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Proteome-wide modulation of degradation dynamics in response to growth arrest

  1. Sina Ghaemmaghamia,b,1
  1. aDepartment of Biology, University of Rochester, Rochester, NY 14627;
  2. bMass Spectrometry Resource Laboratory, University of Rochester, Rochester, NY 14627
  1. Edited by David A. Baker, University of Washington, Seattle, WA, and approved October 18, 2017 (received for review June 6, 2017)

  1. Fig. 2.

    Up-regulation of macroautophagy in quiescent cells contributes to the degradation of long-lived proteins in quiescent cells. (A) Western blots showing the accumulation of LC3-II and depletion of p62 in wild-type quiescent cells. (B) Western blots showing the reduced levels of Ser-2448 phosphorylation of mTOR and phosphorylated S6 kinase in quiescent cells. (C) Western blots indicating the absence of ATG5 protein (normally expressed in the cell complexed to ATG12), depletion of LC3-II, and accumulation of p62 in ATG5?/? cells. (D) Relationship between relative protein degradation rates in quiescent and dividing ATG5?/? cells to kdegradation, in comparison with wild-type cells. Comparisons were conducted by two-sided Mann?Whitney U test. NS (not significant) indicates a P value greater than 0.05. (E) Distribution of kdegradation measurements for wild-type and ATG5?/? cells. The data indicate that the enhancement of kdegradation in quiescent cells is significantly diminished in ATG5?/? cells.

  2. Fig. 3.

    Up-regulation of lysosomal biogenesis in quiescent cells. (A) The scatter plot shows the differential expression of lysosomal genes at protein and mRNA levels as determined by SILAC and RNA-Seq, respectively. Orange dots highlight the lysosomal proteome (51). Three cathepsin genes, analyzed in B, are highlighted. (B) Western blots indicating the up-regulation of cathepsins A, B, and D in quiescent cells. (C) Increased cathepsin D activity in quiescent cell extracts based on a cell-free assay. (D) Increased accumulation of lysosomes in quiescent cells measured by flow cytometry of cells stained with LysoGreen. The box plots indicate the complete range (whiskers), interquartile range (box), and median (white line) of the measurements for replicate experiments. The forward scatter data indicate that cell size was not altered by quiescence. Comparisons were conducted by two-sided Mann?Whitney U test. NS (not significant) indicates a P value greater than 0.05.

  3. Fig. 4.

    Proteasome activity does not contribute to the enhancement of degradation rates in quiescent cells. (A) Proteasome activity is enhanced in quiescent cells based on in vitro chymotrypsin-like activity assays; ?epx (epoxomicin) indicates total chymotrypsin-like activity, +epx indicates chymotrypsin-like activity from nonproteasome sources, and the difference (?) is a measurement of chymotrypsin-like activity from the proteasome. (B) Changes in the mRNA and protein expression levels of proteasome subunits determined by RNA-Seq and SILAC. The data indicate the up-regulation of 11S subunits PSME1 (PA28α) and PSME2 (PA28β) and were validated by (C) qPCR and (D) Western blots. The 20S core subunit PSMB1 is included as a control. (E) Knockdown of PSME1 by shRNA. (F) Knockdown of PSME1 eliminates the enhancement of proteasome activity in quiescent cells based on chymotrypsin-like activity assays. (G) Knockdown of PSME1 does not influence protein degradation rates (kdegradation) in quiescent cells as determined by dynamic SILAC experiments. (H) Expression level of a fluorescent proteasome substrate (pZsProSensor1) is unaffected by quiescence. Fibroblasts were transfected with pZsProSensor1 expression vector in dividing or contact-inhibited quiescent states, with or without subsequent addition of epoxomicin. The fluorescence distribution of cells was analyzed by FACS. Box plots were generated as described in Fig. 3D. Comparisons were conducted by two-sided Mann?Whitney U test. NS (not significant) indicates a P value greater than 0.05.

  4. Fig. 5.

    Regulation of protein dynamics in quiescent cells and its ramification for protein homeostasis. (A) In quiescent fibroblasts, protein synthesis rates are reduced and protein degradation rates are increased. The latter effect is caused by concurrent up-regulation of lysosome biogenesis and macroautophagy and is limited to long-lived proteins. (B) The distribution of the average age of protein populations in quiescent and dividing cells. The average age of a given protein population can be experimentally determined by its clearance rate (Inset). In the theoretical scenario where the quiescence-induced up-regulation of protein degradation is lacking, the age distribution of the proteome would be expected to significantly increase.

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