Altered Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in the Exceptionally Long-Lived Naked Mole-Rat

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life

Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme's inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4-5 and 8-9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8-9 position was necessary to significantly improve the inhibitory potency

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

α1-Proteinase inhibitor (antitrypsin) is a canonical example of the serpin family member that binds and inhibits serine proteases. The natural metastability of serpins is crucial to carry out structural rearrangements necessary for biological activity. However, the enhanced metastability of the mutant Z variant of antitrypsin, in addition to folding defect, may substantially contribute to its polymerization, a process leading to incurable serpinopathy. The metastability also impedes structural studies on the polymers. There are no crystal structures of Z monomer or any kind of polymers larger than engineered wild type (WT) trimer. Our understanding of polymerization mechanisms is based on biochemical data using in vitro generated WT oligomers and molecular simulations. Here we applied atomic force microscopy (AFM) to compare topography of monomers, in vitro formed WT oligomers, and Z type polymers isolated from transgenic mouse liver. We found the AFM images of monomers closely resembled an antitrypsin outer shell modeled after the crystal structure. We confirmed that the Z variant demonstrated higher spontaneous propensity to dimerize than WT monomers. We also detected an unexpectedly broad range of different types of polymers with periodicity and topography depending on the applied method of polymerization. Short linear oligomers of unit arrangement similar to the Z polymers were especially abundant in heat-treated WT preparations. Long linear polymers were a prominent and unique component of liver extracts. However, the liver preparations contained also multiple types of oligomers of topographies undistinguishable from those found in WT samples polymerized with heat, low pH or guanidine hydrochloride treatments. In conclusion, we established that AFM is an excellent technique to assess morphological diversity of antitrypsin polymers, which is important for etiology of serpinopathies. These data also support previous, but controversial models of in vivo polymerization showing a surprising diversity of polymer topography

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