Conformational diseases. When little mistake might cause large troubles

Conformational diseases When little mistake might cause large troubles

Objectives: Understanding protein misfolding diseases Fate of the newly synthesized polypeptide chain: to fold, non-fold or misfold; Abundance of conformational diseases; What can cause protein to misfold? Molecular mechanisms of protein misfolding; Major consequences of misfolding: α-synuclein and Parkinson s s disease as a case study; Nanotools for megaproblems: Nanomedicine and protein misfolding diseases Bioinformatics and protein misfolding diseases

The route from the DNA code to the protein The DNA is responsible for coding for all proteins. Each amino acid is designated by one or more set of triplet nucleotides. The code is produced from one strand of the DNA by a process called "transcription". This produces mrna which then is sent out of the nucleus where the message is translated into proteins. The cartoon to the left shows the basic sequence of transcription and translational events.

Fate of a polypeptide chain Uversky (2003) Cell. Mol. Life Sci. 60 1852 Folding Native coil Non-folding Polypeptide Chain Native PMG Misfolding Native MG

Many proteins have complex 3D shapes which determine their Hemoglobin functions Prion protein Porin Tubulin

Mechanisms of protein folding Folding through the funnels Frame-work model Dobson, C. M. (2003) Nature 426, 884-890 890 Coil PMG MG N O.B. Ptitsyn DAN SSSR 210 (1973) 1213-1215 1215

Protein non-folding problem Protein stays substantially unfolded Protein partially folds to a pre-molten globule-like like state Protein partially folds to a molten globule-like like state

Protein misfolding problem Ordered Native MG Native coil Native PMG Amyloid fibril

Abundance of conformational diseases More than 450 different disorders, which are known to affect all organs; They can be hereditary or sporadic; More than 25 different proteins in amyloidoses only, which prior to fibrillation have different structures (β-sheets, α-helices, β-helices, or contain both α-helices and β-sheets); They may be globular proteins with rigid 3D-structure or be intrinsically disordered (even natively unfolded)

Disease Hypercholesterolaemia Cystic fibrosis Phenylketonuria Huntington s s disease Marfan syndrome Osteogenesis imperfecta Sickle cell anaemia αl-antitrypsin deficiency Tay-Sachs disease Scurvy Alzheimer s s disease Parkinson s s disease Scrapie/Creutzfeldt-Jakob disease, kuru Familial amyloidoses Retinitis pigmentosa Cataracts Cancer Protein Low-density lipoprotein receptor Cystic fibrosis trans-membrane regulator Phenylalanine hydroxylase Huntingtin Fibrillin Procollagen Haemoglobin αl-antitrypsin β-hexosaminidase Collagen β-amyloid/presenilin,, tau protein α-synuclein Prion protein Transthyretin/lysozyme Rhodopsin Crystallins p53 Site of folding ER ER Cytosol Cytosol ER ER Cytosol ER ER ER ER Cytosol ER ER ER Cytosol Cytosol

What can cause protein to misfold? When good proteins go bad DNA mutation causes the production of a protein that cannot fold when initially synthesized by a ribosome; Mutations cause the production of a protein that is destabilized and thus unfolds easier once folded; Stresses during the lifetime of the protein modify it causing it to be destabilized and partially unfolded; Environmental factors (metals, pesticides, toxins) can modify proteins making them more prone to misfold; Posttranslational modifications can change protein structure; Proteolytic cleavages can produce amyloidogenic peptides;

Folding-misfolding alternative w3.dbb.su.se/ ~oliveberg/aims.htm Dobson, C. M. (2004) Sem. Cell Develop. Biol. 15, 3-16.

Protein Aggregation When proteins are partially or completely unfolded they are more susceptible to aggregation; Exposure of hydrophobic segments to water is generally unfavorable; During aggregation, several destabilized proteins bind together; Aggregates are highly stable; Once the polymerized species becomes large enough, it becomes insoluble and falls out of solution;

3D structure and cross-β model for insulin fibrils Jiménez et al. (2002) Proc. Natl. Acad. Sci. USA 99:9196-9201

Protein Aggregates Insoluble; Usually very large; Very difficult for cells to breakdown; Not easy to transport; Continues to grow over time and may even recruit properly folded protein; Often (but not always) toxic to cells;

Multi-Facial Aggregates

Molecular Mechanisms of Protein Misfolding Diseases Altered protein structure and enhanced aggregation AD, PD, prion disease, AL and AA amyloidoses,, type II diabetes, dialysis-related amyloidosis Point mutations and enhanced protein deposition Familial amyloid polyneuropathy, systemic non-neuropathic neuropathic amyloidosis,, familial cases of AD and PD Altered protein structure and impaired protein function SOD1, p53 Changes in supra-molecular structure Muscular dystrophies, cataracts, sickle cell anemia Altered protein structure and changes in cellular/nuclear function Polyglutamine diseases Posttranslational modifications Glycation (cataracts, muscular dystrophies); phosphorylation (AD)

Protein Misfolding Diseases: Alzheimer s s Disease: Aβ protein Over time more and more destabilized protein forms and eventually with age there is enough to aggregate Aggregated protein forms long fibers that wrap themselves around the base of the nerve cells www.ahaf.org/alzdis/ / about/amyloidplaques.htm www.namenda.com

Protein Misfolding Diseases: Mad Cow Disease and Prion Protein Similar to Alzheimer s diseases in that long neurofibrillary tangles are formed by aggregated prion protein; The protein is the infectious particle playing a game of aggregation tag in your cells

Protein Misfolding Diseases: Huntington s s disease and Huntingtin Polyglutamine repeat in the Huntingtin protein of some people causes self association of the protein in neurons; Cytoplsmic inclusions are formed that kill nerve cells

Protein Misfolding Diseases: Amyotrophic Lateral Sclerosis and SOD1 Normal SOD1 performs the crucial role of scavenging potentially damaging superoxide ions. There are 114 SOD1 mutants implicated in familial ALS cases. Mutations in SOD1 causes protein destabilization and enhanced propensity to aggregate. Aggregated SOD1 leads to the death of the motor neurons, which control movement. When this happens, the brain can no longer direct the muscles of the body. This eventually leads to the muscle atrophy.

Protein Misfolding Diseases: Cancer and p53 protein Protein p53 is the tumor suppressor, which is a transcription factor that is at the center of a network of interactions that affect the cell cycle and apoptosis; It is inactivated in virtually all cancers; In about 50% of cancers p53 is directly inactivated by mutation; There Oldfield et al. al. (2005) Biochemistry 44(37):1245444(37):12454-70 are >250 cancerassociated mutations in p53 gene, vast majority is located in DNA-binding domain

Protein Misfolding Diseases: Sickle Cell Anemia and Hemoglobin Aggregate is not toxic to cells, but causes cells to be elongated; Limits cells ability to bind oxygen http://rad.usuhs.mil/sickle/index.html

Protein Misfolding Diseases: Cataract and crystallin proteins Cataract: crystallin proteins The leading cause of blindness world-wide; wide; 50% of people over the age of 80 have cataracts; Likelihood of developing cataracts increases exponentially with age

Protein Misfolding Diseases: AD and tau protein In In norm, tau interacts with tubulin and promote its assembly into microtubules; Regulated by alternative splicing and phosphorylation: Six splicing isoforms,, multiple phosphorylation sites; Hyperphosphorylated tau self-assemble into tangles of paired helical filaments and straight filaments in AD and other taupathies

Protein Misfolding Diseases: Parkinson s s disease and α-synuclein Second most common neurodegenerative disease; An environmental disease (pesticides and metals); Due to death of dopaminergic neurons in the substantia nigra; Pathological hallmark is intracellular proteinaceous inclusions called Lewy bodies and Lewy neurites; Major protein component in LBs and LNs is α-synuclein; Substantial evidence implicates the aggregation of α- synuclein as a key factor in the etiology of Parkinson s disease

The basal ganglia controls movement and balance in the body

Nerve cells in basal ganglia send messages that signal the body to move Nerve cells in basal ganglia send messages that signal the body to move In PD, many nerve cells are damaged and do not produce enough dopamine to carry signals properly.

Where is substantia nigra? Cross-section section of the brain

What is substantia substantia nigra nigra doing? In the substantia nigra, the neurotransmitter dopamine is produced and stored. The substantia nigra is connected to other parts of the brain by nerve cells that transmit dopamine.

Brain regions involved in Parkinson s s disease

Pathology of Parkinson s s disease: Neurodegeneration

Pathology of Parkinson s s disease: Lewy bodies in substantia nigra

Structure of Lewy body Typical LBs appear as intracytoplasmic inclusions, 5-25 μm m in diameter with a dense core of filamentous and granular material that is surrounded by radially oriented filaments.

The relation between α-synuclein aggregation and Parkinson s s disease Parkinson s s disease is a movement disorder first described by James Parkinson in 1817. Symptoms are: paucity of spontaneous movement (bradykinesia, hypokinesia), muscle rigidity, characteristic tremors, shuffling gait, and impaired balance Normally affects people > 60 years of age ( ( 1 M in US) Pathologically, PD is characterized by the presence of Lewy Bodies (cell body) and Lewy neurites (axon) in the substantia nigra In 1997 the gene responsible for familial early onset PD was shown to be α-synuclein.. Only 3 kindreds shown to have familial early-onset PD) α-synuclein shown to be major component of Lewy Bodies and Lewy neurites

α-synuclein and Parkinson s disease α-synuclein is the major fibrillar component of LBs; Point mutations in α-synuclein cause early-onset of PD; Triplication of α-synuclein cause early-onset of PD; Transgenic mice and flies producing α-synuclein develop the motor deficits and neuronal inclusions reminiscent to PD;

Structural features and conformational behavior of α-synuclein [θ] (deg cm 2 dmol -1 ) 0-5000 -10000-15000 ph 7.5 ph 3.0 92 o C 190 200 210 220 230 240 250 [θ] 222 (deg cm 2 dmol -1 ) 520-2000 510 500-2500 490 480-3000 470 0 2 4 6 8 10 λ ANS max (nm) Wavelength (nm) -1000 ph Normalized absorbance ph 7.5 ph 3.0 Fibrils [θ] 222 (deg cm 2 dmol -1 ) -1500-2000 -2500-3000 1700 1650 1600-3500 0 20 40 60 80 Wavenumbers (cm -1 ) Temperature ( o C) Uversky et al.(2001) J.Biol.Chem.. 276, 10737

Fibrillation of α-synuclein in vitro Effect of decrease in ph (A) or temperature increase (B) on fibrillation kinetics of human α-synuclein 400x10 3 ph 4.1 A 57 o C B 250x10 3 ThT fluorescence 300x10 3 200x10 3 100x10 3 ph 2.8 ph 1.9 ph 5.8 ph 7.2 ph 8.5 47 o C 37 o C 25 o C 200x10 3 150x10 3 100x10 3 50x10 3 ThT fluorescence 0 0 20 40 60 80 Time (hours) 0 50 100 150 200 Time (hours) 0 Formation of the partially folded intermediate (low ph or high temperatures) accelerates fibrillation process Uversky et al.(2001) J.Biol.Chem.. 276, 10737

Factors modulating structure and aggregation of α-synuclein Macro-environment: ph, temperature; Amino acid substitutions; PD environmental risk factors (metals, pesticides); Oxidative modification (nitration and methionine oxidation); Crowded environment; Membrane field; Structure of water; Interaction with binding partners;

α-synuclein is a protein- chameleon Uversky (2003) J. Biomol. Struct. Dyn.. 21, 211.

Misfolding or misrecognition? Uversky et al.. (2005) J. Mol. Recognit. 18(5):343

Multifactorial Model for Parkinson s s Disease Genes Genetic susceptibility Environment e.g. A30P A53T e.g. pesticides and metals α-synuclein aggregation Loss of dopaminergic neurons Parkinson s s disease

Conformational prerequisites for amyloidosis.. A model Uversky & Fink (2004) BBA 1698, 131 1a 3a 1b 1c 1d 2 4a 4b 4b There is a unifying mechanism of protein aggregation and fibrillation which involves formation of aggregation-prone partially folded conformation(s). 1e 3b 4c

Nanotools for Megaproblems: Nanomedicine and protein misfolding diseases Why are nanomedicine and nanotools? What are nanotools for? Nano-glasses, nano-toolboxes and nano-containers

Nanomedicine According to Wikipedia, nanomedicine is the medical application of nanotechnology and related research. It covers areas such as nanoparticle drug delivery and possible future applications of molecular nanotechnology. Consider this point wider: To work with misfolded proteins (subcellular( level) one need nano-glasses and nanotools

Nano-glasses: New means to see deeper www.tigerpath.com/ 25X 450X 2,000x 50,000X 100,000X

Active nano-glasses or nanotools? Atomic Force Microscopy Concept of AFM and the optical lever: (left) a cantilever touching a sample; (right) the optical lever.

Measuring the strength of protein- protein interactions by AFM

Fatal attraction: AFM analysis of protein aggregation McAllister et al.. (2005) JMB,, 354(5):1028-42 42 Force, pn Force, pn 1500 1000 500 0-500 1000 500 0-500 Counts 80 60 40 20 0 100 200 Retraction, nm Counts 10 5 ph = 9.8 0 0 400 800 1200 Force, pn -1000 0 100 200 Retraction, nm A B ph = 3.7 0 0 400 800 1200 Force, pn 1500 1000 500 0-500 1500 1000 500 0-500 Counts 30 20 10 ph = 2.0 0 0 400 800 120 Force, pn 0 100 200 Retraction, nm Counts 30 20 10 0 100 200 Retraction, nm C D ph = 1.0 0 0 400 800 1200 16 Force, pn [θ] 220 x 10-3 (deg cm 2 dmol -1 ) Force, pn 300 200 100 ΔpH 0 0 2 4 6 8 10 ph tr -2-3 -4-5 -6-7 -8-9 0 2 4 6 8 10 12 ph ph

Nanotweezers: Probing the inter-filament interaction AFM pulling experiments: The AFM tip was guided to selected points on the α- synuclein fibril (indicated by arrows) and the interaction between filaments within the fibril was measured by pulling the tip away from the surface (see insert (i)). The characteristic saw-tooth pattern indicates the unzipping of the fibril. Kransnoslobodtsev et al. (2005) Nanomedicine. 1(4):300-305

Nano-containers: Systems for the Targeted Drug Delivery Kabanov & Batrakova (2004) Curr Pharm Des.. 10(12): 1355 Protein/polyelectrolyte complex A B PPO block PEO block PEO-PPO PPO-PEOPEO Nanocontainers for protein delivery: the core represents the polyion complex of proteins and synthetic polyelectrolytes of opposite charge. The PEO-PPO PPO-PEO PEO chains grafted to the polyelectrolyte within the core form a micelle-like like structure around the core, with the hydrophobic PPO [poly(propylene[ oxide)] blocks near the core and the hydrophilic PEO [poly(ethylene[ oxide)] blocks forming the exterior shell. Additional non-modified PEO-PPO PPO-PEO PEO chains fill in to optimize the stability of the micelle-like like structure. Using this approach, a model impermeable protein, horse radish peroxidase (HRP), was successfully delivered into the living bovine brain microvessel endothelial cells

Where is the place for bioinformatics in this picture? Database development and maintenance; Development of the informatics tools for the large scale analyses; Classification and systemization; Development of the predictive tools; Systemic analysis; Model development; Drug development; etc., etc., etc.

Understanding the role of charge and hydrophobicity in protein aggregation Mean net charge 0.15 0.10 0.05 Unfold without accumulation of Intermediates. Less prone to aggregate Able to form equilibrium intermediates. More prone to aggregate Many, but not all, globular proteins have been shown to have compact intermediate state(s) under equilibrium conditions in vitro. Those who form intermediates are more prone to aggregate, whereas non-formers are less sticky. 0.00 0.40 0.45 0.50 Uversky (2002) FEBS Lett.. 514, 181 Mean hydrophobicity Charge to hydrophobicity ratio of a polypeptide chain may represent a key determinant in this respect.

Role of charge in aggregation Aggregation is favoured by macromolecules with an opposite charge Amount of precipitation of proteins induced at neutral ph by amyloid fibrils of AβA 25-35 (expressed as ratio of total precipitate and amount of initial AβA 23-35 35 fibrils) plotted against the isoelectric points (pi)) of the proteins. Konno (2001) Biochemistry 40:2148-2154 2154

The Use of Integrated Protein Interactome Data to Mining AD-Related Proteins AD-related proteins were shown to form a highly connected and statistically significant protein interaction sub- network. Chen et al.. (2006) Pacific Symposium on Biocomputing 11:367 Application of the integrated approach to the analysis of human interaction data and Alzheimer disease proteins allowed validation of the existing disease protein targets and predict novel ones not present in the initial list of disease-associated associated proteins.

Bioinformatics tools in treasure hunting for new drugs drugs

Bioinformatics,, intrinsic disorder and drug discovery Chen et al.. (2006) TIB, in press

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