PR3152 IC7 Flashcards
purpose of protein degradation
regulate cell signalling pathways
remove misfolded or damaged proteins
HIF alpha 1 characteristics and degredation
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)
VHL disease
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.
what are the two protein degradation pathways in mammalian cells?
minor: lysosome
major: proteasome
explain lysosome degradation
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
explain proteasome degradation (general)
major pathway, 80-90% by 26s proteasome
SPECIFIC PROCESS
ubiquitinated proteins and SOME non-ubiquitinated proteins
what are the three types of cellular uptake of extracellular proteins?
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)
proteasome structure
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
proteasome function (ubiquitin dependent protein degradation
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)
ubiquitin tag (properties, recycling)
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.
monoubiquitination purpose
form of post-translational modification
- added to transcription factors and histones to regulate transciption
- cell surface receptors to signal for endocytosis and lysosomal degradation
delivery of substrate?
1) adaptor protein between polyubiquitin tag and proteasome
2) directly linked to ubiquitin tag
3) degraded without ubiquitin tag
challenges of using biopharmaceuticals
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.
why do proteins have poor oral absorption?
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.
challenges of oral absorption of protein drugs
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.
innate response of the GI mucosa
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.
SC absorption of protein drugs (structure + methods)
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
SC absorption of small vs large protein drugs?
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)
what are the barriers (and limiting factors) to SC absorption?
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
movement of particles into the lymphatic system vs circulatory system?
lymphatic system
- movement via convection and immune cell mediated transport.
circulatory system
- movement via passive and carrier-mediated transport.
methods of protein distribution
1) protein binding
- to improve the circulation half life
2) (passive transport) tissue distribution
- vascular system > interstitial fluid > tissues
describe the movement of proteins from vascular system to tissues
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.
metabolism of protein drugs via proteolysis. how?
1) in the interstitial fluid
2) cell surfaces
3) intracellularly
purpose of FcRn receptor
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
describe the 2 roles of FcRn receptor
1) cellular recycling of IgG and albumin, therefore increasing the circulation half life of the IgG/albumin
2) transcytosis of IgG and albumin
what is the function of the FcRn receptor in allowing cellular recycling of IgG and albumin?
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.
what is the function of the FcRn receptor in transporting/transcytosis IgG and albumin?
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.
two ways protein drugs are eliminated from the body?
1) glomerular filtration
2) proteolytic degradation
factors affecting glomerular filtration of protein drugs?
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
methods to improve PK profile of protein drugs?
1) glycosylation
2) PEGylation
3) increasing size (MW) via fusion proteins
how does glycosylation improve the PK profile of protein drugs?
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)
why can glycosylation be bad/negatively affect protein drug PK profile?
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.
types of PEG?
structure of PEG?
1) free hydroxyl end
2) methoxylated PEG with hydroxyl at one or both ends
PEG is polymer of repeating units of ethylene oxide.
PEG conjugation to protein drugs?
amino group (lysine, sulfhydryl SH group in cysteine or other nucleophilic groups
how does PEG improve the PK profile of protein drugs?
1) increase size = longer circulation half life
protective barrier against
2) proteolysis
3) elimination by atb and immune cells
how do fusion proteins improve PK profile of protein drugs?
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.
drawbacks of fusion proteins in protein drugs?
may have unwanted effector functions due to presence of the Fc domain = trigger unwanted immune response.
