Molecular Biochemistry II

Protein Degradation

Copyright 2000-2008 by Joyce J. Diwan.

All rights reserved.
There are several classes of
proteolytic enzymes.

Serine proteases include digestive

enzymes trypsin, chymotrypsin, & elastase.
Different serine proteases differ in substrate specificity.
For example:
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.
During catalysis, there is nucleophilic attack of the
hydroxyl O of a serine residue of the protease on the
carbonyl C of the peptide bond that is to be cleaved.
An acyl-enzyme intermediate is transiently formed.
In this diagram a small peptide is shown being cleaved,
while the usual substrate would be a larger polypeptide.
Hydrolysis of the ester linkage yields the second peptide
product.
 PDB 3BTK

Asp102
The active site in each
His57
serine protease includes
a serine residue, a Ser195
histidine residue, & an
aspartate residue.
Catalytic residues in trypsin

During attack of the serine hydroxyl oxygen, a proton is

transferred from the serine hydroxyl to the imidazole
ring of the histidine, as the adjacent aspartate carboxyl is
H-bonded to the histidine.
Aspartate proteases include
the digestive enzyme pepsin
Some proteases found in lysosomes
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 cells
one lysosomal protease.
Some MMPs (e.g., collagenase) are involved in
degradation of extracellular matrix during tissue
remodeling.
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.
 PDB 1YME

A zinc-binding motif at
the active site of a
metalloprotease includes
two His residues whose zinc
imidazole side-chains are
ligands to the Zn++. water oxygen
Colors in Carboxypeptidase
image at right: Zn, N, O.
Carboxypeptidase

During catalysis, the Zn++ promotes nucleophilic attack

on the carbonyl carbon by the oxygen atom of a water
molecule at the active site.
An active site base (Glu in Carboxypeptidase) facilitates
this reaction by extracting H+ from the attacking H2O.
Cysteine proteases have a
catalytic mechanism that involves
a cysteine sulfhydryl group.
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 specificities.
Caspases are cysteine proteases involved in activation &
implementation of apoptosis (programmed cell death).
Caspases get their name from the fact that they cleave on
the carboxyl side of an aspartate residue.
Calpains are Ca++-activated cysteine proteases that
cleave intracellular proteins involved in cell motility &
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 dissociation of an
inhibitory protein.
Activation may occur after a protease is delivered to a
particular cell compartment or the extracellular milieu.
Caspases involved in initiation of apoptosis are
activated by interaction with large complexes of
scaffolding & activating proteins called apoptosomes.
See diagram of apoptosome in a Univ. London website.
Protease Inhibitors:
Most protease inhibitors are proteins with domains
that enter or block a protease active site to prevent
substrate access.
 IAPs are proteins that block apoptosis by binding to
& inhibiting caspases.
The apoptosis-stimulating protein Smac antagonizes
the effect of IAPs on caspases.
TIMPs are inhibitors of metalloproteases that 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 space.
Cystatins protect cells against cathepsins that may
escape from lysosomes.
 Serpins use a unique suicide mechanism to inhibit serine
or cysteine proteases.
A large conformational change in the serpin
accompanies cleavage of its substrate loop.
This leads to disordering of the protease active site,
preventing completion of the reaction.
The serpin remains covalently linked to the protease
as an acyl-enzyme intermediate.
Movie depicting the conformational changes.
(University of Cambridge website)
Serpins are widely distributed within & outside of cells,
and have diverse roles, including regulation of blood
clotting, fibrin cleavage, & inhibition of apoptosis.
Lysosomes contain
a large variety of low Lumen
hydrolytic enzymes internal contains
that degrade proteins & pH hydrolytic
enzymes.
other substances taken
in by endocytosis. Vacuolar ATPase
Materials taken into a H+
ATP ADP + Pi
cell by inward budding
Lysosome
of vesicles from the
plasma membrane may be processed first in an endosomal
compartment and then delivered into the lumen of a lysosome
by fusion of a transport vesicle.
Solute transporters embedded in the lysosomal membrane
catalyze exit of products of lysosomal digestion (e.g., amino
acids, sugars, cholesterol) to the cytosol.
Lysosomes have a
low internal pH due to low Lumen
vacuolar ATPase, a H+ internal contains
pH hydrolytic
pump homologous to enzymes.
mitochondrial F1Fo
ATPase. Vacuolar ATPase
All intra-lysosomal ATP H+
ADP + Pi
hydrolases exhibit Lysosome
acidic pH optima.
Lysosomal proteases include many cathepsins (cysteine
proteases), some aspartate proteases & one zinc protease.
Activation of lysosomal proteases by cleavage 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
(lysosome)

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 macromolecules.
Genetic studies in yeast have identified unique
proteins involved in autophagosome formation.
Protein turnover; selective degradation/cleavage
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 having domains rich in Pro (P), Glu (E), Ser
(S), Thr (T), are more rapidly degraded than other proteins.
 Most autophagy is not a mechanism for selective
degradation of individual macromolecules.
However, cytosolic proteins that include the sequence
KFERQ may be selectively taken up by lysosomes in a
process called chaperone-mediated autophagy.
This process, which is stimulated under conditions of
nutritional or oxidative stress, involves interaction of
proteins to be degraded with:
Cytosolic chaperones that unfold the proteins.
A lysosomal membrane receptor (LAMP-2A) that
may provide a pathway across the membrane.
Chaperones in the lysosomal lumen that may assist
with translocation across the membrane.
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 endoplasmic reticulum membranes.
Activation of SREBP
involves its translocation
to golgi membranes
where sequential
cleavage by 2 proteases
releases to the cytosol a
domain with transcription
factor activity.
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 -helix of the SREBP
precursor within the transmembrane domain.
 Ubiquitin:
Proteins are usually tagged for
selective destruction in proteolytic
complexes called proteasomes by
covalent attachment of ubiquitin,
a small, compact, highly conserved
protein.
ubiquitin PDB 1TBE

However, some proteins may be degraded

by proteasomes without ubiquitination.
An isopeptide bond links the terminal
carboxyl of ubiquitin to the -amino
group of a lysine residue of a "condemned"
protein.
The joining of ubiquitin to a condemned protein is
ATP-dependent.
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 (E2).
 O

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

E3 (Ubiquitin-Protein Ligase)
O

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

A Ubiquitin-Protein Ligase (E3) then promotes transfer of

ubiquitin from E2 to the -amino group of a Lys residue of a
protein recognized by that E3, forming an isopeptide bond.
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

H2 N COO
chain of
ubiquitins

More ubiquitins are added to form a chain of ubiquitins.

The terminal carboxyl of each ubiquitin is linked to the
-amino group of a lysine residue (Lys29 or Lys48) of the
adjacent ubiquitin.
A chain of 4 or more ubiquitins targets proteins for
degradation in proteasomes. (Attachment of a single
ubiquitin to a protein has other regulatory effects.)
 destruction Primary structure of a protein
box targeted for degradation

H2 N COO
chain of
ubiquitins

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.
Ubiquitin Ligases (E3) mostly consist of two families:
Some Ubiquitin Ligases have a HECT domain
containing a conserved Cys residue that participates in
transfer of activated ubiquitin from E2 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.
Regulation of ubiquitination:
Some proteins regulate or facilitate ubiquitin conjugation.
Regulation by phosphorylation of some target proteins has
been observed.
E.g., phosphorylation of PEST domains activates
ubiquitination of proteins rich in the PEST amino acids.
Glycosylation of some PEST proteins with GlcNAc has the
opposite effect,
prolonging half-life
of these proteins.
GlcNAc attachment
increases with elevated
extracellular glucose,
suggesting a role as
nutrition sensor.
 A ubiquitin-like protein called Nedd8 may be attached
to ubiquitin ligases (E3) that have a "cullin" subunit
including a RING finger domain.
De-neddylation (removal of the Nedd8 protein),
catalyzed by a metalloprotease subunit of a complex
called the COP9 signalosome, activates the E3 ligases.
Some disease-causing viruses target host cell proteins
for degradation in the proteasome.
They either activate a host cell Ubiquitin Ligase to
ubiquitinate host proteins, or encode their own
Ubiquitin Ligase.
 20 S Proteasome
(yeast) closed state
Proteasomes:
Selective protein
degradation occurs
in the proteasome,
a large protein
complex in the
nucleus & cytosol
of eukaryotic cells. two views PDB 1JD2

The proteasome core complex, with a 20S sedimentation

coefficient, contains 2 each of 14 different polypeptides.
7 -type proteins form each of the two rings, at the
ends of the cylindrical structure.
7 -type proteins form each of the 2 central 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 subunits,
each having different substrate specificity.
1. One catalytic -subunit has a chymotrypsin-like
activity with preference for tyrosine or phenylalanine
at the P1 (peptide carbonyl) position.
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 position.
Different variants of the 3 catalytic subunits, with
different substrate specificity, are produced in cells of the
immune system that cleave proteins for antigen display.
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 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 surface.
Proteasomal degradation of particular proteins is an
essential mechanism by which cellular processes are
regulated, such as cell division, apoptosis, differentiation
and development.
E.g., progression through the cell cycle is controlled in
part through regulated degradation of proteins called
cyclins that activate cyclin-dependent kinases.
Several subunits of the proteasome are glycosylated with
GlcNAc when extracellular glucose is high, leading to
decreased intracellular proteolysis.
Conversely, under conditions of low nutrition, decreased
modification by GlcNAc leads to increased proteolysis.
Thus protein degradation is responsive to nutrition via
glycosylation of Ubiquitin Ligase & the proteasome itself.
Many inhibitors of proteasome protease activity are
known, some of which are natural products and others
experimentally produced.
E.g., TMCs are naturally occurring proteasome inhibitors.
They bind with high affinity adjacent to active site
threonines within the proteasome core complex.
TMCs have a heterocyclic ring structure derived from
modified amino acids.
Proteasome inhibitors cause cell cycle arrest and
induction of apoptosis (programmed cell death) when
added to rapidly dividing cells.
The potential use of proteasome inhibitors in treating
cancer is being investigated.
Proteasome evolution:
Proteasomes are considered very old.
They are in archaebacteria, but not most eubacteria,
although eubacteria have alternative protein-degrading
complexes.
The archaebacterial proteasome has just 2 proteins,
& , with 14 copies of each.
The eukaryotic proteasome has evolved 14 distinct
proteins that occupy unique positions within the
proteasome (7 -type & 7 -type).
 20 S Proteasome
Regulatory cap (yeast) closed state

complexes:
In crystal
structures of the
proteasome core

alone, there is no

apparent opening
two views PDB 1JD2
to the outside.
The ends of the cylindrical complex are blocked by
N-terminal domains of subunits that function as a gate.
Interaction with a cap complex 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 ATPases.
These are chaperones that carry out ATP-dependent
unfolding of proteins prior to their being threaded into
the core complex.
It is typical of AAA ATPases that they assemble into
hexameric rings
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.
A simpler archaebacterial cap complex called PAN
consists only of a hexameric ring of AAA ATPases,
comparable to the base of the 19S regulatory cap.
PAN, in the presence of ATP, was found to cause opening
of a gate at the end of the 20S proteasome through which
an unfolded protein could enter.
The base of the 19S cap is assumed to do the same,
although high resolution structural evidence is still lacking.
A high resolution structure has been achieved for a complex
of the 20S proteasome with an 11S regulatory cap.
The 11S cap is a heptameric complex of a protein PA28.
 20 S Proteasome
The 11S cap allows (yeast), with
11S Regulator
small, non-ubiquitinated
(Trypanosome)
proteins & peptides to
pass into the core
complex.
This does not require
ATP hydrolysis.
The 11S cap is dome- two views
shaped, with a wide
opening at each end. PDB 1FNT

Binding of the 11S cap alters conformation of N-terminal

domains of core complex subunits, opening a gate into
the proteasome core. For images see a website.
 20 S Proteasome
(yeast), with
There have been many
11S Regulator
structural studies of (Trypanosome)
isolated core complex
with 19S or 11S cap.
Formation of mixed
complexes of
proteasome core
sandwiched between two views
19S & 11S caps has
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 products.
See an animation.
 20 S Proteasome

11S-20S-11S
complex

Compare with Chime the yeast 20S proteasome core

complex, with and without the 11S regulatory cap.