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Protein Degradation and Regulation
Ubiquitin/Proteasome Pathway
Guo Peng, Luo Tong and Yang Kong
2002.12.16
I. Introduction
This pathway is the major non-lysosomal process
responsible for the breakdown of most short and longlived proteins in mammalian cells.
For example, in skeletal muscle, the system is
responsible for the breakdown of the major contractile
proteins, actin and myosins.
In addition, the pathway also controls various major
biological events: cellcycle progression, oncogenesis,
transcriptional control, development and
differentiation, signal transduction, receptor downregulation and antigen processing, via the breakdown
of specific proteins.
two main steps in
the pathway
1.
2.
covalent attachment of
a polyubiquitin chain
to the substrate;
specific recognition of
this signal, and
degradation of the
tagged protein by the
26S proteasome.
Cellular functions of protein degradation

The elimination of damaged proteins:
environmental toxins, translation errors and
genetic mutations can damage proteins.
Misfolded proteins are highly deleterious to the
cell because they can form non-physiological
interactions with other proteins. Repair proteins
called chaperones can, in many instances,
restore the native conformation of misfolded
proteins. However, if a damaged protein is not
repaired, it is degraded in specialized
organelles such as the ysosome, and by the
Mislocalized proteins and stoichiometric
excess
Some proteins are stabilized only when
they are bound to their natural partners.
This ensures that they are present only at
stoichiometric levels. Consequently, the
overexpression of specific ribosomal
proteins can lead to degradation because of
their failure to assemble into the ribosome.
Similarly, proteinsthat are mislocalized may
be degraded because they are unable to
form interactions that normally stabilize
Retro-translocation
Proteins that enter the secretory
pathway and fold improperly in the
endoplasmic reticulum are
transported back to the cytosol where
they are recognized and degraded by
the ubiquitin/proteasome pathway.
Degradation of foreign
proteins
The immune system is a
surveillance mechanism that can
recognize foreign proteins and degrade
them. An essential feature of this
system is the ability to distinguish ‘self’
from ‘non-self’. The MHC class I
antigen presenting cells display
peptide fragments that are derived
from the foreign protein, to cytotoxic T
cells. The generation of these peptides
Degradation of regulators:
Many regulators of cell growth and
development are highly unstable
proteins, whose stability is controlled
by the ubiquitin/proteasome pathway.
Substrates of this pathway include p53,
Rb, cyclins, CDK inhibitors,
transcription factors, and signaltransducing molecules. Distinct
targeting complexes accomplish the
The generation of active
proteins

Enzymes whose activities can be deleterious
to the cell are often expressed as precursors
that are catalytically inactive. The proteolytic
cleavage of the precursor generates an
active enzyme. For instance, proteases that
are present in the digestive tract, and those
that function in the lysosome, are initially
synthesized as precursors. Ubiquitin, and
catalytic subunits of the proteasome are also
expressed as precursors that are
proteolytically processed to yield
catalytically active subunits.
The recycling of amino acids:
Proteases are required for the generation of
free amino acids from short peptides that are
generated by the proteasome and other
intracellular proteases. In many microorganisms
dipeptidases and other proteases that hydrolyze
short amino acid chains are secreted to generate
free amino acids that can be readily imported
into the cell.The availability of free amino acids
and di-peptides can allosterically regulate the
activity of a specific E3 protein, which in turn
controls the levels of a transcription factor that
is required for inducing amino acid biosynthetic
pathway genes.
II. Protein Degradation
Ubiquitin
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Ubiquitin is a highly conserved
protein (3 aa exchanges from
yeast to men)
Ubiquitin is composed of 76 aa
Attachment site to target protein
on ubiquitin is C-terminus
Bond is formed to side chain of
Lys of target protein
Attachment is performed by array
of enzymes (E1, E2, E3, E4)
Subsequently, poly-ubiquitin
chains form via binding of further
molecules to Lys side chains (Lys48
> 6, 11, 29, 63) of primary
ubiquitin
Enzymes of the Ubiquitination

E1:
• ubiquitin-activating enzyme.
• exists as two isoforms of 110- and 117-kDa,
which derive from a single gene and are
found in both the nucleus and cytosol.
Inactivation of this gene is lethal.
• In mammals there is a single E1.

E2:
• Ubiquitin-conjugating enzymes.
• E2s are a superfamily of related proteins.
There are eleven E2s in yeast, and 20-30
E2s in mammals.

E3s:
• Ubiquitin-protein ligases.
• E3s play a key role in the ubiquitin
pathway, as they are responsible for the
selective recognition of protein substrates.
• E3 ligases can be subdivided into at least
six subtypes.

E4:
• catalyzes the efficient polymerization of
very long polyubiquitin chains, it has been
characterized in yeast.
How is ubiquitin activated?
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C-terminus of ubiquitin gets adenylated
Rearrangement to intermolecular thioester with a E1
(activation enzyme)
Transfer of activierted ubiquitin from E1 to E2
(ubiquitin-conjugating enzyme) (thioester bond)
Transfer form E2 via E3(ubiquitin ligase) to target
enzyme
Process of ubiquitin activated
Combinatorial nature of ubiquitination
Modes of recognition of protein
substrates by the different E3s
Which signals lead to ubiquitination?
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Genetic program (amino acids)
• N—end rule
 N—terminal amino acid: D,R,L,K,F (< minutes); A,G,M,S,V
(>10 hours)
• Sequence of significant hydrophobicity(疏水性）
• “PEST” sequences (sequences rich in Pro, Asp, Glu, Ser and Thr)
Phosphorylation of Ser and Thr
Binding to adaptor proteins（衔接蛋白）
Protein damage
• Processing
• Oxidation of Cys and Met
• Age-dependent modifications of side chains: hydrolsis（水解）,
deaminations（脱氨基）, racemizations（外消旋化）, disulfide
bond breaks（二硫键簖裂）, ketoamines(氯氨酮） …
• Wrong folding
Themes and Variations on Ubiquitylation
Pay attention
Ubiquitination is an important and widespread post-translational
modification of proteins, which resembles phosphorylation.
Very importantly, ubiquitination is not only a degradation signal,
but also directs proteins to a variety of fates which include roles in
ribosomal function, in DNA repair, in protein translocation, and in
modulation of structure or activity of the target proteins.
In order to be efficiently degraded, the substrate must be bound to
a polyubiquitin degradation signal that comprises at least four
ubiquitin moieties, These signals are usually determined by short
regions in the primary sequence of the targeted protein.
The nature of the N-terminal amino acid of a protein (N-end rule)
may determine its rate of polyubiquitination and subsequent
degradation.
Monoubiquitination and
multimubiquitination
Deubiquitination enzymes
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Eukaryotic cells also contain DUBs
(DeUBiquitinating enzymes), which are encoded by
the UCH (Ubiquitin Carboxyl-terminal Hydrolases)
and the UBP (UBiquitin-specific Processing
proteases) gene families.
UCHs are relatively small proteins (< 40-kDa);in
contrast, UBPs are 50-250-kDa 8proteins and
constitute a large family.
Genome sequencing projects have identified more
than 90 DUBs .
Possible roles for DUB enzymes
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Editing
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proofread
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Disassembly
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Recycling
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Processing
Basic features of proteasome
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Essential and ubiquitous intracellular protease
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Degrades most of cytoplasmatic, nuclear and membrane , nuclear and
membrane proteins (> 90 %)
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Virtually all target proteins are marked by ubiquitin first
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Ubiquitin is recycled, not cleaved
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Central processes with proteasome involvement are mitosis, antigen
presentation, activation and degradation of transcription factors and
regulation of developmental processes.
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Eukaryotic proteasomes are large protein complexes of ~ 2000 kDa,
consisting of a “core” and a “cap” region
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Prokaryotes lack ubiquitin system and possess no cap region
Schematic representation of the eukaryotic
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Core particle is composed of
four 7-membered rings.
Two types of subunits (25 kDa):
αand β, all differ .
Subunits are similar in structure,
different in sequence.
only only β subunits are
catalytically active .
Cap region regulates activity,
performes the energy
dependent steps.
The structure of proteasome
Processing via the proteasome
•Length of produced peptides: 3-23 amino acids
•Average length of peptides: 7-9 amino acids
•Peptide composition of given protein stays constant
•Protein is completely degraded before import of next protein
•Peptides produced by proteasome are further degraded by
other roteases and aminopeptidases (Tricorn, Multicorn, Thimet,
TPPII)
• Proteasome and immune system function:
•Peptides of 8-9 amino acids in length are transported to the
cell surface via the ER presented on the cell surface via MHC
class I – molecules
Central position of the proteasome
Site of intracellular degradation
Ubiquitin—mediated degradation of cytosolic
and membrane proteins occurs in the cytosol
and on the cytosolic face of the ER
membranes. Although components of the
system have been localized to the nucleus,
conjugation and degradation have not been
demonstrated in this organelle.
Alternative pathways

The 26S proteasome is not an absolute
ubiquitin-dependent proteolytic
enzyme, as it also degrades nonubiquitinated substrates.
protein
c-Fos
lysosomal
ODC
ubiquitination
c-Jun
proteasome
calpai
III. Protein Regulation
(I). General regulation
Alternation of E1, E2s and proteasome
in their activity will affect many substrates.
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One is the up-regulation of the ubiquitin pathway to achieve
bulk degradation of skeletal muscle proteins that occurs in
different pathophysiological conditions such as fasting（禁
食）, cancer cachexia（癌血症）, severe sepsis（脓毒）,
metabolic acidosis（代谢酸中毒）。
The second example of a change in the general components
of the system occurs following treatment with IFN-r. This
cytokine induces changes in the subunit composition of the
20S proteasomal complex. Consequently, the antigenic
peptides that are generated following proteosomal
degradation have higher affinity for the presenting MHC
class I molecules and for the cytotoxic T-cell receptor .
(II). Specific regulation
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A. Regulation by modification of the substrate:
Phosphorylation of many substrates is required for their
recognition by their E3s. Conversley, similar
modification of many other proteins prevents this.
Substrates that require prior phosphorylation include
the yeast G1 cyclins（细胞周期蛋白）, Cln2 and Cln3,
the yeast cyclin—dependent kinase (CDK) inhibitors,
Sic1 and Far1.
Degradation of the proto-oncogene c-mos by the
ubiquitin pathway is inhibited by phosphorylation on Ser.
Interestingly, activation of c-mos leads to
phosphorylation and stabilization of c-fos, another
substrate of the ubiquitin pathway.

B. Regulation by modulation of ubiquitination
activity:
Regulated degradation of specific classes of
substrates could be achieved by modulation of the
activity of the ubiquitination machinery. For
example, it has been shown recently that
degradation of mitotic regulators by the APC（抗原
呈递细胞） is regulated by different activators and
inhibitors and by phosphorylation

C. Regulation by ancillary proteins:
Several viral proteins exploit the ubiquitin system by
targeting for degradation cellular substrates which may
interfere with propagation of the virus. In some instances,
the viral protein functions as a bridging‘ element between
the E3 and the substrate, thus conferring recognition in
trans. The prototype of such a protein is the high risk HPV
oncoprotein(人乳头癌蛋白）E6 which interacts with an E6AP HECT domain E3, and with the tumor suppressor
protein p53. This interaction targets p53 for rapid
degradation and, thus, most probably prevents stress
signalinduced apoptosis and ensures further replication
propagation of the virus . In a different case, the Vpu
protein of the HIV-1 virus is recognized by the F-box protein,
b-TrCP. Vpu also binds to the CD4 receptor in the ER of
Tcells infected by the virus. This leads to ubiquitination and
subsequent degradation of CD4 by the SCFb-TrCP complex,
thus enabling the virus to escape from immune surveillance.
D. Regulation by masking of a
degradation signal:
The presence of either one of two transcription factors,
MATa1 and MATa2, determines the mating type of haploid
yeast cells. The diploid cell expresses both a1 and a2 that
form a heterodimer with distinct DNA-binding specificity. In
haploid cells, the two factors are rapidly degraded by the
ubiquitin system. Degradation of a2 requires two
degradation signals, Deg1 and Deg2. Strikingly, both a1 and
a2 are stabilized by heterodimerization.For a2 at least, it has
been shown that residues required for interaction with a1
overlap with the Deg1 degradation signal and it is possible
that binding of a1interferes with the degradation of a2 by
masking the ubiquitin recognition signal.
IV. Conclusions and future
perspectives
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Only a few targeting signals have been identified, and the
mechanisms that underlie the regulation of the system are still
largely unknown？
While the system has been implicated in the pathogenesis of
several diseases, the underlying mechanisms, as well as its
potential involvement in many other diseases, are still an
enigma？
Why are there so many ubiquitinating enzymes if prior
modifications such as phosphorylation or damage are triggering
events?
Do DUBs show substrate specificity, perhaps by regulating the
levels of ubiquitination of specific subsets of proteins?
What are the binding sites for polyubiquitin chains on the
microtubules and on the proteasome itself?
What is the role of K29-and/or K63-linked polyubiquitin chains
in the cell?
THANK
YOU !