Abstract
Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.
Highlights
Studies of eukaryotic protein turnover have revealed that nearly a third of all newly synthesized polypeptides are targeted for rapid degradation by the ubiquitin-proteasome system [1]
To identify a puromycin concentration range that promotes the production of prematurely terminated polypeptides while maintaining a substantial level of protein synthesis, 293-Kb cells were radiolabeled with [35S]-Met in the presence of varying concentrations of puromycin and radioisotope incorporation was quantified as a measure of total cellular protein synthesis
These results demonstrate that inducing premature termination with low concentrations of puromycin allows the majority of total protein synthesis to be maintained (20 mM) or causes it to slightly increase (0.2–2 mM)
Summary
Studies of eukaryotic protein turnover have revealed that nearly a third of all newly synthesized polypeptides are targeted for rapid degradation by the ubiquitin-proteasome system [1]. Premature termination would be predicted to cause ribosomal dissociation upstream of the termination codon, resulting in a relative decrease in ribosomal density towards the 39 end of transcripts In support of this model, studies of the distribution of ribosomes on mRNAs by both polysome microarrays [14] and ribosomal footprinting [15] indicate higher ribosomal density at the 59 end of mRNAs. Second, specific sequences in the mRNA of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1) regulate the production of prematurely terminated EBNA1 polypeptides, which in turn serve as a source of MHC I peptides [16]. The presence of Pth homologs in eukaryotes [17,18] suggests conservation of the mechanisms for premature translational termination and disposal of the resulting drop-off products
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
