order to quantify the various levels of Cul1, Nedd8 and NAE and determined to be near endogenous levels. There was no difference between control and stably transfected cell lines in viability studies of NAE and CSN inhibitors. Taken together, these results suggest that the NanoBiT assay can be used to monitor Cul1 neddylation specifically and in real time.


Subject(s)
Biological Assay/methods , Cullin Proteins/metabolism , NEDD8 Protein/metabolism , Protein Processing, Post-Translational , Cullin Proteins/genetics , HCT116 Cells , HEK293 Cells , Humans , NEDD8 Protein/genetics
8.
J Am Chem Soc ; 139(23): 8029-8037, 2017 06 14.
Article in English | MEDLINE | ID: mdl-28505435

ABSTRACT

The ability to achieve predictable control over the polarization of strained cycloalkynes can influence their behavior in subsequent reactions, providing opportunities to increase both rate and chemoselectivity. A series of new heterocyclic strained cyclooctynes containing a sulfamate backbone (SNO-OCTs) were prepared under mild conditions by employing ring expansions of silylated methyleneaziridines. SNO-OCT derivative 8 outpaced even a difluorinated cyclooctyne in a 1,3-dipolar cycloaddition with benzylazide. The various orbital interactions of the propargylic and homopropargylic heteroatoms in SNO-OCT were explored both experimentally and computationally. The inclusion of these heteroatoms had a positive impact on stability and reactivity, where electronic effects could be utilized to relieve ring strain. The choice of the heteroatom combinations in various SNO-OCTs significantly affected the alkyne geometries, thus illustrating a new strategy for modulating strain via remote substituents. Additionally, this unique heteroatom activation was capable of accelerating the rate of reaction of SNO-OCT with diazoacetamide over azidoacetamide, opening the possibility of further method development in the context of chemoselective, bioorthogonal labeling.


Subject(s)
Cycloparaffins/chemical synthesis , Sulfonic Acids/chemistry , Cycloparaffins/chemistry , Electrons , Molecular Structure , Quantum Theory
9.
Angew Chem Int Ed Engl ; 56(10): 2619-2622, 2017 03 01.
Article in English | MEDLINE | ID: mdl-28120377

ABSTRACT

Angiogenin (ANG) is a human ribonuclease that is compromised in patients with amyotrophic lateral sclerosis (ALS). ANG also promotes neovascularization, and can induce hemorrhage and encourage tumor growth. The causal neurodegeneration of ALS is associated with reactive oxygen species, which are also known to elicit the oxidative cleavage of carbon-boron bonds. We have developed a synthetic boronic acid mask that restrains the ribonucleolytic activity of ANG. The masked ANG does not stimulate endothelial cell proliferation but protects astrocytes from oxidative stress. By differentiating between the two dichotomous biological activities of ANG, this strategy could provide a viable pharmacological approach for the treatment of ALS.


Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Boronic Acids/pharmacology , Neovascularization, Pathologic/drug therapy , Neuroprotective Agents/pharmacology , Reactive Oxygen Species/metabolism , Ribonuclease, Pancreatic/pharmacology , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Boronic Acids/chemistry , Cell Proliferation/drug effects , Humans , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Neuroprotective Agents/chemistry , Oxidative Stress/drug effects , Ribonuclease, Pancreatic/chemistry
10.
Nucleic Acids Res ; 45(2): 818-831, 2017 01 25.
Article in English | MEDLINE | ID: mdl-27915233

ABSTRACT

Canonical growth factors act indirectly via receptor-mediated signal transduction pathways. Here, we report on an autonomous pathway in which a growth factor is internalized, has its localization regulated by phosphorylation, and ultimately uses intrinsic catalytic activity to effect epigenetic change. Angiogenin (ANG), a secreted vertebrate ribonuclease, is known to promote cell proliferation, leading to neovascularization as well as neuroprotection in mammals. Upon entering cells, ANG encounters the cytosolic ribonuclease inhibitor protein, which binds with femtomolar affinity. We find that protein kinase C and cyclin-dependent kinase phosphorylate ANG, enabling ANG to evade its inhibitor and enter the nucleus. After migrating to the nucleolus, ANG cleaves promoter-associated RNA, which prevents the recruitment of the nucleolar remodeling complex to the ribosomal DNA promoter. The ensuing derepression of rDNA transcription promotes cell proliferation. The biochemical basis for this unprecedented mechanism of signal transduction suggests new modalities for the treatment of cancers and neurological disorders.


Subject(s)
Cell Proliferation , Ribonuclease, Pancreatic/metabolism , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone/metabolism , HeLa Cells , Human Umbilical Vein Endothelial Cells , Humans , Models, Biological , Models, Molecular , Nucleic Acid Conformation , Phosphorylation , Protein Conformation , Protein Kinase C/metabolism , Protein Transport , RNA Cleavage , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Ribonuclease, Pancreatic/chemistry , Signal Transduction , Transcription, Genetic
11.
Chem Biol ; 20(4): 614-8, 2013 Apr 18.
Article in English | MEDLINE | ID: mdl-23601650

ABSTRACT

Endocytosis is a fundamental process of eukaryotic cells that is critical for nutrient uptake, signal transduction, and growth. We have developed a molecular probe to quantify endocytosis. The probe is a lipid conjugated to a fluorophore that is masked with an enzyme-activatable moiety known as the trimethyl lock. The probe is not fluorescent when incorporated into the plasma membrane of human cells but becomes fluorescent upon internalization into endosomes, where cellular esterases activate the trimethyl lock. Using this probe, we found that human breast cancer cells undergo constitutive endocytosis more rapidly than do matched noncancerous cells. These data reveal a possible phenotypic distinction of cancer cells that could be the basis for chemotherapeutic intervention.


Subject(s)
Endocytosis , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Phosphatidic Acids/metabolism , Urea/analogs & derivatives , Cell Line , Esterases/metabolism , Fluoresceins/chemical synthesis , Fluoresceins/chemistry , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , HeLa Cells , Humans , Microscopy, Fluorescence , Phosphatidic Acids/chemical synthesis , Phosphatidic Acids/chemistry , Urea/chemical synthesis , Urea/chemistry , Urea/metabolism
12.
Nucleic Acids Res ; 41(6): 3576-87, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23393192

ABSTRACT

DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box-containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.


Subject(s)
DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Ubiquitination , Binding Sites , DNA/metabolism , DNA Helicases/chemistry , DNA Replication , DNA-Binding Proteins/chemistry , HEK293 Cells , Humans , Protein Structure, Tertiary , Rad51 Recombinase/metabolism
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