The hsp12/hsp26∆ double mutant does not show increased protein aggregation

  Ageing  is  a  complex  process  characterised  by  the  accumulation  of  oxidized,   misfolded   or   aggregated   proteins,   which   have   deleterious   effects   on   cellular   homeostasis   (Gelino   and   Hansen,   2012).   With   replicative   age,   there   is   an   accumulation  of  oxidatively  damaged  proteins,  which  are  retained  within  the  yeast   mother  cell  by  a  Sir2-­‐dependent  process  (Erjavec  et  al.,  2007).  Oxidatively  damaged   proteins   are   not   inherited   by   daughter   cells   during   cytokinesis,   however   in  Sir2∆  

mutants,   damaged   proteins   are   no   longer   retained   in   the   mother   cell   and   are   inherited  by  newborn  daughters  (Aguilaniu  et  al.,  2003).  This  suggests  an  important   mechanism  of  the  yeast  cell  for  dealing  with  oxidative  damage,  which  is  vital  for  the   fitness   of   new   daughter   cells   (Aguilaniu   et   al.,   2003).   It   may   be   that   DR   extends   lifespan   by   affecting   this   Sir2   dependent   mechanism,   however   there   is   no   data   available  on  this  hypothesis.        

  Hsp26,  a  member  of  the  sHsp  family  acts  as  a  molecular  chaperone  and  is   able   to   bind   to   non-­‐native   proteins   during   stress   conditions   preventing   the   formation  of  aggregates  (Haslbeck  et  al.,  2004).  In  contrast,  Hsp12  another  member   of   the   sHsp   family   has   negligible   anti-­‐aggregation   activity   but   instead   acts   as   a   membrane   chaperone,   stabilising   membranes   under   stress   conditions   (Welker   et   al.,  2010,  Herbert  et  al.,  2012).  With  this  in  mind,  it  is  reasonable  to  assume  that  the  

Chapter  4.  Characterisation  of  the  hsp12/hsp26∆  double  mutant     increased  insoluble  protein  aggregates  in  comparison  to  the  wildtype.  Since  glucose   depletion  is  regarded  as  a  stress  and  both  proteins  are  strongly  induced  in  response   to   severe   DR   it   may   be   that   the   double   mutant   also   shows   enhanced   protein   aggregation  with  0.05%  (w/v)  glucose  in  comparison  to  2%  (w/v)  glucose.  Contrary   to   this   hypothesis,   aggregation   assays   did   not   suggest   that   the  hsp12/hsp26∆  

double  mutant  has  increased  levels  of  insoluble  protein  aggregates  when  compared   to  the  wildtype.  This  result  may  be  explained  by  the  presence  of  other  sHsps  and   Hsps,  which  are  able  to  compensate  for  the  loss  of  HSP12  and  HSP26.  Hsp42,  for   example,   is   another   cytosolic   sHsp,   which   has   a   90%   overlap   in   its   substrate   proteins  to  that  of  Hsp26  (Haslbeck  et  al.,  2004).  Unlike  Hsp26,  which  is  induced  in   response  to  stresses,  Hsp42  is  active  under  physiological  conditions  (Haslbeck  et  al.,   2004).  Studies  have  reported  increased  aggregation  of  insoluble  proteins  in  single  

hsp26∆  and  hsp42∆   mutants   which   increases   further   in   an  hsp26/42∆   double   mutant,  suggesting  that  these  proteins  can  compensate  for  one  another  (Haslbeck   et  al.,  2004).  Since  the  hsp12/hsp26∆  double  mutant  did  not  show  increased  levels   of  protein  aggregation  it  could  be  that  the  presence  of  other  chaperones  such  as   Hsp104,  Hsp90,  Hsp70  and  Hsp40  may  also  compensate  for  the  absence  of  HSP12  

and  HSP26  (Glover  and  Lindquist,  1998,  Burnie  et  al.,  2006).  Hsp104  for  example,  is   the   most   crucial   Hsp   of  S.   cerevisiae,   which   enhances   survival   when   exposed   to   extreme   temperatures   and   high   concentrations   of   ethanol   (Glover   and   Lindquist,   1998,  Sanchez  et  al.,  1992,  Sanchez  and  Lindquist,  1990).  Hsp104  is  able  to  refold   denatured  or  aggregated  proteins  with  the  help  of  additional  chaperones  -­‐  Hsp70   and  Hsp40  (Glover  and  Lindquist,  1998).    In  addition,  Hsp90  is  required  for  correct   folding   of   difficult-­‐to-­‐fold   proteins   such   as   Swe1   and   has   a   critical   role   as   a   chaperone   when   the   yeast   is   grown   on   maltose   as   an   alternative   carbon   source   (Burnie  et  al.,  2006,  Bali  et  al.,  2003).    

  An  alternative  interpretation  of  the  aggregation  results  may  be  that  Hsp26  is   a  more  specific  chaperone  and  less  promiscuous  than  its  cytosolic  family  member,   Hsp42  (Haslbeck  et  al.,  2004).  Hsp26  substrates  are  thought  to  range  from  10  to  100   kDa  in  size  and  have  a  pI  range  between  4  to  7  (Haslbeck  et  al.,  2004).  It  may  be  that   these   substrates   do   not   aggregate   during   the   experimental   conditions   tested   and   therefore   we   do   not   see   any   apparent   increase   in   protein   aggregation   for   the  

Chapter  4.  Characterisation  of  the  hsp12/hsp26∆  double  mutant    

hsp12/hsp26∆  double   mutant.   It   may   also   be   that   the  in   vitro  aggregation   assay   utilised  in  this  study  is  not  very  sensitive.  Much  more  precise  indicators  of  protein   aggregation  can  be  achieved  by  using  sophisticated  in  vivo  chaperone  assays  such  as   protein  firefly  luciferase  fused  to  green  fluorescent  protein  (FFL-­‐GFP)  (Abrams  and   Morano,  2013).  The  FFL  protein  is  extremely  sensitive  to  stress-­‐induced  mis-­‐folding   and   aggregation,   from   which   the   luciferase   activity   can   be   monitored   by   an   enzymatic  assay  (Abrams  and  Morano,  2013).  In  addition,  GFP  labeling  of  the  FFL   protein  allows  visualization  of  aggregation  or  solubility  by  microscopy  (Abrams  and   Morano,   2013).   Therefore   using   the   FFL-­‐GFP   method   may   show   that   the  

hsp12/hsp26∆  double  mutant  does  have  increased  insoluble  protein  aggregates  in   comparison  to  the  wildtype.  In  addition  to  performing  FFL-­‐GFP  assays  it  would  also   be   important   to   analyse   the   asymmetry   of   damaged   proteins   in   the   mother   and   daughter   cells   of   the   hsp12/hsp26∆   double   mutants.   It   may   be   that   in   the  

hsp12/hsp26∆  double  mutant  damaged  proteins  are  inherited  by  the  daughter  cells   leading  to  a  reduced  RLS.  To  investigate  this  hypothesis,  old  yeast  cells  would  need   to   be   obtained;   this   could   be   achieved   by   biotin-­‐streptavidin   magnetic   sorting   (Wang   et   al.,   1992).   Oxidized   proteins   could   then   be   analysed   by   in   situ   immunofluorescence  of  carbonylated  proteins,  by  analysing  mitochondrial  structure   by  DiOC6  staining  and  by  detecting  the  presence  of  ROS  by  dihydroethidium  (DHE)  

(Aguilaniu  et  al.,  2003).  Future  work  will  include  both  of  these  experiments  to  help   understand  the  roles  of  HSP12  and  HSP26  in  DR  mediated  lifespan  extension.    

4.4.4   The  hsp12/hsp26∆  double   mutant   does   not   show   a   reduction   in   vacuolar