Lecture 8 Flashcards
what is the proteasome:
- where is it found
- what is its shape like and elements for
- how does it detect misfolded proteins
- The proteasome is an abundant protein complex found in the cytosol and
nucleus (~1% of cellular protein) - the proteasome is a hollow cylinder with a cap at each end and an active site in the core
–> Caps protect cellular proteins from degradation - Proteasome acts on proteins that have been marked for destruction by the addition of a small protein tag named ubiquitin
- Last class: the longer the time to fold, the more change of being degraded
what is the name of the system that adds the ubiquitin to mark protein for destruction?
what are the three enzymes used to do this?
- Ubiquitin is added to proteins by a ubiquitin-conjugating system made up of three enzymes
- E1: an ATP-dependent ubiquitin-activating enzyme is activated by binding to ubiquitin
- E2: ubiquitin-conjugating enzyme (E2 with thiol group) takes the ubiquitin from E1
- ubiquitin conjugated enzyme (E2) with ubiquitin attached exists as a complex with E3, a ubiquitin ligase that selects substrates
- The E2-E3 complex work together.
- the E3 enzyme in the E3-E2 complex binds to specific degradation sequences in substrates
- Ubiquitin is added to a lysine residue on the target protein (ubiquitin from e2 in the e2-e3 complex is transferred to the protein)
- This process is repeated to form a polyubiquitin chain
- Polyubiquitin chain is recognized by specific receptor in the proteasome for degradation
what are other functions ubiquitin additions can have? (4)
- 1, 3, many (1, Lys48, 1, Lys63) ubiquitins
- Ubiquitin modifications can have other functions
- Depends on number of ubiquitin molecules and type of linkage
1 ubiquitin added (monoubiquitylation):
- histone regulation
3 ubiquitin added (multiubiquitylation)
- endocytosis
many ubiquitin added on Lys48 on any green protein lysine:
- proteasomal degradation
many ubiquitin added on Lys63 on any green protein lysine:
- DNA repair
How is the destruction of protein by the ubiquitin ligase (E3) regulated by different substrates which bind to it? how is the destruction of the protein substrate also regulated?
- sometimes the E3 ubiquitin ligase cannot recognize the signal at default and must be changed
- the changes can be that the E3 attached to the E2 can be phosphorylated with ATP, allosterically binded by a ligand or to a protein subunit.
- when these changes are done, then the complex can now regulate protein function by destroying ubiquitin tagged misfolded proteins.
similarly,
- the protein which is misfolded can also not be recognized by the E3 for degradation.
- these proteins can be activated by phosphorylation and ATP, unmasked by a protein which was covering its active binding site, and perhaps create a detablizied the N-terminus, so that the E3 can recognize the protein and degrade it
Step 2 of how proteins undergo steps to become functional –> last lecture
Proteins are covalently modified with chemical
groups (e.g., sugars, phosphate)
can multiple modifications occur on the same protein?
- yes, Multiple modifications can occur on the same protein at the transactication N terminus, tetramerization domain, and c-terminal domain (not the dna binding domain)
- phosphorylation, acetylation, sumo, ubiquitin, methylation etc
- all of these covalent modifications conducts a different prominent function
post-translational regulation by PKA
- what is protein kinase A (PKA) and what small molecule affects PKA activation
- what are the 4 subunits of inactive PKA
- what does the binding of the small molecule to the two specific regions of the PKA?
- Protein Kinase A (PKA) mediated gene expression is a more complete example of post-translational regulation of gene expression.
- Numerous extracellular stimuli result in increased levels of the small molecule cyclic AMP (cAMP)
- cAMP Activates protein kinase A (PKA)
- Inactive PKA has:
- Two regulatory subunits
- Two catalytic subunits
- Binding of cAMP to the regulatory subunits causes a conformational change and release of the active catalytic subunits. inactive –> active
using the example of adrenaline as the ligand, explain how it affects glycogen pathways via cAMP and PKAs in the CYTOSOL
location: cytosol
* substrates that bind to PKA include enzymes involved in glycogen metabolism in skeletal muscle and liver
* Ligand = adrenaline (epinephrine)
* Response = to promote glucose release
how adrenaline activates PKA in the cytosol:
- adrenaline activates a GPCR
- GPCR activates alpha subunit of G protein (Gs)
- this activates adenylyl cyclase protein which creates cyclic AMP (cAMP) via ATP
- cAMP activates protein kinase A (PKA) by binding to the regulatory subunits –> activates catalytic subunits
- by phosphorylation, this can either:
–> inhibit glycogen synthesis
–> promote breakdown of glycogen
explain how active PKA can cause gene transcription and thus protein expression in the NUCLEUS
- activated PKA catalytic subunits translocate to the nucleus from the cytosol (last card)
- activated PKA catalytic subunits phosphorylate specific inactive substrate activator proteins (ex. CREB activator)
- phosphorylated activator binds to the cis regulatory sequence (ex. cAMP response element - CRE) upstream the DNA coding sequence and also to a coactivator (ex. CBP - CREB binding protein – helps activate gene transcription without binding to DNA sequence itself)
- binding of phosphorylated/active activator to the cis sequence causes gene transcription and thus translation and expression of protein (here binding of adrenaline which activates PKA, actually produces more glucose in the blood via transcription and the breakdown of glycogen – energy for fight or flight)
What are the two interactions that proteins are involved in
Proteins usually function in large multi-protein complexes composed of static and transient interactions
- Protein interactions lie at the heart of most biological processes
- Proteins interact with small molecules, nucleic acids, and/or other proteins
what is the interactome map and what is the terminology for a protein and interaction on the interactome map diagram
The interactome map is the complete collection of protein-protein interactions of an organism
on the map, each dot is a protein node, each line is an interaction edge
what is an example of protein interactions interactome map from the previous lesson
E2-E3 protein interaction can lead to so many functions, therefore, many interactions
what is “guilt by association” when analyzing unknown protein interactions
- if you discover a protein and you know what it is interacting proteins do, you have an idea on what the unknown protein does as well, but its not confirmed
thus guilt by association
