Integrated Stress Response

Slide 1 · Integrated Stress Response

Cellular stress sensing • translational control • ER proteostasis

Molecular pathology

Dr Abu Suraih Sakhri

MBBS, MD (Pathology)

www.abusuraihsakhri.com

Slide 2 · Learning objectives

Map diverse stressors to the four eIF2α kinases.

Explain how Ser51 phosphorylation reprograms translation.

Describe ATF4-dependent adaptation and ISR resolution.

Distinguish the ISR from ER unfolded protein response signaling.

Interpret persistent stress signaling in major disease settings.

Slide 3 · Cells continually balance synthesis and repair

Biosynthetic demand

Proteins must be synthesized, folded, trafficked, and degraded.

Energy availability

ATP and nutrient supply impose a limit on cellular production.

Quality control

Chaperones and degradation pathways manage misfolded proteins.

Stress adaptation

Translation and gene expression are reset when demand exceeds capacity.

Slide 4 · A broad range of injuries can activate stress signaling

Metabolic stress

Hypoxia, ischemia, glucose or amino acid deprivation.

Pathogen-associated stress

Infection and abnormal viral RNA.

Proteostasis stress

Misfolded proteins, impaired folding, and ER overload.

Additional injury

Membrane damage, altered redox state, and heme scarcity.

Slide 5 · A cell must choose a response proportional to injury

Protective programs act before irreversible injury develops.

Injury sensed

Metabolic, chemical, or protein-folding disturbance

Resource allocation

Reduce synthesis; Activate repair and recycling

Capacity reassessed

Stress resolves or remains excessive

Cellular outcome

Recovery, persistent dysfunction, or death

Cell fate depends on stress intensity, duration, and cellular context.

Slide 6 · ISR, UPR, and autophagy: Related but distinct

ISR

An eIF2α-centered translational response to many stressors.

UPR

ER-specific pathways that reduce folding load and increase ER capacity.

Autophagy

Lysosome-directed recycling of cytoplasmic material.

Crosstalk

Shared stress inputs and signaling nodes coordinate adaptation.

Slide 7 · What is the integrated stress response?

A conserved signaling network that reprograms protein synthesis and gene expression during cellular stress.

Core event: Phosphorylation of eukaryotic initiation factor 2α (eIF2α).

Slide 8 · Four upstream kinases, one convergence point

Different sensors converge on the same translational-control switch.

PKR

Viral RNA

PERK

ER stress

GCN2

Amino acid scarcity

HRI

Heme deficiency

EIF2α–P (Ser51)

Global translation ↓

ATF4 translation ↑

Slide 9 · PKR links cytoplasmic RNA sensing to translation

Upstream setting

Double-stranded RNA can arise during viral infection.

Interferon signaling can increase PKR expression.

Activated PKR phosphorylates eIF2α.

Functional consequence

Translation suppression limits production capacity during stress.

ATF4-linked gene expression may be induced.

PKR is not a virus-specific marker in every context.

Slide 10 · PERK is an ER-membrane ISR kinase

Location and stimulus

PERK spans the ER membrane.

Its luminal sensor responds to disturbed ER proteostasis.

ER stress activates the cytosolic kinase domain.

Mechanistic linkage

PERK phosphorylates eIF2α (Ser51).

Protein influx into the ER is reduced by translation attenuation.

PERK is the ISR branch of the unfolded protein response.

Slide 11 · GCN2 detects inadequate amino acid availability

Nutritional signal

Amino acid scarcity increases uncharged transfer RNA.

GCN2 is an early sensor of nutrient limitation.

Its regulatory region links tRNA status to kinase activation.

Adaptive logic

Reduce protein production when building blocks are limited.

Favor expression of stress-associated metabolic genes.

Other stressors can recruit GCN2 in particular contexts.

Slide 12 · HRI coordinates translation with heme availability

Canonical setting

Heme-regulated inhibitor (HRI) is especially relevant in erythroid cells.

Heme deficiency activates HRI signaling.

HRI then phosphorylates eIF2α.

Protective consequence

Globin synthesis is restrained when heme is insufficient.

This helps avoid excessive production of unpaired globin.

HRI also responds to some other proteotoxic stresses.

Slide 13 · The four ISR sensors at a glance

Kinase

Representative trigger

Key context

PKR

Double-stranded RNA

Infection-related stress

PERK

Misfolded ER proteins

Endoplasmic-reticulum proteostasis

GCN2

Amino acid scarcity

Nutrient sensing

HRI

Heme deficiency

Erythroid globin–heme coordination

Slide 14 · Why four kinases rather than a single sensor?

Distinct upstream detectors

Different domains recognize different disturbances.

Common catalytic outcome

All phosphorylate the same Ser51 residue in eIF2α.

Cooperative activity

More than one sensor may contribute to a complex insult.

Context-specific duration

Strength and timing shape adaptation versus injury.

Slide 15 · The ISR integrates unrelated injuries

Example inputs can be different, but the downstream translational switch is shared.

Stress signal

ER overload / viral RNA / nutrient deficit / heme deficit

Kinase activation

PERK, PKR, GCN2, or HRI

Common target

EIF2α phosphorylation at Ser51

Cellular response

Broad translation decrease + selective transcriptional program

A single phosphorylation readout cannot reveal which kinase was activated.

Slide 16 · The biochemical core of the ISR

Phosphorylation of eIF2α on serine 51 alters translation initiation.

EIF2 functions in initiation as a GTP-bound carrier of initiator methionyl-tRNA.

Slide 17 · Translation initiation requires eIF2 recycling

EIF2 alternates between active GTP-bound and inactive GDP-bound states.

EIF2–GTP

Binds initiator Met-tRNAᵢ

Ternary complex

Delivers initiator tRNA to the scanning ribosome

GTP hydrolysis

EIF2 leaves in GDP-bound state

EIF2B

Exchanges GDP for GTP; Restarts the cycle

Slide 18 · Stress interrupts a specific point of initiation

Ser51 phosphorylation changes how eIF2 interacts with its exchange factor.

Stress-activated kinase

Phosphorylates eIF2α Ser51

EIF2α–P

Constrains eIF2B nucleotide exchange

Less eIF2–GTP

Ternary complex regeneration slows

Lower initiation rate

Translation of most mRNAs falls

Slide 19 · Why inhibition of eIF2B matters

Normal operation

EIF2B is the guanine-nucleotide exchange factor for eIF2.

It regenerates GTP-bound eIF2 after initiation.

Repeated cycles support ongoing protein synthesis.

During ISR activation

Phosphorylated eIF2α inhibits eIF2B activity.

Less ternary complex is available to initiate translation.

This is an initiation defect, not wholesale destruction of ribosomes.

Slide 20 · The ternary complex becomes rate limiting

EIF2–GTP–Met-tRNAᵢ is essential for productive translation initiation.

EIF2α–P rises

Inhibitory signal increases

EIF2B output drops

Less eIF2 is recharged with GTP

Ternary complex declines

Initiator tRNA delivery becomes limiting

Fewer starts

Overall protein synthesis decreases

Slide 21 · A single switch produces opposite translational effects

The apparent paradox is central to understanding the ISR.

Phosphorylated eIF2α

Most mRNAs

Lower translation initiation.

Reduced biosynthetic energy use.

Selected stress mRNAs

Preferential translation of ATF4 and selected regulators.

Adaptive transcriptional programs are induced.

Slide 22 · Lower translation can be protective

Conserve ATP

Protein synthesis is energetically expensive.

Reduce folding load

Fewer newly synthesized proteins enter stressed compartments.

Limit damaged products

The cell avoids making proteins during resource shortage.

Create repair time

Quality control can catch up before synthesis resumes.

Slide 23 · The pause in synthesis is not universal

Global is relative

Most—not literally all—mRNA translation is attenuated.

Selective exceptions

Some transcripts respond favorably to limited initiation complexes.

Time matters

Persistent inhibition can itself impair cell function.

Context matters

Different tissues tolerate different durations of synthesis arrest.

Slide 24 · ATF4 is a major transcriptional effector

The ISR reduces translation of most proteins while favoring production of ATF4.

ATF4 then changes transcription of genes involved in stress adaptation.

Slide 25 · ATF4: Baseline translation control

Regulatory upstream open reading frames (uORFs) constrain ATF4 expression.

MRNA 5′ region

UORF1

UORF2

ATF4

Basal: Ternary complex abundant

Stress: Ternary complex limited

Unstressed

Reinitiation at an inhibitory upstream ORF limits ATF4 protein output.

Stressed

Delayed reinitiation enables translation of the main ATF4 coding sequence.

Slide 26 · ATF4 preferential translation during ISR

A smaller pool of initiation complexes can paradoxically increase ATF4 translation.

MRNA 5′ region

UORF1

UORF2

ATF4

High initiation availability

Lower initiation availability

Without major stress

Translation resumes at a uORF that restricts downstream ATF4 production.

With eIF2α–P

Ribosomes more often bypass the inhibitory uORF and initiate ATF4 translation.

Slide 27 · ATF4 organizes a coordinated gene program

Amino acid homeostasis

Programs related to transport and nutrient metabolism.

Oxidative stress defense

Genes that support antioxidant responses.

Protein folding and repair

Components supporting cellular proteostasis.

Response remodeling

Regulators that help terminate or reshape the ISR.

Slide 28 · ATF4 couples sensing to transcription

The output is not solely translation inhibition.

EIF2α–P

General initiation rate decreases

ATF4 translated

Selective coding sequence becomes accessible

ATF4 in nucleus

Stress-responsive transcription changes

New proteins

Repair, metabolic adaptation, feedback

Slide 29 · Amino acid programs support adaptation

Supply

Increase availability of amino-acid substrates when possible.

Metabolic routing

Reallocate resources under nutrient stress.

Synthesis restraint

Lower immediate demand for amino acids.

Recovery

Permit biosynthesis to resume if nutrients return.

Slide 30 · Antioxidant defense is one ISR output

Stress context

Hypoxia/reoxygenation and other insults perturb cellular redox balance.

Accumulated reactive species challenge proteins and organelles.

Adaptation

ATF4-responsive programs can contribute to antioxidant capacity.

The exact program depends on the cellular context and additional transcription factors.

Slide 31 · ISR signaling interfaces with protein quality control

Production side

Translation attenuation reduces the quantity of newly synthesized proteins.

This lowers immediate folding demand.

Repair and clearance side

ATF4 supports stress-response gene expression.

ER quality control, proteasomes, and autophagy may cooperate with ISR.

Slide 32 · Do not confuse transcription with translation

Process

ISR-associated event

Interpretive point

Translation

Most initiation rates decrease

Less protein from many pre-existing mRNAs

Selective translation

ATF4 protein increases

Selected mRNAs evade usual suppression

Transcription

ATF4 alters gene expression

New mRNA abundance may rise or fall

Recovery

EIF2α phosphorylation resolves

Normal initiation can resume

Slide 33 · The ISR contains a built-in recovery program

Adaptive responses must be reversible if homeostasis is restored.

Stress

Activates one or more eIF2α kinases

EIF2α–P

Translation attenuates; ATF4 rises

Feedback

ATF4 can induce GADD34-dependent phosphatase activity

Recovery

EIF2α dephosphorylation permits synthesis

Slide 34 · GADD34 and CReP regulate dephosphorylation

Stress-inducible feedback

ATF4-associated transcription can induce PPP1R15A / GADD34.

GADD34 recruits phosphatase activity toward eIF2α.

Constitutive restraint

PPP1R15B / CReP contributes to baseline dephosphorylation.

Both pathways oppose sustained eIF2α phosphorylation.

Slide 35 · Recovery is a dynamic sequence

A successful ISR changes over time rather than remaining permanently “on.”

Detection

Injury activates stress sensors

Early pause

Translation declines; Repair gains priority

Adaptation

Gene programs counter the insult

Resolution

EIF2α dephosphorylated if stress subsides

Slide 36 · When stress is not resolved, adaptation may fail

The same pathway may support survival or contribute to injury.

Duration × intensity × cell state

Manageable stress

Resource conservation and repair.

Gradual restoration of protein synthesis.

Persistent severe stress

Continued dysfunction and maladaptive signaling.

Activation of apoptotic programs may occur.

Slide 37 · Integrated signaling does not guarantee survival

Protective early phase

Translation restraint can buy time to repair injury.

Unresolved insult

Maintained protein misfolding or metabolic deprivation persists.

Maladaptive late phase

Stress transcription can engage proapoptotic effectors.

No universal threshold

Cell fate is not set by a single eIF2α–P level.

Slide 38 · Normal ER proteostasis is an active process

The ER is a synthesis, folding, and quality-control compartment.

ENDOPLASMIC RETICULUM LUMEN

Nascent chain

Chaperone-assisted folding

Folded protein

CYTOSOL

Synthesis

Ribosomes produce secretory/membrane proteins.

Chaperones

Assist protein folding in ER lumen.

Quality control

Abnormal proteins are retained and cleared.

Slide 39 · ER stress means folding demand exceeds capacity

Increased demand

High secretory protein load or altered protein synthesis.

Impaired folding

Mutations, aging, and altered ER redox/calcium state.

Compromised energy

Hypoxia or ischemia limits ATP-dependent foldases.

Accumulated cargo

Unfolded/misfolded proteins strain the ER.

Slide 40 · The unfolded protein response rebalances ER work

An adaptive UPR acts on both protein load and processing capacity.

ER overload

Misfolded proteins accumulate

Sensor activation

PERK, IRE1, and ATF6 signal

Workload reduced

Less translation and ER protein entry

Capacity increased

Chaperones and degradation rise

Slide 41 · Three canonical arms of the mammalian UPR

Sensor

Main proximal output

Adaptive effect

PERK

EIF2α phosphorylation

Reduce translation; Favor ATF4

IRE1

XBP1 mRNA splicing

Increase ER chaperones and ERAD

ATF6

Golgi cleavage; Nuclear ATF6(N)

Induce folding/quality-control genes

Slide 42 · PERK is the direct bridge between UPR and ISR

PERK is both an ER stress sensor and an eIF2α kinase.

ER lumen

Accumulated misfolded proteins

PERK activation

ER-resident kinase signaling

EIF2α Ser51–P

Translation initiation curtailed

ATF4 output

Stress-response transcription

Slide 43 · IRE1 promotes ER quality-control capacity

IRE1 has kinase and RNA-processing activities.

ER stress

IRE1 activation

IRE1 RNase

Unconventional XBP1 mRNA splicing

XBP1s protein

Transcription factor enters nucleus

UPR genes

Chaperones, ERAD, ER capacity

Slide 44 · ATF6 signals through regulated proteolysis

An ER transmembrane precursor generates a nuclear transcription factor.

ER stress

ATF6 activation and trafficking

Golgi processing

Site-1 and site-2 proteases cleave ATF6

ATF6(N)

Active transcription factor released

ER adaptation

Chaperones and ERAD programs

Slide 45 · ER chaperones increase folding capacity

Normal role

Assist maturation of nascent ER proteins.

Reduce inappropriate aggregation.

Support folding of secreted/membrane proteins.

During the UPR

Transcriptional programs increase folding support.

This helps processing catch up with production.

Persistent overload can exceed this adaptation.

Slide 46 · ER-associated degradation removes failed products

ERAD prevents irreparably abnormal proteins from accumulating.

Misfolded protein

Recognized in ER

Retrotranslocation

Transferred toward cytosol

Ubiquitination

Marked for proteasomal destruction

Proteasome

Protein is degraded

Slide 47 · ER proteostasis can be restored by two levers

Homeostasis requires bringing demand back below processing capacity.

ER folding overload

Decrease new input

PERK → eIF2α–P → slower translation.

Fewer nascent polypeptides require folding.

Increase throughput / clearance

IRE1–XBP1 and ATF6 gene programs.

More chaperones and degradation.

Slide 48 · A successful UPR prevents escalation

Conceptual sequence: Stress handling versus terminal injury.

Baseline

Folding demand ≈ capacity

Acute overload

Misfolded proteins accumulate

Adaptive UPR

Translation slows; ERAD/folding rise

Recovery

Misfolded protein burden decreases

Slide 49 · Terminal ER stress can activate apoptosis

If protective measures cannot resolve severe injury, death pathways may dominate.

Persistent folding imbalance

Adaptation effective

Protein quality recovers.

Proteotoxic stress declines.

Adaptation insufficient

Proapoptotic signaling, including BH3/caspase pathways.

Mitochondrial apoptosis may follow.

Slide 50 · ISR versus UPR: Precise distinctions

Feature

ISR

UPR

Primary domain

Many stress inputs

ER proteostasis stress

Core sensors

Four eIF2α kinases

PERK, IRE1, ATF6

Central effect

EIF2α–P / selective translation

Protein folding load and capacity

Overlap

PERK and ATF4 branch

PERK drives part of ISR

Possible outcome

Recovery or dysfunction

Recovery or ER-stress apoptosis

Slide 51 · Autophagy is a partner, not a synonym for ISR

ISR

Modulates translation through eIF2α and transcription via ATF4.

UPR

Reduces ER load and increases quality-control capacity.

Autophagy

Sequesters and degrades selected cytoplasmic cargo via lysosomes.

Integrated protection

Stress pathways coordinate energy conservation and protein turnover.

Slide 52 · Ischemia can recruit multiple ISR mechanisms

Cellular insult

Reduced oxygen and glucose challenge ATP production.

ATP-dependent protein folding becomes less efficient.

Oxidative and organellar stress may accompany reperfusion.

Predicted adaptation

Translation attenuation reduces energy demand.

ATF4 programs may support cellular survival.

Magnitude and persistence of ischemia determine overall injury.

Slide 53 · Infection can generate translational stress

Initiating factors

Double-stranded RNA may activate PKR.

High viral protein production can burden ER folding.

Host immunity contributes additional cell stress.

Integrated cellular response

PKR and/or PERK can phosphorylate eIF2α.

Translation attenuation changes host protein synthesis.

Viral strategies can modulate or evade these pathways.

Slide 54 · Nutrient starvation changes both supply and demand

Nutrient limitation

Amino acid shortages alter charged tRNA availability.

GCN2 can sense the resulting scarcity.

Sustained deprivation impairs normal growth and repair.

Rebalancing response

EIF2α–P restrains protein synthesis.

ATF4 may mobilize nutrient-response transcription.

Recovery requires sufficient restoration of substrates.

Slide 55 · Secretory-cell stress illustrates clinical relevance

High-demand setting

Insulin-resistant states can increase insulin secretory demand.

Plasma cell neoplasms can produce very large protein loads.

Misfolded polypeptides may exceed ER capacity.

What may follow

UPR attempts to expand folding/degradation capacity.

PERK links ER stress to ISR.

Failed adaptation can contribute to dysfunction or cell death.

Slide 56 · Neurodegenerative disease and proteostasis

Pathologic context

Several neurodegenerative disorders involve protein misfolding or aggregation.

Chronic proteostasis disruption may activate stress pathways.

Interpretive caution

A persistent ISR or UPR may be adaptive or harmful by context.

Association does not prove a pathway is the initiating cause.

Cell type and disease stage modify the effects.

Slide 57 · Cancer cells also experience proteotoxic stress

Stress environment

High proliferative demand and altered metabolism raise biosynthetic strain.

Hypoxia and nutrient shortage can coexist in tumors.

ISR/UPR activation has been associated with cancer.

Biologic implications

Stress adaptation can support survival in hostile conditions.

Excessive or manipulated ISR signaling may instead compromise viability.

Tumor-specific interpretation is required.

Slide 58 · Inflammatory and autoimmune disease associations

Why signaling may matter

Infection, immune activation, and increased protein secretion stress cells.

ISR and UPR changes have been linked to immune-mediated disease.

What can be concluded

The pathway may modify cellular responses during inflammation.

Direction and causality differ among cell types and disorders.

A generic ISR marker is not a disease-specific diagnostic test.

Slide 59 · Interpreting common experimental readouts

Readout

What it supports

What it does not establish

Phospho-eIF2α (Ser51)

Activation of an eIF2α kinase pathway

Identity of upstream kinase

ATF4 protein increase

Stress-associated selective translation

Benefit or harm without context

CHOP increase

Stress-associated transcriptional response

Inevitable apoptosis

Global translation assay

Net protein synthesis change

Which stress sensor caused it

Slide 60 · Common mechanistic interpretation errors

Confusing ISR and UPR

UPR is ER-focused; ISR accepts diverse inputs.

Equating phosphorylation with death

EIF2α–P often supports early adaptation.

Treating all mRNAs identically

ATF4 illustrates selective translation.

Ignoring the time axis

Transient and chronic responses can have different effects.

Slide 61 · Case 1: A stressed secretory cell

A highly secretory cell accumulates abnormal proteins within its ER while protein output remains elevated.

Observed cellular context

Misfolded ER proteins accumulate.

Protein folding demand exceeds processing capacity.

ER-resident sensors are plausible responders.

Interpretation task

Predict the earliest protective changes in protein synthesis and quality control.

Which UPR arm directly phosphorylates eIF2α?

Slide 62 · Case 1: Mechanistic interpretation

ER overload can activate several complementary branches.

ER proteostasis stress

Unfolded proteins exceed capacity

PERK / IRE1 / ATF6

Three UPR sensors respond

Integrated adaptation

Less translation; More chaperones / ERAD

Outcome depends

Folding recovers or persistent stress escalates

The UPR is initially adaptive; Its failure may initiate apoptosis.

Slide 63 · Case 2: A virally infected cell

An infected cell develops abundant double-stranded viral RNA and a measurable rise in eIF2α Ser51 phosphorylation.

Observed cellular context

A viral RNA-associated stress input is present.

EIF2α Ser51 is phosphorylated.

Protein synthesis is reduced in a cell-based assay.

Interpretation task

Identify the likely sensing kinase and explain why ATF4 may still increase.

Does phospho-eIF2α alone prove this kinase caused the signal?

Slide 64 · Case 2: Mechanistic interpretation

PKR is a plausible, but not automatically exclusive, stress sensor.

Viral double-stranded RNA

Recognized by PKR

PKR activation

EIF2α Ser51 phosphorylation

EIF2B inhibition

General initiation falls

Selective translation

ATF4 can rise through uORF regulation

Attributing causality requires sensor-specific perturbation or corroboration.

Slide 65 · Clinical reasoning checkpoint

Why can a translation-inhibitory phosphorylation event increase a stress-response transcription factor?

Answer: Selective translation of transcripts such as ATF4 through uORF-dependent regulation.

Slide 66 · Core mechanistic summary

Input

Four kinases respond to distinct cellular stresses.

Switch

All converge on eIF2α phosphorylation at Ser51.

Output

Most translation decreases; ATF4 translation and adaptive transcription rise.

Fate

Homeostasis may recover; Unresolved stress can contribute to dysfunction/death.

Slide 67 · Selected sources and further reading

Robbins, Cotran & Kumar. Pathologic Basis of Disease. 11th ed. Chapter 2, pp. 32–34.

Costa-Mattioli M, Walter P. The integrated stress response: From mechanism to disease. Science. 2020.

Pakos-Zebrucka K, et al. The integrated stress response. EMBO Rep. 2016.

Hiramatsu N, et al. Multiple mechanisms of UPR-induced cell death. Am J Pathol. 2015;185:1800–1808.

Wek RC, et al. Surviving and adapting to stress: translational control and the ISR. Antioxid Redox Signal. 2023.