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.
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.
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.
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 predicates a viable pharmacological approach for the treatment of ALS.
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 -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.
The modulation of interstrand steric clashes can shift the preference of collagen-mimetic peptides from homotrimer to heterotrimer formation, enabling the detection of burn-damaged tissue ex vivo.
SUMMARY
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.
Edited by Karin Musier-Forsyth 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.
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