Title: Protein Degradation and Regulation Ubiquitin/Proteasome Pathway Guo Peng, Luo Tong and Yang Kong 2002.12.16
1Protein Degradation and Regulation
Ubiquitin/Proteasome Pathway Guo Peng, Luo
Tong and Yang Kong2002.12.16
2 I. Introduction
- This pathway is the major non-lysosomal process
responsible for the breakdown of most short and
long-lived 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 down-regulation and antigen processing,
via the breakdown of specific proteins.
3- two main steps in the pathway
- covalent attachment of a polyubiquitin chain to
the substrate - specific recognition of this signal, and
degradation of the tagged protein by the 26S
proteasome.
4Cellular 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
ubiquitin/proteasome pathway.
5Mislocalized 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 them.
6Retro-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/proteasom
e pathway.
7Degradation 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 requires the 26S
proteasome.
8Degradation 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 signal-transducing molecules. Distinct
targeting complexes accomplish the recognition of
these proteins.
9The 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.
10The 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.
11II. Protein Degradation
12Ubiquitin
- 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 gt 6, 11, 29, 63) of primary ubiquitin
13(No Transcript)
14Enzymes 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.
15- 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.
16- How is ubiquitin activated?
- 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
17Process of ubiquitin activated
18Combinatorial nature of ubiquitination
19(No Transcript)
20Modes of recognition of protein substrates by the
different E3s
21Which signals lead to ubiquitination?
- Genetic program (amino acids)
- Nend rule
- Nterminal amino acid D,R,L,K,F (lt minutes)
A,G,M,S,V (gt10 hours) - Sequence of significant hydrophobicity(???)
- PEST sequences (sequences rich in Pro, Asp,
Glu, Ser and Thr) - Phosphorylation of Ser and Thr
- Binding to adaptor proteins(????)
- Protein damage
- Processing
- Oxidation of Cys and Met
- Age-dependent modifications of side chains
hydrolsis(??), deaminations(???),
racemizations(????), disulfide bond
breaks(?????), ketoamines(???) - Wrong folding
22Themes and Variations on Ubiquitylation
23Pay 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.
24- Monoubiquitination and multimubiquitination
25Deubiquitination enzymes
- 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 (lt 40-kDa)in
contrast, UBPs are 50-250-kDa 8proteins and
constitute a large family. - Genome sequencing projects have identified more
than 90 DUBs . -
26Possible roles for DUB enzymes
- Editing
- proofread
- Disassembly
- Recycling
- Processing
27Basic features of proteasome
- Essential and ubiquitous intracellular protease
- Degrades most of cytoplasmatic, nuclear and
membrane , nuclear and membrane proteins (gt 90 )
-
- Virtually all target proteins are marked by
ubiquitin first -
- Ubiquitin is recycled, not cleaved
- Central processes with proteasome involvement are
mitosis, antigen presentation, activation and
degradation of transcription factors and
regulation of developmental processes. -
- Eukaryotic proteasomes are large protein
complexes of 2000 kDa, consisting of a core
and a cap region - Prokaryotes lack ubiquitin system and possess no
cap region
28Schematic representation of the eukaryotic
- Core particle is composed of four 7-membered
rings. - Two types of subunits (25 kDa) aand ß, all
differ . - Subunits are similar in structure, different in
sequence. - only only ß subunits are catalytically active .
- Cap region regulates activity, performes the
energy dependent steps.
29The structure of proteasome
30(No Transcript)
31(No Transcript)
32Processing 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
33(No Transcript)
34(No Transcript)
35Central position of the proteasome
36Site of intracellular degradation
- Ubiquitinmediated 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.
37Alternative pathways
- The 26S proteasome is not an absolute
ubiquitin-dependent proteolytic enzyme, as it
also degrades non-ubiquitinated substrates.
38calpai
c-Fos
protein
lysosomal
ODC
ubiquitination
c-Jun
proteasome
39III. Protein Regulation
40 (I). General regulation
- Alternation of E1, E2s and proteasome in their
activity will affect many substrates.
41- 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(??), cancer cachexia(???), severe
sepsis(??), metabolic acidosis(?????)? - 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 .
42(II). Specific regulation
- 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(??????), Cln2 and
Cln3, the yeast cyclindependent 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.
43- 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(??????) is regulated by different activators
and inhibitors and by phosphorylation
44- 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(??????)E6
which interacts with an E6-AP 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.
45D. 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.
46IV. Conclusions and future perspectives
47- 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?
48THANK YOU !