module 6 mol bio

major post-translational modifications

acetylation, methylation, phosphorylation, ubiquitination

protein modifications can control

stability, activity, protein-protein interactions, localization, specificity, function (essentially everything)

kinase activity is controlled in

level and specificity over cell cycle phases

value of changing kinases/regulators over the cell cycle

target different substrates for kinase activity (establishing ORI, etc.), proceeding into mitosis after replicatoin, proceeding with chromosome segregation, making sure previous steps are inactivated appropriately until next cell cycle

Wee1

adds inhibitory phosphate

Cdc25

phosphatase that removes inhibitory phosphate

G1

cyclin D + Cdks 4,6

transition from G1 to S

cyclin E + Cdk 2

S phase

cyclin A + Cdk2

G2 to M

cyclin B + Cdk1

E1 (2 types)

ATP-dependent form of activation —> adds Ub to cysteine residue of E1

E2 (38 types)

Ub transferred from E1 to E2 —> start to see some specificity

E3 (~ 700 types)

RING and HECT pathways to get Ub to target

RING E3

recruits E2 and transfers Ub from E2 to substrate

HECT E3

recruits E2 and puts Ub on itself and then transfers to substrate

forward and reverse rxns for protein degradation

forward: Ub goes forward through E3

reverse: DUB protease can take it back

cyclins

main regulators of CDKs

cyclins can change within each step of a cell cycle

allows for difference in regulation and PPI

G1/S checkpoint

enough NTPs for DNA recognition?

G2/M checkpoint

was replication carried out correctly or do we have any defects? also checks for size

metaphase/anaphase checkpoint

are chromosomes ready to segregate?

value of changing kinases/regulators over cell cycle

each part of cell cycle targets diff substrates for kinase activity —> kinase that activates something (like the ORI) can also prevent other kinases from doing the same since we only need one

CDK and cyclin relationshp

CDK need cyclin to allosterically bind and change conformation so binding site is open

• sometimes that’s not enough, also need another kinase to come in (like CAK) and phosphorylate binding site to activate

  • example of an “and gate”

ubiquitin

small 76 AA added to proteins (primarily to target them for degradation)

• C-terminal of Ub bonds to target protein’s N-terminal Met or Lys residue

complexity in Ub tagging

mono-Ub —> poly-Ub

• diff linkages are recognized by cell to mean diff things (homotypic linkage of K11 or K48 vs heterotypic linkage of K11 and K48)

UB synthesis

made with ribosomal proteins since cell requires a lot of these —> then DUB just cuts it off from ribosomal protein and both go on to do their own things

monomeric RING E3

perfect adaptor for one substrate —> super simple, E2 binds to one site and substrate to other and Ub is transferredmo

more complex E3

additional modular adaptors bind to substrates —> modular b/c you can switch out adaptor to bind to target diff subtrates

• idea is that you can keep the scaffold the same and allow for greater specificity by changing adaptor

SCF complex (Skp-Cul-Fbox) components:

• Cul = overall scaffold

• F-box = modular adaptor

• Skp = can bind different F-boxes which can target diff substrates

• Rbx1 = brings in E2

PTM intxn with E3 ligases

can promote or inhibit activity

• promote —> PTM to degron so E3 can recognize and Ub and recruit proteasome

• inhibit —> PTM causes degrons to no longer be recognized by E3 leading to protein accumulation

APC

degradation pathway in cell cycle that degrades S-phase cyclins and then M-phase cyclins —> helps ensure M-phase cyclins don’t accumulate prematurely for next cycle and cause the cell to enter mitosis too early

E3 complex for APC

activated by M-Cdk but also is the one that degrades M-phase cyclins and decreases amount of M-Cdks that are active

Cdc20 and Cdh1 —> part of APC

Cdc20 = early mitosis adaptor that starts cleanup by degrading securin (separates sister chromatids) and starts degrading M-cyclins by requires M-Cdk activity to be active

Cdh1 is later switched in to finish M-cyclin degradation as M-Cdk levels drop —> also degrades Cdc20 to prevent process from reversingPT

PTM feed forward mechanism

essentially, one modification helps reinforce the other one and the other modification helps reinforce the initial modification (goes in a cycle)

PRC1 and PRC2

• part of polycomb pathway that silences gene expression

• PRC1 results in monoubiquitination —> this then promotes PRC2 complex which results in trimethylation and gene silencing

• Readers proteins (RYBP) bind to these modifications and bring in writers of PRC1/PRC2 to adjacent nucleosomes

  • helps form long stretches of silenced chromatin instead of isolated patches

• Ultimately it’s a self-reinforcing cycle since PRC1 promotes PRC2 and PRC2 promotes PRC1

situations where Ub can promote situational degradation

1) presence of a degron that’s normally found in a hydrophobic region

2) ribosome stalling

Ub and ribosome stalling pathway

ribosome stalling = cell doesn’t know why but there’s a good chance the protein is only half translated so the answer is to degrade no matter what

• ribosome splits apart

• Listerin binds to large ribosomal subunit and ubiquitinates

• proteasome recruited and degrades polypeptide

any E3 has the ability to degrade a protein in split-open ribosome

pathways for degradation of stalled ribosomes

1) listerin pathway —> NEMF adds poly alanine t-RNAs to expose lysine residue on nascent polypeptide chain, E2 enzyme transfers Ub to lysine and listerin is recruited, degradation

2) if listerin is not working, another E3 will recognize the poly-alanine and result in degradation

Cereblon

Cul4 adaptor that the substrate will bind to

• Cul4 is an E3 that binds E2 and and cereblon/substrate to transfer Ub

  • leads to substrate degradaiton and limb development

thalidomide

inhibit cereblon and changes the substrates it binds to

• new substrates are usually important transcription factors/regulators

  • once Ub is transferred to them instead, it will degrade them and cause cancer/cell deathp

power of thalidomide

we can use it to target specific substrates for degradation

• structure: has cereblon binding site and substrate (that we can change) binding

aka PROTACs

STARR-seq

a technique to find functional enhancers

• essentially, you put an enhancer in as part of the RNA so that it would only be transcribed if the gene itself is active

• this represents a selection because you would only see what works and that level of sequencing gives you how well the enhancer works

proven to work pretty well because past assays have lined up with previously determined enhancer sites