7 research outputs found
PiZ Mouse Liver Accumulates Polyubiquitin Conjugates That Associate with Catalytically Active 26S Proteasomes
<div><p>Accumulation of aggregation-prone human alpha 1 antitrypsin mutant Z (AT-Z) protein in PiZ mouse liver stimulates features of liver injury typical of human alpha 1 antitrypsin type ZZ deficiency, an autosomal recessive genetic disorder. Ubiquitin-mediated proteolysis by the 26S proteasome counteracts AT-Z accumulation and plays other roles that, when inhibited, could exacerbate the injury. However, it is unknown how the conditions of AT-Z mediated liver injury affect the 26S proteasome. To address this question, we developed a rapid extraction strategy that preserves polyubiquitin conjugates in the presence of catalytically active 26S proteasomes and allows their separation from deposits of insoluble AT-Z. Compared to WT, PiZ extracts had about 4-fold more polyubiquitin conjugates with no apparent change in the levels of the 26S and 20S proteasomes, and unassembled subunits. The polyubiquitin conjugates had similar affinities to ubiquitin-binding domain of Psmd4 and co-purified with similar amounts of catalytically active 26S complexes. These data show that polyubiquitin conjugates were accumulating despite normal recruitment to catalytically active 26S proteasomes that were available in excess, and suggest that a defect at the 26S proteasome other than compromised binding to polyubiquitin chain or peptidase activity played a role in the accumulation. In support of this idea, PiZ extracts were characterized by high molecular weight, reduction-sensitive forms of selected subunits, including ATPase subunits that unfold substrates and regulate access to proteolytic core. Older WT mice acquired similar alterations, implying that they result from common aspects of oxidative stress. The changes were most pronounced on unassembled subunits, but some subunits were altered even in the 26S proteasomes co-purified with polyubiquitin conjugates. Thus, AT-Z protein aggregates indirectly impair degradation of polyubiquitinated proteins at the level of the 26S proteasome, possibly by inducing oxidative stress-mediated modifications that compromise substrate delivery to proteolytic core.</p></div
Polyubiquitin pools and load per proteasome.
<p><u>(A). Polyubiquitin pools</u>. 9 sequential extracts were prepared from each 20 mg liver fragment as described in Methods, and an equal fraction of each extract (5 µl our of 40 µl) was analyzed by WBs with antibodies specific to ubiquitin. The total protein contents in WT and PiZ extracts were verified by Ponceau stain to be similar to those shown in Fig. 1A, CB (not shown). (<u>B). Quantitative analysis of polyubiquitin conjugates and proteasomal subunits in detergent-free extracts (extracts - Triton)</u>. Left: Extracts 1-3 identified in A to contain the majority of polyubiquitin conjugates were combined to represent ‘detergent-free extract’, as described in Methods. Left: the indicated amounts of detergent-free WT and PiZ extracts (5, 2.5, and 1.2 µg of total proteins) were analyzed by WB on the same membrane, with multiple membranes used to probe for all proteins shown. Asterisk indicates where a lane was removed from the original image, to match the arrangement of samples in other blots. Right: Ub<sub>n</sub> Western blot intensities were quantitated by Image J and presented as % of the highest intensity. (<u>C). Quantitative analysis of polyubiquitin conjugates and proteasomal subunits in extracts with Triton (extracts + Triton</u>). Experiment like in B except that extract 6 from each set shown in A contained the majority of polyubiquitin conjugates and was used as ‘extract with Triton X-100’ without combining with extracts 7–8, which would dilute this pool (see Methods), and the sensitivity of Western blots was increased by using higher concentration of primary antibodies to ensure detection of lower proteasome levels per similar total protein content. (<u>D). Analysis of polyubiquitin burden per 20S core</u>. The indicated amounts of total proteins in each extract were analyzed side by side on a single membrane to identify the amounts of extracts with similar 20S levels and to estimate the polyubiquitin load per 20S (see text for details). Gray boxes mark samples similar to those marked with gray boxes in panel E. Longer exposure was required for detection of Ub<sub>1</sub> than Ub<sub>n</sub>. <u>(E). Analysis of polyubiquitin burden per 20S core</u>. Experiment like in D except that samples were probed for polyubiquitin (Ub<sub>n</sub>) and the 20S β5 subunit. <u>(F). Quantitation of polyubiquitin burden per 20S core. </u>The indicated WB data from panels D (white bars) and E (colored bars) were quantitated and shown in arbitrary units (AU) and as a percentage of the highest intensity (12,865 AU = 100%). Note that due to vastly different polyubiquitin levels, analysis under less quantitative conditions (Ub<sub>n</sub>, white bars, longer WB exposure) underestimates differences observed under strictly quantitative conditions (Ub<sub>n</sub>, colored bars, short WB exposure). GAPDH (or GAP), and Calnexin (CNX): loading controls. Error bars represent variations observed in similar analysis performed with livers isolated from three WT and three PiZ mice.</p
Reduction-sensitive modifications typical of aging WT mice accumulate prematurely on selected proteasomal subunits in the livers of PiZ mice.
<p><u>(A). Rpt4 Western blot analysis of unreduced, un-boiled liver extracts</u>. 5 µg of the indicated extracts were mixed with Laemmli buffer without (unreduced samples) or with (reduced samples) βME (−/+ βME), separated by SDS-PAGE without prior boiling, and analyzed by Western blot with antibodies specific to Rpt4. <u>(B). Rpt4 Western blot analysis of unreduced, but boiled, samples</u>. Experiment like in A, lanes 1–9, except that extracts were mixed with Laemmli buffer without βME (- βME) and boiled for 4 minutes prior to SDS-PAGE. <u>(C). Analysis of 26S proteasomes co-purified with polyubiquitin conjugates. </u>WT and PiZ 26S proteasomes were co-purified with polyubiquitin conjugates as described in Fig. 4B and analyzed by Western blot after separation by SDS-PAGE with (lanes 1, 2) and without (lanes 3, 4) prior reduction by βME. Serial dilutions of unreduced liver extract from 103 old PiZ mouse are shown as reference (lanes 5–8). Extract prepared from 103 days old WT mice had similar reduction-sensitive modifications (data not shown, see panel A). <u>(D). HPLC of WT and PiZ liver extracts followed by unreduced SDS-PAGE/Western blot analysis</u>. Experiment like Fig. 5B, except that gel filtration fractions were not reduced with βME before SDS-PAGE. Data shown in A-D are representative of at least 3 independent experiments.</p
AT-Z analysis.
<p><u>(A). Histological PASD analysis of liver fragments typical of 78 days old WT and PiZ mice</u>. <u>(B). Model of 20 mg liver fragment</u>. The model was prepared assuming liver density similar to water, which in the case of 20 mg fragment would lead to an approximate volume of 20 mm<sup>3</sup> and a cube root of 2.714 mm. PASD-stained PiZ liver fragments at 50× magnification were fitted into 2.714×2.714 squares with hemacytometer grid. <u>(C). Extraction of AT-Z</u>. Extracts characterized in Fig. 1B and 1C were analyzed by Western blot (WB) for AT-Z. The total protein contents were verified by Ponceau stain to be similar to those observed in Fig. 1A, CB (data not shown). Similar results were obtained with livers isolated from two WT and two PiZ mice, with at least three fragments analyzed from each liver.<u> (D). <i>In vitro</i> de-glycosylation of AT-Z</u>. Extracts adjusted to similar amount of AT-Z were treated with the de-glycosylating enzymes as indicated, followed by separation on 8% SDS-PAGE and WB for AT-Z. <u>(E). Western blot analysis of AT-Z levels</u>. The indicated amounts of extracts prepared from seven randomly selected 20 mg fragments of one perfused and one unperfused liver were analyzed by WB for AT-Z. <u>(F). Quantitation of AT-Z Western blots</u>. Data shown in E were quantitated by Image J and presented as a fraction of the highest signal in each set (1–7). Common background highlights symbols related to one type of extract (purple: extract with SDS; blue: extract with Triton X-100; black stripes: extract without detergent; unfilled symbols: perfused liver; filled symbols: unperfused liver). Arrows show the amount of each extract (10, 1.2, and 0.025 µg) required for 50% of the maximal AT-Z signal.</p
Polyubiquitin conjugates extracted from WT and PiZ livers have similar affinities to ubiquitin-binding domain and associate with similar amounts of catalytically active 26S proteasomes.
<p><u>(A). Psmd4 fragments used in this study.</u> Top: Gst was fused to C-terminal ends of the indicated fragments of mouse Psmd4, as described in methods. FL: full length; UDB: ubiquitin-binding domain; UIM: ubiquitin-interacting motif; Δ: deletion; aa: amino acid. Bottom: proteins retained on 10 µl of G<sup>SH</sup>-Sepharose beads were released by boiling with 20 µl of Laemmli buffer followed by separation of 2 µl by SDS-PAGE and staining with Commassie blue. <u>(B). Binding of polyubiquitin conjugates from detergent-free WT and PiZ extracts</u>. The indicated Gst fusion proteins immobilized on 10 µl of G<sup>SH</sup> Sepharose beads were incubated on ice with 20 µl of undiluted (16 µg/µl) WT or PiZ liver extracts. After 5 minutes, beads were washed 5 times with 1 ml of ice-cold binding buffer, released by boiling with 10 µl of Laemmli buffer, and analyzed by Western blot, as indicated. <u>(C). Binding of polyubiquitin conjugates from serially diluted WT and PiZ extracts</u>. Binding was performed as described in B, except that <sup>Gst</sup>UBD<sup>only</sup> beads were incubated with 20 µl of 2-fold serial dilutions of each extract standardized by total protein content, as indicated. Data shown in lanes 1–8 and 9–16 were derived from separate membranes that were processed and developed side by side. <u>(D). CTL peptidase activity associated with proteins co-purified with polyubiquitin conjugates on <sup>Gst</sup>UBD<sup>only</sup> beads</u>. WT and PiZ liver proteins were isolated on 10 µl of <sup>Gst</sup>UBD<sup>only</sup>, or the control Gst beads, as described in B, followed by suspension in 400 µl reaction mixtures with Suc-LLVY-AMC and incubation at 37°C with gentle mixing. After 7 minutes, beads were sedimented by 10 second spin at 12,000 rpm and supernatants were analyzed for AMC fluorescence, as described in Methods. Error bars show a typical range of variations observed in multiple experiments. <u>(E). Continuous measurements of AMC fluorescence in reaction mixtures separated from beads after the initial 7 minutes of incubation at 37°C</u>. Red symbols: PiZ samples prepared using <sup>Gst</sup>UBD<sup>only</sup> (filled) or Gst (open) bites. Black symbols: WT samples prepared with <sup>Gst</sup>UBD<sup>only</sup> (filled) or Gst (open) bites. The data are representative of 2–3 independent experiments, and error bars indicate a typical range of variations.<u> (F). Western blot analysis of proteins present in reaction mixtures analyzed in E</u>. Proteins recovered from beads (beads after: b.a.) and supernatants (supernatants after, s.a.) separated after the initial 7 minutes of incubation at 37°C were analyzed by Western blot, as indicated. The indicated amounts of purified human 20S (h20S) are shown as reference. <u>(G). EM analysis of the 26S and 20S side views</u>. Samples were prepared like in E, but processed for EM analysis, as described in Methods. Average images of the doubly capped (dc) and singly capped (sc) 26S structures, and the side view (sv) of the 20S include 83–367 individual particles found in multiple samples. Bars show quantitative representation of each type of structure calculated as a percentage of the total number of the proteasomal side views (584 in WT samples, and 370 in PiZ samples). <u>(H). EM analysis of additional particles in WT and PiZ samples</u>. Average images of small rings, large rings, and blob structures include 15–200 particles found in each sample. Bars show quantitative representation of each type of structure, including the side views of the proteasome, calculated as a percentage of the total number of particles (1192 in WT samples, and 1139 in PiZ samples). Error bars represent variations observed in analysis of three independently prepared sets of samples.</p
Rapid extraction of the proteasome pools from snap-frozen WT and PiZ mouse liver fragments.
<p><u>(A). Pilot extractions with increasing KCl concentrations in the absence and presence of Triton X-100</u>. A total of 17 extracts, 40 µl each, were prepared sequentially from a single 20 mg WT or PiZ mouse liver fragment, as indicated and described in text. Equal fraction (5 µl) of each extract was separated by 12.5% SDS-PAGE and analyzed by Western blot (WB) for the 20S subunits alpha, or by Commassie blue (CB) for total proteins. Samples shown in lanes 1–8 and 9–17 were analyzed on separate mini-gels that were processed side-by-side and exposed on a single large X-ray film. <u>(B). Extractions with 50 mM KCl in the absence and presence of Triton X-100. </u>Experiment like in A, except that 9 sequential extracts, 40 µl each, were prepared from a single 20 mg liver fragment using buffers that contained 50 mM KCl and either no detergent, 0.5% of Triton X-100, or 2% SDS, as indicated. Asterisks marks a space without sample loaded that was removed from the original 19S Rpt4 WB image to match the loading used in the 20S β5 WB. <u>(C). The origin of the proteasome pools</u>. Experiment like in B, except that with focus on proteins known to reside in specific subcellular locations: GAPDH and NFκB (cytosol); Cytochrome C (mitochondria); LC3 (autophagic vesicles); Calnexin and BiP (ER); Lamins A and C, and Histone H3 (nucleus). The total protein contents in WT and PiZ extracts were verified by Ponceau stain to be similar to those observed in Fig. 1A, CB (data not shown). Asterisk indicates where a lane without sample loaded (empty space) was removed from the original WB image, to match the arrangement of samples in other blots. The data are representative of four WT and four PiZ mice, with 3 randomly selected fragments extracted from each liver (24 extraction sets total).</p
Gel filtration chromatography reveals similar, albeit not identical, pools of the 26S, 20S, 19S, and unassembled subunits in WT and PiZ mouse liver extracts.
<p>(A). <u>HPLC of freshly thawed, detergent-free WT and PiZ liver extracts</u>. WT (black) and PiZ (red) extracts were separated by HPLC on Superose 6 10/30 and analyzed for CTL peptidase activity (top, 100 µg of total protein input) or Western blot (bottom, 500 µg of total protein input) as described in Methods. Arrows mark the loss of activity after incubation with 1 µM epoxomycin, or apyrase that depletes ATP. (B). <u>HPLC of WT and PiZ liver extracts pre-incubated for 1 hour at 37°C with 200 mM KCl</u>. Experiment like in A, except that extracts were pre-incubated for 1 hour at 37°C with 200 mM KCl, to induce separation of the 20S and 19S complexes. (C). <u>Quantitation of the indicated 20S and 19S subunits in the three major pools separated by HPLC (GF 20</u>–<u>22, GF 25–27, and GF 31–33). </u>The indicated fractions (GF 20–22, GF 25–27, and GF 31–33) were combined and analyzed side-by-side by Western blot, as indicated. Quantitation of WB data is shown on the right, with error bars indicating the range of variations in 3 experiments.</p