ANAT 212 Midterm

• phosphorylation

  - addition of a phosphate group to a hydroxyl

  - kinases = add phosphate

  - phosphates = remove phosphate

  - serine and tyrosine

  - increase size and charge
• types of bonding/interactions for folding

  1. hydrophobic interactions

  - many, strong

  - form tertiary structure

  2. H-bonds

  - many, moderately strong

  - stabilize secondary structure

  3. Van der Waals

  - many, weak

  4. Ionic

  - few, very strong

  - only on outside

  5. disulfide

  - few, very strong

  - covalent
• what determines native state?

  primary structure
• chaperones


  - assist folding and prevent aggregation

  - expression is induced by heat shock response

  - ATP-dependent

  - 3 families

  1. chaperonins/HSP60 (cage)

  2. HSP70 (bind polypep)

  3. HSP90 (open/close at one end)
• heat shock response


  activated by unfolded cytosolic proteins caused by heat stress, oxidative damage, or proteasome inhibition
• HSF1

  - Heat Shock Factor 1

  - mediates heat shock response

  - DNA-binding, regulatory, and transcription activation domains

  - inactive = monomer; mimics an unfolded protein and is therefore bound by HSP90

  - active = trimer; can recognize HSE (heat shock element) promoters; only is activated when there are unfolded proteins circulating to compete with HSF1 for HSP90 binding
• methylation


  add size and some level of hydrophobicity
• HSP70


  - ATP-bound = no substrate peptide bonding; substrate-binding domain is open however

  - ADP-bound = substrate binding domain is closed tightly on the peptide

  - binds short hydrophobic sequences

  - regulated by co-chaperones: DNAJ and NEFs
• HSP70 cycle

  1. DNAJ binds substrate

  2. DNAJ stimulates ATP hydrolysis by HSP70

  3. HSP70-ADP binds substrate

  4. NEFs promote substrate dissociation

  5. Multiple cycles assist folding
• DNAJ

  - co-chaperone

  - regulate HSP70 function

  - conserved J domain (binds transiently to HSP70 to induce it to hydrolyze ATP)

  - specificity achieved through various specific domains

  - can bind substrate directly or not (depends on specific domain)
• substrate-binding DNAJs

  - most-highly conserved DNAJ

  - typically homodimers (originally HSP40)

  - J domain, substrate-binding domain, and dimerization domain

  - bind short hydrophobic sequences

  - transfer substrate to HSP70 during ATP hydrolysis
• NEFs

  - remove ADP from HSP70 and allow ATP to bind by physically opening up HSP70 ATPase domain (weakens interactions with nucleotide)

  - ATP binds when NEF dissociates
• HSP90


  - binds polypeptides at late stages of folding

  - unlike HSP70 and chaperonins, have long "arms" to bind different substrates on different sites

  - homodimer with each subunit (90kDa) joined at the C-terminus

  - dimer can open and close (controlled by ATP)

  - co-chaperone p23 stabilizes closed form
• HSP90 cycle

  1. substrate is bound weakly in the open nucleotide-free state (no ATP/ADP)

  2. ATP-binding allows dimer to close and bind substrate tightly

  3. ATP hydrolysis compacts the dimer and releases substrate
• EEVD


  - HSP70 and HSP90 are not homologous, but have similar C-termini sequence motifs with EEVD

  - TPR domains recognize these motifs

  - can be specific for HSP70 and HSP90 or both
• TPR


  - co-chaperone domain

  - TPR domains are adaptable to HSP70 HSP90 or both

  - TPR proteins often also have domains that directly bind to substrate
• HOP


  co-chaperone with 2 TPR domains (one for HSP70 and one for HSP90)

  - gives smooth transition bc it can bind both HSPs at the same time
• CHIP


  co-chaperone with TPR domain and U-box (for ubiquitin ligase to help degrade proteins)
• FKBP52


  co-chaperone with TPR domain (HSP90 only) and two PPI domains (specific for prolines)
• chaperonins (HSP60)

  - oligomeric complexes with double ring structure

  - GroEL: 2 rings x 7 identical subunits of 60 kDa each

  - GroES cap: 7 subunits of 10 kDa each

  - homologs of human mitochondrial HSP60 and HSP10
• GroEL

  - rings are identical and work in alternating cycles

  1. down position (no nucleotide)

  - subunits bind to hydrophobic polypeptide

  2. up position (ATP-bound)

  - subunits bind to GroES cap instead of substrate

  - large cavity with polar surface is formed

  - substrate is release inside cavity (enclosed but no longer bound)
• GroEL cycle (for one ring)

  1. top ring binds substrate in down position

  2. binds ATP and moves to up position

  3. binds GroES cap and encloses substrate inside cavity

  4. Hydrolyzes ATP, substrate stays inside

  5. Releases ADP, moves to down position, substrate released
• p23


  co-chaperone that stabilized the closed ATP state of Hsp90
• ubiquitin


  - small, 8 kDa

  - can be covalently linked to lysine side chains of other proteins and to itself

  - poly-Ub chains mark protein for degradation
• ubiquitin linkage

  - Ub c-term can be linked to another Lys48 of another Ub

  - E3 ligase attaches first Ub on substrate then extends poly-Ub chain

  - Long Lys48 poly-Ub chains target protein for degradation by proteasome

  - mono-Ub or Lys63 poly-Ub not recognized, but signal other things (endocytosis, autophagy)
• misfolded protein degradation with CHIP

  - CHIP interactions with chaperones are transcient

  - balance between chaperone mediated folding and degradation

  ----> substrates bound by chaperones for long times are more likely to form a complex with CHIP and be ubiquitinated

  ----> substrates bound for short times are likely to escape ubiquitination

  ----> substrate not bound by chaperones are not ubiquitinated
• N-end rule

  1. If N-terminal N, convert to D; if Q, convert to E

  2. If N-terminal D or E, add N-terminal R

  3. If N-terminal R, K, H, F, W, Y, L, or I, ubiquitinate the protein

  4. If not, leave protein alone
• Regulated degradation with SCF E3

  - E3 ubiquitin ligase complex (Skp1/cullin/F-box)

  1. Scaffold binds E2 and substrate-binding (F-box) protein

  2. F-box protein binds phosphorylated substrate

  3. Substrate is presented to the E2 for ubiquitination

  
  

  Skp1: adaptor protein

  cullin: scaffold protein

  F-box: substrate-binding arm
• degradation regulated by phosphorylation

  - many F-box proteins recognize phosphorylated peptide sequences

  - phosphorylation by kinase is used as a signal for degradation

  - de-phosphorylation then prevents degradation

  - SCF ligases degrade native, functional proteins to stop their function
• Ubiquitin-proteasome summary

  1. Ub is activated by E1 and transferred to E2 on Cys side chains

  2. E3 selects substrate polypeptide and transfers Ub from E2 to Lys side chains in the substrate

  3. E2/E3 attaches more Ub onto Lys48 of the previous Ub, to make poly-Ub chain

  4. Poly-Ub is bound by shuttling receptor with UBL domain

  5. 19S cap lid binds poly-Ub, or UBL domain of shuttling receptor

  6. DUBs remove poly-Ub

  7. 19S base ATPase unfolds the substrate

  8. Proteasome core cleaves at basic, acidic, and hydrophobic sites
• important membrane functions

  1. provide an enclosure to cells, and to organelles within the cells

  2. allow regulated transport of materials between compartments

  3. provide sites within cells for biochemical rxns

  4. support contacts with the environment outside the cells
• phospholipids

  - most abundant lipi

  - polar head groups:

  ----> choline (or other charged group)

  ----> phosphate/glycerol

  ----> classification by head groups

  ----> size and charge affect lipid mobility

  - 2 fatty acid tails
• phospholipid head groups

  1. phosphatidyl-ethanolamine (PE)

  - overall neutral

  2. phosphatidyl-serine (PS)

  - overall neg charge

  3. phosphatidyl-choline

  - overall neutral

  - bulkier

  4. phosphatidyl-inositol (PI)

  - doesn't really contribute to structure of membrane, only really a signaling molecule

  5. sphingomyelin

  - overall neutral

  - bulkier

  - not an actual phospholipid but is related
• fatty acid tails

  - double bonds are cis bonds

  - more double bonds, less membrane flexibility and thickness
• glycolipids

  - only found on outside surface of plasma membrane

  - head groups contain different sugar groups (important for cell contacts with environment and other cells)
• cholesterol


  - embeds deeply in the membrane bc only tiny part of its structure is polar

  - helps straighten out fatty acid tails of other lipids

  - structure:

  ---> polar head group

  ---> rigid steroid rings

  ---> hydrocarbon tails
• maintaining membrane asymmetry

  - lipid composition on each side of membrane is different

  - important for function

  - exterior has glycolipids

  - interior has stronger neg charge (more PS)

  - flippase is protein that maintains asymmetry:

  ----> ATP-dependent

  ----> directional

  ----> lipid-specific
• lipid compositions of organelle membranes

  - highest levels of SM, cholesterol in PM

  - highest levels of PC and PE in mitochondrial and ER membranes
• coenzyme A

  - carrier molecule: ADP-linker sulfhydryl

  1. fatty acyl CoA:

  - fatty acid transfer to glycerol-phosphate in ER

  - oxidative breakdown of fatty acid to acetyls in mitochondria

  2. acetyl CoA:

  - citric acid cycle oxidation in mitochondria

  - lysine acetylation
• flippase

  At PM:

  - ATP-dependent

  - directional

  - lipid-specfic

  - new lipids are brought to the PM by vesicles and then flipped to the correct side