Molecules to cells Flashcards
Signal hypothesis
Blobel - proteins have intrinsic signals that govern their transport + localisation in the cell intracellular postcodes
-> signal sequences made up of specific types of a. acids (can be removed after), often hydrophobic side chains.
70% proteins remain in cytosol
Features of nucleus
Envelope - composed of 2 membranes w/ underlying lamina (protein network), continuous w/ ER
Pores - 30+ proteins act ac gateways, small water sol mols diffuse freely through, larger components (RNA, proteins) actively transported across pore complex
Nuclear localisation signal (NLS)
Target proteins to nucleus, common feature is many +ve a. acids (Arg, Lys), short + can be located anywhere in protein, not removed after.
Protein import into nucleus
- Receptor binds proteins w/ NLS in cytosol
- cytosolic fibrils direct receptor to pore + binds pore proteins, cargo proteins moved into nucleus through gel-like meshwork of fibrils
- pores large so folded proteins can be imported
GTP hydrolysis by RAN
Drives nuclear import
Small GTPase RAN is GTP or GDP bound w/ different localisations
- RAN-GAP (reg1) triggers GTP hydrolysis
- GEF (reg2) promotes exchange of GDP for GTP
High Ran GTP in nucleus displaces cargo protein from receptor - receptor recycled back to cytosol
GTP hydrolysed in cytosol so Ran-GDP dissociates
How is NLS exposed?
Nuclear factor of activated T-cells (NFAT) - family of TFs - stimulated to enter nucleus by calcium (changes their conformation).
-> exposes NLS
Cut and paste experiments used to conclude NLS required + sufficient on its own for nuclear import.
Mitochondria features for protein import
Inner membrane impermeable BUT outer membrane permeable to all mols < 5000 Da.
Targeting sequences normally have high Arg(+) & Ser/Thr (nonpolar)
- located N terminus, 20-80 a.acids long
- cleaved after import
-> can form ampiphilic a-helix w/ 2 different sides
Mechanism of import into mitochondria
- Targeting signal recognised by receptor on outer mem.
- Translocator (TOM) channel moves protein into inter-membrane space.
- Signal binds TIM in inner mem.
- Signal sequence cleaved (protein must be open/unfolded to enter matrix)
-> chaperone protein (Hsp70s) pulls protein into matrix + helps refold it.
Protein targeting to ER
Pancreatic cells have extensive rER, hepatocytes have extensive sER.
ER entry point for proteins destine to other organelles/cells, delivered by vesicular transport
Targeting sequence has 8+ hydrophobic a. acids (leu, val, isoleucine) residue near N terminus.
Proteins enter whilst still being synthesised (co-translational)
Mechanism of import into ER
- SRP binds ER signal sequence as it emerges from ribosome (translation paused)
- SRP binds SRP receptor adjacent to translocator protein (Sec61) on ER mem.
- SRP displaced + released for reuse
- ribosome passes through translocator (translation resumed)
Binding of signal sequence by Sec61 opens channel.
Polypeptide threaded though channel as loop.
Signal sequence cleaved by signal peptidase.
Membrane protein insertion
- ER signal sequence binds Sec61 opening channel
- Hydrophobic stop-transfer seq stops polypeptide movement through channel
- Stop-transfer seq released into bilayer forming transmembrane domain
- Protein inserted into bilayer w/ fixed orientation, N terminus in lumen.
- Signal sequence cleaved
Alternating start/stop transfer sequences generate complex multi pass proteins
Post translational modification in ER
Folding assisted by molecular chaperones.
e.g. BiP is an ATPase that binds exposed hydrophobic residues, Calnexin binds N-glycosylated proteins
Disulphide bonds formation: oxidation of cysteine residue -> increases tertiary structure stability
N-linked glycosylation (N=asparagine): lipid donor dolichol donates oligosaccharide to protein, catalysed by OST, only done in specific consensus sequence
Functions of post-translational modifications
- Assist protein folding
- Creation of manmose-6-phosphate tags act as lysosome sorting signal
- Act as ligand for specific cell-cell recognition events.
Glycocalyx (protective layer) made at ER + golgi - used to coat eukaryotic cells
Quality control in ER
Chaperones bind misfolded proteins + stop them leaving ER.
Unfolded protein response (UPR) occurs when build up of misfolded proteins in ER lumen.
- activates ER sensor protein which activates chaperone genes
SDS-polyacrylamide gel electrophoresis
Sodium dodecyl sulphate (SDS) binds protein to give them negative charge, acts as ionic detergent so will unfold proteins
Polyacrylamide gel is mesh like gel that separates charged proteins by size.
They move towards positive anode faster if they’re smaller
Can use coomassie blue, silver stain, radiolabel or antibodies to visualise proteins.
Membrane lipid synthesis
- Catalysed by enzymes on cytosolic face of ER mem.
- Scramblase transporters transfer phospholipids between leaflets non selectively until equilibrium reached
*far more phosphatidyl serine in inner leaflet
-> flippase transporters in golgi flip specific phospholipids from outer -> inner mem
Membranes + proteins retain orientation during vesicular transport -> lumen domain joins extracellular surface
Vesicle formation
- Protein coat deforms membrane into bud captures cargo.
- Dynamin (GTPase) helps bud pinch off.
- Coat proteins (clathrin in endocytic pathway of plasma mem + golgi) removed.
How is cargo selected and vesicle separated?
Adaptins help clathrin attach to mem forming clathrin-coated pit on cytosolic face.
They also bind cargo receptors which recognise specific sorting signals on cargo proteins, recruiting them into vesicle.
-> clathrin cage causes membrane to invaginate, dynamin assembles ring around neck of bud - GTP hydrolysis changes dynamin conformation contricting neck of bud
How does vesicle uncoating occur?
Coat proteins (e.g. clathrin) removed - requires molecular chaperones + ATP
Mechanism of vesicle docking + fusing
- Rab protein binds vesicle, v-SNARE acts as marker
- specific tethering protein on target organelle binds Rab
- v-SNARE & t-SNARE interact + wrap tightly to allow vesicle docking
- bilayers must be close (1.5nm) to fuse, water must be displaced (energetically unfavourable)
Retention & sorting in ER & golgi
Sorting/trafficking signals vital
ER retention signal: KDEL sequence at C terminus of soluble proteins recognised by KDEL receptor in golgi
-> recruited in COPI vesicles + returned to ER
(BiP & PDI contain KDEL sequences)
- short TM domain (18a. acids) retains proteins in golgi
- addition of Mannose-6-phosphate to N linked glycans of some glycoproteins sends them to lysosome.
Golgi apparatus
cis network - entry, carrying vesicles from ER
trans network - exit, carrying proteins onwards
Vesicular transport model in Golgi
Cisternae static components containing specific enzymes. Vesicles bud + fuse through each cisternae .
- cargo mols present in small transport vesicles (100nm)
Cisternal maturation model
Cisternae matures as it migrates outward through the stack (cis -> trans), resident enzymes carried forward are returned to earlier compartment
- transport large proteins like collagen (300nm), too big for typical vesicles
Golgi function
- protein modification - O linked oligosaccharides added to -OH side chains serine + threonine, N-linked oligosaccharides added in ER can be trimmed + rebuilt at golgi
- Protein sorting - at cis network cargo w/ KDEL signal sorted back to ER, other cargo proceeds onwards, at trans network cargo proteins sorted into transport vesicles.
Unregulated/constitutive exocytosis
Constant stream of transport vesicles from trans Golgi.
- supplies proteins for plasma membrane growth, allows protein secretion
*default, no signal required
Regulated exocytosis
Proteins sorted into secretory vesicles + stored until specific signal received
- only in specialised secretory cells
e.g. release of insulin, increase blood glucose -> insulin secretion by pancreatic B-cells
regulated secretion is rapid
3 forms of endocytosis
- Phagocytosis - e.g. protozoa can take up food, macrophages + neutrophils ingest microorganisms
- cells engulf, form phagolysosome
- M. tuberculosis inhibits mem fusion so multiplies in macrophages - Pinocytosis - non-selective mediated by clathrin coated vesicles, small areas of plasma mem + extracellular fluid internalised.
- Receptor mediated endocytosis - selective uptake of molecules, uses cell surface receptors to capture cargo (increased efficiency 1000 fold)
Endocytosis of low-density lipoproteins (LDL)
- Cholesterol transported in blood as LDL, binds LDL receptors - then internalised by clathrin coated vesicles + fuses w. endosome.
- Endosome has acidic pH -> LDL dissociated from receptor
Many materials taken up by receptor mediated endocytosis:
- lipoproteins
- metabolites
- signalling mols
- virus particles
Endosome & lysosome sfeatures
Endosome - cluster of connected tubules + vesicles, most receptors recycled (LDL) but many degraded (EGF), or moved to different domain of plasma mem (transcytosis)
Lysosomes- around 40 hydrolytic enzymes -> degraded macromolecules, optimal at pH 5so inactive in cytosol
-> acidic pH maintained by proton pump
Sorting enzymes to lysosomes
Enzymes made at ER -> Golgi, modified w/ M-6-P.
M-6-P receptor in trans network sorts + packages into vesicles that deliver them
Autophagy
Damaged organelle engulfed by double membrane formed in cytosol, forms autophagosome that fuses w/ lysosome
Biosynthesis of chylomicrons
- PreChylomicrons assembled in ER from triglycerides + large proteins
- packaged into transport vesicles (PCTV)
- mature into chylomicrons in Golgi
- released by exocytosis + enter capillaries
Chylomicron retention disease (CRD)
PreChylomicrons accumulate in ER + unable to reach Golgi
-> caused by defective ER export: COPII coat responsible for cargo exports fails
What causes COPII to not assemble in CRD?
Sar1 GTPase controls formation of COPII vesicles
- regulatory Sar-GEF activates Sar1p turning it on
- Sar1-GTP initiates assembly of COPII proteins
2 isoforms of Sar1: a & b 90% identical but encoded by different genes.
BUT mutations in Sar1b causes CRD -> no Sar1b made + GTP binding site defective
Sar1a still active + mediates transport of most cargo. Sar1b required for preChylomicron transport .
Symptoms + treatment of CRD
Lipid droplets in cell cytoplasm
- Impaired absorption of fats, cholesterol + fat soluble vitamins
- slow growth + weight gain
- GI + nervous system effects
Treatment is low fat diet to minimise accumulation of intracellular preChylomicrons.
Familiar Hypercholesterolaemia
Autosomal dominant disease - can lead to CHD.
Caused by defect in cholesterol uptake so it accumulates in blood -> atherosclerosis.
6 classes of LDL receptor mutations which disrupt cholesterol uptake:
- class 2 disrupt LDL receptor folding
- class 4 disrupt LDL receptor endocytosis (mutated cytoplasmic tail)
-> adapter proteins also affect endocytic uptake of LDL - class 3 disrupt LDL binding (receptor continues circulating)
Treatments for FH
Inhibit cholesterol synthesis - STATINS inhibit HMG-CoA reductase, stimulates LDL receptor expression + increases its uptake -> heterozygotes w/ one wild type copy.
Inhibit dietary cholesterol absorption - EZETIMIBE acts on intestine
Lysosomal storage diseases
Niemann-Pick type C: 95% cases caused by mutations in mem protein NPC1
Gaucher disease: mutations in lysosomal acid B-glucosidase -> misfolded causing ER retention
-> glycolipids accumulate in lysosome prevent cleavage into glucose + seramide
Potential treatments for lysosomal storage diseases
- Enzyme replacement therapy - injecting synthetic enzymes which are taken up via M6P receptors (expensive
- Substrate reduction therapy - reduce amount of glucosylceramide in lysosomes (miglustat inhibits its synthesis) -> treat Gaucher’s
- Pharmacological chaperones can correct folding -> increase amount of enzyme that escapes ER qual control + reaches lysosome.
Protein targeting diseases
Defective mt targeting:
- point mutation in targeting seq of pyruvate dehydrogenase (PDH), R->P has ring structure which break amphiphilic helix.
- inefficient import of protein reduces PDH in mt -> pyruvate accumulates (converted to lactate, lactic acid build up)
-> inherited congenital lactic acidosis
Defective ER targeting:
- point mutation insulin signal seq, R->C, signal seq does not interact correctly w/ Sec61 so not translocated into ER efficiently.
- mutant insulin diverted into cytosol, hydrophobic signal seq at N terminus + Cys forms toxic aggregates causing B cell death
-> less functional insulin made, diabetes
Mechanism to clear misfolded proteins
Proteins localised incorrectly to cytosol degraded by proteasome int a. acids (proteolysis).
- proteins tagged by ubiquitin
- polyubiquitin chain recognised by proteosome cap (ATPase), unfolds protein
- cytosolic proteasome arranged so active side directed toward inwards cavity
hydrophobic regions/unpaired Cys on misfolded proteins -> prone to aggregation so degraded via autophage using lysosome
ER protein misfolding diseases
Mutations in a. acid seq prevent correct folding.
Chaperones can prevent misfolded proteins leaving via exocytosis so lack of functional protein causes disease
e.g. cystic fibrosis
dF508 mutation (90% patients) - > CFTR cannot fold properly so does not reach plasma mem.
-> no Cl- ions out so thick dehydrated mucus + cilia cannot function
Chaperones remove misfolded CFTR to cytosol (proteolysis)
-> ER-associated degradation (ERAD)
Cystic fibrosis therapies
- express CFTR wild type through gene editing
- drugs to enhance folding + escape from ERAD : correctors (chaperones) w/ potentiators (Trikafta) -> effective but expensive
Diseases due to misfolded protein accumulation in ER + treatment
Aggregation of misfolded proteins -> activates stress sensors that trigger unfolded protein response (UPR).
Increases chaperone expression + inhibits protein synthesis to restore homeostasis
-> if it fails apoptosis triggered
Treatment needs to:
- reduce synthesis of mutant protein
- stimulate degradation of mutant protein
- alter UPR signalling to prevent apoptosis
Microtubule structure + nucleation
Hollow cylinders of 25nm diameter assembled by tubulin heterodimers.
Polar structure: +ve end grows rapidly (B tubulin exposed), -ve end grows slowly if at all.
Nucleation - cells use template of gamma-tubulin + other proteins to speed up polymerisation.
-> gamma rings concentrated on specific structures
e.g. ciliated cells have extra set of MTs in cilia nucleated at basal body
MT dynamics
Each grows + shrinks independently of neighbours - can switch between both (dynamic instability)
- unassembled GDP tubulin cannot polymerise
- GTP tubulin can polymerise
- tubulin is a GTPase (switch + timer)
- protein EB1 preferentially binds GTP-tubulin so marks polymerisation
-> if GTP cap lost then MT will depolymerise (catastrophe)
