Fate of Misfolded proteins in Endoplasmic reticulum

1. INDIAN INSTITUTE OF TECHNOLOGY ROORKEE
FATE OF MISFOLDED PROTEINS IN THE
ENDOPLASMIC RETICULUM
Assignment 2
Submitted by Sourik Dey (18610023), M.Sc Biotechnology, 1st Year, 2018-2019
Cell and Developmental Biology (BTN-516)
Submitted to
Dr. R.P. Singh

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The process of ER-associated degradation (ERAD)
can be divided into three steps:-
• Recognition of misfolded or mutated proteins in the
endoplasmic reticulum.
• Retro-translocation into the cytosol.
• Ubiquitin-dependent degradation by the
proteasome.
Mechanism

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• The recognition of misfolded or mutated
proteins depends on the detection of
substructures within proteins such as
exposed hydrophobic regions,
unpaired cysteine residues and
immature glycans.
• In mammalian cells for example, there exists a
mechanism called glycan processing. In this
mechanism, the lectin
type chaperones calnexin/calreticulin
(CNX/CRT) provide immature glycoproteins the
opportunity to reach their native conformation.
• They can do this by way of reglucosylating these
glycoproteins by an enzyme called UDP-
glucose-glycoprotein glucosyltransferase.
• This mannosidase removes one mannose residue
from the glycoprotein and the latter is
recognized by EDEM.
Recognition of misfolded or mutated proteins
in the endoplasmic reticulum

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Retro-translocation into the cytosol
• Because the ubiquitin–proteasome system (UPS) is
located in the cytosol, terminally misfolded proteins
have to be transported from the endoplasmic reticulum
back into cytoplasm.
• Most evidence suggest that the Hrd1 E3 ubiquitin-
protein ligase can function as a retrotranslocon or
dislocon to transport substrates into the cytosol.
• Hrd1 is not required for all ERAD events, so it is likely
that other proteins contribute to this process.

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• Further, this translocation requires a driving force that determines the
direction of transport.
• Since polyubiquitination is essential for the export of substrates, it is
widely thought that this driving force is provided by ubiquitin-binding
factors. One of these ubiquitin-binding factors is the Cdc48p-Npl4p-
Ufd1p complex in yeast.
• Humans have the homolog of Cdc48p known as valosin-containing
protein (VCP/p97) with the same function as Cdc48p. VCP/p97
transports substrates from the endoplasmic reticulum to the cytoplasm
with its ATPase activity.

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Ubiquitin-dependent degradation by the
proteasome
• The ubiquitination of terminally misfolded proteins
is caused by a cascade of enzymatic reactions. The
first of these reactions takes place when
the ubiquitin-activating enzyme E1
hydrolyses ATP and forms a high-
energy thioester linkage between a cysteine residue
in its active site and the C-terminus of ubiquitin.
• The resulting activated ubiquitin is then passed to
E2, which is a ubiquitin-conjugating enzyme.
Another group of enzymes, more specifically
ubiquitin protein ligases called E3, bind to the
misfolded protein.
• Next they align the protein and E2, thus facilitating
the attachment of ubiquitin to lysine residues of the
misfolded protein.

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• Following successive addition of ubiquitin molecules to lysine residues of the
previously attached ubiquitin, a polyubiquitin chain is formed.
• A polyubiquitinated protein is produced and this is recognized by specific subunits in
the 19S capping complexes of the 26S proteasome.
• Hereafter, the polypeptide chain is fed into the central chamber of the 20S core
region that contains the proteolytically active sites.
• Ubiquitin is cleaved before terminal digestion by deubiquitinating enzymes. This third
step is very closely associated with the second one, since ubiquitination takes place
during the translocation event. However, the proteasomal degradation takes place in
the cytoplasm.

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