Download Effect of shRNA knockdown of protein complex subunits on complex

Effect of shRNA knockdown of protein complex subunits on complex formation and
quantitation using SILAC technique.
Mahbod R. Hajivandi; John F. Leite; Xiquan Liang; Antje Taliana; Marieke Svoboda; Marshall Pope;
WP512
Invitrogen, Carlsbad, CA
Results and Discussion
Anti-Arp3 IgG
25.00
Time
35.00
40.00
45.00
50.00
Figure 5. shRNA knockdown of native Arp3 subunit in 293 cells stably transfected with TAPtagged Arp3 results in ~55% knockdown of native Arp3 subunit. Western blots of lysates
from 293 cells stably transfected with TAP-tagged Arp3 show a knockdown of the Arp3
subunit that is consistent with SILAC experiments.
LALETTVLVESYTLPDGR, 1976.05 Da
DITYFIQQLLR, 1408.77 Da
705.4034
100
shRNA
989.0305
B
%
707.3888
%
C
990.9956
Arp3 #1
Target cleavage
Figure 2. shRNA in the RNA interference pathway.
705
707
709
711
m/z
985
987
989
991
993
995
m/z
997
Methods
DNTINLIHTFR, 1342.70 Da
TLESSIQGLR, 1102.62 Da
552.3014
554.2891
%
Figure 6. shRNA knockdown of native Arp3 subunit in 293 cells stably transfected with TAPtagged Arp3 results in knockdown of ArpC2 subunit. Western blots of lysates from 293 cells
stably transfected with TAP-tagged Arp3 show a knockdown of the ArpC2 subunit resulting
from shRNA directed at Arp3. Controls demonstrate that the knockdown is not the result of
shRNA binding to exogenous genes non-specifically or due to non-specific transfection
effects.
E
D
%
674.4113
m/z
678
680
ELLLQPVTISR, 1267.77 Da
605.8733
634.8792
100
100
G
F
Anti-Arp3 IgG
636.8659
%
%
607.8644
604
Light Arg
14 N Arg
4
Heavy Arg
15 N -Arg
4
Cells must be propagated for at least six doubling times to achieve 100% incorporation
For example, the total cell number would be 64 x 105 cells if started with 105 cells
Take small aliquot of cells (such as 108) to examine 100% incorporation in MALDI-TOF
Mix cells at 1:1 ratio
Figure 1. Molecular model of filament extension by the Arp 2/3 complex. (A) A model of the
actin filament branches (in the pink color) was superimposed over the 2D images shown in
(B). An additional representation of the Arp2/3 complex at the filament branch is shown in (C)
so that the protein backbone is exposed.
RNA interference (RNAi) is the preeminent gene
silencing technology currently employed in mammalian
reverse genetics experiments, in which a loss-of-function
phenotype of a gene of known sequence is sought. In
RNAi, the primary effector molecules are double-stranded
short interfering RNAs (siRNAs)1. One strand of each
siRNA molecule is incorporated into a cytoplasmic, multiprotein RNA-Induced Silencing Complex (RISC) and
serves as a guide for locating complementary target RNAs.
A RISC nuclease cleaves the target RNA within the region
basepaired to the siRNA guide. The target is then subject
to degradation by cytoplasmic exonucleases.
Volkmann N, Amann KJ, Stoilova-McPhie S, Egile C, Winter DC, Hazelwood L, Heuser
JE, Li R, Pollard TD, Hanein D. Structure of Arp2/3 complex in its activated state and in
actin filament branch junctions. Science. 2001 Sep 28;293(5539):2456-9.
Cell mix after control shRNA
(-)
Cell mix after shRNA treatment
(+)
TAP-Affinity enrichment of Arp 2/3 complex
shRNA
KO the
endogenous
Arp subunit
to enrich
complex
with biotintagged
transfected
subunit
- +
SDS-PAGE/
Western
tryptic digest
+
LCMS
ShRNA
Quantification via the ratio
of isotopic peptide pairs in
MS spectrum
Figure 3. Experimental strategy of SILAC for quantification. Identical pools of cells
stably transfected with biotin-tagged ArpC2 or Arp3 are differentially labeled with
isotope-enriched lysine. One of the sets of cells is also treated with shRNA targeted to
either native expressed ArpC2 or Arp3. After the complex is enriched by streptavidinagarose, the components are separated by SDS-PAGE followed by MS analysis for
quantitation. Knockdown of the endogenous Arp subunit by shRNA should result in
enhanced efficiency of Arp 2/3 complex affinity-enrichment due to competitive
substitution with biotin-tagged Arp subunit.
m/z
Mock
556
676
ArpC2#4
554
674
LacZ (-)
552
672
pENTRU6(-)
550
LIGNMALLPIR, 1209.73 Da
670
Arp3#2
shRNA
668
Stably Transfected Cells (Biotin-tagged Arp2 or Arp3)
SILAC
Anti-Arp2 IgG
Arp3 #1
Tandem affinity tagged (6xHis coupled to biotin)ArpC2
or Arp3 subunits were stably expressed in mammalian
cells and the tag was used to purify the Arp2/3
complexes from lysates. Cell lines were transfected with
shRNAs U6 Entry clones specifically targeting a second
subunit of the same complex in medium containing
15N-Arg (Ajinomoto). Another culture was treated with
lacZ shRNA U6Entry clones in non-labeled control
medium. The cultures were combined and protein
complexes purified using Streptavidin agarose. The
complex subunits were digested with trypsin and
digested peptides were analyzed by Q-TOF.
672.3736
100
100
Actin related proteins Arp2 and Arp3, along with 5 other
proteins, form a complex (Arp2/3 complex) that binds to the
side of an actin filament and nucleates the formation of a
new filament branch. Filament extension by Arp2 and Arp3
spearheads the molecular mechanism that supports a wide
variety of cellular processes such as cell motility, cell shape
and structure. The mechanism of filament extension is
shown in the figure below from the Pollard laboratory
publication of the high-resolution crystal structure of the Arp
2/3 complex.
30.00
Mock
20.00
100
703
Introduction
15.00
Arp3#2
RISC loading
siRNA unwinding
Target recognition
10.00
ArpC2#4
5.00
Target mRNA
LacZ (-)
%
siRNA (21-23 bp)
pENTRU6(-)
A
Mock
100
LacZ (-)
shRNA expression
Nuclear export
Endogenous Dicer activity
ArpC2#4
shRNA
RNA
Arp3 #1
DNA
Arp3#2
Pol III
pENTRU6(-)
Overview
In combination with mass spectrometry, recent advances in
protein tagging and purification have made it possible to
isolate and characterize native complexes in high
throughput. Stable isotopic labeling in cell cultures (SILAC)
has been used successfully to study the dynamics of cell
signal-dependent protein-protein interactions. Our goal was
to develop short-hairpin RNA (shRNA) knockdown as an
investigative means to study the role of the individual
subunits on the activity and stoichiometry of complexes.
Because many of these knockdown effects are anticipated
to be subtle, we reasoned that conventional methods may
not have sufficient dynamic range to discern small changes
in protein expression. Instead we have employed metabolic
labeling, SILAC, to precisely quantify stoichiometric
changes in complex formation caused by shRNA
knockdown perturbations.
606
608
610
m/z
633
635
637
639
m/z
Figure 7. shRNA knockdown of native ArpC2 subunit in 293 cells stably transfected with TAPtagged ArpC2 results in knockdown of Arp3 subunit. Western blots of lysates from 293 cells
stably transfected with TAP-tagged ArpC2 show a knockdown of the Arp3 subunit resulting
from shRNA directed at Arp3. Controls demonstrate that the knockdown is not the result of
non-specific shRNA or transfection effects.
Figure 5. shRNA knockdown of native Arp3 subunit in 293 cells stably transfected with biotintagged Arp3. Total Ion Chromatogram of digested affinity enriched Arp2/3 complex from a
SILAC labeling experiment (A). Trypsin digestion and RP-LC separation were performed in
the presence of Invitrosol™ LC/MS. MS spectra of peaks correspond to digested peptides
from the different subunits of the complex (B) ARP3 (C) ARP2 (D) AR1A (E) ARPC2 (F)
AR21 (G) AR20, unexpectedly suggest that that recovery of the Arp 2/3 complex via the
tagged Arp3 was knocked down ~55%.
Subunit
APR3.
APR2.
AR1A.
AR34.
AR21.
AR20
Molecular Weight
Da
47341
44732
41557
34311
20533
19654
Calculated
PI
5.61
6.3
8.6
6.84
8.8
8.53
Accession
Number
P32391
O15142
Q92747
O15144
O15145
P59998
Sequence
Coverage
44%
50%
27%
55%
38%
66%
Table 1. showing the Arp 2/3 complex subunits identified and sequence coverage.
Figure 8. Position of 6xHIS Biotin tag in the Arp 2/3 molecular model. Molecular model
representations (left and right images are 90º to eachother) of the Arp 2/3 complex was
generated based on the PDB file and using SPDB program. Both TAP tags (N-terminus for
Arp3 and C-terminus for ArpC2) are located on the protein surface which could lead to
interference of complex formation.
Conclusions
We present a new method for the analysis of protein expression knockdown and how it may be applied to determine protein complex assembly,
stoichiometry and monomer turnover. Specifically, the marriage of two powerful technologies, shRNA and SILAC, combine to produce a
method capable of detecting significant stoichiometric effects under even relatively subtle knockdown conditions. In this study, we focus on the
Arp 2/3 complex assembly. Using differential expression analysis by SILAC, while the introduction of targeted shRNA serves as an
experimental stimulus, we have quantified the degree of knockdown of specific complex subunits. We observe an unexpected reduction in
complex affinity enrichment after introducing an affinity-tagged Arp complex subunit followed by an shRNA treatment to knockdown the native
isomer. We confirm this phenomenon is specific to the Arp complex and validate the effect by SILAC analysis and Western blot analysis. We
hypthesize the charge density associated with the TAP tag disrupts complex formation. We plan to validate this hypothesis using SILAC
analysis to monitor subunit turnover upon shRNA treatment, and a modification in the affinity enrichment design.
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