Protein Degradation
Cellular functions of protein degradation
1. Elimination of misfolded and 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.
– If a damaged protein is not repaired, it is degraded in
specialized organelles such as the lysosome, and by
the ubiquitin/proteasome pathway.
Cellular functions of protein
degradation
2. Regulation of cellular metabolism
– Increase or decrease the number of enzyme
molecules and regulatory substances
3. The generation of active proteins
– The proteolytic cleavage of the precursor generates
an active enzyme – proteases, lysosome
– Ubiquitin and catalytic subunits of the proteasome
are also expressed as precursors that are
proteolytically processed to yield catalytically active
subunits.
Cellular functions of protein
degradation
4. The recycling of amino acids
• Generate free amino acids from short peptides that are
generated by the proteasome and other intracellular
proteases.
• 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.
General Principles
• Proteolysis
The degradation of a protein, usually by
hydrolysis at one or more of its peptide bonds.
Degradation
• Covalent modifications by nonenzymatic chemical
reactions
• Turnover of normal, unmodified proteins
–
–
–
–
Enzymatic hydrolysis, not chemical reactions
Use energy
Use the AAs for the synthesis of new proteins
Regulated during development, the cell cycle and in response
to changes in the environment
• Regulated degradation: Proteolytic degradation of
cyclin
– Cyclin activates a protein kinase, which regulates cell
metabolism (control cell division)
– Degradation of luxury proteins to survive temporary conditions
of starvation
Chemical Aging
• Oxidative modifications by free radicals
– Defense enzymes: SOD, catalase
• Hydrolysis of peptide bonds
–
–
–
–
Asp residue
Deamination of Asn residues
Oxidation of sulfur atoms of Cys and Met
Destruction of disulfide bonds at high pH and temp.
• Covalent modifications by non-enzymatic reactions
between protein amino groups and reducing sugars
– Maillard reaction
– Racemization
Chemical Modifications that Lead to Protein
Degradation
¾ The oxygen rich environment in which proteins exist tend
to produce a variety of chemical reactions in proteins.
¾ ROS react with nucleic acids, lipids, proteins and sugars.
¾ The oxidation of lipids, reducing sugars and amino acids
leads to the formation of carbonyls and carbonyl adducts
such as 4-hydroxy-2-nonenal (HNE).
¾ ROS are also responsible for deamidation, racemization
and isomerization of protein residues.
¾ The oxidatively modified proteins are not repaired and
must be removed - protein degradation.
Production of Reactive Oxygen
Species (ROS)
¾ During normal cellular respiration, oxygen is reduced to
water and highly reactive superoxide (O2 ).
O2 + 4H+ + 4 e-
2 H2O ( about 95% of the time)
2 O2
(about 5% of the time)
¾ These reactive oxygen species (superoxide) react with
nucleic acids, sugars, proteins and lipids - eventually leading
to protein degradation.
Cellular Defense Mechanisms to Prevent
ROS Build-up
¾ Due to the oxygen rich
environment in which
proteins exist, reactions with
ROS are unavoidable.
¾ Superoxide dismutase and
glutathione peroxidase are
natural antioxidants present
in organisms which
eliminate some ROS.
¾ Glutathione peroxidase
catalyzes the reduction of
peroxide by oxidizing
glutathione (GSH) to GSSG.
O
O-
2O2
glutathione
peroxidase
superoxide
dismutase
H2O2 + O2
GSH + H2O2
O
SH
GSSG
H2O + O2 + GSSG
O-
S
2
O
O
H
N
H
N
NH 3
O-
N
H
NH3
O
G SH
O-
N
H
O
G SSG
How Reactive Oxygen Species Lead to
Protein Degradation
‹
ROS can react directly with the protein or they can react with
sugars and lipids, generating products which then can react with the
protein.
‹ Within the protein, either the peptide bond or side chain is targeted
‹ Many of these reactions mediated by ROS result in the introduction
of carbonyl groups into the protein.
‹ This results in:
I) cleavage of protein to yield lower-molecular weight product
II) cross-linkage of protein to yield higher-molecular weight product
III) loss of catalytic and structural function by distorting its secondary
and tertiary structure
These modifications eventually result in the death of the protein
Lipid Peroxidation and Formation of 4hydroxy -2-nonenal (HNE)
¾ Lipid peroxidation is a complex series
of reactions resulting in the
fragmentation of polyunsaturated fats.
¾ One product of lipid peroxidation is 4hydroxy -2-nonenal, which is a highly
reactive alpha, beta unsaturated
aldehyde.
O
H2N
CH
C
O
H
N
CH
C
OH
o
HNE
o
O
HNE
CH2
SH
O
OH
H
H2N
CH
C
CH2
S
O
H
N
CH
C
O
H
HNE
Cys-Gly-
HNE-Cys-Gly-
¾ HNE reacts with nucleophilic side chains of nucleic acids and
proteins via a Michael addition, forming HNE-protein species.
¾ HNE irreversibly alkylates the protein.
¾ This introduces a carbonyl group which results in protein
degradation.
Protein modification via reaction with
reducing sugars
¾ Reducing sugars in the open chain
configuration, such as glucose,
react with amino groups on
proteins to yield Schiff bases.
¾ The Schiff base can oxidize to
release alpha-dicarbonyls or
undergo Amadori rearrangement to
yield Amadori products such as
ketoamine.
¾ This reaction is especially
prevalent when glucose levels are
high. The Amadori products
introduce carbonyl groups into the
protein, which disrupts its structure
and function.
N
CHO
H
HO
NH2
OH
H2O
H
H
OH
HO
H
OH
H
OH
H
OH
H
OH
CH2OH
Glucose
H
CH2OH
Schiff Base
NH
N
H
OH
HO
H
o
Amadori
rearrangement
H
OH
H
OH
HO
H
OH
H
OH
CH2OH
H
OH
Schiff Base
H
CH2OH
Ketoamine
Modified Proteins Which Are Not
Degraded
¾ Not all aberrant proteins are recognized by
degradation systems in the cells
¾ Modified proteins in eye lens are not recognized.
¾ Modified lens proteins accumulate over a
lifetime with deleterious effects to vision.
¾ Chemically modified lens proteins lead to the
formation of cataracts.
Protein Turnover in vivo
• Wide variations in rates
– Ornithine decarboxylase: 11 min half-life
– Eye lens proteins: no degradation
• Degradation of all the molecules of any particular
protein is random
• Posttranslational proteins
– Not reused metabolically after degradation
– Methyl-His: Only occur in actin and myosin, and thus used to
measure their degradation
– Hydroxyproline in urine: measurement for the degradation of
collagens
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.
Š Proteins with N-terminal Met, Ser, Ala, Thr, Val, or Gly have
half lives greater than 20 hours.
Š 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.
Factors that Determine the Rate of
Protein Degradation
•
•
•
•
•
•
•
•
•
Susceptibility to thermal unfolding
The absence of stabilizing ligands
Susceptibility to protease digestion in vitro
Susceptibility of its Cys, His, and Met residues to
oxidation
The presence of attached carbohydrates and
phosphate group
The net negative charge of the protein
The presence of a free a-amino group
Increasing size of the polypeptide chain
The flexibility of the folded conformation as measured
by hydrogen exchange
Proteases Involved in Protein
Turnover
• Calpain: Calcium-activated proteases
– Calpain I: require mM calcium
– Calpain II: require uM calcium
• Lysozomes
– Lysosomes are compartments inside the cell, roughly
spherical and bound by a single membrane.
– They contain proteases known as CATHEPSINS.
– These hydrolytic enzymes degrade proteins and other
substances taken in by endocytosis.
– Lysosomes have low internal pH, hydrolases prefer acidic
medium.
Ubiquitin/Proteasome Pathway
• The major non-lysosomal process responsible for the
breakdown of most short and long-lived proteins in
mammalian cells.
• In skeletal muscle, the system is responsible for the
breakdown of the major contractile proteins, actin and
myosins.
• Controls various major biological events:
–
–
–
–
–
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cell cycle progression,
oncogenesis,
transcriptional control,
development and differentiation,
signal transduction,
receptor down-regulation and antigen processing
Ubiquitin-Mediated Pathway
• The best defined mechanisms of intracellular protein
degradation
• Ubiquitin is abundant in eucaryotes and is highly
conserved
• A small protein of 76 residues with a stable compact
globular conformation (4 b-sheets and a a-helix)
• The 3 C-terminal residues, -Arg-Gly-Gly, are flexible and
extend into the solvent
• Found throughout the cell and can exist either in free form
or as part of a complex with other proteins.
• In complex form, Ub is attached (conjugated) to proteins
through a covalent bond between the glycine at the Cterminal end of Ub and the side chains of lysine on the
proteins.
Ubiquitin-Mediated Pathway
• Ub functions to regulate protein turnover in a cell by closely
regulating the degradation of specific proteins.
• Ub functions in an ATP-dependent fashion.
• Ub itself does not degrade proteins.
• It serves only as a tag that marks proteins for degradation.
• The degradation itself is carried out by the 26S proteasome.
• Specific recognition of this signal, and degradation of the
tagged protein by the 26S proteasome.
• In short, proteins that are to be degraded are first tagged by
conjugating them with Ub and these tagged proteins are then
recognized and shuttled to the proteasome for degradation.
Ubiquitin and Degradation
Three proteins involved: E1, E2 and E3
• C-terminus of ubiquitin gets
adenylated
•
Rearrangement to intermolecular
thioester with a E1 (activation
enzyme)
•
Transfer of activierted ubiquitin
from E1 to E2 (ubiquitinconjugating enzyme) (thioester
bond)
•
Transfer form E2 via E3(ubiquitin
ligase) to target enzyme
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.
Ubiquitin-Mediated Pathway of Protein Degradation
Process
• Ubiqiutin is added to a Lysine
residue of target protein by
conjugating enzymes.
• A series of additional Ubiquitin
molecules is added.
• A multiubiquitin chain is formed.
destruction Primary structure of a protein
targeted for degradation
box
COO−
H2 N
chain of
ubiquitins
• This chain is recognized by a receptor protein in the proteasome must be attached to the Protein for it to be degraded.
• The complex binds to sites on the regulatory particle which recognizes
ubiquitin.
• Unfolded by ATPases using ATP.
• Unfolded protein is translocated into central cavity of core particle.
• Active sites on inner surface break specific peptide bonds of the chain.
• It produces a set of peptides about 8 amino acids long.
• These leave the core and are released for further use.
Proteasome
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•
•
•
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Large complex protein in the
cytosol
The Proteasome contains:
Core Particle: Contains 2 copies
of each of 14 different
polypeptides.
Regulatory Particle: One at each
end of the core particle, each
made of 14 different proteins
same of the subunits have sites
that recognize Ubiquitin.
Responsible for degrading
proteins that have been marked
for destruction by ubiquitination
or other means.
Schematic representation of the
eukaryotic
•
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.
Ubiquitin/Proteasome Pathway Summary