Protein misfolding during biosynthesis

Protein misfolding during biosynthesis

1. Alpha-1-antitrypsin (AAT)

   • normal protein
     • abundant in plasma; 1.75g/L
     • processing
       • 70% synthesised in ER hepatocytes
         • glycosylated
           • processed in Golgi
             • transported to lungs
     • inhibits neutrophil elastase (NE) - a protease
       • too much NE degrades lungs
       • NE binds top of RC
         • RC translocates NE by 70 A towards bottom of molecule
           • RC loop inserted into AAT centre > inactivates AAT > "suicide-substrate mechanism"
           • Destroys NE active site
     • long half-life (5 days)
     • states
       • metastable native state
         • Tm = 55 C
         • so protein is dynamic > moves rapidly as needed
       • cleaved native state
         • most stable native state (Tm > 100 C)
       • thought to form intermediate, but no structure
     • AAT deficiency (conformational disease)
       • Z mutation (E342K) is most severe of 100 mutations
         • 85% protein retained as polymers trapped in liver > liver cirrhosis
           • AAT exported to blood > degrades lungs > COPD (difficult to diagnose)
           • models suggest strand-mediated polymerisation: RC loop from one molecule inserted into other
             • so polymers self-assemble
         • stain liver biopsies using period-acid Schiff staining > AAT goes purple under light microscope
         • folding mechanism
           • studying methods
             • unfold with guanidine
               • fluorescence = tertiary structure
               • circular dichroism = secondary structure
               • Bis-ANS fluorescence = hydrophobic dye only fluoresces when bound to hydrophobic residues
               • CD plateaus and fluorescence peaks (unfolds/polymerises) at 1.2 M guanidine > molten-globule protein
             • difficult to discern folding and misfolding intermediates
               • CD study indicates intermediate = 80% similar to native state
               • similar to misfolding intermediate because can be recapitulated at ~1.5M guanidine (same guanidine concentration when AAT polymerises)
           • AAT folds post-translationally
             • biosynthesis rate slightly faster than folding rate (5found out by using expressing AAT with 35S and taking samples over time during biosynthesis > run on denaturing and native gels
               • Z also shown to be able to synthesise polypeptide
             • same experiment but with cyclohexamide (keeps ribosome intact) > AAT nascent chain showed to stay bound to ribosome
             • Z RNC shown to have shorter lifetime > nascent chain released more quickly than WT
               • Z polymers then form almost instantly in cytosol
                 • ribosomes form higher order assemblies on polymers ("beads on a string polymers" observed in electron microscopy)
                   • limited proteolosis (proteinase K) of RNCs > Western Blot indicates fragments form; smallest fragment = 23 kDa compacted NTD (AAT-191)
                     • Far-UV CD indicates alpha-helix
                     • titrating with more urea > see more protein > so molten globule
                       • lots of secondary structure, little tertiary
                   • Z RNC slightly more proteolytically sensitive than WT
             • mutate residues to cysteines > add PEG-maleimide to see if they are solvent exposed; A332C exposed, I260C in RC buried
               • also study rate of PEGylation >concert to protection factors > WT more protected than RNC (RNC solvent exposed, partially folded)
                 • compare WT and Z protection factors > differences in regions important for folding (A-beta sheet and RC loop)
               • use PEG to trap nascent chains formed off ribosome > WT monomers decrease over time (due to folding); Z monomers last longer (nascent chains trapped)
               • unfolding isolated AAT using urea > PEGylate > find that co-translational intermediate similar structure to post-translational intermediate
             • AAT folding model
               • co-translational intermediate forms on ribosome
                 • post-translational intermediate with Z mutation forms polymers/ proteolyses
         • located at top of A-beta sheet and beginning of RC loop
           • effects = destabilises A-beta sheet; lose salt bridge with K290; lose H-bond with T203
         • only 15% secreted, but once secreted is same as WT
           • CD indicates same thermodynamic stability
           • stop-flow fluorescence indicates Z converts from native to intermediate 1.5x faster than WT
             • so Z kinetically less stable than WT
         • treatments
           • augmentation therapy = inject AAT from blood donations; only 30% efficacy; $50-128k/year
           • double liver and lung transplant
           • repurpose CFTR drug 4-PBA = cheaper; improves Z secretion; binds specifically to breach region of AAT where Z mutation is
           • TTAI mimics RC loop and stabilises AAT
   • serine proteinase inhibitor superfamily
     • 70% homology but <30% sequence identity
     • structure
       • 3 beta sheets (platform) surrounded by 9 alpha helices
       • dynamic reactive centre loop at top
         • vulnerable to forming non-native conformations