Disorders caused by pathological conformation of proteins Jan ILLNER Alice Skoumalová

Disorders Proteins Alzheimer´s disease Amyloid-β Parkinson´s disease α-Synuclein Diabetes mellitus type II. Amylin Amyotrofic lateral sclerosis Superoxide dismutase Cystic fibrosis Cystic fibrosis transmembrane regulator (chloride channel) Sickle cell anemia Haemoglobin Hungtington´s disease Huntingtin Creutzfeldt´s-Jakob´s disease Prion Amyloidoses 10 different proteins

Theoretical model of folding (hierarchic): local secondary structure formation tertiary structure formation (subdomains, domains) stable conformation Thermodynamic model: Protein is folded to gain the lowest levels of his free energy: native state There are other alternative conformations: different protein function pathologic protein

Neurodegenerative diseases Abnormal tertiary structure of specific protein causes pathologic conformation α-helix β-sheet the starting point is the natural protein folded in the native and active conformation normal protein is rich in α-helix conformation (folded structure) the end-point is the same protein adopting prevalent β-sheet structure it is disease-associated protein (misfolded structure) Conformational change Aggregation Gain of toxic activity Neurodegenerative diseases Loss of biological function

Protein with pathologic conformation Mutation (familiar) Error in folding process (sporadic) Identification Degradation (protein quality control system) 1. Chaperones 2. Ubiquitine proteasome system

Molecular chaperones Hsp 70 - new synthetised proteins: (heat shock protein) Chaperonines - misfolded proteins: Hsp 60

Accumulation (Amyloidoses) Gain of toxicity (Alzheimer´s disease) DNA Ubiquitin Ribosome RNA ATP Chaperones Native protein Misfolded protein Aggregate/fibrillar amyloid Chaperones Proteasome Accumulation (Amyloidoses) Degraded protein Gain of toxicity (Alzheimer´s disease) Loss of protein function (Cystic fibrosis)

Protein pathologic conformation consequences: Gain of toxicity neurodegenerative disorders - chronic, progressive diseases loss of neurones pathologic protein accumulation aggregate generation (Alzheimer´s, Parkinson´s, Hungtington´s disease) Loss of biological function cystic fibrosis - gene mutation for chloride channel Accumulation amyloidoses - fibrils are not toxic but they are insoluble

Amyloid fibril structure straight, unbranched, diameters in the range of 8-16 nm composed of two to six protofilaments rich in a type of β-sheet structure (the β-sheets are perpendicular to the fibril axis and bind together by the hydrogen bonds)

Molecular factors in amyloid formation protein misfolding is central to amyloid formation protein stability (the resistance of the folded conformation to misfolding) is an important factor in determining susceptibility to amyloid formation Destabilizing factors: 1. Extreme environments in the body, such as acidic cell compartments 2. Proteolytic removal of portion of a protein by an endogenous protease 3. Mutations that alter primary structure 4. Interaction with lipid bilayers

Amyloid formation

Protein quality control in the cell

Alzheimer´s disease

Therapy: impossible to stop only slow down timely diagnostics Characterization: progressive neurodegenerative disease the most common form of dementia associated with aging loss of cognitive function Diagnosis: histological findings in brain tissue (senile plaques, neurofibrillary tangles) clinical criteria: neuropsychological tests magnetic resonance imaging laboratory problematical diagnostics: symptoms are common for other dementias or are similar with aging Therapy: impossible to stop only slow down timely diagnostics Causation: unclear; β-amyloid peptide accumulation, oxidative stress ??? Aims of research: to produce a laboratory test (from blood or liquor) that could be used for better diagnostics

Types and prevalence of AD in population sporadic familiar most later onset unknown causations less than 2 % onset at the age of 65

Pathological signs of AD Neurofibrillary tangles hyperphosphorylated protein tau Amyloid (senile) plaques protein β-amyloid fibrils (Aβ) abnormal processing of APP (amyloid precursor protein)

Role of amyloid β in pathogenesis of AD Effort in research: decrease Aβ42 accumulation by inhibition of β- or γ-secretase, support α-secretase or increase Aβ42 degradation by specific antibodies

Role of oxidative stress in pathogenesis of AD pathologic changes in the brain oxidative stress

Prions and prion disorders

Prion definition Prions are proteinaceous transmissible pathogenes responsible for a series of fatal neurodegenerative disorders Creutzfeld´s-Jakob´s disease kuru bovine spongioform encephalopathy Prion (proteinaceous infectious particle); analogy for virion It is a type of infectious agent that does not carry the genetic information in nucleic acid! Prions are proteins with the pathological conformation that are believed to infect and propagate the conformational changes of the native proteins into the abnormally structured form

PrPC PrPSc PrPC (cellular) Normal protein Transmembrane glycoprotein (neurons, lymphocytes); its function is unknown; it binds Cu2+ Dominant secondary structure α-helix Easily soluble Monomeric, easily digested by proteases Encoded by a gene designed PRNP located on the chromosome 20 PrPSc PrPSc (scrapie) Abnormal protein, disease-producing protein The same amino acid sequence (primary structure) Dominant secondary structure β-sheet Insoluble Multimeric, resistant to digestion by proteases When PrPSc comes in contact with PrPC, it converts the PrPC into more of itself These molecules bind to each other forming aggregates

Molecular models of the structures of: PrPC PrPSc Predominantly α-helix (3x) β-sheet (40%), α-helix (30%)

Prion aggregates

Prion disorders: rare neurodegenerative diseases sporadic familiar transmissible in 6th or 7th decade rapidly progressive CJD inherited mutations in the PrP gene that favour the transition from the cellular to the pathologic form of PrP rare BSE, kuru Creutzfeldt´s-Jakob´s disease the most common prion disease death in less than a year Kuru disease propagation in New Guinea natives ritualistic cannibalism

Prion transmission Direct contact with infected tissue Consumption of affected tissue from instruments used for brain surgery corneal grafts electrode implants cannibalism, kuru consume material from animal infected with BSE, vCJD How can prion make their way through the gut and into the brain? Hypothesis: Prions circumvent the normal process of intestinal absorption by passing into the GALT (gut-associated lymphoid tissue)