ER stress and disease

introduction

• ER function: an organelle where secretory or membrane proteins are synthesized.

----Nascent proteins are folding with the assistance of ER chaperones located in ER, and only correctly folded proteins are transported to the Golgi apparatus.

----Unfolded or malfolded proteins are retained in the ER, retrotranslocated to the cytoplasm by ERAD.

• ER stress: When cells synthesize secretory proteins in amounts that exceed the capacity of the folding apparatus and ERAD machinery, unfolded proteins are accumulated in the ER. Unfolded proteins exposed hydrophobic amino-acid residues and tend to form protein aggregrates.

Final this evokes ER stress

ER stress response

The mammalian ER stress response consists of four mechanisms

• Attenuation of unfolded proteins• The transcriptional induction of ER chaperone

genes to increase folding capacity;• The transcriptional induction of ERAD

component genes to increase ERAD capacity;• Induction of apoptosis to safely dispose of cells

injured by ER stress to ensure the survival of the organism.

ER stress- inducing chemicals

First group: glycosylation inhibitor N-glycosylation of proteins for folding Tunicamycin: antibiotic produced by

streptomyces lysosuperificus that inhibitors by preventing UDP-GlcNAc-dilichol phosphate GlcNAc-phosphate transferase activity.

2-Deoxy-D-glucose is less efficient than tunicamycin.

Second group: Ca2+ metablism disruptor

• Ca2+ ionophore (A23187) and Ca2+ pump inhibitor (thapsigargin)

• Why: the high concentration of Ca2+ ion in ER should be kept; and ER chaperones such as BiP required Ca2+ ions

• The third group: reducing agents

DTT and β-mecaptoethanol

• The fourth group: hypoxia

ER chaperones

• Molecular chaperones

• Folding enzymes

Molecular chaperones

• BiP/GRP78s : belong to HSP70 BiP binds to the hydrophobic regions of

unfolded proteins via a substrate-binding domain and facilitates folding through conformational change evoked by the hydrolysis of ATP by ATPase domain.

• Oxygen-regulated protein (ORP/GRP170) HSP110 family, the mecanism is similar to

BiP.

• ERdj1, 3, 4, 5, SEC63 and p58IPK belongs to HSP40 family, and modulate the functions of BiP by regulating its ATPase activity as a cocheperone.

• BiP-associated protein (BAP), which is a member of GrpE family and also modulates the function of BiP by enhancing nucleotide exchange.

• GRP94 belongs to HSP90 family.

• FKBP13 is a peptidyl-prolyl isomerase belongs to the FKBP family

• Calnexin and calreticulin are ER chaperones specifically involved in the folding of glycoprotein. They share a similar molecular structure and function, though they are transmembrane and luminal proteins, respectively.

Folding enzymes

• Protein disulfide isomerase(PDI), ERp72, ERp61, GRP58/Erp57, ERp44, ERp29 and PDI-P5.

Functions: oxidize cystein residues of nascent bonds. Reduced folding enzymes are reoxidized by ER oxidoreductin(ERO1),which can use molecularoxygen as teminal electron acceptor.

ERAD

• Unfolded or malfolded proteins are trapped by the ERAD machinery and transported to cytoplasm.

• Retrotranslocated proteins are ubiquitinated and degraded by proteasome in the cytosol.

• The process of ERAD can be divided into four step: recognition, retrotranslocation, ubiquitination and degradation

recognition

• During the calnexin cycle, the oligosaccharide of nascent residue is trimmed by a-mannosidase I, nascent polypeptides with eight mannose residues are released from calnexin or calreticulin and bind to ER degradation-enchaning a-mannosidase-like protein (EDEM), which discriminates unfolded proteins from folded proteins.

• EDEM1(membrane), EDEM2, EDEM3(luminal) contain mannosidase-like domain, which recognite mannose residues.

• Osteosarcoma 9 (OS9) specially binds to unfolded glycoproteins containing eight or five mannose residues and unglycosylated unfolded proteins. OS9 and XTP3-transactivated gene B(XTP3B) contain the mannose—phosphate receptor-like domain, which may be critical to the recognition of mannose residues.

Retrotranslocation

• Nascent glycoproteins recognized by EDEM and OS9 as misfolded are destined for the retrotranslocation. Before retrotranslocation, nascent proteins associate with PDI and BiP to cleave disulfide bonds and to unfold the partially folded structure, respectively.

• Retrotranslocation machinery is elusive• Sec61 , Derlin-1associated with p97 through an

adaptor protein, valocin-containing protein(VCP)-interacting membrane protein1.

• P97/cdc48/VCP is a cytosolic AAA-ATPase and recruits unfolded ER proteins to the cytosol. Ubiquitin fusion degradation protein 1 (Ufd1) and nuclear protein localization 4 (Npl4) bind to p97 as cofactor and help p97 to extract unfolded proteins. The polypeptide portion of unfilded proteins interacts with p97, whereas the poly-Ub chains attached to them are recognized by bothe p97 and Ufd1 and may activate the ATPase activity of p97.

UbiquitinationRetrotranslocated proteins are ubiquitinated by E1-E2-E3 ubiquitin system. Ub is first conjugated to E2 by E1, and then transferred to ERAD substrates by E3.

E1:Ub activating enzyme that is ubiquitously involved in protein degradation.

E2: UBC2 and UBC7.

ERAD-L and READ-C

HMG-CoA reductase degradation protein 1(HRD1,preference for substrate that contain misfolded luminal domain), gp78 and TEB4/Doa10 (prefer transmembrane proteins containing misfolded cytosolic) are membrane-anchored E3;FBX2 (F-box only protein 2) spescially recognizes N-glycosylated proteins located in cytosol.

Parkin: both ERAD-L and ERAD-C

Degradation

Retrotranslocated and ubiquitinated proteins are deglycosylated by peptide-N-glycanase before their degradation by the proteasome,

Why: bulky glycan chains hampering effect

Peptide-N-glycanase is associated with Derlin-1 (cotrotranslocationally)

DSK2 and Rad23 facilited the delivery of substrates into proteasome

Response pathways for ER stress

The mammalian ER stress response has four mechanisms: (1) translational attenuation; and enhanced expression of (2) ER chaperones and (3) ERAD components; (4) induction of apoptosis.

These responses are regulated by the regulatory pathways.

PERK pathwayPERK is transmembrane protein located in ER, which sense the acculumation of unfolded proteins in ER luminal.

PERK: luminal portion

cytosolic portion contains a kinase domains

ER stress(-): BiP binds to luminal PERK luminal domain.

ER stress(+): BiP release from PERK, and PERK is activated through

oligomerization and trans-phosphorylation.

Activated PERK phosphorylates and inactivates the a-subunit of eIF2

ATF6 pathway

• ATF6, on ER membrane, its luminal domain responsible for the sensing of unfolded proteins, its cytosolic domain has bZIP and a transcriptional domain.

ER stress(-): BiP bind to the luminal domain of ATF6 and by hinders the Golgi-localization signal, leading to inhibition of ATF6 translocation

ER stress (+):BiP dissociates from ATF6, and ATF6 is moved to Golgi apparatus by vasicular transport, then cleaved by S2P and S1P. The resultant cytoplasmic portion of ATF6 translocates into nucleus.

pATF6(N) : BiP, GRP94 and calreticulin CCAAT(NF-Y)-(N9)-CCACG (pATF6(N)

IRE1 pathway• The third sensor molecule is IRE1 (inositol requirement 1): IRE1a

and IRE1β• Cytosolic domain contains a kinase domain and Rnase domain• ER stress (-): BiP bind to its luminal domian• ER stress (+): BiP suppression of IRE1 activation is released, IRE1

is activated through dimerization and transphosphorylation.• Activated IRE1a convert XBP1 pre-mRNA into mature mRNA by

cytosolic splicing.• pXBP1(S) not pXBP1(U) : activation of ERAD component genes

such as EDEM, HRD1, Derlin-2 and -3 through a cis-acting element; induce the expression of protein involved in lipid synthesis and ER biogensis, as well as ER chaperons, such as BiP, p581PK, ERdj4, PDI-P5 and HEDJ.

Apoptosis-inducing pathway

• If PERK, ATF6 and IRE1 pathways can not suppress ER stress, an apoptotic pathway is triggering to ensure survival of organism as a last line of defense.

---CHOP/GADD153 pathway

---IRE1-TRAF2-ASK1 pathway

---Caspase 12 pathway

ER stress-related disease

• Neurodegenerative disease AD, PD, polyQ disease, Prion disease,ALS• Bipolar disorder Expression of XBP1 and BiP wane• Diabetes mellitus• Atherosclerosis• Inflammation• Ischemia• Heart disease• Liver disease• Kidney disease• Viral infection