PR3152 IC7

Question 1

purpose of protein degradation

Answer 1

regulate cell signalling pathways

remove misfolded or damaged proteins

Question 2

HIF alpha 1 characteristics and degredation

Answer 2

short half life 5-8 min
oxygen homeostasis involved in stimulating angiogenesis, cell migration, glycolytic pathways

under normoxia, hydroxylated at pro402 and pro564 by oxygen dependent prolylhydroxylases
> ubiquitination > degraded by proteosome 26s (ubiquitin proteosome complex)

Question 3

VHL disease

Answer 3

autosomal dominant mutation
genetic
codes for protein pVHL
part of e3 ligase of ubiquitin proteosome system
in VHL disease, accumulation of HLF-alpha 1 = increased transcription = increased expression of target genes = MMPs, VEGF = increased angiogenesis, metastasis, and invasion = predisposition for tumor types

e.g., pheochromocytomas, hemangioblastomas of the CNS, clear-cell renal carcinomas and retinal capillary angiomas.

Question 4

what are the two protein degradation pathways in mammalian cells?

Answer 4

minor: lysosome

major: proteasome

Question 5

explain lysosome degradation

Answer 5

minor pathway, non specific as long as protein is in lysosome
proteolysis by lysosome

in higher eukaryotes, may only be alien proteins or membrane associated proteins

Question 6

explain proteasome degradation (general)

Answer 6

major pathway, 80-90% by 26s proteasome
SPECIFIC PROCESS
ubiquitinated proteins and SOME non-ubiquitinated proteins

Question 7

what are the three types of cellular uptake of extracellular proteins?

Answer 7

1) phagocytosis (large solid particles)
2) pinocytosis (non-specific, for fluids or solutes)
3) receptor-mediated endocytosis (specific –> internalised into coated vesicles –> either sent to the lysosome or recycled)

Question 8

proteasome structure

Answer 8

33 subunits, large cylindrical protein about 2.5MDa

consists of 20s core particle and two 19s regulatory particle

20s core particle consists of four heptameric rings with two outer (alpha) and two inner (beta) rings
has central cavity contain the proteolytic active sites

19s regulatory particle consists of a cap and base
- 13 a.a entrance

Question 9

proteasome function (ubiquitin dependent protein degradation

Answer 9

ubiquitin tag (selective) recognised by 19s regulatory particle and cleaved by DUB (deubiquitination enzymes)

19s particle contains ATPase, hydrolyses ATP for energy to remove ubiquitin tag and unfold the protein

translocation through the central cavity of 20s core

degraded into smaller peptides (3-25 amino acids long)

Question 10

ubiquitin tag (properties, recycling)

Answer 10

76 residue with 7 lysines
binds to substrate via c terminus of gly of Ub and amino acid of lys on substrate

polyubiquitin tag consists of 4 Ub monomers binded to each other via lys48

ubiquitin tag cleaved by DUBs into monomers and escape proteasome to be recycled.

Question 11

monoubiquitination purpose

Answer 11

form of post-translational modification
- added to transcription factors and histones to regulate transciption
- cell surface receptors to signal for endocytosis and lysosomal degradation

Question 12

delivery of substrate?

Answer 12

1) adaptor protein between polyubiquitin tag and proteasome
2) directly linked to ubiquitin tag
3) degraded without ubiquitin tag

Question 13

challenges of using biopharmaceuticals

Answer 13

1) immunogenicity
- e.g., the presence of impurities (that are non-human) can trigger an immune response.

2) ECF
- proteolysis in the ECF e.g., phagocytes. especially if the molecule is large >200kDa

3) intracellular
- intracellular proteolysis e.g., by lysosomes, ubiquitin-proteasome complex, intracellular proteases.

4) PK
- distribution of the proteins limited by the porosity/permeability of the vasculature.

Question 14

why do proteins have poor oral absorption?

Answer 14

Immunogenicity (impurities of non
human origin contribute to
immunogenicity)
*
Proteins are susceptible to denaturation and protease
degradation in biological fluids (in extracellular fluids) upon
administration.
Generally, when MW of proteins > 200
kDaltons , phagocytosis maybe involved
in their elimination.
*
Proteins are susceptible to degradation by intracellular
degradation systems (lysosomal degradation, intracellular
proteases, ubiquitin proteasomal degradation).
*
Distribution of proteins (macromolecules) to tissues is limited by
the permeability (porosity) of vasculatures.

Question 15

challenges of oral absorption of protein drugs

Answer 15

1) stability
- degraded by enzymes in the GI tract and affected by the low pH.

2) permeability
- tight junctions of epithelial cells
- negative charges on the epithelial cells
- mucosal membrane.

Question 16

innate response of the GI mucosa

Answer 16

the mucosal membrane has innate immune cells present e.g., leukocytes, and neutrophils that target the foreign protein particles.

the higher the molecular weight, the more likely is it to be targeted.

Question 17

SC absorption of protein drugs (structure + methods)

Answer 17

SC absorption: protein enters the hypodermis containing the adipose tissue, ECF, lymphatic and circulatory system.

protein must travel to the lymphatic and circulatory systems.

through:
1) diffusion (mainly circulatory)
- affected by the MW. smaller MW = faster diffusion.

2) convection (mainly lymphatic)
- flux of the fluids
- affected by steric hindrance and charge interactions

Question 18

SC absorption of small vs large protein drugs?

Answer 18

small (<16-20kDa)
- move into both the lymphatic and circulatory system.
- affected by the perfusion rate

large (>16-20kDa)
- movement is slow in the capillaries
- move into lymphatic system > lymph nodes > lymphatic vessels > circulatory system.
- large molecules can enter lymphatic due to the leaky endothelial cells (clefts exist + no basement membrane)

Question 19

what are the barriers (and limiting factors) to SC absorption?

Answer 19

1) perfusion rate
- different sites have diff rate of perfusion.
- some patients may have slower perfusion.
- interstitial fluid and lymphatic transport rate.
- e.g. disease states and old age, like edema, lymphatic disease.

2) ECF/hypodermis contain innate immune cells

Question 20

movement of particles into the lymphatic system vs circulatory system?

Answer 20

lymphatic system
- movement via convection and immune cell mediated transport.

circulatory system
- movement via passive and carrier-mediated transport.

Question 21

methods of protein distribution

Answer 21

1) protein binding
- to improve the circulation half life

2) (passive transport) tissue distribution
- vascular system > interstitial fluid > tissues

Question 22

describe the movement of proteins from vascular system to tissues

Answer 22

plasma <> endosomal space <> interstitial fluid

• endosomal space is porous, part of the endothelium
• contains 2 pores (large and small)
• protein movement into interstitial space via diffusion and fluid phase convection (this is bidirectional)
• affected by MW/size, large molecules tend to be more limited in distribution and therefore slower.

Question 23

metabolism of protein drugs via proteolysis. how?

Answer 23

1) in the interstitial fluid
2) cell surfaces
3) intracellularly

Question 24

purpose of FcRn receptor

Answer 24

transferred to the child via breast feeding and during pregnancy (cross placenta to the fetus)

• acts as an Fc receptor for effector cells
• enables recycling of IgG and albumin by cellular trafficking to escape degradation by lysosomes

Question 25

describe the 2 roles of FcRn receptor

Answer 25

1) cellular recycling of IgG and albumin, therefore increasing the circulation half life of the IgG/albumin
2) transcytosis of IgG and albumin

Question 26

what is the function of the FcRn receptor in allowing cellular recycling of IgG and albumin?

Answer 26

1) IgG/albumin dissolves in neutral pH of blood (7.4)
2) taken up by endothelial cells via pinocytosis, forming endosomes.
3) endothelial cells contain internalised FcRn receptors, which, in the acidic pH fo the endosome (5-6) will fuse with the IgG/albumin to form FcRn-X complexes
4) Exocytosis of complexes
5) Neutral pH of blood causes complexes to dissociate
6) IgG/albumin not bounded to the FcRn will be degraded by the lysosomes.

Question 27

what is the function of the FcRn receptor in transporting/transcytosis IgG and albumin?

Answer 27

1) apical side of the epithelial cell is acidic. this causes FcRn receptors on the cell surface to bind to IgG/albumin
2) endosome formation
3) further processing of FcRn-X complexes
4) endosomes fuse w membrane (transcytosis) of the basolateral side. neutral pH of interstitium causes release of IgG/albumin.

Question 28

two ways protein drugs are eliminated from the body?

Answer 28

1) glomerular filtration
2) proteolytic degradation

Question 29

factors affecting glomerular filtration of protein drugs?

Answer 29

1) molecular weight
- 50kDa cutoff for glomerular filtration
2) charge
- negative charge of glomerular basement membrane
- positive charge more likely to filter
3) size and rigidity
4) tubular reabsorption
- tubular membrane net negative charge, positive charged proteins are reabsorbed more

Question 30

methods to improve PK profile of protein drugs?

Answer 30

1) glycosylation
2) PEGylation
3) increasing size (MW) via fusion proteins

Question 31

how does glycosylation improve the PK profile of protein drugs?

Answer 31

GLYCANs are carbohydrates

increases the circulation half-life of proteins by
1) limiting the glomerular filtration through the increase in size
2) poorer substrates for proteolysis (to glycoprotein receptors)

Question 32

why can glycosylation be bad/negatively affect protein drug PK profile?

Answer 32

e.g., innate immune cells like phagocytes contain pattern recognition receptors on the cell surface/intracellularly like mannose receptors

mannose receptors recognise mannose glycans = recognition and elimination.

Question 33

types of PEG?
structure of PEG?

Answer 33

1) free hydroxyl end
2) methoxylated PEG with hydroxyl at one or both ends

PEG is polymer of repeating units of ethylene oxide.

Question 34

PEG conjugation to protein drugs?

Answer 34

amino group (lysine, sulfhydryl SH group in cysteine or other nucleophilic groups

Question 35

how does PEG improve the PK profile of protein drugs?

Answer 35

1) increase size = longer circulation half life

protective barrier against
2) proteolysis
3) elimination by atb and immune cells

Question 36

how do fusion proteins improve PK profile of protein drugs?

Answer 36

1) increase size = longer circulation half life

2) contains Fc binding domains to bind to albumin or Ig protein in order to utilise FcRn cellular recycling and enhance circulation half life.

Question 37

drawbacks of fusion proteins in protein drugs?

Answer 37

may have unwanted effector functions due to presence of the Fc domain = trigger unwanted immune response.