Supplemental Data

1

• Supplemental Data
• Figure S1. Additional control experiments and
  supportive evidence, related to Figure 1.
• (A) Intracellular accumulation of ubiquitin
  conjugates post-Bortezomib challenge, analyzed on
  SDS-PAGE. NCI-H929 cells were treated with 10 nM
  bortezomib or untreated for 2, 4, 6, 8 and 10
  hours, after which cell lysate was analyzed on
  SDS-PAGE (12.5) and blotted for ubiquitin
  conjugates. The membrane was also probed for
  GAPDH as a loading control. (.B) Effect of
  incubation time and temperature on the degree of
  Bortezomib-mediated inhibition of purified 26S
  proteasomes. 26S proteasomes (purified from yeast
  and from human OPM2 Myeloma cells) were incubated
  at 30oC with 10 nM bortezomib or PBS control for
  2, 4 and 6 hours. The CT-like activity of the
  cells was then measured using Suc-LLVY-AMC, at
  30oC for yeast proteasomes and 37oC for human
  proteasomes. Results were plotted as a percent of
  DMSO control. An important technical point is
  that prolonged incubation at 30oC (but not at
  4oC, see Figure1B) gradually destroyed
  proteasomes integrity in the test tube, as
  evinced by declining activity in the DMSO control
  samples (for incubation times 2, 4 and 6 hrs
  human 26S control activity declined from 100 to
  85.6 to 68.4, and yeast 26S control activity
  declined from 100 to 88.5 to 77.5). However,
  as this figure and Fig1B show, relative degree of
  Bortezomib-mediated inhibition was stable and
  independent of the length of time Bortezomib was
  pre-incubated with proteasomes (n2 per value).
• Figure S2. Additional control experiments and
  supportive evidence, related to Figure 1.
• (A) Inhibition of each type of active-site in
  bortezomib-treated NCl-H929 lysate. The effect of
  a Bortezomib concentration range on proteasome
  activity in cell lysate was tested, using
  fluorogenic peptide substrates against each of
  the three types of active-sites. Measured as a
  percentage of DMSO treated control. Cell lysate
  from NCI-H929 cells was used. Proteasome
  substrates were Suc-LLVY-AMC, Ac-RLR-AMC,
  Ac-GPLD-AMC. (n2 per value).(.B) NCl-H929 Cell
  Viability after addition of 10nM Bortezomib. Cell
  viability measured by Annexin-V staining of 10nM
  bortezomib treated NCl-H929 cells. Measured as a
  percentage of DMSO control at 24 hours. (C)
  CT-Like activity in cell lysate of NCl-H929 cells
  treated with 10nM bortezomib, measured with and
  without 0.015 SDS added to the assay reaction,
  in order to test for proteasome destabilisation.
  The entrance to the lumen of free CP is occluded,
  and thus not accessible to fluorogenic
  substrates adding a low dose of SDS opens access
  to the lumen and thus visualises proteasome
  activity of free CP. NCl-H929 cells were treated
  with 10nM bortezomib for different periods of
  time. Cells were harvested, and the CT-like
  activity of the cells was then measured using
  Suc-LLVY-AMC, either with or without 0.015 SDS
  added to the assay reaction to visualise activity
  of any free CP particles. Results were plotted as
  a percent of DMSO control. (n2 per value).
  Because the presence of SDS in the assay did not
  change the result, the severe decrease in
  proteasome activity is not due to
  Bortezomib-triggered destabilisation of the
  proteasomes and disruption of the CP-RP
  interaction. (D) CT-Like activity of 10nM
  bortezomib-treated sensitive in-sensitive cell
  lines. Cell viability measured by Annexin-V
  staining of 10nM bortezomib treated cells.
  Measured as a percentage of DMSO control from
  0-10 hours. Cell lines which are insensitive to
  10nM Bortezomib (T-cell line Jurkat lung
  carcinoma cell line A549) retain around 20 of
  CT-like activity remaining, whereas the myeloma
  cell lines (RPMI-8226 NCI-H929) that die
  experience an even more severe drop in CT-like
  activity. (n2 per value). (E) T-Like activity of
  10nM bortezomib treated NCl-H929 cells. NCl-H929
  cells were treated with 10nM bortezomib for
  different periods of time. Cells were harvested,
  and the T-like activity of the cells was then
  measured using Ac-RLR-AMC. Results were plotted
  as a percent of DMSO control. Note that although
  bortezomib does not inhibit the T-Like site its
  activity starts to decrease around 6h post
  treatment (n2 per value)
• Figure S3. Blots of proteasomal subunits after
  incubation with or without PDE1S1, related to
  Figure 2.
• (A) Rpn11-tagged proteasomes were purified from
  cell lysate by capture on NiNTA Agarose beads.
  The beads were then treated with PDE1S1
  combination for 45min at 20oC. Proteasomes were
  then washed to remove enzymes and eluted in
  PBS250mM imidizole and run on SDS-Page gel. Note
  changes to banding patterns and intensities
  including relative intensities between control
  and treated lanes. (B) Coomassie stained SDS-PAGE
  gel showing relative amounts of substrate to
  PDE1S1 treated and non treated purified
  proteasomes. Run as proteasome/substrate x3 ratio
  as seen in figure 3B.

2

• Supplemental Data
• Figure S4. In vitro ubiquitination and
  proteasome-mediated degradation system, related
  to Figure 3.
• (A) Domain topology of model G3P-cyclin
  substrate. Domain topology of model G3P-cyclin
  substrate. Points of interest His6 and strep-tag
  domains used for purification, Lysine residue of
  G3P domain from which ubiquitination occurs, PY
  domain recognised by Rsp5 (E3) enzyme. (B)
  Purification and ubiquitination of model
  G3P-cyclin substrate. Coomassie-stained SDS-PAGE
  gels showing i) purification of substrate and
  ubiquitination enzymes (Substrate 15.6kDa, E1
  118.4kDa, E2 17.1kDa, E3 67.6kDa) . (C) in
  vitro Ubiquitination conditions. Combinations of
  enzymes, substrate and ubiquitin incubated on ice
  and at 37oC. Note that only with addition of all
  the required enzymes/substrate/ubiquitin/ATP and
  incubation at 37oC is efficient ubiquitination
  observed. (D) Degradation of ubiquitinated
  substrate Western blot against strep-tag of
  substrate. After incubation of yeast proteasomes
  and substrate. Note loss of signal consistent
  with degradation of substrate in last lane. Note
  that, under these in vitro conditions, ubiquitin
  chains on the substrate are released intact and
  not subject to proteasome-mediated hydrolysis,
  leading to a downward shift in molecular weight
  seen in the a-ubiquitin blot and on Coomassie.

3
Figure S1
a
4 hrs
6 hrs
8 hrs
2 hrs
10 hrs
-

-

-

-

-

Bortezomib (10 nM)
120
Ubiquitinated conjugates
90
50
a-ubiquitin conjugates (FK2 MoAb)
a-GAPDH
(SDS-PAGE, 12.5)
b
2
2
4
6
6
4
Hours of incubation (at 30oC), before measuring
degree of inhibition
4
a
Figure S2
d
b
e
c
5
Figure S3
a
NiNTA captured Tag-Rpn11 proteasomes Incubated
for 45min at 20oC
-
-
-
-
PDE1S1
100 70 55 40 35 25
a-Rpt4 Long exposure
a-Strep Rpn11
a-Rpt4
a-a7
NiNTA captured Tag-Rpn11 proteasomes Incubated
for 45min at 20oC
-
-
-
-
-
PDE1S1
100 70 55 40 35 25
a-S5a-Rpn10
a-ß5i
a-Rpn12
a-Rpt2
a-Rpt5
b
NiNTA Proteasome
Substrate
PDE1S1
-
100
70
55
40
35
25
15
6
Figure S4
a
G3P-cyclin substrate
K
His6 G3P
ssra cyclin PY
Thrombin

Strep-tag
c
b
d