Macromolecule ADME + Recombinant Proteins Flashcards
How is HIF-1α normally degraded?
Hydroxylated HIF-1α is recognised and targetted for ubiquitination, which is then degraded by 26s proteasome (ubiquitin-proteasome system)
What happens in Von Hippel-Lindau disease?
pVHL cannot degrade HIF-1α, increasing VEGF, migration, invasion, metastasis and angiogenesis, predisposing to highly vascularised tumours
What are the two means of protein degradation in mammalian cells?
Lysosomal degradation (10%)
Proteolysis (90%)
The proteasome in mammalian cells is the ___S proteasome
26s
The proteasome is made up of a ___s degradation chamber with two ___s caps, and a channel that is ___Å wide
20s, 19s, 13Å
What is attached to the 19s subunit caps?
ATP subunits (energy dependent)
What does ubiquitin do for proteolysis
Polyubiquitin tag (at least 4 x Ub) acts as a label for the 19s cap to recognise and bind to
What enzyme cleaves the polyubiquitin tag?
deubiquitinising enzymes (DUB)
What are the 3 methods by which proteins can be delivered to the proteasome?
- binding directly to the 19s subunit
- brought to the proteasome by 3rd party adaptor proteins
- degraded without ubiquitin
What are the 5 challenges of BCPs?
- Immunogenecity (CHO cell pdts if not well purified)
- Susceptibility to denaturation (leak out from cell to ECF)
- If MW > 200kDa, eliminated via phagocytosis
- Susceptible to degradation in cells by lysosomes and proteasomes
- Distribution limited by permeability and porosity of the vasculature
What are the two main methods of movement after administration?
Diffusion and convection (bulk movement with fluid)
Where do large proteins > 16-20 kDa enter?
Lymph mainly
Where do small proteins < 16-20 kDa enter?
Both lymph and blood
What are the 2 main factors that affect distribution?
Size/MW and protein binding
How are macromolecules usually degraded?
Proteolysis
