Premature translational termination products are rapidly degraded substrates for MHC class I presentation.

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.

Tnni3k modifies disease progression in murine models of cardiomyopathy.

The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.