L10- Protein Homeostasis 2

what are the 2 main protein degradation pathways

1. Proteasomal degradation and ubiquitination 

2. lysosome 

what are proteasomes

Proteasomes are large protein complexes- not organelles, as doesn't have a membrane 

• Located in the nucleus and cytoplasm- Possibly proteasomal subunits in the mitochondria 

• Work by proteolysis – a chemical reaction that breaks peptide bonds.

• Yields peptides about 7-8 amino acids long, which are then further degraded into amino acids.

• Regulates concentration of particular proteins and removes misfolded proteins.

what is the structure of the 20S proteasome

Like a barrel with a pore 

Enzymatic activity is localised in the centre 

Protein disassembled, unfolded and fed through 

• alpha-subunits maintain a gate through which substrates enter

• beta-subunits contain the protease active sites on interior

can proteasomes degrade aggregates

no

Cant degrade aggregated proteins but can prevent proteins from being aggregated by removing those with a tendency to be misfolded 

what is the structure of the 26S proteasome

• alpha-subunits of 20S proteasome bind to the 19S regulatory cap.

• The 19S cap contains ATPase active sites and ubiquitin binding sites.

• Substrates must be tagged by ubiquitin to be recognised

what allows for the proteasome to detect ubiquitinated proteins

• The Proteasome contains regulatory ends/subunits that provide specificity for recognising ubiquitinated proteins.

• These subunits can use ATP to bind ubiquitin chains attached to target proteins.

• The proteasome recognises ubiquitinated proteins, unfolds them using ATP, and feeds them through a central pore.

• The protein is then degraded into peptides inside the proteasome.

what does S refer to (eg in 26S proteasome)

S refers to the Svedberg sedimentation coefficient which is used to characterise the behaviour of a particle type in ultracentrifugation.

what is ubiquitin

• Small regulatory protein (8.5kDa)

Single ubiquitin molecules or chains of ubiquitin are added to substrate proteins

• Dependent in how chain is built- the signal will mean something different to the machinery 

Ubiquitination (also called Ubiquitylation)

what is the effect of ubiquitination on the substrate

1. Degradation

2. Change in cellular location

3. Change in protein activity

what are the 3 ubiquitin proteins

Ubiquitin-activating enzyme (E1 enzyme)

Ubiquitin-conjugating enzymes (E2 enzymes)

Ubiquitin ligases (E3 enzymes)

what is Ubiquitin-activating enzyme (E1 enzyme)

Only one type in mammalian cells

• It activates ubiquitin (ATP-dependent reaction)

what are Ubiquitin-conjugating enzymes (E2 enzymes)

Several enzymes

• They bind to activated ubiquitin

what are Ubiquitin ligases (E3 enzymes)

Many different enzymes e.g. Mdm2

• Each enzyme has specific substrate proteins

• They interact with E2 enzymes (E2 pick it up, relay it and pass it onto E3 )

how does ubiquitination occur

• E1 uses energy from ATP to attach ubiquitin and make bond with sulphide group of cysteine 

• As it interacts with E2, it passes the Ub onto cysteine of E2, reaction continues to E3 

• Ub passed on via isopeptide bond- using lysine on protein to make initial single Ub chain 

• Can be done with single Ub, or straight away with chain of Ub

what are deubiquitinating enzymes (DUBs)

Large group of proteases that cleave ubiquitin molecules

• The majority are cysteine proteases e.g:

• Ubiquitin-specific protease family (USPs)

• Ubiquitin C-terminal hydrolases (UCHs)

what are the 2 functions of DUBs

1. Reverse the action of ubiquitination- remove chain of ubiquitin or chop chains into individual ubiquitin 

2. Recycling ubiquitin back into cell

how does ubiquitination change with age

• Ubiquitin levels and ubiquitin enzymes remain relatively stable with age.

• However, ubiquitinated proteins accumulate with age, suggesting impaired proteasomal degradation.

• This may reduce the pool of free available ubiquitin because ubiquitin becomes trapped on undegraded proteins.

how does the proteasome change with age

• Some proteasomal components may decline with age

Proteasome activity decreases with age in many different tissues

• Indirect reference to potentially impaired proteosome (with age) 

  • If ubiquitin all attached to undegraded proteins- reduced ability to ubiquitinate other proteins  

what is the evidence that proteasome activity declines with age in human epidermis

levels of oxidised proteins increased with age

what happens to aggregated proteins containing ubiquitin in the ageing retina

increased % of patients with alpha-synuclein aggregates and those containing ubiquitin

• Implies that proteasomal degradation is less efficient with age  

Therefore see accumulation of proteins and protein aggregates 

• In peripheral tissues, skin and organs

what are the 3 types of autophagy

1. macroautophagy

2. microautophagy

3. chaperone-mediated autophagy

what is macroautophagy

Intracellularly- Lysosome serves to degrade proteins by autophagy 

• Can degrade proteins carbs, lipids, nucleotides etc 

• The lysosome is surrounded by a single membrane.

• Cellular material is enclosed within a double-membrane vesicle called an autophagosome.

• The autophagosome fuses with the lysosome to deliver its cargo for degradation.

• This allows cytoplasmic material to become accessible to lysosomal enzymes and be recycled by the cell.

what is microautophagy

Allows proteins to be taken in by invagination of membrane lysosome and then be degraded once budded in 

what is chaperone-mediated autophagy

chaperone system- allows proteins to be translocated to lysosomal membrane 

what is the role of macroautophagy and nutrient response pwathways

Nutrient sensing- everything in cell needs to be sensed- eg how much protein , amino acid etc to know if functions can occur like translation 

• Converge on mTOR complex – 2 key functions (autophagy or quality control)

autophagy-

1. Promote anabolic pathway- activated when enough nutrients 

2. starvation- switched off, activate catabolic pathway 

how is the autophagosome organelle unlike any other vesicle in the cell

• Formed de novo , borrows lipiss from ER 

• like a flattened balloon that stretches- double membrane 

• When fuses with lysosome, second membrane can fuse and pop and lysozymes can degrade the contents  

Discovered in budding yeast 

what are the 2 key functions of macroautophagy

• Converge on mTOR complex – 2 key functions 

1. autophagy- to feed cell from inside- provide nutrients- cascade with many proteins in process

2. quality control

how does microautophagy ensure quality control

through selective macroautophagy pathways

• Can pick up things that are undesirable  

• Broken or dangerous things- difunctional chromosomes and mitochondria etc 

• Can trigger apoptosis 

what is the relationship between macroautophagy and ageing

Hallmark of ageing 

• Fluorescence- lysosomes with undegraded material- though to be mitochondrial components 

how does suppressed basal mitophagy drive cellular ageing phenotypes

Select autophagy pathways may be effected  

Signal less with age so picked up less by autophagy machinery  

• See accumulation of mitochondria in ageing, but are less active  

• Don’t get moved when damage and start leaking intracellular mitochondrial components sending a signal that could lead to innate immune system response creating an inflammatory effect in cell and neighbouring cells 

how does chaperone-mediated autophagy (CMA) change with ageing

Process that becomes less efficient with ageing 

 

• Uses chaperones, help assist degradation 

• Signal means exposure of specific motif, so when unfolded motif can be recognised at lysosomal surface 

• Protein passed to receptor lamp-2a 

• Pore formed and can pass through 

• Chaperones help form pore and disassemble it 

 

• Reduced in ageing due to reduction of lamp-2a itself 

what happens to LAMP-2A in CMA with age

Decline in LAMP-2A receptors with age (due to increase in its degradation)- System also susceptible to clogging - Misfolded proteins can block the pore 

Mutant proteins bind to LAMP-2A receptors and block them e.g.

• α-synuclein, UCHL1 (Parkinson’s disease)

• Tau (Alzheimer’s disease)

Insufficient degradation can be caused directly by the misfolded proteins 

what happens regardless of if lysosomal or proteasome pathways become inhibited first

Both lysosomal and proteasomal pathways decline with age

• Regardless of which system becomes inhibited first:

• Lysosomes (from their burden of defective mitochondria and lipofuscin) or

• Proteasomes (from their burden of damaged proteins)

Both systems may become overwhelmed leading to a loss in protein homeostasis.

how is protein homeostasis altered with age

normal- Proteins degraded by both systems, mitochondria only by lysosomes 

aged- Not degraded efficiently- causing impairment of homeostasis 

what are some theories of disturbed protein homeostasis in ageing

1. Chaperone overload

2. Quality control of proteins depends on cross-talk between chaperones and proteolytic pathways. Failure of any part of the system leads to aggregation

3. Garbage catastrophe theory of ageing

what is the protein triage model for quality control

Failure in any part of the quality control system will lead to an increase in aggregation

what is the garbage catastrophe theory of ageing

More generic take on the theory that ageing is a build up of damage 

• Imbalance between oxidative damage and renewal of biological structures may lead to a progressive loss of functionally effective elements and accumulation of waste products with age.

but- Don’t know if it’s a result of ageing or a cause of ageing still 

How does cellular plasticity and protein homeostasis change with ageing or cell state?

• Young/healthy cells:

  • High capacity > load

  • Rely on fast, flexible mechanisms (e.g. chaperones)

  • Less need for last-minute/emergency responses

• Older or altered-state cells (e.g. differentiated, quiescent):

  • Reduced plasticity and efficiency

  • Loss of rapid-response mechanisms

  • Greater reliance on slower systems (e.g. autophagy)

  • More “last-ditch” responses to maintain homeostasis

what is the effect of protein aggregation on the proteostasis network

If not degraded/folded 

• Potentially may be assembled into aggregates  

• Happens increasingly in age related diseases 

what is the definition of a protein aggregate

Change in secondary/tertiary structure

• Poor solubility in aqueous or detergent solvents

• Aberrant sub-cellular or extracellular localisation

what are the features of a protein aggregate

Protein misfolds and becomes clumped together 

• Preceded by change in structure of protein 

• Can be inside or outtside cell 

• Triggered by hydrophobic residue- proteins likely to stick to each other 

what happens to hydrophobic regions of aggregates

they are internalised

can all proteins aggregate

Not all proteins aggregate prone and some more likely than others 

• Some basically pre destined to become aggregates 

what does the propensity of a protein to aggregate depend on

• The secondary structure

• Stability of the tertiary structure

• Degree of disorder (intrinsically disordered proteins)

how are aggregates formed

Once something is misfolded, causes aggregation of other things  

what is chaperones cannot remove aggregates

If process is inefficient, cause inclusion- hallmark of various diseases 

how are misfolded proteins propagated

aggregates can be transported around  

• Can toxify cell form within but can also spread 

what is the aggresome

• Coalescence of inclusion bodies by active retrograde transport of misfolded proteins along microtubules

• Protective vs damaging function

what is the function of the aggresome

Formation of aggresome within the cell is a protective mechanism 

• Try to contain aggregates together 

• Near nucleus as aggregates travel along microtubules 

• Forms a depot of unwanted things 

• Aggresomes are something that ultimately kills the cells 

• Can become so big that they trap organelles, can disrupt processes of the cell 

• Maybe suffocate the cell from inside  

describe the sequestration of cellular components with aggregates

both loss-of-function phenotype and gain-of-function toxicity can contribute to the detrimental effect of protein aggregates on cell function

what are some interventions that improve proteostasis and extend lifespan

If we upregulating selectively and meaningly 

• Could be selective to the degradation fo age related things 

1. HSP16 overexpression in c. elegans

2. ATG5 overexpression in mice

3. Rpn11 overexpression in drosophila