Student day 2012 4 1-2012

Angela SanchezMentor: Pamela Larsen

Pro-Aging Branches

of the Unfolded Protein

Response

• Short-lived

• Inexpensive

• Tractable model

• Considerable discovery potential

C. elegans as a model system for longevity

C. elegans as a model system for longevity

• Identify a long-lived intervention

• Use worm to understand mechanism

Cool Temperature Doubles Lifespan in C. elegans

Cool temperature correlates with longevity

Caloric Restriction

(Lane 1996)


Ames Dwarf

(Hunter 1999)


Long-lived human

(Roth 2002)

Why does cool temperature improve health and survival?

Underlying mechanism not understoodHow can we use C. elegans to gain insight?

Classic Genetic Approach for Mutants

Screen for mutants that fail to be normal (WT)

Mutants that fail to increase lifespan due to cool temperature

daf-2 mutants!

Qa

daf-2: Candidate for cool temperature mediated longevity

Homologous to human insulin/IGF receptorFound to have increased lifespan

daf-2 25˚C

Wild type 25˚C

daf-2 15˚C

Wild type 15˚C

Similar to benefit from cooler temperaturesAttenuated benefit at 15˚C for daf-2mutants

Qa

daf-2: Candidate for cool temperature mediated longevity

daf-2 25˚C

Wild type 25˚C

daf-2 15˚C

Wild type 15˚C

SUGGESTS DAF-2 AND COOL TEMPERATURE ARE LINKEDOVERLAPPING MECHANISM OF LONGEVITY

Understanding cool mediated longevityMicroarray

• Affymetrix C. elegans array

• Unbiased genome wide snapshot

• Comparing daf-2 mutants and wild type

• Comparing response to temperature (15C vs 25C)

• Look for +/- correlations with longevity

UNFOLDED PROTEIN RESPONSE GENESNEGATIVELY CORRELATE WITH LONGEVITY

• Synthesis of

• Membrane

• Secreted

• Glycosylation

•Trimming

•Post-translational

Endoplasmic Reticulum Synthesizes Membrane and Secreted Proteins

P

•Sorted to

• Membrane

• Organelle

• Secreted

Endoplasmic Reticulum Synthesizes Membrane and Secreted Proteins

SECRETED

LYSOSOME

MEMBRANE

BiP

Unfolded Protein Response

IRE-1PEK-1

BiP

Translation

eIF2

Transcription of UPR Genes

ATF-5

Misfolded Protein

ATF-6BiP

Site 2Protease

XB P-1 CHOP

daf-2 has lower levels

Temperature and Genotype Regulation of S2PM

icro

arra

y E

xpre

ssio

n Le

vels

S2P

WT daf-2

CHOP

WT daf-2

hsp-3

WT daf-2


Long-lived human

(Roth 2002)

Reduced Bip Levels in Offspring of Long-lived Individuals

Reduced Bip in other longevity promoting interventions?


Caloric Restriction

(Lane 1996)

Reduced Bip levels in Dietary Restricted Mice


Ames Dwarf

(Hunter 1999)

Reduced Bip levels in Growth Hormone Deficient Mice

Canonical Unfolded Protein Response

IRE-1PEK-1

BiP

ATF-5

ATF-6

Site 2Protease

CHOP

PRO-AGING BRANCHES of UPR

Two BiP Isoforms in C. elegans: hsp-3 and hsp-4

IRE-1PEK-1 ATF-6

hsp-4hsp-3

Heatshock InducibleConstitutive/Non-Heatshock

BiP

•93% Identical 607/658 amino acids•hsp-4 is heatshock-inducible (>29°C)•hsp-3 constitutive

hsp-3 and hsp-4 RNAi increase lifespan at 25˚C in wild type

Inhibition of UPR genes increases lifespan at 25˚C in wild type%

Sur

viva

l

Adult Age (days)

Knockdown increases lifespan under warm conditions

Inhibition of Pro-Aging UPR and Cool Temperature:Same Mechanism of Longevity?

Can we identify signaling pathways underlie cool longevity?

IRE-1PEK-1

Translation

eIF2ATF-6

Site 2Protease

CHOPATF-5

BiP BiP BiP

Inhibition of Pro-Aging UPR and Cool Temperature:Same Mechanism of Longevity?

RNAi of UPR genes does not alter lifespan at 15˚C

Cool temperature promotes longevity byINHIBITION OF PRO-AGING UPR

Pro-Aging Branches of the Unfolded Protein Response

IRE-1PEK-1 ATF-6

Site 2Protease

CHOPATF-5

BiPBEFORE

AFTER

?

Pro-Aging Branches of the Unfolded Protein Response

IRE-1PEK-1 ATF-6

Site 2Protease

CHOPATF-5

BiP

?

pek-1, ire-1, atf-6 deletion mutantsire-1 (C41C4.4)

ok799Deletion

atf-6 (F45E6.2)

ok551Deletion

pek-1 (F46C3.1)

ok275Deletion

ire-1 deletion reduces lifespan at 25˚C

ire-1 is reduces for wild type life span

hsp-3 and hsp-4 RNAi rescues ire-1 lifespan

hsp-3 and hsp-4 longevityDOWNSTREAM of ire-1

hsp-3and hsp-4 RNAi rescues ire-1 to hsp-3/4 RNAi levels

hsp-3 and hsp-4 RNAi longevity is INDEPENDENT of ire-1

pek-1 and atf-6 deletion increases lifespan at 25˚C

pek-1 and atf-6 are PRO-AGING

IRE-1PEK-1 ATF-6

hsp-4 hsp-3

PRO-AGING PRO-AGING

hsp-4

Model of Pro-Aging Branches of the UPR

pek-1 deletion increases lifespan and is hsp-3 dependent

hsp-4 RNAi promotes longevity by pek-1 inhibition

atf-6 deletion increases lifespan and is hsp-3 independent

Different response to hsp-3 RNAi in atf-6 and pek-1 deletion mutants

atf-6 deletion increases lifespan and is hsp-3 independent

hsp-4 RNAi promotes longevity by inhibition of atf-6

hsp-3 interacts with pek-1 to regulate longevity

atf-6 and hsp-3 inhibition have an additive affect on lifespanpek-1 mutant longevity partially dependent on hsp-3

hsp-4 RNAi promotes longevity by inhibition of atf-6

Can structural details of hsp-3 and hsp-4 predict UPR sensor Interaction?

Amino Acid Differences Cluster In Two Distinct Regions

SubstrateBindingDomain

hsp-4 more hydrophobic

UPR SensorBindingDomain

LID SubstrateBindingDomain

UPR SensorBindingDomain

hsp-4 more hydrophobic

Different charges

Model of Pro-Aging Branches of UPR

IRE-1PEK-1 ATF-6hsp-4hsp-3

PRO-AGING PRO-AGING

+ +

PRO-LONGEVITY

DAF-2

Cool TemperatureWarm Temperature

Future Directions

• Identified a novel gene involved in physUPR

• Identified novel genes with strong longevity signatures

• Identified novel genes with strong aging signatures

AcknowledgementsCommittee MembersPast Larsen Lab Members

CollaboratorsFunding

Dr. Shelly Buffenstein

Dr. Ellen Kraig

Dr. Merry Lindsey

Dr. Jim Lechleiter

Dr. Syuichi Takano

Wendy Lee

Dr. Hal Boylston

Dr. Pamela Larsen

NIA Training GrantGlenn Medical FoundationEllison Medical Foundation

Dr. Simon Tavaré

Dr. Christina Curtis

Student day 2012 4 1-2012

Science

Transcript of Student day 2012 4 1-2012