2. Assisted Protein Folding by Chaperons
3. Enzymes involved in Folding Pathways
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4. Protein Misfolding and related Diseases
Introduction
? Proteins are synthesized in ribosome as a linear sequence of amino acids (aa).
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? Newly synthesized polypeptide folds into its characteristic and functional 3-D
structure via a physical process known as protein folding.
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? Interactions among aa lead to formation of a folded 3-D structure known asnative protein.
? 3-D structure is determined by its aa sequence.
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Protein folding in sequential manner
1. Newly synthesized polypeptide
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chain emerges from theribosome, short segments fold
into secondary structural units.
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(Image by MIT Open Course Ware, adapted from image by Professor Jonathan King)
Cont--
2. Folding proceeds via an initial clustering among side chains of hydrophobic
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residue which prefer to be aloof from an aqueous environment.
? Clustering due to non-specific interaction among the hydrophobic residues that
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lead to the formation of a compact arrangement (molten globule state).? Hydrophobic residues of proteins gather inside the collapsed forms within the
core.
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? Collapsed state favors the formation of secondary structure and encourages
tertiary interaction among the residues.
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Assisted Protein Folding? Most Proteins fold spontaneously to their native.
? Cells have a variety of mechanism to ensure proteins are folded correctly by
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specialized proteins.
? Molecular chaperones interact with partially folded or improperly folded
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polypeptides, facilitating correct folding pathways.? Chaperones provide microenvironments in which folding can occur.
Cont--
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Chaperones: Hsp70 family of chaperones binds short sequences of hydrophobic aathat emerge while a new polypeptide is being synthesized, shielding them from
solvent.
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? Protein chaperones aid protein folding in two basic ways:
a) Assisted folding and assembly.
b) Binding of unfolded proteins to prevent aggregation.
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Cont--2. Chaperonins: Hsp60 acts as chaperonins in the folding process, together with an
Hsp70 chaperone.
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? Central cavity of the Hsp60 chaperone provides a sheltered environment in which
a polypeptide fold until all hydrophobic regions are buried in its interior, thus
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prevent protein aggregation.Chaperone-assisted protein folding
Fig.8.23: The Cell, 4th ediiton
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Enzymes involved in Protein Folding Pathways1) Protein disulfide isomerase (PDI):
? Protein-suflhydryl oxidase catalyzes oxidation of cysteine residues to form
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disulfide bonds.
? Cysteine form a disulfide bond with cysteinyl residue.
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? By catalyzing disulfide exchange, the rupture of an S-S bond and its reformationwith a different cysteine.
? Protein disulfide isomerase facilitates the formation of disulfide bonds that
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stabilize a protein's native conformation.
Cont--
? Eukaryotic sulfhydryl oxidases are flavin-dependent, dietary riboflavin deficiency
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accompanied by an increased incidence of improper folding of disulfide-
containing proteins.
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Cont--2) Peptide prolyl cis-trans isomerase (PPI):
? All X-Pro peptide bonds--where X represents any residue-- are synthesized in
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the trans configuration.? X-Pro bonds of mature proteins, ~6% are cis. Cis configuration is common in
turns.
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? Interconversion of the cis and trans-isomers of peptide bonds with the proline by
Cyclophilins.
? Cyclophilins participate in the folding of proteins expressed by viral invaders.
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? Cyclophilins are pursued as targets for development of drugs such as
cyclosporine.
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? Alisporivir for the treatment of HIV, hepatitis C and other virally transmitteddiseases.
Protein Misfolding and Diseases
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Incompletely and incorrectly folded proteins present two serious problems to the
cells:
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1. Loss of function due to absence of correctly folded protein:? Cystic fibrosis
Cystic Fibrosis (CFTR)
? Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane
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protein and encoded by CFTR gene.
? CFTR gene codes for an ABC transporter-class ion channel protein that conducts
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chloride ions cross epithelial cell membranes.? Mutations of CFTR gene affect chloride ion channel function leads to
dysregulation of epithelial fluid transport in lung, pancreas resulting in cystic
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fibrosis.
Cont--
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? Caused by deletion of a 3 nucleotides which results in a loss of aa (Phe) residue at508th position, causes improper protein folding.
? Improved understanding of protein folding may lead to new therapies for CFTR.
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2. Aggregation of incorrectly folded proteins:? Beta-Thalassemias
? Alzheimer 's disease (Amyloid beta)
? Mad Cow Disease (Prion)
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Alzheimer 's disease (Amyloid beta)? Refolding or misfolding of -amyloid protein endogenous to human brain tissue.
? Senile plaques and neurofibrillary bundles contain aggregates of the protein -
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amyloid.
? A 4.3-kDa polypeptide produced by proteolytic cleavage of a larger protein
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known as amyloid precursor protein.Cont--
? Levels of -amyloid become elevated.
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? This protein undergoes a conformational transformation from a soluble -helix to-sheet and prone to self-aggregation.
? Apolipoprotein E is a potential mediator of this conformational transformation.
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Mad Cow Disease (Prion)
? Prion diseases, are fatal neurodegenerative diseases characterized by spongiform
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changes, astrocytic gliomas, and neuronal loss resulting from the deposition ofinsoluble protein aggregates in neural cells.
? They include Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine
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spongiform encephalopathy (mad cow disease) in cattle.
Cont--
? Human prion-related protein (PrP), a glycoprotein encoded on the short arm of
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chromosome 20, monomeric and rich in -helix.
? Normal form of protein is PrPC, while the infectious form is PrPSc.
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? PrPSc has a higher proportion of -sheet in place of -helix structure.? Aggregation of these PrPSc form highly structures fibers, accumulates to form
plaques.
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Beta-Thalassemias
? Caused by genetic defects that impair synthesis of one of the polypeptide
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subunits of hemoglobin (Hb).? During the burst of Hb synthesis, a specific chaperone called -hemoglobin-
stabilizing protein (AHSP) binds to free Hb -subunits awaiting incorporation into
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the Hb multimer.
? In absence of AHSP, free -Hb subunits aggregate, and resulting precipitate has
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cytotoxic effects on the developing erythrocyte.Summary
? In protein folding steps, regions of secondary structure may form, fol owed by
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folding into supersecondary structures.? Large ensembles of folding intermediates are rapidly brought to a single native
conformation.
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? For many proteins, folding is facilitated by Hsp70 chaperones and by chaperonins.
? Disulfide bond formation and the cis-trans isomerization of Pro peptide bonds are
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catalyzed by specific enzymes.Group Discussion
? Subtopics of previous class discussed in group discussion.
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Reference Books1) Harper 's Il ustrated Biochemistry-30th edition
2) Biochemistry 7th edition by Jeremy M. Berg, John L. Tymoczko and Lubert Stryer
3) Lehninger Principles of Biochemistry
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26
Thank you