Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from <i>Methanoculleus marisnigri JR1</i> in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min^–1 × 10^–3), affinity and Michaelis constant (<i>K</i>_m) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (<i>K</i>_i) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail

Silver_fox_dataset_DRYAD

mtDNA sequences for 3 breeding lines of russian silver foxe

p53 depletion does not suppress but rather exacerbates Spns1 deficiency.

<p>(<b>A</b>) Effect of <i>p53</i> knockdown on embryonic senescence and autolysosome formation in <i>spns1</i> morphants. The impact of transient <i>p53</i> knockdown on SA-β-gal (SABG) induction, as well as on EGFP-LC3 and LysoTracker (LysoT) puncta, was determined in <i>spns1</i> morphants at 84 hpf, followed by the MO (4 ng/embryo) injections. Inverse-sequence <i>p53</i> MO (inv. <i>p53</i> MO) was used as a negative control for the original <i>p53</i> MO. Scale bar, 250 µm in the SABG images. Scale bar, 10 µm in the fluorescence images. (<b>B</b>) Quantification of the SA-β-gal intensities in MO-injected animals, as shown for the SABG images in (A). Quantification of data presented in the top row (SABG) in B (n = 12) is shown; the number (n) of animals is for each morphant. (<b>C</b>) Quantification of EGFP-LC3 and LysoTracker puncta in MO-injected animals shown in (A) (n = 9); the number (n) of animals is for each morphant. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (<b>D</b>) Effect of a <i>p53</i> mutation on embryonic SA-β-gal activity in the <i>spns1</i> mutant. The heritable impact of p53 and Spns1 on SA-β-gal induction was tested in each single gene mutant [<i>spns1^hi891/hi891</i> (<i>spns1^−/−</i>) or <i>tp53^zdf1/zdf1</i> (<i>p53^m/m</i>)] and double mutant <i>spns1^hi891/hi891;tp53^zdf1/zdf1</i> (<i>spns1^−/−;p53^m/m</i>) compared with wild-type (<i>wt</i>) animals at 84 hpf. Scale bar, 250 µm. (<b>E</b>) Quantification of the SA-β-gal intensities in <i>wt</i>, <i>tp53^zdf1/zdf1</i>, <i>spns1^hi891/hi891</i> and <i>spns1^hi891/hi891;tp53^zdf1/zdf1</i> animals, shown in (D). Quantification of data presented in panel D (n = 12) is shown; the number (n) of animals is for each genotype. (<b>F</b>) Quantitative RT-PCR analyses of senescence marker and/or mediator expression as well as p53-downstream target genes in <i>wt</i>, <i>tp53^zdf1/zdf1</i>, <i>spns1^hi891/hi891</i> and <i>spns1^hi891/hi891;tp53^zdf1/zdf1</i> at 72 hpf. Data are mean ±SD [n = 4 samples (3 embryos/sample) per genotype]. Asterisks denote significant changes compared to <i>wt</i> values. *<i>p</i><0.05. (<b>G</b>) LC3 conversions in <i>p53</i> and <i>spns1</i>-mutant animals. Protein detection for the conversion/accumulation of LC3-I to LC-II was performed in the described mutant background animals in comparison with <i>wt</i> fish at 84 hpf. Western blot analysis using anti-LC3 antibody shows endogenous LC3 protein levels, which can confirm an increase of the total amount of LC3 in the <i>p53</i> mutant compared with <i>wt</i> fish. Increased LC3-II conversion/accumulation was detected in <i>p53</i> and <i>spns1</i> double-mutants as well as in <i>spns1</i> single-mutant fish. (<b>H</b>) The blotting band intensities of LC3-I, LC3-II and β-actin were quantified (n = 6), and the relative ratios between LC3-II/actin and LC3-I/actin are shown in the bar graph; the number (n) of animals is for each genotype. (<b>I</b>) <i>wt</i>, <i>tp53^zdf1/zdf1</i>, <i>spns1^hi891/hi891</i> and <i>spns1^hi891/hi891;tp53^zdf1/zdf1</i> embryos injected with <i>beclin 1</i> MO or control MO (12 ng/embryo) were assayed for SA-β-gal at 84 hpf. <i>beclin 1</i> MO-mediated suppression of SA-β-gal in <i>spns1^hi891/hi891</i> animals was attenuated in the <i>p53</i> mutant background. Scale bar, 250 µm. (<b>J</b>) Quantification of the SA-β-gal intensities shown in (I). Quantification of data presented in H (n = 12) is shown; the number (n) of animals is for each genotype with MO. Error bars represent the mean ± S.D., *<i>p</i><0.005; ns, not significant.</p

Schematic model for Spns1 function under the control of the network module of autophagy-senescence signaling cascades differentially regulated through Beclin 1 and p53.

<p>(<b>A</b>) Beclin 1 is essential for the early stage of autophagy and its depletion suppresses the Spns1 defect by blocking the ‘autophagic process’ and its progression. BafA can decelerate ‘lysosomal biogenesis’, which subsequently presumably prevents autophagosome-lysosome fusion, through the inhibition of the v-ATPase, and contributes to amelioration of the Spns1 defect at least temporarily. Basal p53 activity may suppress the intersection between the ‘autophagic progress’ and ‘lysosomal biogenesis’ where the Beclin 1 depletion was not sufficient, but the v-ATPase inhibition was still effective enough, to compete with the p53 loss to suppress the Spns1 deficiency. By switching the basal p53 state to the activated version with UV irradiation, p53 can promote autophagy. Spns1 might be a gatekeeper of autolysosomal maturation followed by lysosomal biogenesis. It remains unknown how p53 can mechanistically be linked to the lysosomal ‘efflux’ function of Spns1 as well as the lysosomal ‘influx’ function of v-ATPase, and further investigations will be required to explore this connection. (<b>B</b>) Roles of Spns1, p53 and Beclin 1 in senescence equilibrium. Loss of Spns1 leads to an imbalance in homeostasis and increased senescence. This effect can be ameliorated by concurrent knockdown of Beclin 1. p53 has a comparatively less dramatic impact on Spns1-loss-induced embryonic senescence. When in the “basal” state, p53 helps retain equilibrium. When p53 is “activated” by UV irradiation, a modest increase in senescence is observed. The higher level of senescence is seen during loss of Spns1 in the absence of basal p53 or in the presence of activated p53. During loss/knockdown of all three genes (<i>spns1</i>, <i>p53</i> and <i>beclin 1</i>), a state of moderate senescence is observed. An increase in senescence is accompanied by a p53-dependent decrease in cellular proliferation.</p

Knockdown of <i>beclin 1</i> suppresses abnormal autolysosomal puncta formation and embryonic senescence caused by Spns1 deficiency in zebrafish.

<p>(<b>A</b>) Effect of <i>beclin 1</i> knockdown on EGFP-LC3 puncta formation in <i>spns1</i>-depleted zebrafish embryos. Injection of control (water) injection, <i>spns1</i> MO (4 ng/embryo) or coinjection of <i>spns1</i> MO (4 ng/embryo) and <i>beclin 1</i> MO (12 ng/embryo) into <i>Tg(CMV:EGFP-LC3)</i> fish was performed to assess whether the <i>beclin 1</i> knockdown reduces or eliminates aggregated LC3 puncta induced by Spns1 depletion at 84 hpf. Scale bar, 10 µm. Quantification of data presented in panel A (n = 12) is shown in the right graph; the number (n) of animals is for each morphant or water-injected control. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (<b>B</b>) Effect of <i>beclin 1</i> knockdown on EGFP-GABARAP as well as mCherry-LC3 puncta formation in <i>spns1</i>-depleted zebrafish embryos. Injection of control (water), <i>spns1</i> MO or coinjection of <i>spns1</i> MO and <i>beclin 1</i> MO into <i>Tg(CMV:EGFP-GABARAP;mCherry-LC3)</i> fish was performed to evaluate whether the <i>beclin 1</i> knockdown reduces or eliminates the aggregation of GFP-GABARAP puncta in comparison with those of LC3 caused by the Spns1 depletion at 84 hpf. Scale bar, 10 µm. Quantification of data presented in the top row (green; EGFP) (n = 9), middle row (red; mCherry) (n = 12), and bottom row (yellow; merge of EGFP and mCherry) (n = 9) in panel B is shown in the right graphs; the number (n) of animals is for each morphant or water-injected control. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (<b>C</b>) Effect of <i>beclin 1</i> knockdown on embryonic senescence in <i>spns1</i> morphant. By using the same injection samples [injection of control (water), <i>spns1</i> MO or coinjection of <i>spns1</i> MO and <i>beclin 1</i> MO into <i>Tg(CMV:EGFP-GABARAP;mCherry-LC3)</i> fish], SA-β-gal staining was performed to assess whether the <i>beclin 1</i> knockdown has any impact on the embryonic senescence caused by Spns1 depletion at 84 hpf. Representative images of individual fish by bright field (BF, live samples) and SA-β-gal (SABG) staining are shown in the upper and middle panels, respectively. Scale bar, 250 µm. Lower panels are larger magnification images of corresponding SA-β-gal samples shown in the middle panels and the fluorescence images of nuclei counterstained with DAPI. Scale bar, 10 µm. Quantification of data presented in the middle row (SABG) in panel C (n = 12) is shown in the right graph; the number (n) of animals is for each morphant or water-injected control. Error bars represent the mean ± S.D., *<i>p</i><0.005.</p

Knockdown of <i>beclin 1</i> suppresses the Spns1 deficiency in zebrafish.

<p>(<b>A</b>) Schematic representation of the zebrafish <i>beclin 1</i> (<i>zbeclin 1</i>) gene, its mRNA and protein products. A splice-blocking <i>beclin 1</i> MO was designed to overlap the intron-exon boundary at the 5′-splice junction of exon 4 in the zebrafish <i>beclin 1</i> gene. To detect aberrantly spliced RNA products, two forward primers were designed for exon 3 (EX3 primer) and exon 4 (EX4 primer), and one reverse primer was designed for exon 7 (EX7 primer) within the <i>beclin 1</i> gene. The zebrafish <i>beclin 1</i> gene has a total of 11 exons having three unique domains [BH3 domain, coiled-coil (CCD) domain, and evolutionarily conserved (ECD) domain], and the <i>beclin 1</i> MO was anticipated to disrupt the BH3 domain encoded by exon 4 and exon 5. (<b>B</b>) Splicing detection of <i>zbeclin 1</i> mRNA by RT-PCR. Amplified PCR fragments show the intact sizes of the two amplicons for EX3-EX7 and EX4-EX7 following control (water) injection or only <i>spns1</i> MO injection. Either <i>beclin 1</i> MO (12 ng/embryo) injection or coinjection of <i>spns1</i> MO (4 ng/embryo) and <i>beclin 1</i> MO (12 ng/embryo) generated a skipping of exon 4 (<i>beclin 1Δexon4</i>). This was detected by the presence of an altered EX3-EX7 amplicon and undetectable EX4-EX7 product. The deletion of exon 4 was confirmed by sequencing. Injected embryos were harvested for total RNA isolation at 54 hpf. (<b>C and D</b>) Rescue of the <i>spns1</i> morphant by <i>beclin 1</i> knockdown. (C) The yolk opaqueness phenotype appearance in control-injected (water), <i>spns1</i> MO-injected, and <i>spns1</i> and <i>beclin 1</i> MOs-coinjected embryos was followed through 72 hpf. At 24 hpf, opaqueness commenced from the yolk extension region, which had almost disappeared or was severely damaged (more than 95% of <i>spns1</i> MO-injected animals) with an extension of opacity to the entire yolk at 48 hpf. By 72 hpf, yolk opaqueness became highly dense throughout most of the <i>spns1</i> MO-injected embryos, which usually died within another 24 h. Scale bar, 250 µm. (D) Clarification of the yolk opaqueness phenotype in <i>spns1</i> morphants at 72 hpf. As described in (C), more than 95% of the <i>spns1</i> MO-injected embryos showed a ‘mostly opaque’ yolk at 48 hpf, and such embryos subsequently died. Animals showing a ‘partially opaque’ yolk could sometimes be recovered and subsequently survived 96 h and beyond. <i>beclin 1</i> MO coinjection dramatically increased (more than 10 times) the animal numbers with the partial yolk opaque phenotype. (<b>E</b>) Survival curve for <i>spns1</i> morphant and <i>spns1;beclin 1</i>-double morphant larvae (log rank test: χ² = 162.5 on one degree of freedom; <i>p</i><0.0001).</p

Acidity-dependent lysosomal biogenesis is rate limiting in <i>spns1</i>-mutant zebrafish.

<p>(<b>A</b>) Effect of bafilomycin A₁ (BafA) on the yolk opaque phenotype (BF; bright field) and embryonic senescence (SABG; SA-β-gal) in the <i>spns1</i> mutant in the presence or absence of <i>p53</i> at 48 hpf. Normal wild-type (<i>spns1^+/+;p53^+/+</i>), <i>tp53^zdf1/zdf1</i> (<i>p53^m/m</i>), <i>spns1^hi891/hi891</i> (<i>spns1^−/−</i>) and <i>spns1^hi891/hi891;tp53^zdf1/zdf1</i> (<i>spns1^−/−;p53^m/m</i>) embryos at 36 hpf were incubated with BafA (200 nM) for 12 h, and stained with LysoTracker at 48 hpf, followed by SA-β-gal staining at 60 hpf. Scale bar, 250 µm. (<b>B</b>) Quantification of the SA-β-gal intensities shown in (A). Quantification of data presented in panel A (n = 12) is shown; the number (n) of animals is for each genotype with DMSO or BafA. (<b>C</b>) Gross morphology, EGFP-LC3 and LysoTracker intensities in wild-type (<i>wt</i>) and <i>spns1</i>-mutant animals treated with BafA shown at 48 hpf (12 h treatment starting at 36 hpf). Scale bar, 250 µm. (<b>D</b>) Quantification of the EGFP-LC3 and LysoTracker fluorescence intensities shown in (C). Quantification of data presented in the middle and bottom rows (green; EGFP, red; mCherry) in panel C (n = 12) is shown; the number (n) of animals is for each genotype with DMSO or BafA. (<b>E</b>) Intracellular autolysosome formation and lysosomal biogenesis in the BafA-treated <i>spns1</i> mutant. The samples analyzed in (C) were observed by using confocal microscopy at high magnification (×600). Scale bar, 10 µm. (<b>F</b>) Quantification of the EGFP-LC3 and LysoTracker fluorescence intensities shown in (E). Quantification of data presented for EGFP (green) and mCherry (red) signals in panel E (n = 6) is shown; the number (n) of animals is for each genotype with DMSO or BafA. Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (<b>G</b>) Insufficient intracellular acidity constituent in the <i>spns1</i> mutants. Using two different acidic-sensitive probes, LysoSensor 189 and neutral-sensitive LysoSensor 153 (green), in combination with LysoTracker (red), <i>wt</i> and <i>spns1</i>-mutant animals showed detectable signals when stained at 72 hpf. In <i>spns1</i>-mutant animals, autolysosomal and/or lysosomal compartments were more prominently detectable by LysoSensor 153 than by LysoSensor 189, at the cellular level with enhanced signal intensity of these enlarged compartments. In stark contrast, the cellular compartments in <i>wt</i> fish treated with pepstatin A and E-64-d (P/E) (12 h treatment from 60 hpf through 72 hpf) were more prominently detectable by LysoSensor 189 than by LysoSensor 153 under the identical LysoTracker staining conditions. Of note, these autolysosomal and lysosomal compartments in <i>spns1</i> mutants, as well as in <i>wt</i> animals treated with pepstatin A and E-64-d, may still retain some weak (higher pH) and strong (lower pH) acidity, respectively, as short-term BafA treatment (for 1 h between 71 and 72 hpf) can abolish the acidic compartments stained by both LysoSensor and LysoTracker (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004409#pgen.1004409.s017" target="_blank">Figure S17C and D</a></b>). Scale bar, 10 µm. (<b>H</b>) Quantification of the LysoSensor (189 and 153) and LysoTracker fluorescence intensities shown in (G). Quantification of data presented for LysoSensor (green) and LysoTracker (red) signals in panel G (n = 6) is shown; the number (n) of animals is for each genotype with DMSO or pepstatin A and E-64-d (P/E). Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. Error bars represent the mean ± S.D., *<i>p</i><0.005; ns, not significant.</p

Genes located inside or within 50,000 bp from start and end of the multi SNP clusters in the dog genome.

<p>Cluster numbers refer to the numbering from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127013#pone.0127013.t002" target="_blank">Table 2</a>.</p><p>Genes located inside or within 50,000 bp from start and end of the multi SNP clusters in the dog genome.</p

SNP minor allele frequency and heterozygosity in tame and aggressive populations.

<p>SNP minor allele frequency and heterozygosity in tame and aggressive populations.</p

Estimation of linkage disequilibrium (r²) in tame and aggressive fox populations.

<p>Distributions of r^<b>2</b> values between pairs of SNPs separated by different distances are compared between tame (green) and aggressive (red) populations. SNP pairs were divided into 14 sets (bins) using the estimated distances between SNPs in the fox genome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127013#pone.0127013.s007" target="_blank">S2 Table</a>). Each bin is represented by a doubled bar (green and red) on the graph. The range of distances between SNPs in each bin is indicated on the x-axis. The width of the bar represents the relative number of SNP pairs in that bin for that population after a log transformation (wider bars have more pairs of SNPs). Exact numbers of SNP pairs in each bin are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127013#pone.0127013.s007" target="_blank">S2 Table</a>. The y-axis indicates r^<b>2</b> values for pairs of SNPs. The yellow diamonds correspond to the mean r^<b>2</b> for all SNPs in that bin in the population. The white circles correspond to the median values. The thin black line within each bar represents r^<b>2</b> values in that bin in the population in the interval from the 25th to 75th percentile. The horizontal line corresponds r^<b>2</b> = 0.2.</p