ER stress and disease

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ER stress and disease
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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.

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• 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

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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.

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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.

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• 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

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• The third group reducing agents
• DTT and ß-mecaptoethanol
• The fourth group hypoxia

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ER chaperones

• Molecular chaperones
• Folding enzymes

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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.

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• 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.

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

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• 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.

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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.

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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

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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.

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• 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 mannosephosphate receptor-like domain, which
  may be critical to the recognition of mannose
  residues.

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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.

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• 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.

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Ubiquitination
Retrotranslocated 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. E1Ub 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 E3FBX2 (F-box
only protein 2) spescially recognizes
N-glycosylated proteins located in
cytosol. Parkin both ERAD-L and ERAD-C
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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 (cotrotranslocational
ly) DSK2 and Rad23 facilited the delivery of
substrates into proteasome
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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.
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PERK pathway
PERK 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. Activa
ted PERK phosphorylates and inactivates the
a-subunit of eIF2
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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)

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IRE1 pathway

• The third sensor molecule is IRE1 (inositol
  requirement 1) IRE1a and IRE1ß
• 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.

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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

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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