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Cellular Protein turnover, the proteasome and autophagy BMS2021 - 2012

Dr Janet Macaulay Janet.macaulay@monash.edu

All Proteins have a limited lifetime within the cell


Examples: enzyme degradation

Some enzymes have lifetimes ranging from several days to months - Constitutive Enzymes Constitutive Enzymes - are present in constant concentration in the cell p - concentration does not change with changes in metabolic state or environment of cell - are usually enzymes of central metabolic pathways (glycolysis, citric acid cycle, etc.).
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Metabolism in Fasting and starvation Diabetes mellitus Cellular Protein turnover, the proteasome and autophagy Intracellular trafficking of proteins I Intracellular trafficking of proteins II Intracellular trafficking and disease 34. Revision
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Cellular Protein turnover, the proteasome and autophagy


Learning objectives:
At

All Proteins have a limited lifetime within the cell


Examples: enzyme degradation

the completion of this lecture students should be able to: Discuss the functions and importance of cellular protein turnover Discuss the signals for protein degradation Explain the molecular mechanisms of cellular protein turnover

Some enzymes have very short lifetimes of minutes to hours. They are destroyed by proteases. Their short lifetimes are the consequence of either particular amino acid sequences which are very susceptible to attack by proteolytic enzymes or ubiquitination which makes them targets for specific proteases. Many of these short lived enzymes are inducible/repressible enzymes.

References:

Alberts et al Molecular Biology of the cell 5th ed (2008) 39 396, 782-786 B ology ( 008) 391-396, 78 786 Nelson and Cox, Lehninger Principles of Biochemistry, 5th ed (2008) 1107-1109

Why do proteins need to turnover be destroyed?


All proteins have a limited lifetime within the cell Removal of abnormal proteins Denatured Damaged by oxidation etc. Mutated, therefore not in correct tertiary/quaternary structure No enzyme-catalysed repair mechanism for proteins Cellular adaptation to altered conditions
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All Proteins have a limited lifetime within the cell


Examples: enzyme degradation

Inducible/repressible enzymes Enzymes for which the rate of synthesis can be E f h h h f h b increased (induction) or decreased (repression) by specific substances (metabolites, hormones, growth factors, etc.).

Half-Lives of Some Proteins in Mammalian Cells Rapidly degraded


Signals for degradation: Ubiquination


Molecular mechanism of how ubiquitin is added to target proteins

Half-life (h)
0.5 1.3 2.0 2.6 2 6 5.0

c-myc, c-fos, p53 oncogenes RNA polymerase 1 -Hydroxyl--methylglutarl coenzyme A reductase Deoxythymidine kinase Phosphoenolpyruvate carboxykinase

Slowly degraded
Aldolase Cytochrome b5 Glyceraldehyde-3-phosphate dehydrogenase Lactate dehydrogenase (isoenzyme 5) Cytochrome c 118 122 130 144 150

Alberts et al 5th ed Fig 6.92

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How are proteins marked for destruction?


Signals for degradation: q Ubiquination Amino-terminal amino acid residues PEST regions Abnormal proteins

Signals for degradation: Ubiquination


Molecular mechanism of how ubiquitin is added to target proteins Step 1 Activated E1 bound ubiquitin formed

Alberts et al 5th ed Fig 6.92

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Signals for degradation: Ubiquination


The structure of ubiquitin

Signals for degradation: Ubiquination


Molecular mechanism of how ubiquitin is added to target proteins Step 2 The E2 enzymes act in conjunction with accessory E3 The E2-E3 complex is called ubiquitin ligase

Alberts et al 5th ed Fig 6.92A 9 Alberts et al 5th ed Fig 6.92 12

Signals for degradation: Ubiquination


Molecular mechanism of how ubiquitin is added to target proteins Step 3 E3 binds to specific degradation de radati n signals helping E2 to for a polyubiquitin chain

Signals for protein degradation:


Amino-terminal amino acid residues

Alberts et al 5th ed Fig 6.92

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Lehninger Principles of Biochemistry

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Signals for protein degradation:


PEST regions

PEST (Pro, Glu, Ser, Thr), internal regions 10-60aas, rich in PEST create presumably structural domains which are recognised by specific proteases

half-life: < 2 h
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Figure 6-94b Molecular Biology of the Cell ( Garland Science 2008)

How are proteins marked for destruction?


Signals for protein degradation: q Ubiquination Amino-terminal amino acid residues PEST regions Abnormal proteins

Signals for protein degradation:


Abnormal Proteins

Denatured Damaged by oxidation etc. Mutated, therefore not in correct tertiary/quaternary structure
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Signals for protein degradation:


Misfolded proteins

Digestion of Ubiquitin marked protein

Unfolded U f ld d protein response pathway

Chaperones, Ch folding helper enzymes

19 From: Dobson (2003) Nature 426, 884-890.

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Figure 6-90 Molecular Biology of the Cell ( Garland Science 2008)

A giant machine for degrading ubiquitinylated proteins

The proteasome.

The proteasome resides in the cytosol and nucleus. An ATP dependent protease It is completely distinct from lysosomal systems for protein Alberts et al 5th ed degradation.

Fig 6.89 20
Figure 6-91b Molecular Biology of the Cell ( Garland Science 2008)

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Protein turnover in lysosomes

Proteasome structure

Mr 106 ATP-dependent proteolytic system

From: Whitby et al.(2000) Nature 408, 115 - 120

Figure 13-42 Molecular Biology of the Cell ( Garland Science 2008)

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Protein turnover in: lysosomes


Lysosmes are:
Membrane bound organelles Filled with soluble acid hydrolases (hydrolytic (h d l ti enzymes) i l di proteases ) including t

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Figure 13-36 Molecular Biology of the Cell ( Garland Science 2008)

Protein turnover: Autophagy

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Figure 13-41 Molecular Biology of the Cell ( Garland Science 2008)

Protein turnover: Autophagy

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Figure 13-42 Molecular Biology of the Cell ( Garland Science 2008)