Cellular Stress Responses

Cellular stress responses
Jeffrey L. Brodsky, Ph.D.
Department of Biological Sciences
University of Pittsburgh
Renal cells are subject to
profound stresses (I):
•  Renal cells must normally function under conditions of
high osmotic flux and in the presence of denaturants
•  Protein synthesis and turnover rates are high in the kidney
•  In chronic kidney disease, cells are exposed to uremic
toxins, inflammatory signals, reactive oxygen species, and
infections
•  Links between heat shock protein release and
inflammatory signals
The heat shock response
Renal cells are subject to
profound stresses (II):
•  The stress associated with the accumulation of misfolded
and potentially toxic proteins in the secretory pathway
•  Mutated, secreted proteins linked to nephrotic syndrome
(e.g., nephrin and podocin) are retained in the ER and
induce the unfolded protein response (UPR)
•  Glomerular disease, proteinuria, and tubular injury also
result in UPR induction or misregulation
Early: ER folding machinery, protein transport, ERAD Late: Apoptosis (via ATF4-­‐dependent CHOP inducBon) XBP-­‐1 protein ATF4 ATF6-­‐p50 Golgi export XBP-­‐1 mRNA TranslaBon inhibiBon ProteolyBc acBvaBon (unspliced spliced) ATF4 synthesis The unfolded protein response
(UPR)
IRE1 PERK ATF6 cytoplasm ER Brodsky & Skach, 2011
and/or BiP Duckhour & Krepinsky, 2009
Renal cells are subject to
profound stresses (III):
•  ER associated degradation (ERAD) plays a key role in
mitigating the toxic effects of aberrant protein
accumulation in the secretory pathway and acts in a
complementary fashion to the UPR
ER-­‐Associated DegradaBon (ERAD)
Vembar and Brodsky 2008 Nat Rev Mol Cel Biol
Diseases associated with the ERAD pathway
Guerriero and Brodsky, SubmiYed Physiol. Rev. Our favorite ERAD substrates in yeast…
Substrate pαF, CPY*, and AT-­‐Z cytoplasm ER cytoplasm ApoB ER cytoplasm ENaC ER α
β
cytoplasm CFTR Ste6p* Kir2.1 & ROMK NCC ER cytoplasm ER cytoplasm ER cytoplasm ER x 4
γ
How can we identify
the components of a
“misfolded protein
stress response”?
CFTR
CIS1, !
CUE4, SSA1, UBA1, !
UBC13, ULP4 !
ATG17, APJ1,!
HSP12,HSP30,!
ATG8, UGX2 !
HSP42, HSP78,!
HSP82, SSA4,!
SSE2,!
HSP26, HSP32,!
GRE3!
AT822,!
FMP43, DDR2,
ECM21, GAC1,
XBP1!
UIP4!
GRE2, AFT1,
ADD37, SSA3, UBX6!
USV1
DER1, ERO1, JEM1,
MPD1, SIL1, ULI1!
AFT1, ARN1, ARN2, ATX1, BNA2,
GRE1, SIP18 !
CCC2, COT1, ENB1, FET3, GET4,
FET5, FIT2, FIT3, FMP23, FRE1, FRE2,
FRE3, FRE4, FRE5, FRE6, FRE8, FTH1,
FTR1, HAP1, HMX1, MMT2, MRS4,
SMF3, TIS11
Kir2.1
ENaC
Deletion of the small heat shock proteins blocks the ERAD
of CFTR in yeast
A
0 20 40 60 90 min
HSP26
B
0 20 40 60 90 min
HSP26 HSP42
hsp26Δ hsp42Δ
CFTR remaining
CFTR remaining
hsp26Δ
1
0.5
1
0.5
0
0
0
40
80
time [min]
0
40
80
time [min]
Ahner et al., 2007
ΔF508-CFTR remaining
Over-expression of a human sHsp hastens the ERAD
of ΔF508-CFTR in HEK293 cells
*
1
**
αA-crystallin
0.5
mock
***
****
0
0
1
2
3
time [h]
Ahner et al., 2007
Deletion of the small heat shock proteins also slows the ERAD
of ENaC in yeast
Kashlan et al., 2007
Deletion of the small heat shock proteins also slows the ERAD
of ENaC in yeast
And, sHsp over-expression increases ERAD in an
oocyte expression system
Kashlan et al., 2007
CFTR
CIS1, !
CUE4, SSA1, UBA1, !
UBC13, ULP4 !
ATG17, APJ1,!
HSP12,HSP30,!
ATG8, UGX2 !
HSP42, HSP78,!
HSP82, SSA4,!
SSE2,!
HSP26, HSP32,!
GRE3!
AT822,!
FMP43, DDR2,
ECM21, GAC1,
XBP1!
UIP4!
GRE2, AFT1,
ADD37, SSA3, UBX6!
USV1
DER1, ERO1, JEM1,
MPD1, SIL1, ULI1!
AFT1, ARN1, ARN2, ATX1, BNA2,
GRE1, SIP18 !
CCC2, COT1, ENB1, FET3, GET4,
FET5, FIT2, FIT3, FMP23, FRE1, FRE2,
FRE3, FRE4, FRE5, FRE6, FRE8, FTH1,
FTR1, HAP1, HMX1, MMT2, MRS4,
SMF3, TIS11
Kir2.1
ENaC
Brodsky lab
Jen Goeckeler
Teresa Buck
Annette Chiang
Cristy Gelling
Chris Guerriero
Patrick Needham
Karen Hecht
Sarah Herrle
Alex Kolb
Joseph Tran
Luci Zacchi
Support
NIH (GMS, NIDDK, HL)
Cystic Fibrosis Foundation Therapeutics
American Heart Association
Alpha-1 Foundation
Dystonia Medical Research Foundation