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Expanding the Scope of Protein Synthesis Using Modified Ribosomes.

ABSTRACT: The ribosome produces all of the proteins and many of the peptides present in cells. As a macromolecular complex composed of both RNAs and proteins, it employs a constituent RNA to catalyze the formation of peptide bonds rapidly and with high fidelity. Thus, the ribosome can be argued to represent the key link between the RNA World, in which RNAs were the primary catalysts, and present biological systems in which protein catalysts predominate. In spite of the well-known phylogenetic conservation of rRNAs through evolutionary history, rRNAs can be altered readily when placed under suitable pressure, e.g. in the presence of antibiotics which bind to functionally critical regions of rRNAs. While the structures of rRNAs have been altered intentionally for decades to enable the study of their role(s) in the mechanism of peptide bond formation, it is remarkable that the purposeful alteration of rRNA structure to enable the elaboration of proteins and peptides containing noncanonical amino acids has occurred only recently. In this Perspective, we summarize the history of rRNA modifications, and demonstrate how the intentional modification of 23S rRNA in regions critical for peptide bond formation now enables the direct ribosomal incorporation of d-amino acids, ?-amino acids, dipeptides and dipeptidomimetic analogues of the normal proteinogenic l-?-amino acids. While proteins containing metabolically important functional groups such as carbohydrates and phosphate groups are normally elaborated by the post-translational modification of nascent polypeptides, the use of modified ribosomes to produce such polymers directly is also discussed. Finally, we describe the elaboration of such modified proteins both in vitro and in bacterial cells, and suggest how such novel biomaterials may be exploited in future studies.

SUBMITTER: Dedkova LM

PROVIDER: S-EPMC6530915 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Expanding the Scope of Protein Synthesis Using Modified Ribosomes.

Dedkova Larisa M LM Hecht Sidney M SM

Journal of the American Chemical Society 20190405 16

The ribosome produces all of the proteins and many of the peptides present in cells. As a macromolecular complex composed of both RNAs and proteins, it employs a constituent RNA to catalyze the formation of peptide bonds rapidly and with high fidelity. Thus, the ribosome can be argued to represent the key link between the RNA World, in which RNAs were the primary catalysts, and present biological systems in which protein catalysts predominate. In spite of the well-known phylogenetic conservation ...[more]

PMID: 30901982

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Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).

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Expanding the Scope of Protein Synthesis Using Modified Ribosomes.

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Expanding the Scope of Protein Synthesis Using Modified Ribosomes.

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