Chymotrypsin- and trypsin-like activities, but not the caspase-like activity were higher in the whole cell lysates from naked mole-rat than in mouse lysates.

<p>In each assay 50 µg of whole cell liver lysates from physiologically age-matched young mice (4 mo) and naked mole rats (2 yr) were used. The samples were incubated with 100 µM of substrate specific for the type of active center of the proteasome being measured. A saturating concentration of proteasome inhibitor N-(benzyl-oxycarbonyl) leucinyl-leucinal (MG132), determined by titration, was added to parallel samples. The difference of the fluorescence released with and without inhibitor was used as a measure of the specific peptidolytic activity of proteasome. Hatched lines indicate the amount of non-specific protease activity in excess of net specific proteasome activity. Values are means ± SE. Significant p-values are indicated in the figure.</p

Levels of markers of an inflammatory response were higher in naked mole-rat than in mice.

<p>We quantitated protein levels in Western blot analyses of liver tissue lysates probed with anti NFκB and TNFα antibodies. Both NFκB and TNFα protein levels were more than two-fold higher (p≤0.01) in naked mole-rats. Samples from three different individuals of each species were used and the experiment was repeated with lysates from different animals several times to verify the outcome. The blots shown are representative of these experiments. Actin was used as a loading control and lysates from mouse spleen tissue (MsSp) represented a positive control for these immune-related markers.</p

The largest species differences observed were the contribution of the cytosolic fraction for both ChT-L and T-L activities.

<p>Percent contribution of the total activity was calculated using the values of specific activities presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035890#pone-0035890-g004" target="_blank">Figure 4</a>. In both species proteasome activity was highest in the microsomal fraction, but the microsomal contribution to the total activity within the lysate was greater in mouse samples (76%) than in naked mole-rat samples (50%) for ChT-L and this difference in % contribution was even greater for TL-activity (87%, 53% respectively). Nuclear fractions, regardless of the catalytic activity, only contributed 7% or less to the total activity in both species. PGPH activity showed a similar distribution within the subcellular fractions in both species. In sharp contrast the cytosolic fraction of ChT-L activity of naked mole-rats showed more than double (46%) the proportionate contribution to that of mice (18%) and this species difference was even greater for T-L activity (32% to 7%) in revealing that distributional differences in the observed total activity between species could be explained by interspecific differences in cytosolic activity.</p

Analysis of proteasome subunit composition showed that naked mole-rats had higher protein content of 19S and immunoproteasome subunits than mice.

<p>(A) Representative Western blots with PVDF-transferred proteins were probed with antibodies specific for 20S, 26S and immunoproteasome subunits. Different content of various subunits revealed an upregulation of particular proteasome subassemblies. Samples from three different animals from each species were used per experiment and the experiment was repeated with samples from different animals at least one additional time to verify the outcome. The blots are representative of these sets. Actin was used as a loading control for our analyses. For immunoproteasome subunits, lysates from mouse spleen tissue (MsSp) were also used as a positive control. (B) Quantitation of Western blots grouped by 20S, 19S or immunoproteasome. Not only did naked mole-rats have higher content of constitutive non-catalytic subunits, but they also tended to have more immunoproteasome components (β2i, β5i, PA28α) than did mice. Naked mole-rats also had increased protein content of two critical 19S subunits (RPT5, RPN10). Values represent the mean ± SE with significant p-values highlighted in the figure.</p