The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion

Background: Cytotoxic T cells detect intracellular pathogens by surveying peptide loaded MHC class I molecules (pMHC I) on the cell surface. Effective immune surveillance also requires infected cells to present pMHC I promptly before viral progeny can escape. Rapid pMHC I presentation apparently occurs because infected cells can synthesize and present peptides from antigenic precursors called defective ribosomal products (DRiPs). The molecular characteristics of DRiPs are not known. Methodology/Principal Findings: Here, using a novel method for detecting antigenic precursors and proteolytic intermediates, we tracked the synthesis and processing of Epstein-Barr Virus encoded nuclear antigen 1 (EBNA1). We find that ribosomes initiated translation appropriately, but rapidly produced DRiPs representing,120 amino acid truncated EBNA1 polypeptides by premature termination. Moreover, specific sequences in EBNA1 mRNA strongly inhibited the generation of truncated DRiPs and pMHC I presentation. Significance: Our results reveal the first characterization of virus DRiPs as truncated translation products. Furthermore

Non-conventional sources of peptides presented by MHC class I

Effectiveness of immune surveillance of intracellular viruses and bacteria depends upon a functioning antigen presentation pathway that allows infected cells to reveal the presence of an intracellular pathogen. The antigen presentation pathway uses virtually all endogenous polypeptides as a source to produce antigenic peptides that are eventually chaperoned to the cell surface by MHC class I molecules. Intriguingly, MHC I molecules present peptides encoded not only in the primary open reading frames but also those encoded in alternate reading frames. Here, we review recent studies on the generation of cryptic pMHC I. We focus on the immunological significance of cryptic pMHC I, and the novel translational mechanisms that allow production of these antigenic peptides from unconventional sources

A Distinct Translation Initiation Mechanism Generates Cryptic Peptides for Immune Surveillance

MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition.

Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3'-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin

A Health Threat to Bystanders Living in the Homes of Smokers: How Smoke Toxins Deposited on Surfaces Can Cause Insulin Resistance.

Thirdhand smoke (THS) is the accumulation of secondhand smoke on environmental surfaces. THS is found on the clothing and hair of smokers as well as on surfaces in homes and cars of smokers. Exposure occurs by ingestion, inhalation and dermal absorption. Children living in homes of smokers are at highest risk because they crawl on the floor, touch parents' clothing/hair and household objects. Using mice exposed to THS under conditions that mimic exposure of humans, we show that THS increases cellular oxidative stress by increasing superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) levels while reducing the activity of antioxidant enzymes catalase and glutathione peroxidase (GPx) that break down H2O2 into H2O and O2. This results in lipid peroxidation, protein nitrosylation and DNA damage. Consequences of these cell and molecular changes are hyperglycemia and insulinemia. Indeed, we found reduced levels of insulin receptor, PI3K, AKT, all important molecules in insulin signaling and glucose uptake by cells. To determine whether these effects on THS-induced insulin resistance are due to increase in oxidative stress, we treated mice exposed to THS with the antioxidants N-acetyl cysteine (NAC) and alpha-tocopherol (alpha-toc) and showed that the oxidative stress, the molecular damage, and the insulin resistance, were significantly reversed. Conversely, feeding the mice with chow that mimics "western diet", which is known to increase oxidative stress, while exposing the mice to THS, further increased the oxidative stress and aggravated hyperglycemia and insulinemia. In conclusion, THS exposure results in insulin resistance in the form of non-obese type II diabetes (NODII) through oxidative stress. If confirmed in humans, these studies could have a major impact on how people view exposure to environmental tobacco toxins, in particular to children, elderly and workers in environments where tobacco smoke has taken place

Correction: A Health Threat to Bystanders Living in the Homes of Smokers: How Smoke Toxins Deposited on Surfaces Can Cause Insulin Resistance.

<div><p>Thirdhand smoke (THS) is the accumulation of secondhand smoke on environmental surfaces. THS is found on the clothing and hair of smokers as well as on surfaces in homes and cars of smokers. Exposure occurs by ingestion, inhalation and dermal absorption. Children living in homes of smokers are at highest risk because they crawl on the floor, touch parents’ clothing/hair and household objects. Using mice exposed to THS under conditions that mimic exposure of humans, we show that THS increases cellular oxidative stress by increasing superoxide dismutase (SOD) activity and hydrogen peroxide (H₂O₂) levels while reducing the activity of antioxidant enzymes catalase and glutathione peroxidase (GPx) that break down H₂O₂ into H₂O and O₂. This results in lipid peroxidation, protein nitrosylation and DNA damage. Consequences of these cell and molecular changes are hyperglycemia and insulinemia. Indeed, we found reduced levels of insulin receptor, PI3K, AKT, all important molecules in insulin signaling and glucose uptake by cells. To determine whether these effects on THS-induced insulin resistance are due to increase in oxidative stress, we treated mice exposed to THS with the antioxidants N-acetyl cysteine (NAC) and alpha-tocopherol (alpha-toc) and showed that the oxidative stress, the molecular damage, and the insulin resistance, were significantly reversed. Conversely, feeding the mice with chow that mimics “western diet”, which is known to increase oxidative stress, while exposing the mice to THS, further increased the oxidative stress and aggravated hyperglycemia and insulinemia. In conclusion, THS exposure results in insulin resistance in the form of non-obese type II diabetes (NODII) through oxidative stress. If confirmed in humans, these studies could have a major impact on how people view exposure to environmental tobacco toxins, in particular to children, elderly and workers in environments where tobacco smoke has taken place.</p></div