Protein Degradation and Regulation UbiquitinProteasome Pathway Guo Peng, Luo Tong and Yang Kong 2002

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Protein Degradation and Regulation
Ubiquitin/Proteasome Pathway Guo Peng, Luo
Tong and Yang Kong2002.12.16
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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.

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• 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.

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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
  ubiquitin/proteasome pathway.

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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 them.

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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/proteasom
  e pathway.

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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 requires the 26S
  proteasome.

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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 signal-transducing molecules. Distinct
  targeting complexes accomplish the recognition of
  these proteins.

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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.

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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.

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II. Protein Degradation
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Ubiquitin

• 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

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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.

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• 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.

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• 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

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Process of ubiquitin activated
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Combinatorial nature of ubiquitination
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Modes of recognition of protein substrates by the
different E3s
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Which 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

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Themes and Variations on Ubiquitylation
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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.

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• Monoubiquitination and multimubiquitination

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Deubiquitination 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 .

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Possible roles for DUB enzymes

• Editing
• proofread
• Disassembly
• Recycling
• Processing

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Basic 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

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Schematic 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.

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The structure of proteasome
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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

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Central position of the proteasome
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Site 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.

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Alternative pathways

• The 26S proteasome is not an absolute
  ubiquitin-dependent proteolytic enzyme, as it
  also degrades non-ubiquitinated substrates.

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calpai
c-Fos
protein
lysosomal
ODC
ubiquitination
c-Jun
proteasome
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III. Protein Regulation
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(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(??), 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 .

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(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.

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• 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

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• 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.

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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.

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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?

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