humanpriondiseases(part1)structure,functions,andgeneticsofprions.pdf

Tinjauan Pustaka

Maj Kedokt Indon, Volum: 57, Nomor: 8, Agustus 2007

Human Prion Diseases (Part 1)Structure, Functions, and Genetics of Prions

Ginus Partadiredja

Department of Physiology Faculty of Medicine Gadjah Mada University, Yogyakarta

Abstract: Prion diseases, or transmissible spongiform encephalopathies (TSE), are a group ofneurodegenerative disorders, which affect humans and animals. Human prion diseases appearas a result of inherited, sporadic, and acquired manifestations. The main pathological feature ofthese disorders implicates the structural alteration of prion protein (PrP). It is believed thatthese disorders are associated with the accumulation of an abnormal isoform (PrPSc) of thenormal cellular prion protein (PrPC). Several aspects of these disorders, including the classifi-cation, structure, functions, and genetics, were reviewed.Keywords: Creutzfeldt-Jakob disease, transmissible spongiform encephalopathies

Penyakit Prion pada Manusia (Bagian 1)Struktur, Fungsi, dan Genetika Prion

Ginus Partadiredja

Bagian Fisiologi Fakultas Kedokteran Universitas Gajah Mada, Yogyakarta

Abstrak: Penyakit-penyakit Prion atau transmissible spongiform encephalopathies (TSE) adalahsekelompok penyakit neurodegeneratif yang mengenai manusia dan hewan. Penyakit Prion padamanusia muncul sebagai manifestasi keturunan, sporadik, dan didapat. Gambaran patologisutama penyakit ini melibatkan perubahan struktur protein prion (PrP). Penyakit ini diperkirakanberkaitan dengan akumulasi isoform abnormal (PrPSc) protein prion seluler normal (PrPC).Berbagai aspek penyakit ini, meliputi klasifikasi, struktur, fungsi, dan genetik, dibahas dalammakalah ini.Kata kunci: Penyakit Creutzfeldt-Jakob, transmissible spongiform encephalopathies

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Human Prion Diseases (Part 1)

Introduction

In the past 10 years, prion diseases have attracted pub-lic and government attention, due to their role as the caus-ative agents of ‘mad cow disease’.1,2 Prion diseases, or trans-missible spongiform encephalopathies (TSE), are a group ofhuman and animal neurodegenerative disorders. These dis-orders share common etiology and pathogenesis, which in-volve the structural alteration of prion protein (PrP).3,4 Thesechronic and fatal diseases consist of inherited, infectious,and sporadic subtypes. In human, prion diseases manifestin four subtypes, including Creutzfeldt-Jakob disease (CJD),Gerstmann-Sträusler-Scheinker syndrome (GSS syndrome),kuru, and fatal familial insomnia (FFI).5 These disorders dem-onstrate clinical features such as dementia (except FFI), aki-netic mutism, ataxia, motor dysfunction, sight loss, and othervariable symptoms of central nervous system distur-bances.1,2,6,7 Electroencephalograms may show generalized,synchronous, and periodic waves. Deaths may occur rap-idly within months or even weeks. Unfortunately no effec-tive therapy for this fatal disease has been found.6

It has been suggested that prion diseases are causedby an agent termed “prion” (proteinaceous infectious par-ticles). This agent is an unconventional infectious patho-gen that could not be classified as virus or viroid.8,9 Regard-less the routes of the prion agent in order to manifest inhuman, either through inheritance, sporadic, or iatrogenic/acquired transmission, these disorders are characterized withthe accumulation of an abnormal isoform (PrPSc) of the nor-mal cellular prion protein (PrPC).1,3,10 It is thought that PrPC isconverted to PrPSc through the unfolding of alpha-helicesand refolding into beta-sheets.2,5,11. Up to present, however,the nature and the pathophysiology of this nucleic acid de-ficient agent2,12 remain to be the conundrum of many stud-ies.1,3

This essay describes several aspects of prion diseaseswith the focus on the overview classification, the structure,the characteristic, the function and the genetics of priondiseases. In this essay, the term PrPSc is used interchange-ably with the term PrPres (protease ‘resistant’ prion protein),referring to the term used by the original articles, althoughthis might be inappropriate for depicting the protease resis-tance characteristic of PrPSc.9 Another review on the patho-genesis of prion diseases will follow in a separate manu-script.

Classification of Prion Diseases

Prion diseases affect both humans and animals. In ani-mals, it has been reported that these diseases only appearamong mammalian species.12 The animals prion diseases in-clude six varieties, namely scrapie (in sheep and goats), trans-missible mink encephalopathy (mink), bovine spongiformencephalopathy (BSE) or “mad cow disease” (cattle), chronicwasting disease (mule deer, elk), feline spongiform encepha-lopathy (cats), and exotic ungulate encephalopathy (ante-

lopes). The humans prion diseases fall into four major cat-egories, i.e. Creutzfeldt-Jakob disease (CJD), which is broughtabout by iatrogenic, sporadic, familial, and variant causes;kuru; Gerstmann-Sträusler-Scheinker syndrome (GSS syn-drome); and fatal familial insomnia (FFI).1,5,6.8,12,13

Human prion diseases appear as a result of inherited,sporadic, and acquired manifestations. Sporadic Creutzfeldt-Jakob disease appears as the most frequent form of humanprion disease. Autosomal dominant inherited forms occur in15% of all cases. Less frequent forms of prion diseases mani-fest as a consequence of iatrogenic routes, such as treat-ment with duramater or corneal transplantation, growth hor-mone of the pituitary human cadaver, and exposure to non-sterile neurosurgical equipments. The vast majority of priondiseases are the sporadic form with unknown etiology. It isthought that somatic mutation of PRNP (prion protein gene)or spontaneous transformation of PrPC (normal prion pro-tein) to PrPSc (abnormal scrapie isoform) underlies the mani-festation of the sporadic forms.1,6,14,15

Prions Structure and Characteristic

In the early 1980s, a new protein agent has been re-ported to be associated with scrapie infections. These smallproteinaceous infectious particles, which was later termed“prion”, differ from viruses and viroids since they do nothave any nucleic acids, neither DNA nor RNA.1,2,7,8,12,16 Theseprions are very small in dimension, the smallest ones of whichare 100 times smaller than the smallest virus.12

Prions are mainly composed of a proteinase-resistantsialoglycoprotein which has molecular size of 27-30 kDa (PrP27-30).2,4,5,12 PrP comprises two forms: PrPC and PrPSc. PrPC isprotease sensitive protein with a molecular weight of 33-35kDa, while PrPSc is composed of a protease-resistant protein(PrP 27-30). PrPSc is an insoluble derivation of an N-terminalcleavage of PrPC. 2,4,5,7,12,16 PrPSc is formed from PrPC by post-translational modification.11,16 Both PrPC and PrPSc have beenobserved to have identical amino acid sequence, which con-sists of N-terminal sequence formed by the loss of a 22-amino-acid signal peptide (see Figure 1).11,17 This, in spite ofthe sequence of PrP, could be various in different species oreven in the same species. There are two variants in humans,the M type that contains methionine, and the V type that ismade up of valine in position 129.7,8,12,18 It is predicted thatthe tertiary structure of PrPC is a four-helix bundle with twoparallel pairs of alpha-helical overlapping segments.19 Animportant difference between the normal and the abnormalisoform can be seen in their tertiary structure. PrPC containsmostly alpha-helical structure (42%) with a small percentageof beta-pleated sheets (3%). In contrast, PrPSc and PrP 27-30are formed with higher percentages of beta-sheet-like form,namely 43% and 54%, respectively (see Figure 2). The alpha-helix constitutes 30% of PrPSc structure.2,4-7,11-13

PrPC is a normal membrane-bound sialoglycoprotein ofcell surface, which is constantly recycled and can be found

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in various tissue but it is prominent in the central nervoussystem and in lymphoid tissue. It can also be observed inthe liver, pancreas, kidney, mammary gland, and skeletalmuscle.1,3,5,7,8,11,16,18

This normal isoform is produced in the endoplasmicreticulum, and distributed through the Golgi apparatus tothe cell membrane, where it is engaged to a glycophos-phatidylinositol (GPI) and a cell surface proteoglycan. PrPC

is formed slowly, degraded rapidly, released after treatmentwith phosphatidylinositol-specific phospholipase, and en-tirely disintegrated by proteinase K digestion. Its half-life isvery short, approximately 3-6 hours.5,11-13,16 PrPC is trans-ported along axons, and it is piled up at the neuromuscularjunction.9,19 On the other hand, PrPSc is produced slowly(but probably more rapid than the formation of PrPC) by apost-translational process. PrPSc is degraded slowly and isnot released following phospholipase exposure. It is alsopartially cleaved and liberates PrP 27-30 after protease treat-ment by removing 66 NH

2-terminal amino acids. PrPSc is be-

lieved to be more stable than PrPC. It is stored mainly in thesecondary lysosomes. PrPSc can be found in endosomalvesicles and on the cell surface. Neuronal degeneration mightbe due to the deposition of these proteolytic-enzyme-resis-tant PrPSc in synapses.6,12,16,21 The rate of PrPSc formationseems to correlate with the degree of PrPC expression,whereas the rate of PrPSc deposition does not correspond toPrPC level.9

Figure 1. Tertiary structure of cellular prion protein whichis inserted into a membrane lipid bilayer (E). (A) analpha helix, (B) a beta sheet, (C) N-terminal, (D)glycosyl phosphatidylinositol (GPI) anchor. Repro-duced from Aguzzi & Heikenwalder (2006)3 with permission from the publisher, with a slight modifica-tion.

B

A C

E

D

Figure 2. The structural conversion of normal prion protein (PrPC) into its abnormal isoform (PrPSc). PrPC con- sists of four alpha helices. During the conversion, two of the helical structures are loss and are changed into the linear beta-pleated sheets. Reproduced from http://www.bseinquiry.gov.uk/report/volume2/fig2 _1. htm.19

fore they can survive in gastrointestinal tract and penetratethe lymphoreticular systems); DNase or RNase, psoralens,hydroxylamine, and photochemical reactions (ionization andultraviolet or gamma radiation). However, they can be para-lyzed with a strong base liquid or proteinase.1,3,5,7,12,16

The Function of Prions

To date the exact function of PrPC remains unknown.1-

3,5-7,11,13 It has been thought that this protein is involved inactivating lymphocyte, acts as a growth factor for neurons,assists synaptic functions, and induces or protects cellsagainst apoptosis. It may also play a role in metal ions traf-ficking, cell adhesion, cellular protection against oxidativestress, copper and zinc metabolism, and signal transduc-tion.2,3,11,12 It has been shown that in mice lacking PrP gene,the neuromuscular junction functioning is normal, but theneuronal and synaptic functions in hippocampus may bedisrupted. However, other studies on hippocampus and cer-ebellum failed to demonstrate these abnormalities.19

PrP is likely to play a pivotal role in the functioning ofinhibitory synapses, especially in GABA transmission. Thisis based on the finding that in mice with inactivated geneencoding PrP, demonstrated an attenuated GABA

A receptor-

mediated fast inhibition in hippocampus. This suggests a

Prions are characterized with their high resistanceagainst heat, many chemical substances, such as formalin,alcohols, detergents, disinfectants, stomach enzymes (there

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possible postsynaptic function of PrPC. Another finding insimilar mice revealed extensive degeneration of Purkinje cellsin cerebellum, which are believed to produce large quantityof PrP and use GABA in inhibitory signaling. The Purkinjecells loss is probably a consequence of persistent over exci-tation due to the absence of PrP. The alterations of circadianrhythms in this type of mice substantiate this notion. Circa-dian rhythms are considered to be regulated bysuprachiasmatic nucleus of the hypothalamus, and the pro-duction of GABA receptor subunits is high in this nucleus.21

However, the importance of PrPC is questioned since it hasbeen shown that mice lacking PrP gene underwent a normaldevelopment5,12, although they died prematurely withoutsigns of specific disease.18

Genetics of Prion Diseases

PRNP, the human PrP gene, which is engaged in theshort arm of chromosome 20, provides a single copy encod-ing the 253-amino acids PrPC. The mutations of PRNP in thefamilial subtypes of CJD, GSS, and FFI (all of which are char-acterized with autosomal dominant inheritance) involve twoways: a) point mutations6,18, and b) insertions which inte-grate the enhanced number of octapeptide repeats.12 Stud-ies on PRNP reported mutations at codons 102, 105, 117, 145,178, 180, 183, 198, 200, 208, 210, 217, 232, and insertions of 2,4, 5, 7, 8, or 9 octarepeats.14,19,22 The mutations are also asso-ciated with insertions at 144-base-pair (six extra octapeptiderepeat) and 96-bp (additional four octapeptide repeat) in theprion protein gene.23 It is believed that one point mutationsat codons 105, 117, 198, and 217 are always related to valine129, whereas codon 102 correlates with the methionine 129.22

All of the mutations are considered to give rise to structuralalterations of PrP protein from alpha-helical configuration tobeta-pleated sheet.8

Familial CJD is signified with the insertion of octarepeatsequences or point mutations at codon 178, 200, 208, or 210.The point mutation at codon 200 brings about a replacementof a glutamate (E) to lysine (K) (E200K).7,8,10,12 The mutationat the second nucleotide of codon 208 substitutes guanineto adenine, which results in a non conservative replacementof arginine to histidine at residue 208 of the protein (R208H).19

Since there is no particular PRNP mutation observed insporadic CJD, it is unknown how this subtype manifests.Nevertheless, the majority of sporadic CJD patients sharethe feature of PrP polymorphism of the amino acids methion-ine (Met) and valine (Val) at codon 129. The increased riskfor CJD manifestation and the reduced duration of the dis-ease compared to the heterozygous form are thought to beassociated with the homozygosity of Met/Met. This resultsin the conversion of PrPC to PrPSc. This occurs in all cases ofthe new variant of CJD. The homozygosity of valine at PrPcodon 129 has also been observed in iatrogenic CJD cases.1,6-

8,12,15,24,25 Both iatrogenic and sporadic subtypes of CJD arelikely to appear in subjects with certain genetic predisposi-

tion, related to ordinary polymorphism at codon 129. Theproportion of homozygosity of methionine allele is approxi-mately 38% among Caucasians, while that of valine alleleand heterozygosity are about 11% and 51% respectively.Homozygous subjects, either with Met or Val at the polymor-phic codon 129, were associated with the largest proportionof sporadic and pituitary-hormone related iatrogenic CJD.This suggests that the homozygosity might be a predispos-ing factor.14,19,24 Nevertheless, in the Japanese population,the proportion of the Val allele is much lower. CJD cases withheterozygosity at codon 129 (M/V) are more common (18%)in the population, which has the proportion of 0% for V/V,92% for M/M, and 8% for M/V.9

Inherited disease with autosomal dominance and fullpenetrance were found in nearly all GSS syndrome patients.12

The most ordinary genetic subtypes of GSS are identifiedwith the replacement of proline to leucine at codon 102 of thePrP gene (ataxic GSS).7-9 Other mutations occur at codons117 (telencephalic GSS), 145, 198, and 217.7

On the other hand, a rapid progressive autosomal domi-nant inheritance with incomplete penetrance was observedin FFI cases.12 Although FFI differs from familial CJD in neu-ropathological and clinical findings, FFI resembles familialCJD in the point mutation at PrP gene codon 178 (asparticacid to asparagine mutation). FFI is characterized with eitherpolymorphic homozygosity or M/V heterozygosity at codon129. This disorder involves a PrP allele with methionine atcodon 129 and asparagine at codon 178 (D178N-129M); whilefamilial CJD shows a combination of a PrP allele with valineat codon 129 and asparagine at codon 178. FFI with homozy-gosity at codon 129 is associated with short duration dis-ease, whereas that with heterozygosity corresponds withlong duration disease.7,8,16,18 It should be noted however, thatthe clinical and pathologic characteristic of FFI may varywidely, and this disorder cannot be discriminated easily fromsporadic CJD on clinical basis only. The diversity of clinico-pathologic features of FFI designates the less tight associa-tion between genotype and phenotype. It seems that thisvarious manifestation correlates with other factors, ratherthan the non-pathogenic polymorphism at codon 129. Forinstance, it was reported that pathogenic lysine mutation atcodon 200 associates with pronounced insomnia.26

It seems that the classical classification of human priondisease, covering the four subtypes (CJD, GSS, FFI, and kuru),is not sufficient in delineating the more complex variability ofneuropathological changes. For instance, in the cases ofmutation at codon 183 involving an adenine to guanine tran-sition at nucleotide 547 and bringing about non-conserva-tive substitution of threonine by alanine in the PrP protein,the clinico-pathological features could not be categorizedinto the available subtypes.22

Conclusion

Several facets related to the characteristics of the caus-

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ative agent of prion diseases have been described, leaving anumber of unsolved puzzles. Many problems associated withthe fundamental aspects, such as the function of prion pro-teins and the relationship between genetic mutation andpathological manifestations of prion diseases remain unan-swered.

Acknowledgment

This manuscript has been prepared mainly under thesupport of NZODA Scholarships.

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