MHC class I peptides are products of endogenous cellular protein degradation. Their prompt presentation, after rapid degradation of their newly synthesized source proteins, is needed to alert the immune system during pathogen infection. A possible source for such rapidly degrading proteins can be defective ribosome products (DRiPs), which include polypeptides produced as part of the pioneer round of translation, premature translation termination, and proteins failing to fold properly or to assemble into their multisubunit protein complexes. However, the identities and relative contribution to the MHC peptidome of these mature or newly synthesized and rapidly degraded cellular proteins is not well understood. To clarify these issues, we used dynamic stable isotope labeling by amino acids in cell culture to define the relative rates of synthesis of the HLA class I peptidomes and the source proteomes of three cultured human hematopoietic cell lines. Large numbers of HLA class I peptides were observed to be derived from DRiPs, defined here as HLA peptides that shift from their light to heavy isotope forms faster than their source proteins. Specific groups of proteins, such as ribosomal and T-complex protein 1 (TCP-1), contributed a disproportionately large number of DRiPs to the HLA peptidomes. Furthermore, no significant preference was observed for HLA peptides derived from the amino terminal regions of the proteins, suggesting that the contribution of products of premature translation termination was minimal. Thus, the most likely sources of DRiPs-derived HLA peptides are full-sized, misassembled, and surplus subunits of large protein complexes.

A new pseudo-disaccharide NB23 with a 3′,4′-methylidene protection was designed and its properties were evaluated in comparison to other two structurally related pseudo-disaccharides. The basicity of the 2′-amine was found to be well correlated to acute toxicity data in mice: the increase in the basicity is associated with the toxicity increase. Based on these data, a new pseudo-trisaccharide NB45 was constructed. NB45 exhibited significant antibacterial activity while at the same time retained low acute toxicity.

Bactericidal properties were recently shown to emerge from hydrophobicity and charge buildup in oligo-acyl-lysine (OAK) peptide mimetics. Toward understanding the attributes that govern the activity of this novel antimicrobial system, we compared the functional and mechanistic properties of a known octamer and a newly generated hexamer analog. The data provide strong evidence for multiple similarities that included high tissue stability, low hemolysis, large-spectrum antibacterial activity in vitro, and the ability to prevent Escherichia coli-induced mortality in vivo. Despite these similarities, however, the octamer mode of action involved membrane disruption, unlike the hexamer, which acted predominantly through inhibition of DNA functions with characteristically slower bactericidal kinetics. Collectively, the data support the view that the analogous OAKs induced bacterial death by distinct mechanisms and further suggest that relatively minor differences in the sequence of host defense peptides are responsible for selecting one mechanism over another, possibly in conjunction with differential binding affinities to the external and/or cytoplasmic membrane.

We describe peptidomimetic oligomers that show rapid, nonhemolytic, broad-spectrum bactericidal properties in mice and do not induce the emergence of resistance. The oligomers contain acyl chains, which prevent the formation of stable secondary structure. This design appears advantageous over conventional antimicrobial peptides with respect to in vivo efficacy and safety, and may provide a convenient platform for the development of peptide antibiotics.