lecture 3.5 - protein turnover

What determines the concentration of a protein within a cell?

The balance between its rate of synthesis and its rate of degradation.

What are the two main pathways of cellular protein degradation?

Lysosomal degradation by acidic hydrolases and cytosolic degradation by proteasomes or other proteolytic enzymes.

What is the main function of lysosomes?

They are digestive organelles that degrade both intracellular and extracellular molecules using hydrolytic enzymes.

What is the structure of a lysosome?

A single-membrane-bound organelle containing numerous hydrolytic enzymes and an acidic interior.

How is the acidic environment of the lysosome maintained?

A vacuolar H+ pump transports protons into the lysosome, maintaining a pH of approximately 4.5.

Why is an acidic lysosomal pH important for protein degradation?

Lysosomal hydrolases function optimally at low pH, which also promotes protein unfolding and makes substrates easier to degrade.

Which hydrolytic enzymes are found in lysosomes?

Proteases, lipases, glycosidases, nucleases and amylases.

How are lysosomal enzymes targeted to lysosomes?

They are tagged with mannose-6-phosphate, which directs their transport from the secretory pathway to lysosomes.

What are the two main processes by which material enters lysosomes for degradation?

Heterophagy and autophagy.

What is heterophagy?

The degradation of material taken into a cell from its external environment, such as bacteria engulfed by phagocytosis.

How are engulfed bacteria degraded by heterophagy?

The bacterium is enclosed in a phagosome, which fuses with a lysosome so its hydrolytic enzymes can digest the contents.

What is autophagy?

The lysosomal degradation of a cell's own components, including damaged organelles and aggregated proteins.

What types of cellular material can be degraded by autophagy?

Damaged mitochondria, endoplasmic reticulum, aggregated proteins and other intracellular components.

Which conditions can stimulate autophagy?

Nutrient deprivation, low cellular energy, cellular damage, hormonal signalling and environmental stress.

How does nutrient deprivation stimulate autophagy?

A shortage of nutrients increases autophagy, allowing cellular components to be degraded and recycled.

How can cellular damage trigger autophagy?

Damaged or dysfunctional components, such as mitochondria, are targeted for lysosomal degradation.

How does ageing affect autophagy?

Autophagy tends to decline with age, contributing to the accumulation of damaged cellular material.

What are the main stages of autophagy?

Initiation of a phagophore, expansion into an autophagosome, fusion with a lysosome and degradation of the enclosed material.

What is a phagophore?

An isolation membrane that forms during the initiation of autophagy and expands around material targeted for degradation.

What is an autophagosome?

A double-membrane-bound vesicle that encloses cellular components destined for lysosomal degradation.

How does an autophagosome deliver its contents for degradation?

It fuses with a lysosome, exposing its enclosed cargo to lysosomal hydrolytic enzymes.

What is chaperone-mediated autophagy?

A pathway in which proteins are transported directly into lysosomes for degradation rather than being enclosed in an autophagosome.

What is autolysis?

The destruction of a cell by the release of lysosomal contents into its cytoplasm.

When can autolysis occur?

In injured cells or dying tissues when lysosomal enzymes are released into the cytoplasm.

How is autolysis associated with fetal development?

Incomplete removal of tissue between developing digits can result in webbed fingers or toes.

What is the proteasome?

A large multienzyme complex that degrades proteins into short peptides, particularly proteins targeted by ubiquitination.

Where are proteasomes located in eukaryotic cells?

In the cytosol and nucleus.

What is the function of the 20S proteasome core?

It contains the catalytic chamber in which proteins are degraded into short peptides.

How is the 20S proteasome core organised?

It contains four stacked rings: two outer alpha rings and two inner beta rings.

How many polypeptide subunits make up the 20S proteasome core?

28 subunits, consisting of two copies each of 14 different polypeptides.

What is the arrangement of alpha and beta subunits in the 20S proteasome?

Each outer ring contains seven alpha subunits, while each inner ring contains seven beta subunits.

What are the functions of the alpha and beta rings of the proteasome?

The alpha rings form the outer ends and regulate access, while catalytic beta subunits degrade proteins inside the central chamber.

Why does protein degradation occur inside the proteasome's central cavity?

The catalytic sites face the interior, allowing proteins entering the chamber to be degraded in a controlled environment.

Which amino acid is responsible for catalysis in proteasomal beta subunits?

A conserved N-terminal threonine residue.

Why are proteasomal beta subunits initially synthesised as inactive precursors?

Their catalytic threonines are initially blocked by N-terminal sequences that must be removed to activate the enzymes.

How are the catalytic beta subunits of the proteasome activated?

Their N-terminal propeptides are removed, exposing the catalytic threonine residues on the inner surface of the core.

What is the role of the 19S regulatory cap?

It recognises polyubiquitinated proteins, removes ubiquitin, unfolds substrates and allows their entry into the 20S core.

How is a polyubiquitinated protein degraded by the proteasome?

The 19S cap recognises the ubiquitin tag, removes ubiquitin and unfolds the protein before threading it into the 20S core for proteolysis.

Why must proteins be unfolded before entering the proteasome core?

Unfolding allows the polypeptide chain to pass through the narrow entrance into the catalytic chamber.

What happens to ubiquitin before a tagged protein enters the proteasome core?

Ubiquitin is removed by the regulatory cap before the substrate is threaded into the core.

What is the typical length of peptide fragments produced by the proteasome?

Approximately 7–9 amino acid residues.

What happens to peptide fragments after proteasomal degradation?

Other proteases break them down further into amino acids that can be reused for protein synthesis.

What are the three catalytic specificities of the proteasome's beta subunits?

Chymotrypsin-like, trypsin-like and post-glutamyl proteolytic activities.

Which amino acids are preferred by the chymotrypsin-like proteasomal activity?

Aromatic residues, particularly phenylalanine and tyrosine, at the P1 position.

Which amino acids are preferred by the trypsin-like proteasomal activity?

Positively charged residues, particularly lysine and arginine, at the P1 position.

Which amino acids are preferred by the post-glutamyl proteasomal activity?

Glutamate and other acidic amino acid residues at the P1 position.

What do the P1 and P1′ positions represent in proteolysis?

They are the amino acid residues immediately before and after the peptide bond being cleaved.

How does proteasomal degradation regulate the cell cycle?

Controlled degradation of cyclins changes their concentrations and allows progression through different stages of the cell cycle.

What are cyclins?

Regulatory proteins whose controlled degradation helps regulate progression through the cell cycle.

Why can proteasome inhibitors cause cell cycle arrest?

They prevent the degradation of cyclins and other regulatory proteins needed for normal cell cycle progression.

Why are proteasome inhibitors potentially useful as anticancer drugs?

They can cause cell cycle arrest and induce apoptosis in rapidly dividing cells.

How does high extracellular glucose affect proteasome activity?

High glucose promotes glycosylation of proteasome subunits with GlcNAc, decreasing intracellular proteolysis.

How does low extracellular glucose affect proteasome activity?

GlcNAc groups are removed from proteasome subunits, increasing proteolysis.

What is GlcNAc and how does it regulate the proteasome?

N-acetylglucosamine is a sugar modification that reduces proteasomal proteolysis when attached to certain proteasome subunits.

Why does proteasome regulation by glucose link protein degradation to metabolism?

Changes in glucose availability alter proteasome glycosylation and therefore the rate of intracellular protein breakdown.

What are proteolytic enzymes or proteinases?

Enzymes that hydrolyse peptide bonds, breaking proteins into shorter peptides and eventually amino acids.

How are proteases classified according to their catalytic mechanisms?

Serine, threonine, cysteine, aspartate, metalloproteases and glutamic acid proteases.

What determines the classification of a protease as a serine or cysteine protease?

The amino acid side chain responsible for the catalytic chemistry in its active site.

What determines which peptide bonds a protease preferentially cleaves?

The enzyme's specificity for amino acid side chains near the cleavage site.

Which amino acid residues does chymotrypsin preferentially recognise?

Aromatic residues, particularly phenylalanine and tyrosine.

Which amino acid residues does trypsin preferentially recognise?

Positively charged residues, particularly lysine and arginine.

Which amino acid residues does elastase preferentially recognise?

Small hydrophobic residues, particularly alanine.

What is the catalytic triad of serine proteases?

Aspartate, histidine and serine.

How does histidine activate serine during serine protease catalysis?

Histidine removes a proton from the serine hydroxyl group, making serine more reactive towards the peptide bond.

What is the role of serine in the catalytic triad?

Activated serine participates directly in the chemical reaction that cleaves the substrate peptide bond.

What is the overall biological purpose of complete protein degradation?

To remove unwanted proteins and recycle their constituent amino acids for synthesis of new proteins.