Chemical Modifications that Lead to Protein Degradation

1
Chemical Modifications that Lead to Protein
Degradation

• Biochemistry
• James Mignone
• December 6, 2002

2
Introduction

• The oxygen rich environment in which proteins
  exist tend to produce a variety of chemical
  reactions in proteins. Reactive oxygen species
  (ROS), which are products of cellular
  respiration, react with nucleic acids, lipids,
  proteins and sugars. 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). In addition to
  forming carbonyl groups, ROS are responsible for
  deamidation, racemization and isomerization of
  protein residues. These oxidatively modified
  proteins are not repaired and must be removed,
  this is known as protein degradation.

3
Production of Reactive Oxygen Species (ROS)

• During normal cellular respiration, oxygen is
  reduced to water and highly reactive superoxide (
  ).
• These reactive oxygen species (superoxide) react
  with nucleic acids, sugars, proteins and lipids -
  eventually leading to protein degradation.

4
Cellular Defense Mechanisms to Prevent ROS
Buildup.

• Due to the oxygen rich environment in which
  proteins exist, reactions with ROS are
  unavoidable.
• Superoxide dismutase and glutathione peroxidase
  are natural antioxidants present in organisms
  which eliminate some ROS.
• Glutathione peroxidase catalyzes the reduction of
  peroxide by oxidizing glutathione (GSH) to GSSG.

5
How Reactive Oxygen Species Lead to Protein
Degradation

• Reactive Oxygen Species 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
  sidechain 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

6
Oxidation of Proteins via Formation of Carbonyl
Adduct Products Lipid Peroxidation and Formation
of 4-hydroxy -2-nonenal (HNE)

• Lipid peroxidation is a complex series of
  reactions resulting in the fragmentation of
  polyunsaturated fats.
• One product of lipid peroxidation is 4-hydroxy
  -2-nonenal, which is a highly reactive alpha,
  beta unsaturated aldehyde.
• HNE reacts with nucleophilic side chains of
  nucleic acids and proteins via a Michael
  addition, forming HNE-protein species.
• HNE irreversibly alkylates the protein. This
  introduces a carbonyl group which results in
  protein degradation.

7
Oxidation of Proteins via Formation of Carbonyl
Adduct Products Protein modification via
reaction with reducing sugars

• Reducing sugars in the open chain configuration,
  such as glucose, react with amino groups on
  proteins to yield Schiff bases.
• The Schiff base can oxidize to release
  alpha-dicarbonyls or undergo Amadori
  rearrangement to yield Amadori products such as
  ketoamine.
• 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.

8
Radical Mediated Cleavage of Peptide Bonds

• Instead of forming carbonyl adduct products, ROS
  can directly cleave and oxidize the peptide bond.
• Table 1 illustrates the four most common types of
  radical mediated cleavages and the corresponding
  products.
• Table 1

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Deamidation, Racemization and Isomerization of
Protein Residues

• Besides introducing carbonyl groups into the
  protein, ROS are also responsible for
  deamidation, racemization and isomerization of
  residues.
• Gln and Asn residues deamidate and racemize about
  their C alpha atoms to the D-isomers.
• Asymmetric side chains of Thr and Ile residues
  convert from the L-isomer to the D-isomer.
• Spontaneous prolyl cis-trans isomerization
  occurs.

10
Modified Proteins Which Are Not Degraded

• The previous slides dealt with chemical
  modifications which lead to protein degradation,
  but not all aberrant proteins are recognized by
  degradation systems in the cells.
• For example, modified proteins in eye lens are
  not recognized.
• Therefore, modified lens proteins accumulate over
  a lifetime with deleterious effects to vision.
• Chemically modified lens proteins lead to the
  formation of cataracts.

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Conclusion

• Reactive oxygen species are kept to a minimum
  concentration by an organisms natural supply of
  antioxidants. Since proteins exist in an oxygen
  rich environment, reactions with ROS are
  unavoidable. Carbonyl and carbonyl adducts are
  the result of ROS reacting with lipids, sugars
  and amino acids. In addition to oxidizing
  proteins, ROS are responsible for deamidating,
  racemizing and isomerizing residues. These
  chemical modifications result in protein
  cleavage, aggregation and loss of catalytic and
  structural function by distorting the proteins
  secondary and tertiary structure. Chemical
  modifications that significantly alter the
  structure of a protein usually lead to its
  degradation, because most cells have degradation
  mechanisms which recognize modified proteins.

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References

• Amarnath, V., Montine, T., Neely, M., Picklo, M.
  (2002). Carbonyl toxicology and alzheimers
  disease. Toxicology and Applied Pharmacology.
  184, 187-197.
• Creighton, T. (2002). Chemical aging. Proteins
  structure and molecular properties. 2nd edition,
  464-465.
• Levine, R. (2002). Carbonyl modified proteins in
  cellular regulation, aging, and disease. Free
  Radical Biology Medicine. 32, 790-796.
• Levine, R., Stadtman, E. (2001). Oxidative
  modification of proteins during aging.
  Experimental Gerontology. 36, 1495-1502.
• Squier, T. (2001). Oxidative stress and protein
  aggregation during biological aging. Experimental
  Gerontology. 36, 1539-1550.