Disease related to misfolding (Protein misfolding diseases)

Yun-Ru Chen, Ph.D.

Protein folding in biological system

Chaperone assisted folding

In vivo protein folding (pulse-chase, location/trafficking, proyl isomerase, disulfide isomerase)

Disease related protein misfolding

Protein aggregations Protein degradation Chemical chaperone © 1995-2007 by Michael W. Davidson

and The Florida State University.

crowding

In vitro folding without chaperones

Definition of molecular chaperone:

A functional class of unrelated proteins that assist the correct noncovalent assembly of other polypeptide containing structures in vivo, but are not components of these assembled structures when they are performing their biological functions

Pro-domain or pro-sequence act like chaperone to guide mature protein (ex:subtilisin) toward the native stateChemicals act like chaperone

Pro Eu

TF family Trigger factor

NAC, GimC

Hsp70 family (ATPase)

DnaK Hsc/Hsp70, Saa, Sab

Hsp40

Family

J domain (DnaJ)

Hsp40

Hsp60 family (ATPase)

GroEL TRiC/CCT

Hsp10 family GroES

Small chaperone IbpA/B,

Hsp17

Hsp25,

A/B crystallin

GroEL-ES system in E.Coli

Chaperonin catalytic cycle

Cell, Volume 125, Issue 5, 2 June 2006, Pages 903-914

Structural Features of the GroEL-GroES Nano-Cage Required for Rapid Folding of Encapsulated Protein

•the structural features of the chaperonin cage are critical for rapid folding of encapsulated substrates. •Modulating the volume of the GroEL central cavity affected folding speed in accordance with confinement theory. •The chaperonin cage provides a physical environment optimized to catalyze the structural annealing of proteins with kinetically complex folding pathways.

Figure 1. Altering the Size or Charge of the GroEL Folding Cavity Can Have a Profound Influence on Protein Folding (Left) Wild-type GroEL/GroES with a folding substrate (green) is shown. (Middle) Changing the cavity size by deleting or replicating the motifs at the C terminus of each GroEL subunit affects substrate folding. (Right) Alteration of the surface charge of the cavity in a version of GroEL (named SR1) that contains only a single ring can also affect the folding of some substrates.

Cell. 2006 Jun 2;125(5):831-3.

Hsp70 multiple cellular location: cytosol, nu

cleus, ER, Mitochondria Soluble or tethered to ribosome, to

membrane

Trends in Biochemical Sciences Volume 32, Issue 8, August 2007, Pages 372-380

Small heat shock proteins

Enzyme that catalyzes folding

Peptidyl prolyl isomerase

(overcome kinetic barriers presented by incorrectly oriented peptide bonds)

(Cyclophilin, FK506-binidng proteins, parvulins)

Protein disulfide isomerase (PDI)

(thioredoxin, Dsb family)

Regulation of protein folding in the ER

Protein degradation pathway

Ubiquitin-Proteosome system Macroautophagy

Before they are targeted for proteasomal degradation, most proteins are covalently modified with ubiquitin (Ub). Typically, three enzyme types are involved in this process — ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes. Proteins tagged with chains of four or more ubiquitins are shuttled to the the proteasome by various proteins such as CDC48/p97. In the proteasome, proteins are reduced to peptides, which are then released into the cytosol and further broken down by peptidases. See the text for more details.

Nature. 2006 Oct 19;443(7113):780-6

Autophagy begins with the formation of double-membrane-bounded autophagosomes. The origin(s) of the autophagosome membranes are unclear. The mammalian target of rapamycin (mTOR) is a negative regulator of autophagosome formation, although how mTOR regulates this process in mammals is not clear. When mTOR is inhibited by rapamycin, autophagy is stimulated.Autophagosomes fuse with lysosomes to form autophagolysosomes, a process that is governed by a number of factors, including dynein activity. The contents of autophagolysosomes are finally degraded by acidic lysosomal hydrolases.The green dots represent the protein LC3 (also known as ATG8). This is the only known marker that specifically localizes to autophagosome and autophagolysosome membranes and not to other membranes. LC3 localizes to these structures after it has been processed and conjugated to phosphatidylethanolamine.

Protein misfolding problemProtein misfolding problemProtein dysfunction

Protein misfolding is no longer viewed as an experimental artifact.

From gene mutation to underpins diseases

Studies of protein folding have revolutionized our ideas of disease transmission

Protein misfolding causes diseases

I. Diseases associated with the loss of active protein

Sickle-cell hemoglobin anemia (2 point mutations out of 574aa.)

Cystic fibrosis ( 囊性纖維化 ) (508, CFTR)

P53 tumor suppression (mutation leads to half of all reported cancers)

Lysosomal storage diseases

II. Diseases associated with accumulation of aggregate

Amyloidosis

Sickle cell hemoglobinHemoglobin (oxygen-transport protein): single site mutation (E6V) in beta subunit,

Protein Structure The structure of normal deoxy hemoglobin is shown in Figure 1. The four gray clusters are noncovalently bonded heme groups. Each heme serves as a contact site for binding one molecule of oxygen. The two gold spheres near the top of the molecule are phosphate groups. The green and blue chains are alpha chains, and the gold and aqua chains are beta chains. The red box highlights the general region of two beta chains where the sixth glutamic acid residue is located. A genetic variant associated with this amino acid is the most common cause of sickle cell anemia. Figure 2 shows the clumping together of two deoxy hemoglobin molecules that contain this genetic variant.

Normal red cells maintain their shape as they pass through the capillaries and release oxygen to the peripheral tissues (upper panel). Hemoglobin polymers form in the sickle red blood cells with oxygen release, causing them to deform. The deformed cells block the flow of cells and interrupt the delivery of oxygen to the tissues (lower panel).

Cystic fibrosis

Mutations in one gene, called the cystic fibrosis transmembrane conductance regulator (CFTR), cause the body to make nonfunctional CFTR protein, which leads to the disease.

CFTR as a Cl Ion channel lost its function by single deletion at F508

most patients with CF would be to use a ligand(s) of CFTR that acts a pharmacological chaperone to correct the folding defect

Imaging shows the normal CFTR fragment (top) curling into a helix, while the mutant fragment unravels into random shapes, blocking it from migrating to the cell surface, where it would prevent symptoms.

http://www.hopkinsmedicine.org/hmn/F99/mu_8.html

RESEARCH

PRE-PHASECLINICAL

PHASE PHASE TO

1 2 3 PATIENTS

Gene Therapy

Protein Assist/Repair

 

Restore Salt Transport 

 Mucus Treatment

 Anti-Inflammatory

 

Anti-Infective

 

Transplant Drugs

Nutritional Supplements

P53 mutation and Cancer Half of all known cancers

involve some mutation in p53, the so-called guardian of the cell.

P53 is a tumor suppressor which signals for cell death if their DNA gets damaged. If these cells didn't die, their damaged DNA would lead to the strange and unusual growths found in cancer tumors and this growth would continue unchecked, until death.

When p53 breaks down and does not fold correctly (or even perhaps if it doesn't fold quickly enough), then DNA damage goes unchecked and one can get cancer.

http://folding.stanford.edu/English/FAQ-Diseases#ntoc2

http://p53.free.fr/Database/p53_cancer_db.html

Newly synthesized proteins in the living cell must go through a folding process to attain their functional structure. To achieve this in an efficient fashion, all organisms, including humans, have evolved a large set of molecular chaperones that assist the folding as well as the maintenance of the functional structure of cellular proteins. Aberrant proteins, the result of production errors, inherited or acquired amino acid substitutions or damage, especially oxidative modifications, can in many cases not fold correctly and will be trapped in misfolded conformations. To rid the cell of misfolded proteins, the living cell contains a large number of intracellular proteases, e.g. the proteasome, which together with the chaperones comprise the cellular protein quality control systems. Many inherited disorders due to amino acid substitutions exhibit loss-of-function pathogenesis because the aberrant protein is eliminated by one of the protein quality control systems. Examples are cystic fibrosis and phenylketonuria. However, not all aberrant proteins can be eliminated and the misfolded protein may accumulate and form toxic oligomeric and/or aggregated inclusions. In this case the loss of function may be accompanied by a gain-of-function pathogenesis, which in many cases determines the pathological and clinical features. Examples are Parkinson and Huntington diseases. Although a number of strategies have been tried to decrease the amounts of accumulated and aggregated proteins, a likely future strategy seems to be the use of chemical or pharmacological chaperones with specific effects on the misfolded protein in question. Positive examples are enzyme enhancement in a number of lysosomal disorders.

Metabolic diseases: lysosomal storage diseases A genetic disease that occurs when the cata

bolism of glycocojugates is impaired and result in accumulation of the products in lysosome.

More than hundred kinds of single site mutants effect correct folding of the enzymes which leads to deficient lysosomal enzyme activity.

Fabry: -galactosidaseGaucher: -glucosidase(Pombe: :-glucosidase)

Gal4GalCer

GlcCer

Ceramide

Gal3GalNAc4

Gal4GlcCerNeuAc3

GalNAc4

Gal4GlcCerNeuAc3

GM1

-galactosidaseGM1 Ganliosidosis

-HexATay-Sachs Disease

GM2

GM3Gal4GlcCer

NeuAc3

-galactosidaseFabry Disease

Gal4Gal4GlcCer

GalNAc3Gal4Gal4GlcCer

Globoside

-HexA,BSandhoff Disease

-glucocerebrosidaseGaucher Disease

-galactosidaseneuraminidase

Deficient Enzyme : Deficient Enzyme : Lysosomal Storage DiseasesLysosomal Storage Diseases

Fabry disease - causes kidney and heart problems, pain and a skin rash Gaucher disease - causes the spleen to enlarge, anemia and bone lesions if untreated Hurler syndrome - causes deformities of the skeleton and facial features, enlargement of the spleen and liver, joint stiffness, clouding of the cornea, mental retardation and deafness Niemann-Pick B disease - leads to enlargement of the spleen and liver, as well as lung disease Pompe disease - an often fatal storage disease in which glycogen builds up in the liver, heart and muscle, especially during infancy (also known as acid maltase deficiency) Tay-Sachs disease - a lysosomal storage disease that occurs more commonly in people of Eastern European Ashkenazi descent and causes degeneration of the brain in infants

Deficient Enzyme :Deficient Enzyme : Glycogen storage disease

Type II: Pompe disease, deficient -glucosidase Type I: von Gierke disease, deficient glucose-6-phosphat

ase (100% mis-sense), mouse and dog model available. (Director YT Chen, IBMS)

Structure, Folding, Protein not available

Glucose-6-phosphatase converts glucose-6-phosphate into free glucose and is active in the lumen of the endoplasmic reticulum, where it is bound to the membrane

Glucose-6-phosphatase

Faces of GSD1

-galactosidaseFabry

GaucherPombe

Current therapy: Enzyme replacement (costly and not the best yet)Chemical chaperones were shown to stabilize the enzyme against misfolding, increasing proper trafficking from ER.

Yu, Zhanqian, Sawkar, Anu R. & Kelly, Jeffery W.

Pharmacologic chaperoning as a strategy to treat Gaucher disease.FEBS Journal 274 (19), 4944-4950.

Selectivity of inhibitors with related hydrolases. Inhibitors were tested on GC, α-glucosidase (α-Gluc), α-galactosidase (α-Gal), and β-N-acetylglucosaminidase (HEX). Data represent the results of three independent experiments performed with three replicates per sample. (A) Compound 1, an aminoquinoline derivative. (B) Compound 2, a sulfonamide derivative. (C) Compound 3, a triazine derivative. (D) Nonyl-DNJ (compound 5), an iminosugar.

Proc Natl Acad Sci U S A. 2007 August 7; 104(32): 13192–13197.

Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease

Cell-permeable chemical chaperones (dark red triangles) act as a template for variant lysosomal enzymes as they fold in the ER. Some of these lysosomal enzymes would otherwise misfold and be retained in the ER and ultimately be degraded by the proteasome. Chemical chaperones facilitate folding in the ER lumen, which is necessary for proper trafficking to the lysosome where a high concentration of substrate (red triangles) exists to compete with the chemical chaperones that bind to the active site. Although the chemical chaperones are crucial for folding at pH 7 (for example, in the ER), the variant lysosomal enzymes might not need the chaperones when they arrive in the lysosome (where the pH is about 5) because their sequences are stably folded at this pH.

transthyretin