Pathophysiology of Misfolded Proteins

8/13/2019 Pathophysiology of Misfolded Proteins

http://slidepdf.com/reader/full/pathophysiology-of-misfolded-proteins 1/12

Pathophysiology of Misfolded Proteins

Infection? We don't know

Comes from misfolded β amyloid protein○

Doesn't involve prion proteins○

β amyloid misfold into highly hydrophobic proteins → aggregate together and direct misfolding of other β amyloids →

to plaques and neuronal death

○

No holes in brain but there are plaques that prevent neuronal function○

Alzheimer's Disease:-

You get plaques that leads to neuronal death (sponge like)□

Prion proteins acts as chaperones - once one is misfolded, the direct others to misfold so the disease spreads

Primarily prion disease○

Human version of mad cow disease○

Brain is a solid structure

In the spongiform encephalopathy the brain looks like a sponge - has holes

Occur naturally in neuronal tissue - necessary for neuronal function□

Results of prion proteins

Infectious protein□

Problem occurs when these prion proteins become misfolded

Spongiform encephalopathy - your brain as it sits in your brain right now has consistency of raw egg○

Prion diseases can be caused by: genetics, result from spontaneous mutation in prion protein (that generates misfoldin

come from eating infected material

○

You can cook the meat and you won't destroy the protein - it doesn’t get broken down by enzymes in your stom

(radiation, ethanol, chemicals - nothing destroys the prion diseases)

You can't destroy the prion diseases - they are hydrophobic○

Cruzfeldt-Jacob Disease:-

Used as matrix for bone

Connective tissue of skin and eyes = collagen

Collagen○

Defects in collagen○

Genetic defect○

Don't result in misfolding of protein but they prevent collagen from assuming it's stable, native structure○

Osteogenesis Imperfecta:-

Alzheimer’s Disease & Cruzfeldt-Jacob Disease, Osteogenesis Imperfecta•

Friday, October 21, 2011

Cruzfeldt-Jacob Disease“Mad Cow Disease,” Scrapies•

Misfolded Proteins Drive Neurodegeneration•

Necessary for proper neuronal function○

Prion proteins = naturally occurring-

Prion diseases : spongiform encephalopathies•

PrPC is normally found on the extracellular face of the cell membrane.

YvonBiochem I T

Pathophysiology of Misfolded ProteinsWednesday, October 19, 2011

Biochem 1 - Test 2



Found on extracellular face of cell membrane-

On extracellular face and held to membrane by being anchored into the membrane (by a hydrophobic lipid)-

PrPc = normal prion protein•

Undergo endocytosis1.

Lysosomes = acidic (denatured)

Protein is degraded

Endocytotic vesicle goes to lysosome2.

How is the protein degraded at the end of its limited lifetime?-

Proteins have limited lifetime•

Alter physical ionization state of protein-

When protein is endocytosed, transferred to a lysosome, and is put in an acidic environment = acidic environment is denaturing•

Structure of PrPC and PrPSc Proteins

Picture on left: PrPc Picture on right : PrPSc

Normal Diseased

"Sc" = scrapies originally discovered in sheep)Primarily α helical Primarily β sheet

Hydrophilic Hydrophobic

PrPSc = one of the possible stable structures-

Prion protein enters lysosome, acidic environment denatures the prion protein and the prion protein refolds into another stable stru•

When PrPC refolds into PrPSc → the transformation from alpha helix to beta sheets expose a lot of nonpolar molecules on surface of •

Can be genetics○

Can be a spontaneous mutation in prion○

Or you can eat tissue that contains prion protein○

What causes PrPC to refolds into PrPSc?-

PrPSc can aggregate due to hydrophobic effect•

Figure 6-49A represents the native form (P

the prion protein and Figure 6-49B represe

the misfolded, pathogenic prion protein (Pr

Biochem 1 - Test 2



Prion = infectious protein

PrPSc Acts as a Chaperone for Folding of PrPC

Since they are hydrophobic, they aggregate due to hydrophobic effect○

Leads to formation of plaques (fibrils) of PrP Sc

Aggregation = irreversible○

Self propagated - once you have a misfolded prion protein, the number of misfolded prion proteins will continue to inleads to pathophysiology

○

PrPSc will act as template to fold any unfolded protein into PrPSc-

When PrPC gets unfolded it goes to a degraded state and you get PrPSc folding•

Isoforms of PrPSC lead to different prion pathophysiologies.

Isoforms act as templates to fold denatured PrPC protein - gives us different isoforms of fibrils of PrPSc○

Different isoforms leads to different diseases○

You can have different conformational ioforms of PrPSc-

PrPSc = primarily β sheets but they can have different conformations on how they are arranged in space•

It is hard to study these diseases because PrPSc = insoluble in water so you have to work with it in organic solvents (where yostudy what drive conformational changes in aqueous solutions)

-

Why do they assume different isoforms? We do not know•

Biochem 1 - Test 2



Progression of disease = rapid-

Aggregation○

Fibrils hit critical point (you feel good one day then over the next couple of days you won't anymore) - rapid progression-

Once fibrils start forming they lead to cell death•

PrPSc Aggregate into Rod-Like Clusters

Figure 6-48. Electron micrograph of prion rods. The black dots are colloidal gold beads coupled to anti-PrP antibodies used to identifprotein in the rod-like structure. Rod-like structures lead to formation of amyloid-like vacuoles that are the cause of neurodegenerat(Left side)

BOX 4-6 FIGURE 1 Stained section of cerebral cortex from autopsy of a patient with Creutzfeldt-Jakob disease shows spongiform (vadegeneration, the most characteristic neurohistological feature. The yellowish vacuoles are intracellular and occur mostly in pre- anpostsynaptic processes of neurons. The vacuoles in this section vary in diameter from 20 to 100 μm. (Right side)

White spots = fibrils-

Black dots = gold label antibodies-

Aggregates are intracellular - occurs when prion protein comes into cell and when clusters get to a certain size it leads to cell(and degeneration)

-

The rod (fibril) that the antibodies are attached to is a plaque-

Left picture:•

Caused by localized cell deaths by formation of the fibrils/plaques inside the cell○

White holes = spongie holes-

Right picture:•

Alzheimer’s Disease

Changes in Secondary Structure Become Deadly•

Amyloid-β is derived from amyloid precursor protein due to site-specific proteolysis by the enzyme γ-secretase.

Biochem 1 - Test 2



Origins = similar to prion disease-

Alzheimer's = is not a prion disease•

Carboxy terminal fragment-

Where do the misfolded proteins for Alzheimer's come from? - APP : amyloid precursor protein•

Amino terminus = intracellular•

Prion protein = purely extracellular protein that was anchored into protein-

Spans lipid bilayer and has a segment in extracellular fluid and segment in cytosol○

Degradation is different○

These enzymes break peptide bonds at specific regions in a transmembrane protein

Presenilin complex = ubiquitous

Necessary for normal functions

Enzymes in presenilin complex break down APP at three specific sites : epsilon, zeta, gamma site

All of the transmembrane like proteins are degraded by complex of enzymes called presenilin complex○

This protein is a transmembrane protein-

Protein has limited lifetime - at end of lifetime it gets degraded•

Presenilin at epsilon site = essential (if you disrupt it you will disrupt other internal processes in cell and cause trouble)-

At epsilon site it will yield fragment that is released intracellulary - can go and be a molecule in cellular signaling processes•

Zeta site ends up in segments in interior membrane - seem to be unimportant (they get degraded)•

Amyloid β protein is the one that misfolds and leads to Alzheimer's-

γ-cleavage site is performed by γ secretase-

Gsap = gamma secretase activating protein○

Not only for amyloid precursor proteins

Gsap binds to presenilin complex and stimulates γ secretase to cleave protein at γ site○

γ secretase = controlled by protein called Gsap-

γ-cleavage site : yields amyloid β protein•

Really big chemotherapeutic agent-

Tyrosine kinase inhibitor-

Slow down in formation of β amyloid plaques (inhibits formation)○

Slow down amyloid proteins = slow down plaque formation

Formation of amyloid plaques in Alzheimer's = directly related to number of amyloid β proteins being produced○

Gleevac binds to Gsap and inhibits Gsap activity-

Gleevac:•

Structure of Helical Fragment of β-Amyloid Protein

FIGURE 4-31b Formation of disease-causing amyloid fibrils. (b) The amβ peptide, which plays a major role in Alzheimer’s disease, is derived fro

larger transmembrane protein called amyloid-β precursor protein or APThis protein is found in most human tissues. When it is part of the largeprotein, the peptide is composed of two α-helical segments spanning th

Biochem 1 - Test 2



Hydrophilic•

Has alpha helices•

On opposite faces of helix (they do not see each other)○

Phenylalanine residues-

Two key residues on alpha helices:•

Proteolysis Yields Aggregating β-Sheet Structure

FIGURE 4-31c Formation of disease-causing amyloid fibrils. It can then assemble slowly into amyloid fibrils (c), which contribute to thcharacteristic plaques on the exterior of nervous tissue in people with Alzheimer’s. Amyloid is rich in β-sheet structure, with the β st

arranged perpendicular to the axis of the amyloid fibril. In amyloid-β peptide, the structure takes the form of an extended two-layer

β sheet. Others may take the form of left-handed β-helices (see Figure 4-21).Figure 6-50. X-ray crystal structure of amyloid fibril. Arrowheads indicate the path of the sheet but not necessarily the direction of thcomponent β-strands.

It doesn't stay alpha helix-

When amyloid β proteins misfold they misfold into a β sheet structure•

It can be a genetic predisposition-

Can be a spontaneous mutation in protein-

Can just happen-

What drives the misfolding?•

Once it happens, the β sheet structure is hydrophobic•

Like prions, proteins will aggregate•

Aggregate together and precipitate out = responsible for Alzheimer's disease-

How do β proteins aggregate? - they use the two Phe that can now interact with each other in the β sheet structure as a nucleation for aggregation

•

Formation of One β-Sheet Fragment Drives Formation of β-Sheet Structure in Other Fragments

larger transmembrane protein called amyloid-β precursor protein or APThis protein is found in most human tissues. When it is part of the largeprotein, the peptide is composed of two α-helical segments spanning thmembrane. When the external and internal domains (each of which havindependent functions) are cleaved off by dedicated proteases, the remand relatively unstable amyloid-β peptide leaves the membrane and losα-helical structure.

Formation of amyloid fibrils is driven by the hydrophobic effecFIGURE 4-31a Formation of disease-causing amyloid fibrils. (aProtein molecules whose normal structure includes regions ofsheet undergo partial folding. In a small number of the molec

before folding is complete, the β-sheet regions of one polypep

associate with the same region in another polypeptide, formin

the nucleus of an amyloid. Additional protein molecules slowl

Biochem 1 - Test 2



Water insoluble-

Once they form, they stay formed (not destroyed)-

Act as templates to direct folding of other β amyloid proteins that are cleaved off -

Misfolded β amyloid = very stable•

Once you start having β amyloids misfolding into β sheet structures - they drive the misfolding of other β amyloid proteins that are coff by gamma secretase

•

Amyloid plaque formation•

Reside on surface-

You don't have a rapid cell death - you do have decrease in neuronal function-

Alzheimer's disease = long, slow decline-

Prions = intracellular•

Usually people with Alzheimer's disease dies of causes other than Alzheimer's-

Eventually number of plaque reaches a number where you have cell death•

Osteogenesis ImperfectaCollagen Mutations Can Be Fatal•

Mutations in collagen•

Collagen forms a matrix-

Matrix = nucleation point for calcium phosphate participation and its structural integrity-

All of our bones have different structures but there is always collagen matrix behind them-

Bone = precipitate of calcium phosphate•

Tendons, Cartilage, Cornea, Bone Matrix

before folding is complete, the β-sheet regions of one polypep

associate with the same region in another polypeptide, formin

the nucleus of an amyloid. Additional protein molecules slowl

associate with the amyloid and extend it to form a fibril.

Figure 6-47. Photomicrograph of brain tissue fromindividual with Alzheimer’s Disease. The two circu

objects are plaques that consist of β-amyloid prot

surrounded by a halo of neurites from dead and d

neurons.

Biochem 1 - Test 2



FIGURE 4-11 Structure of collagen. (Derived from PDB ID 1CGD) (a) The α chain of collagen has a repeating secondary structure uniq

this protein. The repeating tripeptide sequence Gly –X –Pro or Gly –X –4-Hyp adopts a left-handed helical structure with three residue

turn. The repeating sequence used to generate this model is Gly –Pro –4-Hyp. (b) Space-filling model of the same α chain. (c) Three o

helices (shown here in gray, blue, and purple) wrap around one another with a right-handed twist. (d) The three-stranded collagen

superhelix shown from one end, in a ball-and-stick representation. Gly residues are shown in red. Glycine, because of its small size,

required at the tight junction where the three chains are in contact. The balls in this illustration do not represent the van der Waals

the individual atoms. The center of the three-stranded superhelix is not hollow, as it appears here, but very tightly packed.

Collagen forms: connective tissue in skin, tendon, cartilage, cornea (reason for blue sclera)•

Collagen = α helix-

Left handed helix-

Figure A:•

Collagen doesn't exist as single helix - forms more complex structure (coil of 3 helices of collagen)-

Ending helix (made up of three helices) = right handed helix-

Close packing - in order for left handed helices to fit together there has to be really close packing-

Figure C:•

Turn the coil on its side and look down the axis-

Symmetry of axis has close packing-

X can sometimes be lysine (doesn't always have to be)

-Gly-Pro-X-○

To have close packing, Gly are found in the center○

Proline and the other amino acid are on the exterior of the coil and doesn't matter about the size

In order for collagen triple helix to form, this is essential because only glycine is small enough to give us this close pack

other amino acid won't give us the close packing)

○

Primary structure of collagen has repeating sequence of amino acids-

Figure D:•

Biochem 1 - Test 2



Heads and tails line up-

How heads and tails line up gives us striated pattern-

Collagen fibril is composed of triple helix of collagen•

You have -Gly-Pro-X○

Prolines are converted to hydroxyprolines○

In a position where they H bond with each other - which stabilizes collagen triple helix○

H-bonding between hydroxy proline residues on surface-

Needs glycine for close packing and hydroxyproline to form hydrogen bonding within helix to stabilize it-

What is stabilizing the fibril?•

Amino termini and carboxyl termini line up to give striations- In between the collagen triple helices = cross links involving lysine residues-

Collagen triple helices associate to form a fibril•

Cross links (covalent bond) residues-

What stabilizes the fibril?•

FIGURE 4-12 Structure of collagen fibrils.

Collagen (Mr 300,000) is a rod-shaped moleculeabout 3,000 Å long and only 15 Å thick. Its threehelically intertwined α chains may have

different sequences; each chain has about 1,000

amino acid residues. Collagen fibrils are made

up of collagen molecules aligned in a staggeredfashion and cross-linked for strength. The

specific alignment and degree of cross-linking

vary with the tissue and produce characteristic

cross-striations in an electron micrograph. In

the example shown here, alignment of the head

groups of every fourth molecule produces

striations 640 Å (64 nm) apart.

Biochem 1 - Test 2



BOX 4-3 FIGURE 2 Reactions catalyzed by prolyl 4-hydroxylase. (a) The normal reaction, coupled to proline hydroxylation, which doerequire ascorbate. The fate of the two oxygen atoms from O2 is shown in red. (b) The uncoupled reaction, in which α-ketoglutarate oxidatively decarboxylated without hydroxylation of proline. Ascorbate is consumed stoichiometrically in this process as it is convertdehydroascorbate. 4-Hyp residues crosslink individual collagen helices within the right-handed superhelic by hydrogen bonds.

Iron is involved as an electron tranferate○

Hydroxyproline gets oxidized, succinate gets reduced, and iron is redox reagent○

In that reaction, it will take a proline residue, and alpha ketoglutarate residue and makes hydroxyproline and succinate-

Hydroxyproline = generated by enzyme called prolyl hydroxylase•

Succinate is being reduced but something has to get oxidized to the iron goes to iron +3 and that results in an inactive enzym-

If that happens, the proline in collagen can't be converted to hydroxyproline, H-bonding can't occur in triple helix, triple helix

becomes unstable and unfolded

-

Prolyl hydroxylase will take alpha ketoglutarate and spontaneously convert it into succinate•

Symptoms: bleeding gums (collagen = connective tissues)-

Vitamin C is necessary-

Scurvy = lack of vitamin C•

Vitamin D increases uptake of calcium•

An example of one type of cross-link between collagen molecules in a collagen fibril. Increasing the number of cross-links between cmolecules results in increased tensile strength and rigidity of the collagen fibril.

It involves two modified lysine residues-

Lysine residues are enzymatically modified and then join together to from the crosslink-

Dehydrohydroxylysinonorleucine = name of crosslink (CH=N)•

Biochem 1 - Test 2



Cross links covalent bond between the triple helices of collagen involving lysine residues•

Wednesday, October 26, 2011SDS-PAGE analysis of patient’s collagen and normal collagen.

C = control-

P = patient's-

α1 has 2 subunits

α2 has 1 subunit

Subunit composition of collagen in bones:○

There are no differences in control collagen and patient's collagen (in the reducing condition)○

What do the gels tell us about the patient's collagen?-

In picture:•

Most commonly fatty acids○

Reagent = detergent-

Uniform negative mass to charge ratio-

Makes everything similar in shape because it denatures the stuff ○

Denature-

SDS-PAGE analysis : sodium dodecyl sulfate polyacrylamide gel electrophoresis•

Reducing disulfide bonds-

Has something like having mercaptoethanol or dithiothreitol (common reducing agents for disulfide bonds)-

Originally you have -S-S- and they break to -SH HS--

-

Reducing conditions (picture on the left):•

Don't break disulfide bonds-

Still denaturing protein but you do not break bonds - they are still intact-

The gel is telling us that there is a difference○

"X" is twice the molecular weight of "2X" (in picture above)○

Non-reducing conditions:-

But on the gel you can't see weight difference because the gel can't detect the different in molecular weight beamino acids (they are close)

Substitution weighs more than the normal collagen○

Glycine has been switched for something normal collagen doesn't have-

Glycine is replaced by cysteine because it can form disulfide bonds

The one at twice the molecular weight has cysteine and is linked by disulfide bonds○

Some of the α1 must have cysteine substituted for glycine (the ones at the top of the gel has cysteine and is linked togby disulfide bonds - therefore are twice the molecular weight)

○

α1 = half at normal collagen, half at twice molecular weight-

α1 : some must be cross linked together-

The bigger the amino acid, the more severe the mutation because the triple helix is prevented more from formfrom (you need close packing)

Type of amino acid that replaces glycine○

(Disrupts the helix more or less)

Carboxy terminus = beginning

Big mutation near carboxy terminus - prevent helix from wrapping together□

If mutation occurs near amino terminus - part of helix are stuck together and you may have weakened f□

Mutations at carboxy = more severe than mutations at amino terminus

Where glycine mutation occurs○

Severe vs. non severe mutation:-

Non-reducing conditions (picture on the right):•

Biochem 1 - Test 2



Once helix is wrapped together and transported where they belong - peptides are cleaved off anhave active form of collagen

collagen but it would still be somewhat viable

Pathophysiology of Misfolded Proteins

Documents

Transcript of Pathophysiology of Misfolded Proteins