Protein turnover

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36 Terms

1

What is protein concentration a balance between?

Synthesis and degradation

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2

What are the 2 types of protein degradation?

  • Lysosomal

    • Proteases in an acidic organelle

  • Cytosolic

    • In proteasomes, multienzyme complexes in the cytoplasm after ubiquitination

    • Or by proteolytic enzymes (trypsin, chymotrypsin, pepsinā€¦)

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3

What are lysosomes?

Major digestive organelle for both cytosolic and extracellular molecules

Single membrane

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4

How do lysosomes work?

  • Acidification needed for proper activity. Low pH environment

  • Vacuolar H+ pump to keep pH at ~4.5 (helps unfold target proteins)

  • Most proteins will be unfolded ā€“ acidic

  • Primarily through hydrolytic activity ā€“ add water to amide bond to break it

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5

How do targets for degradation enter lysosomes?

Either heterophagy or autophagy

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6

What do lysosomes contain?

Contains many hydrolytic enzymes (hydrolases)

o Proteases, lipases, glycosidases, nucleases, amylase

o Can digest lipids, sugars, mRNA ect

Work optimally at low pH

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7

What targets proteins into lysosomes from ER?

Mannose-6-phosphate tag

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8

How are proteins to be degraded delivered to lysosomes?

By endocytosis

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9

What is heterophagy?

Digestion within a cell of a substance taken in by phagocytosis from the cell's environment. e.g. bacteria

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10

How does heterophagy work?

  • Receptor recognizes bacteria

  • Create invagination

  • Phagosome forms

  • Lysosome combines with phagosome to form phagolysosomes to degrade

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11

What is autophagy?

ā€¢ Degradation of cell's own components using lysosomes

ā€¢ Lysosomes fuse with vesicles and degrade contents

ā€¢ Stimulated by starvation

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12

How does autophagy work?

  1. Autophagosome forms

  2. Substrate sequestered

  3. Fuses with lysosome to for autolysosome

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13

What is autolysis?

ā€¢ Cell destroys itself by releases lysosome contents into cytoplasm

ā€¢ Occurs in injured cells or dying tissue

ā€¢ Webbed fingers or feet occur due to incomplete autolysis in a fetus

ā€¢ Sometimes when proteins donā€™t go into autolysis they become cancerous

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14

What is the proteasome?

Machine for degrading proteins located in the nucleus and cytosol of eukaryotic cells

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15

What is the structure of the core particle (20S proteasome)

  • 4 stacked rings, forming a cylinder

  • Each ring made of 7 polypeptides

  • 2 outer rings made of Ī±-subunits, and the inner two rings consist of Ī²-subunits, which are involved in the proteolytic activity.

  • The inner cavity of the proteasome contains the proteolytic sites, where protein degradation occurs.

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16

What are the beta subunits of the proteasome?

  • Proteasome hydrolases - threonine proteases

  • Conserved N-terminal threonine is involved in catalysis at each active site

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17

Why are the threonine proteases not active all the time?

have a sequence at the end that caps the OH of the N terminus

When removed, threonine proteases are active

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18

How are the beta subunits synthesised and activated?

  • Synthesised as inactive pre-proteins

  • Activated when N-terminal removed, making threonine the N terminal residue

  • Catalytic threonines are then exposed at the inner surface

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19

What do the threonine proteases do and then what happens?

  • Threonine proteases chops the protein substrate into 7-9 residue peptides

  • Another set of enzymes makes them 3 aa long

  • Another set chops them into single aa

  • Step process to make sure we donā€™t degrade the wrong protein or at the wrong time

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20

What happens to a ubiquitin tagged protein in a proteasome?

An ubiquitin-tagged protein is unfolded and de-ubiquitinated in the cap, then threaded through the core, where it is digested into peptides

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21

What is the regulatory particle (19S proteasome)

  • The regulatory particle is composed of additional subunits that control the entry of substrates into the proteasome.

  • It contains a lid that helps recognize polyubiquitinated proteins (those tagged for degradation) and a base that unfolds and translocates the protein substrate into the core for degradation.

  • Opens the 20S so a substrate can enter

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22

What is protease activity done by?

3 of the beta subunits - each with different specificities

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23

What are the specificities of the beta subunits?

ā€¢ One catalytic b subunit has a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 (peptide cabonyl) position.

ā€¢ One has a trypsin-like activity with preference for arginine or lysine at the P1 position.

ā€¢ One has a post-glutamyl activity with preference for glutamate or other acidic residue at the P1 position.

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24

How is progression through the cell cycle controlled?

Regulated degradation of cyclins

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25

How can proteasome inhibitors cause cell cycle arrest?

  • Stop cyclins being degraded

  • Cause cell cycle arrest and induction of apoptosis when added to rapidly dividing cells

  • Potential anti-cancer drug

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26

How is proteasome controlled by glucose?

When extracellular glucose is high, several subunits of the proteasome are glycosylated with GlcNAc (N-acetylglucosamine. Intracellular proteolysis then decreases.

Conversely: low extracellular glucose leads to removal of GlcNAc which increased proteolysis

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27

What is rhabdomyolysis?

o When we starve for a long time, so body starts eating at protein from muscles

o Releases toxins into the blood

o Can be reversed when start eating protein again

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28

What are proteinases?

Enzymes that break proteins into shorter fragments (peptides) and eventually into their component amino acids

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29

How are proteinases classified?

Classified by how they work:

o Serine proteases

o Threonine proteases

o Cysteine proteases

o Aspartate proteases

o Metalloproteases

o Glutamic acid proteases

where Serine etc. is the side chain in the active site that does the chemistry

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30

What is the preferred side chain (r group) of chymotrypsin?

Aromatic - Phe and tyr

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31

What is the preferred side chain (r group) of trypsin?

Positive charge - lys and arg

cleaves peptide bonds at the C-terminal side of lysine or arginine residues

Serine protease

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32

What is the preferred side chain (r group) of elastase?

Small hydrophobic eg ala

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33

How do proteinases work?

ā€¢ Different enzymes are specific for different side chains (R groups)

ā€¢ Can work in combination with metal

ā€¢ Add water and break a peptide bond

ā€¢ Usually not active on own, need triad

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34

How do serine proteases work?

  • Active site contains catalytic triad - serine, histidine and aspartate

  • To activate serine, histidine removes hydrogen so have a negative oxygen

  • Aspartate takes away hydrogen from histidine which becomes positive and takes the hydrogen away from serine

  • Attach the peptide bond and break it

  • Ultimately, proteins are chopped up into their amino acids, ready to be used again to make new proteins

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35

What does each member of the catalytic triad do?

  • Serine (Ser): The nucleophilic residue that attacks the peptide bond.

  • Histidine (His): Acts as a base, accepting a proton from serine, making serine a better nucleophile.

  • Aspartate (Asp): Stabilizes the positive charge on histidine, aiding in its role as a base.

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36

More detailed serine protease digestion

  1. Substrate Binding:

    • The substrate protein (the peptide or protein to be cleaved) binds to the enzyme's active site.

    • The scissile bond (the peptide bond to be cleaved) is positioned near the active site, and specific binding interactions help orient the substrate for catalysis.

  2. Nucleophilic Attack:

    • The serine hydroxyl group (OH), activated by the histidine residue, performs a nucleophilic attack on the carbonyl carbon of the scissile peptide bond.

    • This attack forms a tetrahedral intermediate, which is a temporary structure with four bonds around the carbonyl carbon.

  3. Formation of Acyl-Enzyme Intermediate:

    • The tetrahedral intermediate quickly collapses, leading to the breakage of the peptide bond and the formation of an acyl-enzyme complex. The leaving group is typically the amino portion of the substrate.

    • The carboxyl group of the cleaved peptide (the part after the scissile bond) is released as a free product.

  4. Water Attack:

    • A water molecule is then activated by the histidine residue, and the water molecule attacks the acyl-enzyme complex.

    • This step regenerates the active serine hydroxyl group and releases the remaining cleaved peptide as a product.

  5. Regeneration of Active Site:

    • After the hydrolysis of the acyl-enzyme intermediate, the active site is regenerated, ready to cleave another peptide bond.

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