Unit II- Protein Misfolding and Disease

Question 1

In vivo folding with chaperones

Answer 1

• the chaperones are a family of proteins that prevent aggregation during folding
• there are 60- and 70- kDa heat-shock proteins (Hsp60 and Hsp70) are protein folding molecules
• expression is upregulated when cells are exposed to high temperatures (fever) or in other conditions where aggregation could be a problem

Question 2

Hsp70 proteins

Answer 2

• binds to proteins when they are in intermediate, but not native conformations
• they protect against aggregation by covering up sticky hydrophobic patches

Question 3

Hsp60 class

Answer 3

• 14 Hsp60 monomers associate to form a large hollow double donut structure
• misfolded proteins enter the cavity GroEL
• once inside the groES “cap” seals the cavity, changes interior residues from nonpolar to polar and ATP hydrolysis is used to physically unfold the misfolded protein
• they can refold properly while protected inside the cavity
• if they don’t fold by the time the GroES cap dissociates, the protein is expelled back into the cell and given another change to fold on its own
• proteins can fold faster inside the box than in solution

-ATP binding to GroEL allows the GroES cap to bin, which induces the conformational change in the cavity that simulataneously unfolds the misfolded protein and changes the residues lining the cavity from hydrophobic to hydrophilic. The GrosES has little or no affinity for the ADP-bound form of GroEL

Question 4

Protein degradation

Answer 4

• turnover is necessary; many cellular processes are regulated by ubiquitin/proteasome pathway
• proteasome inhibitors in clinical trials for cancer, HIV, cardiovascular disease
• many proteins are supposed to be only transiently active
• also plays a role in immunological response (MHC class I)

Question 5

Proteins targeted for destruction

Answer 5

• E1 enzyme activates ubiquitin in an ATP driven reaction that creates a high energy, covalent, thioester E1-ubiquitin bond
• one of several different E2 enzymes then transfer the activated ubiquitin to the target protein bound to a specific E3 enzyme, again via a thioester E2-ubiquitin intermediate
• E3 then catalyzes the final transfer to the epsilon amino group of one of more specific lysine residues on the target protein
• causes polyubiquitin chains of various length

Question 6

Degredation by proteasome

Answer 6

• proteasome is 26S particle (20S core particle and 19S regulatory protein caps)-there is six AAA-ATPase subunits of the 19S cap
• polyubiquitinated proteins bind to some of the non ATPase subunits of cap (need at least 4)
• the AAA-ATPase subunits then use ATP to unfold the bound protein
• the unfolded protein is fed through the small channel of the 20S core (De-ubiquitinated to be able to reuse)
• the 20S particle cleaves the protein into peptides 3-30 aa.
• peptides can be transported through ER for antigen presentation by MHC class I proteins are just recycled to build new proteins

Question 7

Disease of the ubiquitin-proteasome pathway

Answer 7

1) Cancer
- increased growth rates make cancer cells more dependent on proteasome
- increased degradation of tumor supressors (p53, p27)

2) Neurodegenerative diseases
- Alzheimer, Parkinson, Huntington diseases
- observe accumulation of ubiquitinated proteins in plaques, Lewy bodies
- not clear whether cause or by-product, but some disease causing mutations have been IDed

3) Cystic fibrosis
- clears misfolded DF508 CFTR

4) Autoimmune disease
- improper processing of peptide antigens

Question 8

Molecular basis of disease

Answer 8

• what is the protein’s function
• what is the defect, if any, that causes the disease
• what property of protein (structure, stability, function) does the defect change)
• how does this change lead to the symptoms
• how might a small molecule interact with the protein to fix the defect

Question 9

Mechanisms that can effect protein structure/function

Answer 9

• Direct knock out
• Destabilization
• Toxic conformation

Question 10

Direct knockout

Answer 10

• mutation of a residue that is essential for function
• the structure and stability of the proteins are essentially unchanged; it simply cannot function because a critical side chain has been altered

Question 11

Destabilization

Answer 11

• pushes the equilibrium toward the unfolded state
• the protein is traumatized by this mutation that it cannot muster enough energy to fold
• an example would be side chain in the tightly packed, hydrophobic core being changed to one of a substantially different size, shape or charge
• deletion of a stretch of amino acids

Question 12

Toxic conformation

Answer 12

• occurs when a mutation shifts the conformational equilibrium not to the unfolded state, but to an incorrectly folded state
• e.g. mutating a surface charged residue to a hydrophobic one which causes the protein to aggregate
• mutations can also cause the conformation of the protein to change more substantially, as in the amyloid disease

Question 13

p53 related cancers

Answer 13

• most frequently mutated protein in cancer
• ~50% of all tumors have point mutations in p53
• over 15,000 mutations to date
• TF
• activated by DNA damage or other insult
• triggers cell cycle arrest or apoptosis
• prevents accumulation of chromosomal mutations
• mutations in DNA occur frequently under natural circumstances- errors in replication, UV lught

Question 14

Mutations in p53

Answer 14

• p53 has a short C-terminal domain, a large central domain responsible for DNA binding, and N-terminal activation domain facilitates transcription by binding to other proteins and recruiting them to appropriate sites
• over 90% of mutations are in the DNA binding domain
• extensive B strands arranged in beta clamp gold
• binding site consists of alpha helix and loops

Question 15

Effects of mutations on p53 stability

Answer 15

• DNA contact mutants alter side chains that directly bind to DNA
• contact mutants act by simply reducing DNA binding without changing overall protein structure or stability
• stability mutants do not change DNA binding residues, often very distant from binding site
• stability mutants decrease protein stability by disrupting hydrophobic,electrostatic, H-bonding, or van der Waals interactions
• less stable p53 leads to faster degredation by the ubiquitin/proteasome pathway- not enough p53 around to do the job
• can also form aggregates

Question 16

Small molecule treatment for p53

Answer 16

• look at the solvent accessible surface area rendering
• try to find a cavity or pocket to which a small molecule can be tailored to fit
• this would only stabilize the native structure; the small molecule will in theory stabilize the folding of that protein and that protein only
• PhiKan083 binds the Y220C tumorigenic mutation of p53

Question 17

Inhibit the p53 MDM2 interaction

Answer 17

• block the interaction between the p53 and MCM2, the E3 ubiquitin ligase that recognizes p53
• these molecules can bind from either the MDM2 side or the p53 side
• the idea is to allow mutant p53 to accumulate in cells, so even though the specific activity of the mutant p53 is low, overall activity can be restored by elevating total p53 levels

Question 18

Cystic fibrosis overview

Answer 18

• fatal disease- ~30 years
• characterized by thick, sticky mucus in lung, pancreas, intestine
• also affects sweat, tear, and salivary glands
• inability to absorb nutrients- high infant mortality
• buildup of fluids in lungs- infection and lung degeneration
• 70% of cases caused by deletion of Phe508 in cystic fibrosis transmembrane conductance regulator
• most lethal mutation in Caucasian population
• gated chloride channel of unknown structure

Question 19

Structure of CFTR

Answer 19

• member of membrane proteins called ABC transporters
• pump various solutes in and out of the cell
• involved in multidrug resistance
• only nucleotide binding domain is known
• deletion of F508 has little effect on the function properties of native CFTR. Mutant can bind nucleotide and function just as well as wild-type
• folding pathway is changed
• takes much longer to fold, increasing chances of aggregation
• nearly all delta-F508 CFTR get stuck in ER- gets processes and degraded by ubiquitin/proteasome machinery
• much less CFTR makes it to the native state-not enough to do the job

Question 20

Treatment options for delta-F508

Answer 20

• 25 degrees -try to stop aggregates
• small organic molecules (glycerol, myoinositol, benzofalavones- not specific)
• overexpressing chaperones
• inhibiting degradation by ubiquitin/proteasome pathway

Question 21

Alpha1-antitypsin (Alpha1-AT) deficiency

Answer 21

• characterized by lung disease (emphysema) and liver disease)
• 20+ year decrease in life span if smoker
• 30% of southern Europeans harbor one of two mutations: Z-type or S type
• serine protease inhibitor- they are enzymes that bind and cleave the polypeptide chain at specific locations
• serpins bind to target protease and prevent it from binding substrate
• serpin is then cleaves but does not readily dissociate from enzyme- cleaved serpin cannot re-bind.
• one of major proteins present in plasma
• principle target in neutrophil elactase, which is released at sites of inflammation

Question 22

Conformational states of serpins

Answer 22

• 394 amino acid protein synthesized in liver
• serpin fold consists of three beta-sheets and nine-alpha helices
• reactive center is a stretch of 20 amino acids on the surface. The target protease cleaves an internal site in this segment
• X-ray crystal structure of cleaved alpha1-AT revealed a puzzle: ends of cleavage site were on opposite ends of ends of protein
• reactive center (region N-terminal to the cleave site) formed center sixth strand of large Beta-sheet
• solution of the uncleaved alpha1-AT structure showed reactive center forms a solvent-exposed alpha-helix that is far away from the Beta sheet which has five strands

Question 23

Mechanism of serpins

Answer 23

• mechanism: a molecular mousetrap, e.e. alpha1-AT uses stored energy to trap its target
• target protease binds the reactive center loop (RCL) of alpha1-AT and cleavees it
• the RCL is a frustrated beta strand- it would prefer to be in the middle of the alpha1-AT beta-sheet. Once cleaved, it can do so
• The RCL inserts into the beta-sheet and drags the protease along with it. In order for the loop to insert, the sheet has to split in the middle and open up

Question 24

Alpha1-AT mechanism

Answer 24

• active, inhibitory forms of the serpin. The enzyme target binds to the RCL and forms the michaelis complex
• P1 refers to the residue in the RCL that is primarily responsible for specificity
• after cleavage, the RCL integrates into beta sheet A and forms an additional strand. The catalytic Ser195 side chain remains covalently bonded to the carbonyl group of residue P1
• thus the protease is dragged to the opposite end of a1-AT. A recently solved X-ray structure of the alpha1-AT/protease complex shows that alpha1-AT forces the protease to partially unfold and become disorded
• the partially disordeded protease is now efficiently attacked by other cellular proteases which quickly degrade it

Question 25

Structural changes affects function

Answer 25

• antithrombin (serpin family) has novel anti-angiogenesis and tumor suppressing activity when it is cleaved or locked form
• thus the structural change causes function to swtich from protease inhibitor to angiogenesis inhibitor

Question 26

S type mutation in alpha1-AT

Answer 26

• the central beta sheet must be flexible enough to accept the Reactive Center loop (RCL) as part of normal inhibitory process
• if the beta sheet is structurally weakened, then it may be unusually prone to strand insertion, at premature or inopportune times
• polymerization occurs when the central beta sheet aberrantly opens and allows part of the reactive loop of a second protein to insert into the lower portion of the sheet
• beads on string structures in liver

Question 27

Mechanism of polymerization can be blocked

Answer 27

• can be blocked and even reversed by various peptides that correspond to portions of the RCL
• P1-P14 indicate the amino acid numbers of the RCL
• P2-P8 peptides are especially effective
• they bind in the pocket of the beta sheet where part of the RCL from a second antitrypsin molecule would occupy
• peptides as short as four amino acids inhibit polymerization in vitro
• the P3-P8 peptide from antithrombin and P2-P7 peptide from antitrypsin can partially reverse polymerization of WT antithrombin and Z-type antitrypsin respectively

Question 28

Prion diseases

Answer 28

Scrapie and Human Creutzfeld-Jakob disease, Kuru

Question 29

Scrapie (sheep), bovine spongiform encephalopathy (BSE), elk/deer wasting disease

Answer 29

• characterized by incessant rubbing,wasting, loss of coordination
• invariably fatal
• brains exhibit spongiform degeneration, nerve death
• found to be transmissible when sheep vaccine became contimainated
• amyloid fibers found in damaged areas of the brain. Diagnosed by staining with congo red and thioflavin T

Question 30

Human Creutzfeld-Jakob disease (CJD), Kuru

Answer 30

-similar spongiform degeneration, nerve death
-amyloid fibers detected in brain
-CJD can be transmitted to primates by direct imlpantation
-other sources of TSE’s in humans
+genetic
+sporadic
+cornea, dura mater grafts
+iatrogenic means
+human growth hormone, gonadotropin from cadavers

Question 31

PrPc

Answer 31

• normal cellular protein
• covalently linked to a glycolipid, which serves to anchor it to the outside of the cell membrane, and to sugar moieties called glycans
• expressed on brain and also many other tissues

Question 32

What is the infective particle of TSE

Answer 32

• infectivity not abolished by many conventional techniques (irradiation, heat)
• infectively reduced by protein denaturants (NaOH, sodum dodecyl)
• genetics: >20 mutations known to cause inherited forms of prion disease (Fatal familial insomnia and CJD share common primary site mutation
• proteins induce, no nucleic acid
• prion protein: 208 residue glycoprotein of unknown function, expressed in brain and many other organs

Question 33

Protein only hypothesis

Answer 33

1) There are two covalently identical forms of PrP:
PrPc-
-non pathogenic
-soluble
-protease sensitive
-40% alpha-helix, little Beta sheet

PrPsc-

• pathogenic
• insoluble
• protease insensitive
• 30 % helix, 45% heet

2) The two forms interconvert, equilibrium favored towards PrPc
3) When several molecules of PrPsc come in contact (very rare), they can bind via a beta sheet interaction
4) The resulting complex greatly stabilized beta sheet structure which does not dissociate readily. It serves as a nucleus; subsequent addition of converted monomers is rapid

Transgenic mouse studies also support protein-only model

Question 34

Model of PrPsc

Answer 34

• a-the beta helix motif is the building block for the amyloid fibril. The beta-helix consists of a trimer of beta sheets fromed by the N-terminal residues. Large aggregates are formed by vertically stacking PrP trimers along the beta-helical axis. The C-terminal region of PrP molecules is proposed to mostly retain its native structure
• b-the N terminal residues convert to beta strands and the C-terminal residues do not change their structure. However instead of forming the beta-helix trimer the N terminal residues form a continupis beta-sheet backbone to which the PrP subunits attach and spiral around.
• c- PrPsc as a stackof parallel, in register beta-strands
• PrP completely converting to an extended sheet

Question 35

Potential therapies for TSE- siRNA silencing of PrPc

Answer 35

• transgenic mice bearing 60 copies of PrPc gene

- made chimeric mice using stem cells expressing shRNA against PrPc

Question 36

Potential therapies for TSE- stabilizing PrPc: computer design and high throughput screening

Answer 36

-prevent PrPc from converting to the toxic species
-the idea is to develop compounds that bind specifically to the PrPc conformation and thus pull the equilibrium from the toxic PrPsc form.
Like the PhiKan083 binding to p53

Question 37

Potential therapies for TSE- immunotherapy

Answer 37

• immune system shows no ration to PrPc or PrPsc
• immunize using PrP-PrP dimer
• generates CD4 and CD8 T-cell response in mice
• biochemical manipulation of PrP-PrP structure (refolding conditions, ionic strength, pH) improves immunogenicity)

Question 38

Amyloid Beta-peptide

Answer 38

• produced by cleavage of APP by alpha, beta, gamma secretases
• 90-95% of Amyloid Beta in brain is AB40, 5-10% is AB42
• correctly cleaved APP eliminates possibility of AB42
• Alzheimer plaques: amyloid fibers virtually all AB42
• triplication of APP gene, either alone or trisomy 21, leads to AD
• > 100 mutations in catalytic subunit of gamma-secretase associated with early onset AD
• mutations increase ratio of AB40:AB42

Question 39

Structure of AB42

Answer 39

• in its amyloid fibril form consists of parallel, in-register beta sheet
• four copies of AB42 are shown
• each side chain on a single peptide is contacting the corresponding side chain on adjacent peptides
• the strands are also parallel, as they all point in the same orientation
• the fibril structure is stabilized by side chain-side chain interactions between strands within the same peptide, as well as between adjacent peptides
• most of these interactions are hydrophobic

Question 40

Alzheimer treatments on horizon

Answer 40

• mature amyloid fibers might not be as harmful
• the pre-fibrillar aggregates are real toxic and aggregate
• drug studies fail, damage was already done-maybe have to give medication earlier

Question 41

Islet amyloid polypeptide (IAPP)

Answer 41

• 37 residue peptide co-secreted with insulin by Beta cells in islets of Langerhans
• increased insulin production leads to increased levels of IAPP
• in late stages of diabetes, B-cell crisis due to large scale apoptosis
• IAPP amyloid observed in 90% cases post-mortem
• rodents and pigs do not develop diabetes
• transplanting pig Beta cells into humans is showing some promise

Question 42

Linkage between IAPP, obesity and diabetes

Answer 42

• in beta cells are producing normal amounts of insulin, and no amyloid is detected
• overeating leads to high levels of insulin production which leads to increased IAPP levels

Question 43

Structure of IAPP

Answer 43

• structureless insolution, but has helical tendencies
• it is known to bind lipid bilayers and lipid bilayers are reported to accelerate the rate of amyloid fibril assembly
• as helical wheel a hydrophobic face is apparent

Question 44

How does amyloid cause cell death

Answer 44

• soluble oligomers of IAPP are more toxic than mature fibrils
• IAPP disrupts integrity of cell membranes
• similar results for PrP, AB, alpha-synuclein
• hypothesis: pre-fibrillar species disrupt cell membranes

Question 45

Peptide inhibitors of AB and IAPP fibril formation

Answer 45

• targeting formation of amyloid fibrils-rational drug design
• all fibrils are beta sheets and therefore grow by forming both side chain-side chain interactions and peptide-peptide hydrogen bonds, from one peptide to the next
• extension can be blocked by peptides that share a similar amino acid sequence, so that side chain interactions can form to the endogenous peptides
• if the peptide nitrogens on the growing face of the beta strand are blocked (such as by methylation) then hydrogen bonds cannot form the next peptide

Question 46

Where folding goes wrong

Answer 46

• unfolding conformation to native conformation (destabilization from p53, slow folding from CFTR)
• native conformation to alternate conformation (destabilization for alpha1AT)
• alternate conformation to aggregate (protein deposition, AB42, IAPP, PrP, alpha1AT
• native conformation to direct knockout (active/binding site mutation (p53)