the proteasome inhibitor ( pi ) bortezomib is used as a first- and second - line treatment of multiple myeloma ( mm ) ( anderson et al . , 2011 ) .
2012 ) main function is to degrade ubiquitinated proteins in a controlled manner ( bedford et al .
proteasomes are comprised of a cylindrical core particle ( cp ) capped at each end by a regulatory particle ( rp ) ( lander et al . , 2012 , he et al . , 2012 ) .
the rp captures and denatures ubiquitin - marked protein substrates , and translocates their unfolded polypeptide chains towards proteolytic active - sites in the cp 's lumen ( finley , 2009 ) .
cp contains three types of active - sites , each of which comprises a peptide - docking area and an exposed catalytic threonine .
pis including bortezomib prevent protein hydrolysis by forming covalent adducts with the catalytic threonines of the active - sites ( groll et al . , 2009 ,
some proteasome activity is necessary for any cell to live ( heinemeyer et al . , 1997 ) , not just mm cells ( craxton et al . , 2012 ,
although the bortezomib concentrations at which cells of different cancers die differ widely , from low nanomolar to high micromolar ic concentrations , cells of the ( incurable ) b - cell malignancy mm are exquisitely sensitive ( shabaneh et al . ,
2013 ) , hence bortezomib 's success in treatment of mm ( anderson et al . , 2011 ) .
intriguingly , bortezomib at its low ic concentration causes only a small reduction in proteasomes ' ability to degrade proteins ( kisselev et al .
the reduction is small because bortezomib preferentially inhibits the chymotrypsin - like ( ct - like ) active - site , but  at ic  does not inhibit the caspase - like and trypsin - like active - sites ( fig .
s2a ) ( kisselev et al . , 2006 ) ; however , protein substrates can be hydrolysed by any of the three types of active - sites ( kessler et al . , 2001 , kisselev et al . , 2006 ) .
thus , the question arises why minimal inhibition of proteasome function suffices to induce apoptosis in mm but not in bortezomib - insensitive cells .
several explanations have been proposed , including high proteasome workload in mm cells ( bianchi et al . , 2009 , meister et al .
we report that , surprisingly , a ( low ) ic bortezomib challenge  which in - vitro minimally inhibits proteasomes  in living mm cells severely inhibits proteasomes ' hydrolytic activity .
our data suggest that , in living mm cells , a bortezomib - induced structural change in the proteasome ( pitcher et al . , 2014 ) is responsible for this severe degree of proteasome inhibition .
antibodies from enzo life sciences : -ubiquitin ( fk2 , pw8810 ) , -cleaved caspase 3 ( pab , adi - aas-103 ) , -20s 7 ( moab ln43 , bml - pw8110 ) , -rpn12/s14 ( pab , bml - pw8815 ) , -rpn10/s5a ( moab s5a-18 , bml - pw9250 ) , -rpt5/s6a ( moab tbp1 - 19 , pw8770 ) , -rpt4/s10b ( moab p42 - 23 , pw8830 ) , -rpt2/s4 ( pab , bml - pw8305 ) , and -5i / lmp7 ( pab , pw8355 ) .
antibodies from other sources : -streptag ( qiagen , moab 34850 ) , -parp ( cellsignalling , pab , # 9542 ) and -procaspase3 ( cellsignalling , pab , # 9661 ) .
additional reagents : ada - k(biotin)-ahx3-l3-vs , epoxomicin ( enzo lifesciences ) , bortezomib ( millennium / takeda ) , streptactin resin ( qiagen , 30004 ) , ninta resin ( sigma - aldrich , his select hf nickel affinity gel , h0537 ( fig . 
annexinv/7aad apoptosis staining ( ebioscience 88 - 8007 - 74 ) was used to assess cell viability .
enzymes : dnase1 , micrococcal nuclease , rnasea / t1 , rnaseh , s1 nuclease and rnase1 ( fermentas / thermo ) , pde1 from crotalus adamanteus venom ( sigma , p3243 - 1vl ) .
caspase inhibitor set iii ( enzo life sciences , alx-850 - 227-ki01 ) , used at 1:500 dilution  =  4  m .
antibodies were diluted in pbs  +  0.5% tween20 for western blotting , pvdf membranes were re - probed multiple times ( yeung and stanley , 2009 ) .
myeloma cell lines nci - h929 , kms12-bm , rpmi-8226 , opm2 , and the t - lymphocyte jurkat cell line , were grown in rpmi ( sigma ) , supplemented with 10% v / v fbs , and 1% v / v pen / strep .
for fractionation , a cytosol extraction ( ce ) buffer ( 25  mm tris 
hcl [ ph  7.8 ] , 5  mm mgcl2/edta , 1  mm atp / adp , 2  mm dtt , 150  mm nacl , 0.1 or 0.5% np-40/igepal ) and nuclear extraction ( ne ) buffer ( bakondi et al . , 2011 ) ( 20  mm hepes [ ph  7.4 ] , 420  mm nacl , 0.5  mm edta , 0.5  mm egta , 1  mm dtt , 1 tablet of inhibitor cocktail ( roche , 11 - 873 - 580 - 001 ) per 50  ml ) were used . when using the ne for subsequent affinity - purification , 2 volumes of h2o
were added to 1 volume ne to reduce the nacl concentration to physiological levels . for non - fractionated
lysate , cell pellets snap - frozen in ln and stored at   80  c were resuspended and combined in 10   volume of ce buffer and passed through a nanodebee ( bee international ) homogeniser at 19,000 psi .
proteasome activity was measured basically as described ( kisselev and goldberg , 2005 , vilchez et al . , 2012 ) , using fluorogenic proteasome substrates suc - llvy - amc , ac - rlr - amc , ac - gpld - amc ( enzo life sciences ) for the three types of active - site .
hcl , ph  7.5 , 250  mm sucrose , 5  mm mgcl2 , 0.5  mm edta , 2  mm atp and 1  mm dtt ) by passing cells through a 29  g needle ten times .
fluorescence ( 380  nm excitation , 460  nm emission ) was monitored continuously on a microplate fluorometer for 1  h at 37  c . to measure for the presence of free 20s proteasomes ,
a second was set up with the addition of 40  mm bortezomib ; any activity in the bortezomib wells was subtracted from corresponding wells to compensate for unspecific protease activity . the ubiquitinated substrate ( g3p ) ( matyskiela et al . , 2013 )
was kindly provided by dr andreas martin , and used as described . in - vitro degradation with purified yeast ( fig .
3c , d ) proteasomes was done in pbs supplemented with 2.5  mm atp , 2.5  mm mgcl2 , 2.5  mm dtt , and 10% dmso in a total volume of 20  l .
reaction was performed for 1  h either at 30  c or 37  c ( fig . 
3c , d ) , after which the reaction was stopped by addition of 20  l 2   sds sample buffer and boiled .
data plotted were mean of replicates , with error bars plotted of the standard error of mean ( sem ) .
research was supported by leukaemia lymphoma research uk ( llr grant10016 to mfk , llr gordon piller studentship award grant11043 to mfk , ak , dp ) , by an mrc - imperial confidence - in - concept ( icic ) grant to mfk , and by the nihr biomedical research centre at imperial college nhs trust , london .
nci - h929 mm cells were challenged with a lethal ( 10  nm ) bortezomib concentration for varying lengths of time .
cells were then lysed and an artificial fluorogenic peptide substrate was used to measure proteasomes ' hydrolytic activity ( fig . 
only when it was nearly absent ( <  10% ) , after 68  hour incubation , was an increase in ubiquitin conjugate levels observed ; intracellular accumulation of ubiquitinated proteins indicates that proteolytic workload exceeds proteasome capacity , accumulation being a phenotype which integrates other regulatory mechanisms in the cell including deubiquitinating enzyme activity ( fig . 
s1a ) . however , when cells were first lysed and then challenged with 10  nm bortezomib , only a 40% reduction in proteasomal ( ct - like ) activity was observed ( fig .
s2a : 61.5% ct - like activity left , and as previously reported ( kisselev et al . , 2006 ) ) .
a much stronger - than - expected in - vivo inhibitory effect was also observed for the other types of proteasomal active - sites ( figs .
predictable explanations for bortezomib 's severe inhibition of proteasomes ' activity in mm cells did not apply : ( 1 ) severe inhibition was not due to prolonged incubation , because exposing purified proteasomes in the test tube to 10  nm bortezomib for longer periods of time still showed modest inhibition in line with fig .
furthermore , we identified a mm cell line , rpmi-8226 , in which ct - like proteasome activity dropped very rapidly , within 2  h , to <  10% ( fig . 
1e ) ; this time - frame approaches that of the in - vitro test using cell lysate ( fig .
( 2 ) although 10  nm bortezomib induced apoptosis , and apoptosis is known to inhibit proteasomes via caspase activation ( sun et al . , 2004 ,
adrain et al . , 2004 ) , caspase activation was not responsible for the observed proteasome inhibition : inhibition preceded caspase activation ( fig . 
1d ) , cells were alive until 10  h post - challenge as measured by annexin - v staining ( fig .
s2b ) , and there was a non - myeloma , t - lymphocyte cell line that did not die with 10  nm bortezomib but in which proteasome activity still dropped well beyond expectation to ~  20% ( figs .  1f , s2d ; see also lung carcinoma a549 cells : fig .
( 3 ) severe inhibition was not because mm cells actively pumped in and/or retained bortezomib to establish a 10100 fold higher ( fig .
we used the irreversible biotinylated inhibitor ada - k(biotin)-ahx3-l3-vs ( kessler et al . , 2001 ) to simultaneously measure inhibition of activity and the degree to which active - sites were inhibited , i.e. biotinylated ( kessler et al . , 2001 ) .
we observed in - vivo a discrepancy between active - site inhibition and reduction in hydrolytic activity of those active - sites ( fig . 
we now report a structural change in the proteasome which does correlate with bortezomib - triggered early shutdown of proteasomal activity : bortezomib - triggered early changes in posttranslational modifications on proteasomal subunits .
we recently reported that human nuclear proteasomes carry a constitutive , ctab - page - detectable , posttranslational modification that , in some respects , resembles poly(adp)-ribose ( pitcher et al . , 2014 ) .
we reported that exposing mm cells to bortezomib induced changes in these ctab - page - detectable proteasome modifications , exactly at bortezomib 's ic concentration and above ( pitcher et al . , 2014 ) .
2b  ctab - page analysis of total cell lysate  shows that changes in these modifications of the proteasomal rpn12 subunit occur between 1  h ( rpmi-8226 ) and 4  h ( h929 ) of pi challenge . to examine these bortezomib - triggered changes in finer detail , we combined cell fractionation with affinity - purification of human proteasomes .
( purification is a technical trick that enables modified subunits to also become compatible with , and visible on , sds - page ( pitcher et al . , 2014 ) . )
we generated retrovirally - transduced opm2 mm cells , which expressed a rpn11 subunit tagged n - terminally with a his6strepii  strepii  tev - affinity cassette .
cell fractionation and then affinity - purification ( using streptactin or ninta ) showed that subunits from specifically nuclear proteasomes are extensively modified , as we reported previously ( pitcher et al .
, 2014 ) ( fig .  2c : rpn11 subunit , detected with anti - streptag antibody ) . repeating this fractionation - then - affinity - purification procedure , but this time after opm2 cells had been treated with 20  nm ( lethal ) bortezomib for up to 57  h , showed that patterns of modified subunits changed ( fig . 
generalising , these changes involved ( a ) a reduction in complexity for nuclear proteasomes ( e.g. rpt5 , rpn12 ) , and/or ( b ) the appearance of modified subunit species in the cytosol fraction ( e.g. 5i , rpn12 ) .
( a ) and ( b ) raise the possibility that severe inhibition of proteasome activity in total lysate ( fig . 
1 ) may result from changes in proteasome structure which happen both in the cytosolic and nuclear compartments of the cell after cells are challenged with bortezomib .
we are currently investigating the exact nature of the proteasome modifications . in order to test directly if changes in these proteasome modifications affect proteasome function , we first searched for commercial enzyme preparations that can digest the modifications of nuclear proteasomes .
we discovered that a combination of venom phosphodiesterase-1 and s1 nuclease was efficient in trimming these modifications and collapsing modified rpt2 species into its correct subunit - size species ( fig . 
next , we combined this enzyme protocol with an in - vitro proteasome - mediated proteolysis assay , which uses a ubiquitinated model protein as substrate ( matyskiela et al . , 2013 )
we scaled up cell growth and affinity - purified proteasomes from total ( i.e. including nuclear ) lysate .
proteasomes bound to affinity - resin were mock - treated or treated with pde1/s1 at a sub - optimal ( 20  c ) temperature , after which resin was washed to remove enzymes .
most subunits of pde1/s1-treated versus untreated proteasomes were very similar , but for example rpn12 showed pronounced differences ( fig .
we then incubated the proteasome preparations with a ubiquitinated model protein ( matyskiela et al . ,
s4 ) in order to assess the ability of these proteasome preparations to process a ubiquitinated substrate . at low proteasome / substrate ratio at 30  c ,
however , increasing the ratio overcame this defect , with both preparations degrading substrate equivalently . at even higher proteasome / substrate ratio , and at 37  c , proteasomes processed the substrate towards deubiquitination rather than degradation , and again both preparations behaved equivalently ( fig . 
in addition , we found that changes in the redox state of the reaction conditions uncovered qualitative differences between enzyme - treated and untreated proteasomes ( fig . 
3d ) : in the oxidizing conditions minus dtt , enzyme - treated proteasomes removed streptag epitope ( i.e. substrate ) more efficiently than untreated proteasomes but did not shift down the ubiquitin signal correspondingly , indicating that these enzyme - treated proteasomes only partially digested the substrate protein  starting from the tagged carboxyterminus  before premature release .
in contrast , under reducing conditions , untreated proteasomes were more efficient in degrading substrate than enzyme - treated proteasomes ( see also fig . 
our data indicate that , under certain experimental conditions , changes in proteasome modifications affect proteasome function , thereby strengthening the case that bortezomib - triggered changes in proteasome modifications within cells also affect proteasome function .
in summary , our data reveal a dramatic inhibition of proteasome activity in mm cells after a ( low ) ic bortezomib challenge , and suggest that this inhibition is the compound result of , first , inhibition of a subset of active - sites , and , second , structural changes in the proteasome which further impair hydrolytic activity ( fig . 
engagement of pis with active - sites changes proteasome conformation and stabilizes the ( distant ) cp  rp ( kleijnen et al . , 2007 , park et al . , nov 28 2008 ) and rp - hplic / ubiquilin ( kleijnen et al . , 2000 ) interactions , thus providing a possible signalling mechanism into the cell that may enable active - site inhibition to directly trigger activation of the cellular machinery that then changes the posttranslational modifications of the proteasomes .
whereas it has been very difficult to explain why mm cells die from a nanomolar ic bortezomib challenge when assuming that the modest level of proteasome inhibition observed in - vitro holds true in - vivo ( bianchi et al . , 2009 , kisselev et al . , 2006 , shabaneh et al . , 2013 ) , it is not surprising that a myeloma cell with over 95% ct - inhibition and observable proteasome stress ( i.e. accumulation of ubiquitin conjugates , fig . 
in addition , our data show that bortezomib , in cancer cells which are bortezomib - resistant , does not achieve the same degree of proteasome inhibition as in ( bortezomib - sensitive ) mm cells ( figs . 
1f , s2d ) , thus providing a molecular mechanism explaining what differentiates mm from most other cancers which bortezomib can not treat . please note that our data indicate that a high proteasome workload in mm cells ( bianchi et al . , 2009 ,
meister et al . , 2007 , shabaneh et al . , 2013 ) can not be the primary reason for mm cells ' sensitivity to bortezomib : for this explanation to work , all proteasomes in a cell would need to be fully engaged  with no spare capacity left  in order for a minimal inhibition of proteasomes to produce proteasome stress ; instead , we observed that mm cells have much spare proteasome capacity , and that reducing capacity even to 20% still did not yield proteasome stress ( fig . 
its effect on proteasome function may enable future intervention to re - sensitize bortezomib - resistant cells to treatment .
 the proteasome inhibitor bortezomib is used to treat multiple myeloma ( mm ) . 
 bortezomib inhibits protein degradation by inactivating proteasomes ' active - sites . 
 mm cells are exquisitely sensitive to bortezomib  exhibiting a low - nanomolar ic50  suggesting that minimal inhibition of degradation suffices to kill mm cells . 
 instead , we report , a low bortezomib concentration , contrary to expectation , achieves severe inhibition of proteasome activity in mm cells : the degree of inhibition exceeds what one would expect from the small proportion of active - sites that bortezomib inhibits . 
 our data indicate that bortezomib achieves this severe inhibition by triggering secondary changes in proteasome structure that further inhibit proteasome activity . comparing mm cells to other , bortezomib - resistant 
 , cancer cells shows that the degree of proteasome inhibition is the greatest in mm cells and only there leads to proteasome stress , providing an explanation for why bortezomib is effective against mm but not other cancers . 
Introduction
Experimental procedures
Results
Discussion
Authorship contributions
Disclosure of conflicts of interest
Supplementary data