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

Small Molecule Modulation of Proteasome Assembly

The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S–20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes

Comparison of α₁-PI topography generated with AFM, the AFM simulator and the crystal structure model.

<p>The AFM generated topography of the wild type α₁-PI is in a good agreement with the shape and dimensions of particles obtained with the AFM simulator and calculated from a model of the protein surface occluded by a water shell. In contrast, the waterless Van der Waals protein surface model was too rich in structural details to be efficiently compared with AFM topographs. The models were based on the crystal structure of α₁-PI WT monomer (PDB ID: 1qlp [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151902#pone.0151902.ref031" target="_blank">31</a>]). The length of a space-filled crystal structure model of the kidney-shaped antitrypsin molecule was 7–8 nm and its width up to 4–5 nm. (A) a tilted AFM image (side plot) of a typical WT monomer particle; (B) a surface model generated with the Microscope Simulator using the cone-sphere tip model of a radius 7 Å and a cone angle 25°; (C) a Van der Waals protein surface model generated with the 3V program using a probe of 10 Å radius that adds the water shell; (D) a protein surface model obtained as in (C) by applying a probe radius of 0 Å (“waterless”). Models presented in B-D are structurally aligned.</p

<i>In vitro</i> polymerization of led to formation of structurally diverse oligomers observed with AFM.

<p>Top panels: AFM height images of representative fields of WT α₁-PI particles polymerized by: (A) incubation with 1.4M GuHCl (37.5 hrs, 25°C, pH = 7.4); (B) exposure to a low pH (pH = 4.1, 2 hrs, 25°C); (C) treatment with the elevated temperature (55°C, 4 hrs, pH = 7.4). Bottom panels: arrays of zoomed-in images of oligomers representative for each polymerization method.</p

Native PAGE analysis of α₁-PI monomers and <i>in vitro</i> formed polymers.

<p>Polymers were formed from purified WT plasma-derived α₁-PI monomers (lane 1) by incubation at pH 4.1 (lane 2), incubation with 1.4M guanidinium chloride (lane 3) or incubation at 55°C (lane 4). Lane 5 presents purified human Z variant monomer. Quantification of the bands indicated that approximately 87% of the protein was migrating as a monomer in lane 1 (WT), markedly more than in lane 5 (69%; Z variant). M–monomer; D–dimer. Approximated molecular weights are indicated on the left. Lanes 2 to 4 were run concurrently on the same gel.</p

AFM Imaging Reveals Topographic Diversity of Wild Type and Z Variant Polymers of Human α₁-Proteinase Inhibitor - Fig 4

<p><b>Volume distributions of WT α</b>_<b>1</b><b>-PI (A) and Z variant (B) particles.</b> The distributions revealed a prevailing content of objects, which size was consistent with monomers. Surprisingly, the presence of larger molecules was also detected. Among the larger objects, particles with volume approximately twice as big as monomers were the most abundant. The content of dimers and bigger particles was more pronounced in the Z variant (B) than in the wild type (A). The volume of particles was calculated with the grain analysis of SPIP software. Relative count of particles is shown. Total of 847 and 639 particles were analyzed for the WT and Z variant, respectively. Histogram bin size was 5 nm. Fitted normal distribution curves are shown in green for monomers and in blue for dimers, whereas total frequency distribution is represented by red traces (OriginPro).</p

AFM height images of human Z mutant α₁-PI preparations obtained from a PiZ mouse liver.

<p>(A) Images of two representative fragments of fields with α₁-PI particles. The presence of long, linear, often tangled strands of polymers is striking. Arrows in the top image point at the types of particles presented in zoomed-in B to E panels on the right. The examples are marked b to e, respectively. (B)–(E) galleries of zoomed-in images of diverse polymer and oligomer types. In particular: (B) fragments of long polymer fibers with straight, smooth and compact arrangement of units; (C) fragments of long polymer fibers with grainy appearance and loose arrangement of well discernible units; (D) linear, short and thin oligomers with compact arrangement of units; (E) oligomers built from large globular units arranged as “large packed beads”.</p