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

The oxygen rich environment in which proteins exist tend to produce a variety of chemical reactions in proteins.

ROS 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).

ROS are also responsible for deamidation, racemization and isomerization of protein residues.

The oxidatively modified proteins are not repaired and must be removed - protein degradation.

Production of Reactive Oxygen Species (ROS)

During normal cellular respiration, oxygen is reduced to During normal cellular respiration, oxygen is reduced to water and highly reactive superoxide ( ).water and highly reactive superoxide ( ).

These reactive oxygen species (superoxide) react with These reactive oxygen species (superoxide) react with nucleic acids, sugars, proteins and lipids nucleic acids, sugars, proteins and lipids -- eventually leading eventually leading to protein degradation.to protein degradation.

O2

O2 + 4H+ + 4 e- 2 H2O ( about 95% of the time)

(about 5% of the time)2 O2

Cellular Defense Mechanisms to Prevent ROS Build-up

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

H2O2 + O2 GSSG

superoxidedismutase

glutathioneperoxidase

2O2

GSH + H2O2 H2O + O2 + GSSG

NH3 NH

HN

O-

O-OO

O

SH

GSH

NH3 NH

HN

O-

O-OO

O

S

GSSG

2

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 productII) cross-linkage of protein to yield higher-molecular weight productIII) 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 4- hydroxy -2-nonenal (HNE)

Lipid peroxidation is a complex series Lipid peroxidation is a complex series of reactions resulting in the of reactions resulting in the fragmentation of polyunsaturated fats.fragmentation of polyunsaturated fats.One product of lipid peroxidation is 4One product of lipid peroxidation is 4--hydroxy hydroxy --22--nonenal, which is a highly nonenal, which is a highly reactive alpha, beta unsaturated reactive alpha, beta unsaturated aldehyde.aldehyde.

o

OH

HNE

H2N CH C

CH2

O

SH

HN CH C

H

O

O

Cys-Gly-

o

O H

H N E

H2N CH C

CH2

O

S

HN CH C

H

O

O

HNE HNE-Cys-Gly-

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.

Protein modification via reaction with reducing sugars

Reducing sugars in the open chain Reducing sugars in the open chain configuration, such as glucose, configuration, such as glucose, react with amino groups on react with amino groups on proteins to yield Schiff bases.proteins to yield Schiff bases.

The Schiff base can oxidize to The Schiff base can oxidize to release alpharelease alpha--dicarbonyls or dicarbonyls or undergo Amadori rearrangement to undergo Amadori rearrangement to yield Amadori products such as yield Amadori products such as ketoamine.ketoamine.

This reaction is especially This reaction is especially prevalent when glucose levels are prevalent when glucose levels are high. The Amadori products high. The Amadori products introduce carbonyl groups into the introduce carbonyl groups into the protein, which disrupts its structure protein, which disrupts its structure and function.and function.

NH2

CHOOHHHHOOHHOHH

CH2OH

H2O OHH

HHO

OHH

OHH

CH2OH

N

Schiff BaseGlucose

OHH

HHO

OHH

OHH

CH2OH

N

Schiff Base

OHH

HHO

OHH

OHH

CH2OH

oNH

Ketoamine

Amadorirearrangement

Modified Proteins Which Are Not Degraded

Not all aberrant proteins are recognized by degradation systems in the cells

Modified proteins in eye lens are not recognized..

Modified lens proteins accumulate over a lifetime with deleterious effects to vision.

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

•

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.

Protein turnover: selective degradation/ cleavage

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: – cell cycle progression,– oncogenesis, – transcriptional control, – development and differentiation, – signal transduction, – receptor down-regulation and antigen processing

• 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 C-

terminal end of Ub and the side chains of lysine on the proteins.

Ubiquitin-Mediated Pathway

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 (ubiquitin- conjugating 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.

• 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.

H2N COO−

destruction box

chain of ubiquitins

Primary structure of a protein targeted for degradation

Proteasome•

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