macromolecules Flashcards
Why do proteins undergo degradation?
- Different lifespan across proteins
- Structural tend to have longer lifespans (low turnover)
- (most) Regulatory tend to have shorter lifespans
- To ensure proper regulation of cell signalling pathways through maintaining normal protein turnovers
- To remove misfolded (no native conformation) and damaged (eg. oxidised) proteins that can lead to abnormal cellular activities
What if proteins cannot undergo degradation?
If no degradation —> accumulation of toxicity —> body cannot function properly —> disease
what is an example of the consequences of a protein not being able to undergo degradation?
Short-lived protein: Hypoxia inducible factor 1 alpha (HIF-1 alpha)
what normally happens to HIF-1 alpha in normoxia conditions?
- A transcription factor that is produced during hypoxia (insuff O2 in tissues for adequate homeostasis) to maintain oxygen homeostasis
- MOA: Induces expression of genes involved in angiogenesis (form new blood vessels), cell migration, glycolytic (glucose is broken down to form pyruvate) pathway
- In normoxia (normal O2 levels), HIF-1 alpha maintained at low levels (negligible) as not needed —> so half life is only 5-8 mins
- Short half-life due to prolyhydroxylases, which is only active when there is O2 (normoxia) —> hydroxylated HIF-1 alpha —> recognised and targeted for ubiquitination —>
- Ubiquitinated HIF-1 alpha degraded by 26S proteasome (aka degraded by ubiquitin-proteasome system)
- pVHL: Part of the ubiquitin-proteasome system that recognises HIF-1 alpha (which is a substrate of pVHL) —> HIF-1 alpha gets ubiquitinated —> Proteasomal degradation
what if HIF-1 alpha cannot be degraded?
- If HIF-1 alpha cannot be degraded —> Von Hipel-Lindau (VHL) disease
- Mutated pVHL —> Cannot degrade HIF-1 alpha —> HIF-1 alpha accumulates (so this happens despite being in normoxia) —> increase in HIF-1 alpha transcriptional activity —> increase expression of target genes: Increase matrix metalloproteinases (MMPs: enzymes that encourage cell migration and invasion), increase vascular endothelial growth factor (VEGF: promote angiogenesis) —> increase invasion, metastasis (spread of cancer cells), angiogenesis
what are the clinical outcomes if HIF-1 alpha cannot be degraded?
- Clinical outcomes: Tumour like pheochromocytomas, hemangioblastomas of CNS, clear-cell renal carcinomas, retinal capillary angiomas ] all have high degrees of vascularisation due to angiogenesis and high HIF-1 alpha transcriptional activity 😖
why does healthy HIF-1 alpha have a short half life?
Short half-life due to prolyhydroxylases, which is only active when there is O2 (normoxia) —> hydroxylate two proline residues (Pro402 and Pro564) in HIF-1 alpha —> hydroxylated HIF-1 alpha —> recognised and targeted for ubiquitination
what is VHL?
- VHL: Heriditary disease, caused by autosomal dominant mutation in one of the alleles of gene VHL which encodes for VHL protein (aka pVHL)
what are the Types of protein degradation in mammalian cells?
- Proteasomal degradation (80-90%)
- Lysosomal degradation
note: Therapeutic proteins (from drugs) will also get degraded by either pathways —> Either identified as alien (since exogenous and taken up by lysosomes) or recombinant protein (eg. recombinant insulin and taken up by proteasome)
who is proteasomal degradation for?
- Normally for ubiquitinated proteins (exceptions for very small minority)
- By 26S proteasome
who is lysosomal degradation for?
Only for membrane-associated proteins and alien proteins (extracellular proteins) —> internalised into a vesicle in lysosomes by endocytosis
which degradation type is a specific process?
proteasomal degradation!
Specific process: Unique for each protein
lysosomal degradation: Not specific: Will be degraded regardless of identity
how does lysosomal degradation occur?
When proteins enter lysosomes that has acidic interior of pH 4.5 —> proteolysis occurs (cleavage of peptide bond)
what is the Structure of Proteasome?
- Very huge cylindrical (core is hollow) protein in cells
- Consist of min 33 subunits, with total molecular weight ~2.5MDa
- Major proteasome: 26S proteasome
- Found in all cells
- Degrades and removes regulatory proteins
- Composed of a 20S core particle: Capped by a 19S regulatory particle at lid and base
- 20S core particle is made up of 4 heptameric rings assembled to form cylindrical structure —> 2 outer rings = 2 alpha subunits AND 2 inner rings = 2 beta subunits
- Inner rings contain proteolytic active sites
how is proteasomal degradation a Selective process?
- Narrow entry into 20S core particle for selectivity
- Partially unfolded proteins need to assume a primary polypeptide chain to fit through —> Upon entry, will totally unfold to stretch along channel to reach/ be translocated to inner rings —> Gets hydrolysed to short peptides of 3-25AA —> released from opposite end of channel
- 19S regulatory particles contain ATPase subunits to gate entrance, remove Ub, unfold and transfer unfolded protein to chamber
- Gate normally closed
- Only allows ubiquitinated proteins into channel
- But ubiquitin is a very small molecule, so need multiple of it to act as a tag on protein to be recognised by proteasome —> Polyubiquitin chain (min 4 ubiquitin molecules that is linked through Lys48)
- Prior to entry, deubiquitinating enzymes (DUBs) will cleave tag into monomers
- Monomers escape proteasome into cytoplasm and are recycled to tag other protein substrate
- Proteasome engages protein substrate —> Polypeptide unfolds to enter and gets translocated into 20S core to be degraded
What is ubiquitin (Ub)?
- Peptide, only has 76 residues with high lysine content of 7Lys
- Amino group of Lys gets utilised to form polyubiquitin chain to be attached to protein substrate
what is Monoubiquitination?
- Attachment of one Ub to protein
- Not targeted for degradation
- Post-translational modification event: Activated or not to carry out cellular function
what is an example of monoubiquitination?
- Monoubiquitination of histones and transcription factors to allow activation of transcription
- Monoubiquitination of surface cell receptors to activate them to participate in endocytosis and degradation in lysosomes
what are the Types of ways that protein substrates reach proteasome?
- Close proximity: Substrates bind directly to proteasome by interacting with 19S regulatory particle subunits (which can recognise ubiquitin tag)
- Substrates brought to proteasome by adaptor proteins that bind to both proteasome and polyubiquitin chain
- Minority: Substrates gets degraded without being ubiquitinated
what are the Types of endocytosis?
- Phagocytosis (cell eating)
- Pinocytosis (cell drinking)
- Receptor-mediated endocytosis
what is endocytosis: Phagocytosis (cell eating) under lysosomal degradation?
- By cytotoxic T cells, innate immunity (eg. macrophages)
- Substance is quite big: Cell debris, dead cells, protein aggregates, pathogenic microorganisms, dust particles, particulate non-living matter
- Membrane bound upon being eaten up
what is endocytosis: Pinocytosis (cell drinking) under lysosomal degradation?
- Substance is extracellular fluid
- Non specific as applies for any solutes that are dissolved in extracellular fluid
what is endocytosis: Receptor-mediated endocytosis under lysosomal degradation?
- Substance is ligands that are recognised by receptors expressed on cell membrane —> Bind —> Folding up of plasma membrane which internalises both receptor and ligands into the vesicles —> Coated vesicles fuse with endosomes intracellularly —> Contents in endosomes are sent to lysosomes for degradation or recycles to plasma membrane
- Eg. Insulin, cytokines, hormones, proteins, metabolites
what are Biopharmaceutical products?
- Macromolecules
- Mainly protein products: Recombinant proteins, monoclonal antibodies, nucleic acid-based products etc
compare between traditional chemical-based drugs and biopharmaceutical products/ biologics
- Traditional chemical-based drugs —> More artificial
- Molecular weight: Smaller, <1000Da
- Can be chemically synthesised and purified to homogeneity
- Can chemically modify to enhance activity
- Off-target effects due to small size
- Biopharmaceutical products/ biologics —> increasingly prescribed as it is more natural (protein-based)
- Molecular weight: Larger, thousands of Da
- Not easily characterised and refined to high degree of purity due to polypeptide complexity —> So derive from living sources
- Modifications in amino acid residues —> Same name still used
- More predictable and lesser side effects
what are the Challenges of Biopharmaceutical products?
- Immunogenicity*: Due to deriving non-human proteins —> Might not be properly purified
- Proteins are susceptible to denaturing and protease degradation in biological fluids (in extracellular fluids) upon administration due to interaction with body’s defence system
- MW of proteins > 200kDaltons (outlier, quite rare) —> undergoes phagocytosis as too large so want to kill it
- Proteins are susceptible to degradation by intracellular degradation systems
- Distribution of proteins (macromolecules) to tissues is limited by varying permeability (porosity) of vasculatures (blood and lymphatic vessels) across patients
what kind of absorption property do macromolecules like proteins have ?
Poor oral systematic absorption oral F ~2%
why do macromolecules have poor oral systemic absorption?
- Poor protein stability —> Get degraded (eg. acidity of gastric fluids, digestive enzymes)
- Poor permeability (mucus layer lining entire GIT, intestinal epithelial overall carry -ve charge + tight junctions exist between mucosal epithelial cells restrict absorption of hydrophilic peptides/ proteins)
- Mucosal epithelial are also present in respiratory, digestive, urinary, reproductive tract
- Have immune cells of innate immunity —> Ready to attack —> Degrade proteins
whats the solution for targeting the problem of macromolecules having poor oral systemic absorption?
-
SC or IM administration —> Bloodstream
- SC: Upon delivery to hypodermis/ subcutaneous tissues (made of adipose tissue, network of extracellular matrix (ECM), nerves (thus feel pain), blood capillaries) —> Proteins diffuse and convect through ECM (elderly: loose ECM so quick) to reach blood or lymphatic capillaries
what are the methods of subcutaneous admin of macromolecules into the bloodstream?
Diffusion
Convection
how does diffusion occur?
- Movement of single/ smaller particles
- High to low conc (slow down after awhile as equilibrium is established)
- Enters blood cap directly
- Inversely related to MW of proteins (easier for small proteins to diffuse) —> Limited for large proteins
how does convection work?
- Movement of large mass of particles in fluid
- Enters lymphatic cap first before going into blood cap
- Not so limited by MW unless really very enormous and get trapped in ECM
- Limited by steric hinderance and charge interactions
whats the molecular weight of proteins and how does this affect absorption?
- Larger proteins: >16-20kDa (mAb)
- Diffusion slow so absorption due to convection —> Enter larger lymphatic capillaries (blood cap too small) —> Drain into lymph nodes (has T cells, B cells which can attack proteins)
- Smaller proteins: <16-20kDa (Eg. insulin, cytokines)
- Absorption mostly due to diffusion, can be via circulatory and lymphatic systems
- Perfusion (blood flow throughout tissue) influence capillary absorption
what are the Rate limiting factors that can change absorption rates of proteins (could be due to diseases or demographics)?
- Interstitial fluid transport rate
- Lymphatic transport rate —> Due to disease where fluids trapped in lymphatic cap
how is the distribution like for macromolecules?
Protein drugs can be unbounded or bounded in circulation
- Binding: Normally binds to plasma proteins (albumin found in blood) —> Improves circulation half-life —> more efficient delivery to target tissues
- Albumin is quite large
- Unbound: Can attract attention of immune cells —> Destroyed!!
how do proteins move in the body?
- Movement from circulation —> Interstitial fluid of tissues —> Tissues or vice versa
- Movement is across or between endothelial cells
- Passive movement is via convection or diffusion
is there an instrument that reps how proteins move in the body?
-
Two pore model
- Characterise tissue level protein disposition
- Endosomal space: Rep porous tissue microvascular endothelium aka blood cap endothelial wall
- Has small and large pores
- Passive movement: Small proteins can pass through both, large proteins only large to gain entry to interstitial space from plasma space
- J is convection, PS is diffusion
how are proteins metabolised?
- Metabolism of protein drugs is by proteolysis by proteolytic enzymes
- ECF contains proteases, immune cells ready to perform phagocytosis and proteolysis
- Can happen in (i) interstitial fluids (aka ECF), (ii) on cell surfaces, (iii) intracellularly via lysosomal (non-protein specific) or proteasomal (protein specific) degradation
are proteins metabolised by the liver?
No metabolism of protein by liver
what is FcRn?
Neonatal Fc receptor
FcRn is an ADDITIONAL (dk if part of the 6) lg-binding Fc receptor,
where there are 6 normal lgG-binding Fc receptors on effector cells for Fc of lg to bind to —> activate effector cells
imagine:
effector cell has Fc receptors x6 that can bind to lg to activate effector cells
sudd effector cells has an additional receptor called FcRn
what is a characteristic of FcRn?
FcRn is structurally similar to MHC Class I (which recognises endogenous antibodies)
FcRn has separate binding sites for lgG and serum albumin (produced by liver) —> can bind simultaneously
how was FcRn discovered?
during preggo
FcRn was found to be responsible for transport of maternal lgG (i) across placenta to foetus and (ii) from milk across intestines of newborn babies
what is the function of FcRn?
- Regulates lg turnover* (lg homeostasis) and activates effector cells when FcRn expressed on effector cells binds to Fc receptor on lgG
- Involved in the recycling of antibodies and serum albumin
where is FcRn expressed?
FcRn receptor is expressed in a wide range of cell types, eg. endothelial and epithelial cells
how does FcRn help in the recycling of antibodies and serum albumin in ENDOTHELIAL CELLS?
note: endothelial cells (aka blood cap cells)
- In bloodstream (pH 7.4): Contains dissolved (and hence soluble) endogenous lgG and albumin —> Taken up by endothelial cells by pinocytosis (cell drinking) to form endosomes (pH 5-6) that contains FcRn
- Acidic pH in endosomes causes binding of albumin and lgG to FcRn —> FcRn-albumin complex
- Complex can be recycled to cell surface —> Release (exocytosis) albumin and lgG as free entities, FcRn re-expressed on cell surface of endothelial cell again —> Lengthens circulation half life of lgG antibody & albumin
how does FcRn help in the recycling of antibodies and serum albumin in EPITHELIAL CELLS?
note: epithelial cells (in GIT) —> Idea for delivery by oral route
- 3 small squares illustrate the tight junctions
- Express FcRn receptor at apical end (facing intestinal contents) which is acidic —> allows FcRn receptor to bind to free lgG and albumin —> taken up by epithelial cells —> house in acidic endosomes —>
- Transcytosis occurs whereby endosomes crosses to fuse with basolacteral side (face bloodstream) —> exposure to neutral pH causes FcRn to dissociate from albumin and lgG —> free albumin and lgG released into interstitial (bloodstream)
how are proteins eliminated?
- Proteolytic degradation
- Renal excretion: Glomerular filtration
what are the factors affecting elimination of proteins?
- Proteins >50kDa cannot be renally eliminated as too big to get filtered
- Net positive charged proteins have higher renal filtration for the same size, since glomerular basement membrane carries neg charges
- Shape and rigidity of protein
- Tubular reabsorption: Pos charge proteins gets reabsorbed more as tubular epithelial have net neg charge (abit contradictory with point 2, but need to see net loss)
what are the strategies to improve pk of protein therapeutics?
- Glycosylation of proteins
- PEGlyation of proteins
- Increase in size (MW) via fusion proteins
what happens if protein cannot be filtered?
Technically if protein cannot be filtered, will be recirculated back into bloodstream —> Half life lengthened
what is glycosylation of proteins in helping to improve its PK?
- Addition of glycan groups (carbohydrates) to protein
- Types of glycan groups can vary and hence affect activity: straight vs branched chain, size, position of attached in AA seq of protein
- MOA: N-linked glycosylation of proteins —> Increase size —> increase half-life or modify binding to glycoprotein receptors
how can glycosylation of proteins help in improving its PK?
- Glycoengineering can occur in antibodies: Double-edged sword (this portion abit confusing but im sick)
- Human lgGs contain N-linked glycans —> remove fucose attached to Asn297 (defucosylated, refer to IC5) —> improve affinity of binding of Fc in lgG to Fc receptor 😄
- Want mannose glycans to be high in content in Fc —> to be more recognised by mannose receptor —> trigger innate immunity to rapidly remove antibodies —> shorten half life 😖
how can PEGlyation of proteins help in improving its PK?
- Polymer with lots of hydroxyl groups —> make water-compatible —> conjugate to protein drugs —> increase size >50kDa to not be filtered —> lengthen half life
- Decrease elimination by proteolysis due to formation of protective layer upon conjugation —> shield from immune cells and proteolytic enzymes —> reduced immunogenicity
how can Increasing size (MW) via fusion proteins help in improving its PK?
- Fc domain of antibody or albumin gets bound to FcRn —> Increase circulation half life
- Eg. Etanercept
- Note albumin has many binding pockets —> bind to fatty acids, drugs, proteins —> competition —> compromise FcRn binding and hence recycling —> hence experimental strategy
