11 - Post-translational regulation

function of molecular chaperones

assist protein folding to lead to the correct (and most stable) form

how do Molecular chaperones work

• Chaperones repeatedly bind and release partially folded regions

• An exposed hydrophobic region can be bound by molecular chaperones to prevent aggregation until the protein has had a chance to fold correctly

Where should regions rich in hydrophobic amino acids mostly be found in a correctly folded protein?

towards the inside

Expression of molecular chaperones increases in response to heat. Why?

more heat = denature of proteins = need more help from molecular chaperones

Proteasomes

abundant sites for protein degradation and are found in the cytoplasm and nucleus

The protease activity is isolated inside a hollow cylinder

Why is protease activity isolated? Doesn’t that make it less efficient to access proteins?

specificity; you dont want to accidently cleave a properly functioning protein

“Unfoldase” enzymes

unfold target proteins and thread them into the cylinder

signals for degradation in the proteasome

• Polyubiquitin & misfolded regions are signals for degradation in the proteasome

• Proteins can be marked for destruction by covalent addition of a polyubiquitin chain

why it could be said that proteasome compete with molecular chaperones

• If chaperones fail to fold a protein, it’s targeted to the proteasome for degradation.

• Proteasome protects the cell from toxic aggregates.

• both systems act on the same pool of misfolded proteins, but with different outcomes:

  • Chaperones: "Let me try to fix it."

  • Proteasome: "Too far gone—destroy it."

 role of ubiquitin in targeting proteins for degradation

a small protein that can be covalently added to different sites on proteins

 role of  ubiquitin ligases in targeting proteins for degradation

combination of enzymes that covalently adds Ubiquitin

 identify possible mechanisms through which degradation process could be regulated

activation of a ubiquitin ligase

activation of a degradation signal

activation of a ubiquitin ligase

• Signals in the cell can activate a ubiquitin ligase complex (E2 and E3 shown here).

• Once activated, the ubiquitin ligase will bind to and ubiquitinate specific amino acid target sequences

• Proteins containing the ubiquitinated target amino acid sequence are then sent to the proteasome for degradation

activation of a degradation signal

• Signals in the cell result in creating a site that is recognized by a ubiquitin ligase

• Once the recognition site is created (ex. by phosphorylation or a change in conformation or complex dissociation) a ubiquitin ligase can target the protein for degradation

signal transduction

The signal is amplified and diversified (signaling branches to different pathways/ cell functions)

post-translational modification

chemical modification after translation that alters protein activity, stability, localization.

post-translational modification examples

importance of Intercellular signaling

allows coordination between different cells/tissues

Types of cell -to -cell communication

paracrine, autocrine, juxtracrine, synaptic and endocrine

Endocrine signaling

ex. hormones; travel long distances in the blood

Paracrine signaling

involves secreted factors with a limited range (signal within a localized region)

Juxtacrine signaling

contact dependant

is mediated by contact NOT secreted, diffusible factors

autocrine

If the cell producing the signal is the same type as the cell receiving the signal, then the signaling can also be said to be autocrine

intracellular receptors

signaling receptors are located inside the cell

roles for Nuclear receptors

are usually transcription factors. Ligand binding results in a change in control of transcription (example: glucocorticoid receptor).

May initially be in cytosol but translocate to nucleus after ligand binding

 describe roles for cell surface vs nuclear receptors

Cell surface receptors: bind extracellular ligands.

Nuclear receptors: bind ligands inside the cell (often transcription factors).

what effects cell signal

• The receptors expressed by a particular cell determine which signals it can receive

• The amount, duration, and combination of signals received together determine what effect a signal has on cell function

• The type of cell also influences the effect of receiving a particular signal

importance of signals

Some signals are required for the growth and survival of cells

Integrins

are transmembrane proteins that help cells adhere to the extracellular matrix (ECM)

Anoikis

a term for apoptosis that occurs in response to loss of adhesion (death due to lack of integrin signaling)

 evidence that integrin can influence cell function/survival

Integrins connect cells to extracellular matrix (ECM).

Transmit survival and differentiation signals.

Loss of adhesion → anoikis (cell death).

Morphogens

are signaling molecules that diffuse from a localized source and form a concentration gradient across a developing tissue.

how can concentration of a signaling molecule influence cellular signaling responses

The amount of signal that reaches a cell is shaped by the synthesis/source, transport, and degradation of the paracrine factor.

Different concentrations of morphogen can activate different sets of genes in responding cells.

Why might morphogens be useful for building tissues?

to coordinate forming distinct tissues and building a multicellular orginism

are Signaling responses fast or slow

both

Many cellular signaling responses occur through regulation of gene expression (transcription) This takes time as the signal must be transmitted to the nucleus, then impact transcription and then impact translation

The fastest signaling responses involve direct modification of existing proteins to rapidly alter cellular processes

are Signaling pathways positive or negative feedback

Signaling pathways can include positive & negative feedback

“Molecular switches” in signal transduction: GTP binding

GTPases have different activity depending on whether they are bound to GTP or GDP

Generally, the GTP-bound form is active (”on”) in signal transduction pathways

Hydrolysis of GTP creates GDP and turns signaling “off

what promote GTP hydrolysis

GTP-ase activating proteins (GAPs)

what stimulate release of GDP so that GTP can bind.

Guanine nucleotide exchange factors (GEFs)

Mutations impacting GTPase signaling

are common in cancer (ex. in RAS GTPases) and create oncogenes with uncontrolled signaling

What types of changes could promote aberrant increased signaling of a GTPase?

• deletion of gap

• a mutation that impaired or slowed down the ability of KRas to hydrolyze GTP

“Molecular switches” in signal transduction: Phosphorylation

Phosphorylation can change protein conformation, catalytic activity, and create/destroy binding sites for other interactions

Kinases add phosphate group from ATP to a particular amino acid.

Phosphatases remove the phosphate group.

Tyrosine kinases

phosphorylates tyrosine amino acids.

Serine/threonine kinases

phosphorylate serine and threonine amino acids

predict what types of mutations will produce oncogenes

• Ras mutation that prevents GTP hydrolysis → always active.
  
  

• Receptor tyrosine kinases mutated to be active without ligand.
  
  

• Overactive kinases → excessive cell division.