Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation

[Image: see text] Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from Escherichia coli to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNA(Ile) in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNA(Ile) from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids

Exploiting Oligo(amido amine) Backbones for the Multivalent Presentation of Coiled-Coil Peptides

The investigation of coiled coil formation for one mono- and two divalent peptide–polymer conjugates is presented. Through the assembly of the full conjugates on solid support, monodisperse sequence-defined conjugates are obtained with defined positions and distances between the peptide side chains along the polymeric backbone. A heteromeric peptide design was chosen, where peptide K is attached to the polymer backbone, and coiled-coil formation is only expected through complexation with the complementary peptide E. Indeed, the monovalent peptide K-polymer conjugate displays rapid coiled-coil formation when mixed with the complementary peptide E sequence. The divalent systems show intramolecular homomeric coiled-coil formation on the polymer backbone despite the peptide design. Interestingly, this intramolecular assembly undergoes a conformational rearrangement by the addition of the complementary peptide E leading to the formation of heteromeric coiled coil–polymer aggregates. The polymer backbone acts as a template bringing the covalently bound peptide strands in close proximity to each other, increasing the local concentration and inducing the otherwise nonfavorable formation of intramolecular helical assemblies

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity

Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This “bounce-back” response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis