L9- Protein Homeostasis 1

what is homeostasis

Homeostasis is the property of an open system, especially living organisms, to regulate its internal environment to maintain a stable, constant condition by means of multiple feedback controls, regardless of the external conditions.

• controlled by feedback loops

what is proteostasis

the maintenance of the correct amounts of functional proteins and organelles in and outside the cell

what is the proteostasis network (PN)

serves to ensure that correctly folded proteins are generated at the right time and cellular location. Additionally, it prevents proteins from misfolding and aggregating and ensures that superfluous and misfolded protein species are removed

what are the key effectors of the PN

molecular chaperones, which ensure proper protein folding and conformational maintenance and cooperate with the degradation machinery

how are protein levels maintained

protein turnover

• Need to maintain the correct number of each type of protein in the cell.

give an example of protein regulation by negative feedback

p53 tumour suppressor

• transcription factor

• Involved in cell cycle arrest, DNA repair and apoptosis

• Normally kept at low basal level- Otherwise stops cells from proliferating  and completing normal functions

how is P53 kept low at basal level

maintained by negative feedback loop with mdm2

• Mdm2 is an E3 ligase. Mdm2 binds to p53, and targets p53 for degradation by the proteasome

• Only when p53 detects damage is it phosphorylated and prevented from target by mdm2 

• High levels of p53 lead to an increase in Mdm2.

• High levels of Mdm2 leads to a decrease in p53.

So, we have a negative feedback loop.

how can p53 negative feedbacl loop be visualised

fluorescence microscopy

In nucleus of one single cell 

• Wave of one protein then the other 

• When one activated the other inactivated 

what are the other aspects of proteostasis apart from protein amount

From synthesis, folding and acquisition of naïve state of protein is controlled 

Steps after that- eg misfolding, can return to normal state 

Aggregation and degradation 

• A lot controlled by chaperones  

what is the life cycle of a protein

1. Synthesis

2. Folding

3. Transport

4. Modifications

5. Function

6. Degradation

what is transcription

• Transcription is regulated by transcription factors which can either promote or inhibit transcription

• Aged and senescent tissues display overall reduced transcription, while the fraction of transcripts with sequence errors and splicing errors increase

what are 3 age-related changes in transcritption

1. Stalling RNA polymerase as result of increased DNA damage  

2. truncated mRNA- When RNA polymerase binds in gene itself instead of promoter

3. accelerated RNAPII- RNA pol works faster- makes errors and mutations that affect quality of proteome downstream 

what is the relationship between translation and ageing

• Dysregulation in signalling pathways (e.g. mTOR) will lead to changes in translation

• Activity of some translation factors declines with age

how can reduced translation with age be visualised

Toxin that binds to new proteins from ribosome- tells how many copies being translated 

• Can see the rate of overall protein translation 

• less protein labelled in old cells- rate of translation becomes slower

describe protein folding

for small proteins, happens naturally as exit the ribosome

• for larger proteins- need help from chaperones

1. Chaperone-independent folding. The protein folds as it is synthesized on the ribosome (green)

2. Hsp70-assisted protein folding. Hsp70 (gray) binds to nascent polypeptide chains as they are synthesized and assists their folding.

3. Folding assisted by Hsp70 and chaperonin complexes

how are proteins transported from the endoplasmic reticulum

Chaperones prevent misfolded or incompletely assembled proteins from exiting the ER

what is the unfolded protein response (UPR)

Caused by an increase in misfolded proteins in the ER or other organelles

• A failed response may lead to apoptosis and contribute to ageing

what are protein modifications

Many reversible covalent modifications affect protein function e.g.

• Phosphorylation

• Acetylation

• Glycosylation

• Ubiquitination

Also undesired modifications due to reactive oxygen or reactive nitrogen species

what are the 3 major classes of oxidative damage

1. conformational

2. covalent

Both kinds of damage can drastically affect protein function.

what is conformational protein damage

Conformational damage refers to unfolding of the protein

• caused by heating, attack by free radicals, chemicals, pH changes, etc

what is covalent protein damage

Covalent damage is a chemical change in the amino acids that make up the protein

• E.g. oxidation, isomerization, carbonylation or formation of isoaspartate.

• changes may occur spontaneously or may be induced and/or accelerated by environmental factors.

what are advanced glycation end-products (AGEs)

Type of protein modification 

• Advanced glycation end-products (AGEs) are harmful protein modifications formed when sugars react non-enzymatically with proteins, lipids, or DNA.

• Glycation is promoted by high glucose levels, unhealthy diet, ageing, and diseases such as diabetes.

• Glucose chemically reacts with proteins to form:

  • a Schiff base

  • then an Amadori product

  • which can develop into irreversible AGEs

• These modified proteins cannot function normally.

• AGEs cause cross-linking and clumping of proteins inside and outside cells.

• AGE accumulation in blood vessels and tissues contributes to:

  • plaques

  • vessel stiffening

  • cardiovascular disease

  • age-related tissue dysfunction

what does repair of protein damage depend upon, what if it is unsuccessful

the ability to recognise a change in a protein as abnormal,

• a means of reversing the change.

If repair is unsuccessful damaged protein needs to be removed to prevent:

• cellular dysfunction

• protein aggregation

what molecules are key in repairing conformational damage

Chaperones are key to repairing misfolding or removing the protein if acn’t be repaired 

describe repair/removal of different covalent damage

what is another name for molecular chaperones

Many chaperones are also known as heat shock proteins e.g. Hsp90. (Heat shock triggers misfolding in the cell)

• Present in cells under normal conditions.

• Upregulated after stress.

Many functions, e.g.

• Folding/refolding

• Prevent protein aggregation

• Assist in targeting proteins for degradation

give 5 examples of chaperones and their function

Classified based on protein size, and increasing in complexity

what regulates the transcription of heat shock genes

heat shock transcription factor 1 (HSF1)

Normally present as inactive monomers.

• In stressed cells HSF1 forms homotrimers.

• Trimers are activated by phosphorylation and translocate to the nucleus where they bind to the heat shock element (HSE).

• This results in synthesis of heat shock proteins (known as the “heat shock response” but stress does not have to be thermal).

what is the heat shock response cycle

1. Normally HSF1 as monomer in cytoplasm as bound to chaperones in abundance, as not enough misfolded proteins in cell 

2. If misfolded proteins detected, chaperones sequestered to deal with them 

3. With additional signalling, HSF1 escapes and goes to the nucleus to form trimeric assembly with additional co factors bind to DNA and synthesize a number of needed chaperones  

4. Increased chaperone level slows down activation as can trap more monomers of HSF1 

5. Trimeric form of HSF1 is recycled to monomeric state or degraded by proteolytic systems

how do chaperones prevent protein aggregation

• Misfolded proteins expose hydrophobic regions which tend to “stick” together

• Chaperones bind to hydrophobic region to prevent misfolding protein “sticking” together

how are chaperones involved in protein degradation and signal transduction

Chaperone-mediated autophagy

• target some proteins for degradation via ubiquitin-proteasome system

• Hsp70 and Hsp90 interact with many

different signalling proteins:

• Nuclear hormone receptors

• Protein kinases

• Cell cycle regulators

• Cell death regulators

how does Hsp90 regulate glucocorticoid receptor (GR) signalling

1. Hsp90 binds the Glucocorticoid receptor (GR) in the cytoplasm and keeps it in an inactive but stable state.

2. GR signalling only occurs when chaperone exchange/remodelling permits receptor activation.

3. When a glucocorticoid hormone binds GR, the chaperone complex is rearranged/displaced.

4. The GR complex is transported to the nucleus by the dynein motor system.

5. In the nucleus, the chaperone complex must disassemble so GR can function as a transcription factor (TF).

6. Activated GR then binds DNA and regulates gene transcription.

how do chaperones change with ageing

Decline in the heat shock response with age

• Possibly due to decline in HSF1 transcriptional response with age.

• Overexpression of HSF1 and HSPs has been shown to extend lifespan in C. elegans and D. melanogaster.

• Cells from human centenarians display preserved upregulation of HSPs during stress

what happens to HSF1 transcriptional response with age

it declines

Evidence of downregulation 

• After exercise  

what does overexpression of HSF1 in C. elegans cause

extended lifespan

what happens to HSP in human centenarians

Cells from human centenarians display preserved upregulation of HSPs during stress

how does proteostasis decline with age

what is the chaperone overload hypothesis

Based on narrow understanding  

An increase in misfolded proteins:

• increased oxidation, protein damage, impaired protein degradation

A decrease in available chaperones:

• chaperone damage, impaired synthesis, trapped substrates

causes:

Defects in:

• signal transduction, protein transport, immune function, cellular organization