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

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

Treatment with puromycin increases the fraction of rapidly degraded polypeptides.

<p><i>A.</i> 293-K^b cells were labeled with [³⁵S]-Met for 10 minutes in the presence of 0 to 40 µM MG132. [³⁵S] incorporation was measured as in Fig. 1A and normalized to controls without proteasome inhibitor (<i>n</i> = 3; mean ± s.e.m.) <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met +/−20 µM puro and +/−20 µM MG132 for 5 minutes, then chased from 0 to 50 minutes in the presence of excess cold methionine, CHX and +/−20 µM MG132. DMSO is a solvent control for MG132. The chase was terminated at the indicated time points by the addition of TCA to cell suspensions to precipitate polypeptides. TCA precipitates were solubilized and [³⁵S] was measured by liquid scintillation counting (<i>n</i> ≥4; mean ± s.e.m.) <i>C</i> and <i>D</i>. Solubilized TCA precipitates from cells radiolabeled in the absence (<i>C</i>) or presence (<i>D</i>) of 20 µM puro were separated by tricine SDS-PAGE on 10% gels. Gels were dried and exposed to a PhosphorImager plate overnight. Note that for <i>D</i>, the darkness of the image has been enhanced in order to see the contrast in degradation rates between <i>C</i> and <i>D</i> more clearly.</p

Puromycin treatment leads to increased levels of polyubiquitinated proteins.

<p>293-K^b cells were treated for 4 hours with media alone, 20 µM MG132, or the indicated concentrations of cycloheximide and puromycin (µM). Lysates were subjected to Western blotting with FK2 (<i>upper panel</i>), a monoclonal antibody specific for mono- and polyubiquitinated protein conjugates. Beta-tubulin was probed as a loading control (<i>lower panel</i>). Results are representative of three independent experiments.</p

Puromycin stimulates the production of truncated polypeptides in a dose-dependent manner.

<p><i>A.</i> 293-K^b cells were radiolabeled with [³⁵S]-Met for 10 minutes in the presence of a linear range of puromycin concentrations from 0 to 20 µM. Radiolabeled polypeptides were visualized as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone-0051968-g001" target="_blank">Fig. 1B</a>. In the later panels, we analyze the [³⁵S] signals from the four regions indicated to the right of the gel. <i>B</i>. PhosphorImager signal intensities (arbitrary units) from selected lanes in <i>A</i>. The left side of the graph corresponds to the top of the gel while the right side of the graph corresponds to the bottom of the gel at the dye front. The highlighted regions correspond to the parts of the gel indicated in <i>A</i>. <i>C</i>. The effects of puromycin concentration on [³⁵S] signal for each of the highlighted gel regions in <i>A</i> and <i>B</i>. Results are representative of three independent experiments.</p

MHC class I-peptide complex recovery assay using a fluorescent reporter encoding antigenic peptides.

<p><i>A.</i> Schematic of the modified NP-SIINFEKL-eGFP reporter (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Princiotta1" target="_blank">[2]</a>) containing eight additional tandem repeats of SIINFEKL (nine total) and its five flanking amino acids from the native ovalbumin sequence, NP-[SIINFEKL]₉-eGFP (Tandem Repeat x9 or TRx9). <i>B.</i> Validation of MHC I peptide stripping and recovery in TRx9-expressing cells. Biexponential scatter plots show single cell profiles of the mean fluorescence intensity (MFI) for eGFP on the <i>x</i>-axis and 25-D1.16 on the <i>y</i>-axis. Plots show fluorescence profiles immediately pre- (<i>left</i>) and post- (<i>middle</i>) peptide stripping, and after a 4 hour recovery (<i>right</i>).</p

Rapid degradation of premature translational termination products.

<p><i>A.</i> Denaturing immunoprecipitation of peptidyl-puromycins. 293-K^b cells were radiolabeled for 30 minutes with [³⁵S]-Met, 20 µM puromycin, and 20 µM MG132. Polypeptides were precipitated with TCA, solubilized, then subjected to a denaturing immunoprecipitation using either non-specific rabbit serum (negative IP control) or anti-puromycin serum. Results are representative of three independent experiments. <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met and 20 µM puro and chased as described in Fig. 3B. Solubilized TCA precipitates were subjected to denaturing immunoprecipitation using anti-puromycin serum. [³⁵S] in the anti-puromycin immunoprecipitates was measured by liquid scintillation counting (<i>n</i> = 4; mean ± s.e.m.).</p

Time-dependent inhibition of MHC class I pathway function following puromycin treatment.

<p><i>A</i> and <i>B.</i> 293-K^b cells were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface K^b was conducted in the presence of varying concentrations of puro for one (<i>A</i>) and four (<i>B</i>) hours. During the final 30 minutes of the recovery, cells were treated with either distilled water ((−) peptide) or 5 µM SIINFEKL peptide ((+) peptide) to promote the export of K^b to the cell surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Day1" target="_blank">[31]</a>. Flow cytometry was used to measure total cell surface K^b and the MFI was normalized to untreated cells in the absence of exogenous SIINFEKL peptide (<i>n</i> = 3; mean ± s.e.m.; * <i>p</i><0.05 for (−) peptide vs. (+) peptide).</p

Effects of puromycin on the recovery of cell surface MHC class I-peptide complexes.

<p><i>A</i>–<i>E</i>. 293-K^b cells expressing the TRx9 reporter were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface MHC class I-peptide complexes was conducted in the presence of varying concentrations of puro from 0 to 180 minutes. Flow cytometry was used to measure reporter eGFP fluorescence (<i>A</i>), as well as cell surface K^b-SIINFEKL complexes (<i>B</i>) and total cell surface K^b (<i>D</i>). To quantitate differences in the kinetics of MHC class I-peptide complex recovery, we normalized the MFI values of puro-treated cells to untreated cells for K^b-SIINFEKL (<i>C</i>) and total K^b (<i>E</i>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Qian1" target="_blank">[4]</a> (<i>n</i> = 5; mean ± s.e.m.) For <i>C</i> and <i>E</i>, * <i>p</i><0.05 compared to untreated samples. <i>F.</i> MHC class I-peptide complex recovery after 1 hour in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.) <i>G</i>. MHC class I-peptide complex recovery after 4 hours in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.).</p