Protein Degradation (PowerPoint) by ert554898


             Degradación de proteínas

• Procesos Lisosomales
     Proteínas Extracelulares

• Procesos no-lisosomales
     Proteínas intracelulares
            Degradación de Proteínas

• Los niveles de proteínas están determinados por las
  velocidades de síntesis y degradación.

• Así, altos niveles de una proteína se alcanzarán por una
  alta velocidad de síntesis o por una baja velocidad de

• A la inversa, una proteína que no es abundante puede ser
  porque tiene una síntesis lenta o que se degrada muy
La cantidad de una proteína en la célula esta
dada por la siguiente relación:

                   ks        kD
    aminoácidos         P             Degradación

              d[P]/dt = ks – kD [P]

La constante de velocidad de degradación se
puede calcular a partir de la diferencia entre
la velocidad de síntesis (ks) y la velocidad del
incremento de P en la célula (d[P]/dt)
                Degradación de proteínas
  Las proteínas no se degradan a la misma velocidad. La
        cinética de degradación es de primer orden.

ENZIMA                        vida media
Ornitina descarboxilasa       11 minutos
-Aminolevulinato sintetasa   70 minutos
Catalasa                      1,4 dias
Tirosina aminotransferasa     1,5 horas
Triptófano oxigenasa          2 horas
Glucoquinasa                  1,2 dias
Láctico deshidrogenasa        16 dias
HMG CoA reductasa             3 horas
Se ha observado y no confirmado que la velocidad
de degradación depende de los siguientes factores:

-Susceptibilidad a la desnaturación térmica
-Ausencia de ligandos que estabilizan
-Susceptibilidad a la proteolisis in vitro
-Velocidad intrínseca de deaminación de Gln y Asn
-Susceptibilidad a la oxidación de Cis,His y Met
-Presencia de carbohidratos o fosfatos en P
-Grupo alfa-amino libre
-Carga neta negativa de la proteína
-Aumento del tamaño de la cadena polipéptidica
-Flexibilidad de la estructura nativa (H-D intercam)
            Degradación de proteínas

• Puede depender de la distribución en los tejidos
      Ejemplo: Láctico Deshidrogenasa
                   Tejido             Vida media
                   Corazón            1,6 dias
                   Músculo            31 dias
                   Hígado             16 dias
• La degradación de proteínas es un proceso regulado
     Ejemplo: Acetyl CoA carboxylasa
                 Estado nutricional Vida media
                 Alimentado          48 horas
                 Ayunado             18 horas
Proteolytic enzymes
                                                serine (Ser, S)

                                              H3N+   C     COO
There are several classes of
proteolytic enzymes.                                 CH2

Serine proteases include digestive
enzymes trypsin, chymotrypsin, & elastase.
Different serine proteases differ in substrate specificity. For
  Chymotrypsin prefers an aromatic side chain on the
    residue whose carbonyl carbon is part of the peptide
    bond to be cleaved.
  Trypsin prefers a positively charged Lys or Arg residue at
    this position.
                                        aspartate (Asp)
Aspartate proteases include                   H

                                       H3N+         COO
   the digestive enzyme pepsin               C

   Some proteases found in lysosomes         CH2

   the kidney enzyme renin
   HIV-protease.
Two aspartate residues participate in acid/base
catalysis at the active site.
In the initial reaction, one aspartate accepts a
proton from an active site H2O, which attacks the
carbonyl carbon of the peptide linkage.
Simultaneously, the other aspartate donates a
proton to the oxygen of the peptide carbonyl group.
Zinc proteases (metalloproteases) include:
 digestive enzymes carboxypeptidases
 matrix metalloproteases (MMPs), secreted by
 one lysosomal protease.
Some MMPs (e.g., collagenase) are involved in
degradation of extracellular matrix during tissue
Some MMPs have roles in cell signaling relating
to their ability to release cytokines or growth
factors from the cell surface by cleavage of
membrane-bound pre-proteins.

                                      H3N+   C     COO

Cysteine proteases have a
catalytic mechanism that involves
a cysteine sulfhydryl group.            cysteine

Deprotonation of the cysteine SH by an
adjacent His residue is followed by nucleophilic
attack of the cysteine S on the peptide
carbonyl carbon.
A thioester linking the new carboxy-terminus
to the cysteine thiol is an intermediate of the
reaction (comparable to acyl-enzyme
intermediate of a serine protease).
Cysteine proteases:
 Papain is a well-studied plant cysteine protease.
 Cathepsins are a large family of lysosomal
  cysteine proteases, with varied substrate
 Caspases are cysteine proteases involved in
  apoptosis (programmed cell death).
  A caspases cleave on the carboxyl side of an Asp.
 Calpains are Ca++-activated cysteine proteases that
  cleave intracellular proteins involved in cell motility
  and adhesion.
  They regulate processes such as cell migration and
  wound healing.
Activation of proteases:
 Most proteases are synthesized as larger pre-
  proteins. During activation, the pre-protein is
  cleaved to remove an inhibitory segment.
 In some cases activation involves removal of an
  inhibitory protein.
 Activation may occur after a protease is delivered
  to a particular compartment within a cell or to the
  extracellular milieu.
Protease Inhibitors:
 Many protease inhibitors are proteins with
  domains that enter or block a protease active
  site to prevent substrate access.
 Serpins utilize a suicide mechanism to inhibit
  serine or cysteine proteases.
  A conformational change induced by serpin
  cleavage prevents completion of the reaction,
  leaving the serpin covalently linked as an acyl-
  enzyme intermediate.
  Extracellular and intracellular serpins have
  diverse roles, including regulation of blood
  clotting, fibrin cleavage, and inhibition of
 IAPs are protein inhibitors of caspases that
  regulate apoptosis.
 TIMPs are inhibitors of metalloproteases. They
  are secreted by cells. A domain of the inhibitor
  protein interacts with the catalytic Zn++.
 Cystatins are inhibitors of lysosomal
  cathepsins. Some (also called stefins) are found
  in the cytosol, and others in the extracellular
  Cystatins protect cells against cathepsins that
  may escape from lysosomes.

Structure - membrane bound bag containing hydrolytic
        - hydrolytic enzyme = (water split biological
          i.e. using water to split chemical bonds
Function - break large molecules into small molecules by
          inserting a molecule of water into the chemical
Lysosomes contain
a large variety of                                  Lumen
hydrolytic enzymes                low
                                internal          contains
that degrade proteins             pH            hydrolytic
& other substances                               enzymes.
taken in by
endocytosis.                               Vacuolar ATPase
Lysosomes have a                       H+ ADP + Pi
low internal pH due
to vacuolar ATPase.
All lysosomal hydrolases exhibit acidic pH optima.
Lysosomal proteases include many cathepsins (cysteine
proteases), some aspartate proteases and one zinc
Activation may be catalyzed by other lysosomal enzymes
or be autocatalytic, promoted by the internal acidic pH.
One model
for autophagic
vacuole                 autophagosome        autophagic
formation                                      vacuole

  In autophagy, part of the cytoplasm may become
   surrounded by two concentric membranes.
  Fusion of the outer membrane of this
   autophagosome with a lysosomal vesicle results in
   degradation of enclosed cytoplasmic structures and
  Genetic studies in yeast have identified unique
   proteins involved in autophagosome formation.
  Autophagy is not a mechanism for selective
   degradation of individual macromolecules.
                           Schematic depiction of autophagy

(a, b) Cytosolic material is
sequestered by an expanding
membrane sac, the phagophore,
(c) resulting in the formation of a
double-membrane vesicle, an
autophagosome; (d) the outer
membrane of the autophagosome
subsequently fuses with a
lysosome, exposing the inner
single membrane of the
autophagosome to lysosomal
hydrolases; (d) the cargo-
containing membrane
compartment is then lysed, and
the contents are degraded.
Protein turnover
Protein turnover:
Individual cellular proteins turn over (are degraded
and re-synthesized) at different rates.
E.g., half-lives of selected enzymes of rat liver cells
range from 0.2 to 150 hours.
N-end rule: On average, a protein's half-life
correlates with its N-terminal residue.
 Proteins with N-terminal Met, Ser, Ala, Thr, Val,
  or Gly have half lives greater than 20 hours.
 Proteins with N-terminal Phe, Leu, Asp, Lys, or
  Arg have half lives of 3 min or less.
PEST proteins, rich in Pro (P), Glu (E), Ser (S), Thr
(T), are more rapidly degraded than other proteins.
Selective degradation or cleavage of particular
proteins may occur in response to internal & external
signals. e.g.:
  Degradation of metabolically important enzymes
   may be modulated in response to metabolite
   concentrations or hormonal signals.
  Protein cleavage may be involved in generating or
   degrading signal molecules.
Intramembrane-cleaving proteases (I-CLiPs) cleave
regulatory proteins such as transcription factors from
membrane-anchored precursor proteins.
  E.g., precursors of SREBP (sterol response element
   binding protein) transcription factors are integral
   proteins embedded in ER membranes.
Pathway selective gene activation by
         SREBP-1 and -2
                             lumen         SCAP-activated
Activation of SREBP                        S1P cleavage
involves its translocation
to golgi membranes
where sequential                               membrane
cleavage by 2 proteases                            S2P cleavage
releases to the cytosol a                          releasing
domain with transcription                          SREBP
                                       C       N
factor activity.             cytosol
The released SREBP can then translocate to the cell
nucleus to regulate transcription of genes for enzymes
involved, e.g., in cholesterol synthesis.
S2P (site 2 protease, an I-CLiP) is a membrane-
embedded metalloprotease that cleaves an a-helix of
the SREBP precursor within the transmembrane
The SREBP regulatory pathway
Non-lysosomal cytoplasmic
   protein degradation
                  Non-lysosomal pathway

• Two step process
  1. Tagging of the protein by attachment of ubiquitin molecules
  2. Degradation of the tagged protein by the proteosome.

• What is ubiquitin?
  Small protein (8.5 kDa)
  Present in all eukaryotic cells
  Hence the name: it is ubiquitous
                       Proteins are tagged for selective
                       destruction in proteolytic
                       complexes called proteasomes
                       by covalent attachment of
                       ubiquitin, a small, compact
                       protein that is highly conserved.

ubiquitin   PDB 1TBE                        H3N+    C      COO

     An isopeptide bond links the                   CH2
     terminal carboxyl of ubiquitin to              CH2
     the e-amino group of a lysine
     residue of a "condemned" protein.
                                                    NH
The joining of ubiquitin to a condemned protein is
Three enzymes are involved, designated E1, E2
& E3.
 Initially the terminal carboxyl group of
  ubiquitin is joined in a thioester bond to a
  cysteine residue on Ubiquitin-Activating
  Enzyme (E1). This is the ATP-dependent step.
 The ubiquitin is then transferred to a sulfhydryl
  group on a Ubiquitin-Conjugating Enzyme

ubiquitin   C   S   Cys   E2   +   H2N    Lys      protein to be degraded

                                   E3 (Ubiquitin-Protein Ligase)

ubiquitin   C   N   Lys   protein to be degraded     + HS     Cys    E2


 A Ubiquitin-Protein Ligase (E3) then transfers activated
 ubiquitin to the e-amino group of a Lys residue of a
 protein recognized by that E3, forming an isopeptide
 There are many distinct Ubiquitin Ligases with differing
 substrate specificity. One E3 is responsible for the N-end
 rule. Some are specific for particular proteins.
             destruction Primary structure of a protein
                 box        targeted for degradation

       H2N                                       COO
                      chain of

More ubiquitins are added to form a chain of
The terminal carboxyl of each ubiquitin is linked to the
e-amino group of a Lys residue of the adjacent ubiquitin.
A chain of 4 or more ubiquitins (linked via Lys29 or
Lys48) targets proteins for degradation in proteasomes.
             destruction Primary structure of a protein
                 box        targeted for degradation

       H2N                                       COO
                      chain of

Some proteins (e.g., mitotic cyclins involved in cell cycle
regulation) have a destruction box sequence recognized by
a domain of the corresponding Ubiquitin Ligase.
Proteins have been identified that regulate or facilitate
ubiquitin conjugation.
Regulation by phosphorylation of some target proteins has
been observed.
Ubiquitin Ligases (E3) mostly consist of two
  Some Ubiquitin Ligases have a HECT domain
   containing a conserved Cys residue that
   participates in transfer of activated ubiquitin to a
   target protein.
  Some Ubiquitin Ligases contain a RING finger
   domain in which Cys & His residues are ligands to
   2 Zn++ ions.
   A RING (Really Interesting New Gene) finger is
   not inherently catalytic. It stabilizes a
   characteristic globular domain conformation that
   serves as a molecular scaffold for residues that
   interact with E2.
  N-end rule: code and generation of N-degrons

Tomado de Mogk et al., 2007. TRENDS in Cell Biol. 17: 165-172
                   N-end rule substrate binding

Tomado de Mogk et al., 2007. TRENDS in Cell Biol. 17: 165-172
                   N-rule pathway in pro and eukaryotes

Tomado de Mogk et al., 2007. TRENDS in Cell Biol. 17: 165-172
                       Other Factors

• PEST sequences
• All short-lived proteins examined to date contain a
  region enriched with Pro, Glu, Ser and Thr
• Not yet shown to represent a consensus proteolytic
  targeting signal
• Misfolding
• Due to stress
  – e.g., heat shock
• Due to mutation
  – e.g., CFTR protein
                                          20 S Proteasome
Proteasomes:                             (yeast) closed state
Selective protein
degradation occurs
in the proteasome,
a large protein
complex in the
nucleus & cytosol      a
of eukaryotic cells.
                                       two views     PDB 1JD2

The proteasome core complex, with a 20S sedimentation
coefficient, contains 2 each of 14 different polypeptides.
  7 a-type proteins form each of the two a rings, at the
   ends of the cylindrical structure.
 7 b-type proteins form each of the 2 central b rings.
                                20 S Proteasome
                               (yeast) closed state
                             two views     PDB 1JD2

The 20S proteasome core complex encloses a cavity
with 3 compartments joined by narrow passageways.
Protease activities are associated with 3 of the b
subunits, each having different substrate specificity.
     Proteosome proteolitic activities

1. One catalytic b-subunit has a chymotrypsin-like
   activity with preference for tyrosine or
   phenylalanine at the P1 (peptide carbonyl)
2. One has a trypsin-like activity with preference for
   arginine or lysine at the P1 position.
3. One has a post-glutamyl activity with preference
   for glutamate or other acidic residue at the P1
                     threonine (Thr)

                     H3N+   C     COO

                            CH OH


The proteasome hydrolases constitute a unique family of
threonine proteases. A conserved N-terminal threonine
is involved in catalysis at each active site.
The 3 catalytic b subunits are synthesized as pre-
proteins. They are activated when the N-terminus is
cleaved off, making threonine the N-terminal residue.
Catalytic threonines are exposed at the lumenal
TMC-95s are naturally occurring proteasome
inhibitors that bind with high affinity adjacent to
active site threonines within the core complex.
These inhibitors have a heterocyclic ring structure
derived from modified amino acids.
Proteasome evolution: Proteasomes are
considered very old. They are in archaebacteria, but
not most eubacteria.
  The archaebacterial proteasome has just 2
   proteins, a & b, with 14 copies of each.
  The eukaryotic proteasome has evolved 14
   distinct proteins that occupy unique positions
   within the proteasome (7 a-type & 7 b-type).
                                        20 S Proteasome
                                       (yeast) closed state
Regulatory cap     a
complexes:                           two views     PDB 1JD2

 In crystal structures of the proteasome core alone, there
 is no apparent opening to the outside.
 The ends of the cylindrical complex are blocked by
 N-terminal domains of a subunits that function as a gate.
 Interaction with a regulatory cap causes a
 conformational change that opens a passageway into
 the core complex.
The 19S regulatory cap complex recognizes
multi-ubiquitinated proteins, unfolds them,
removes ubiquitin chains, and provides a
passageway for threading unfolded proteins into
the core complex.
The 19S cap is a 20-subunit 700 kDa complex,
also referred to as PA700. When combined with
a 20S core complex, it yields a 26S proteasome.
Only low-resolution structural information,
obtained by electron microscopy, is available for
the 19S cap.
Location and roles of some constituent proteins
have been established.
 The outermost "lid" of the 19S cap is a ring of
  eight proteins.
 The innermost "base" of the 19S cap includes a
  ring of six members of the AAA family of
  These are chaperones that carry out ATP-
  dependent unfolding of proteins prior to their
  being threaded into the core complex.
 Isopeptidases in the 19S cap disassemble
  ubiquitin chains. Ubiquitins can then be re-used.
  At least one deubiquitylating enzyme is located
  between the lid & base regions of the 19S cap.
11S Regulatory cap:                   20 S Proteasome
a heptameric complex                    (yeast), with
of a protein PA28.                     11S Regulator
It allows small, non-
ubiquitinated proteins
& peptides to pass
into the core complex.
This does not require
ATP hydrolysis.
11S cap is dome-
shaped, with a wide                      two views
opening at each end.
                                               PDB 1FNT

Binding of the 11S cap alters conformation of N-
terminal domains of core complex a subunits,
opening a gate into the proteasome core.
                                        20 S Proteasome
There have been                            (yeast), with
many structural                          11S Regulator
studies of isolated                       (Trypanosome)
core complex with
19S or 11S cap.
Formation of mixed
complexes of
proteasome core
sandwiched between
19S & 11S caps has                          two views
been shown by EM.
                                                  PDB 1FNT

In vivo a 19S cap may recognize, de-ubiquitinate, unfold
& feed proteins into a core complex, while an 11S cap at
the other end may provide an exit path for peptide
The COP9 signalosome is a complex of 8 distinct
proteins homologous to lid proteins of the 19S
proteasome cap complex.
The COP9 complex has roles in regulating ubiquitin-
proteasome-mediated protein degradation.
A subunit of the COP9 signalosome (designated
Jab1) has metalloprotease activity.
Ubiquitin ligases (E3) with a "cullin" subunit having
a RING finger domain are modified by attachment of
a ubiquitin-like protein called Nedd8.
COP9-catalyzed de-neddylation (removal of the
Nedd protein) activates the E3 ligase and may
have a role in assembly of the functional E3
Quantification of
structural dynamic of
proteosome 20S by

Nature Vol 445, 8
February 2007, 618-622.
      Interaction of 11S activator with 20 S

Kd = 12 ± 10 M
Attachment of the ubiquitin peptide to
proteins targets them for proteolytic
degradation by a complex cellular
structure, the proteasome. The regulated
proteolysis of proteins by proteasomes
removes denatured, damaged or
improperly translated proteins from cells
and regulates the level of proteins like
cyclins or some transcription factors. E1
and E2 enzymes prepare ubiquitin chains
that are then attached to proteins by the E3
enzyme. The sequence of ubiquitin and
the basic structure and function of the
proteasome are highly conserved. The
core proteasome in man (20S proteasome)
consists of four rings each with 14
subunits stacked on top of each other that
are responsible for the proteolytic activity
of the proteasome. The PA700 regulatory
complex is stacked on the ends of the
cylindrical core to form a 26S proteasome.
Proteins that are tagged with ubiquitin are
recognized and bound by the regulatory
subunits, then unfolded in an ATP-
dependent manner, and inserted into the
core particle, where proteases degrade the
protein, releasing small peptides and
releasing the ubiquitin intact. The PA28
regulatory complex is alternative
regulatory complex that appears to play a
role in antigen processing for presentation
of peptides to immune cells in the MHC I

complex   .
                       Proteosome regulation

Tomado de Kraut et al., 2007. TRENDS in Cell Biol. 17: 419-421
                Controlled proteolysis in bacteria

The outer "unfolding"
chamber of bacterial
proteosome picks up a
protein from the
surrounding with the help of
its 'moving arms' and
passes it on to the inner
chamber for further
processing after unfolding.
Tomado de Science (1999) 286, 1881-1893
                     Regulated proteolysis

Tomado de Mogk et al., 2007. TRENDS in Cell Biol. 17: 165-172
In order to dis-assemble the protein chain, it is sometimes necessary
to unfold it prior to cleavage of the linkage between amino acids

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