Physical Address

304 North Cardinal St.
Dorchester Center, MA 02124

Proteins


Prion Disease, A Disease of Protein Conformational Change

The prion diseases in man are Creutzfeldt–Jakob, Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia, kuru, and Alpers syndrome. Creutzfeldt–Jakob disease (CJD) is of interest, especially in view of the outbreak in Britain of the mad cow disease (bovine spongiform encephalopathy). Other important prion diseases of animals are chronic wasting disease in mule deer and elk and scrapie in sheep. These diseases can be contracted by the ingestion of diseased brain or lymphoid tissue even if cooked. The brain is the major site of the pathology of this disease and the major concentration of the diseased form of the prion protein (scrapie, PrP Sc ). The first description of this disease was in sheep, the scrapie disease. Consequently, the diseased conformation of the normal cellular prion (PrPc) is designated PrP Sc . The function of the normal prion protein is still not understood, although there are characteristics of the protein (discussed later) that suggest a normal cellular function. The ingested PrP Sc can be absorbed through the intestine at Peyer’s patch ( Fig. 4.1A and B ).

Figure 4.1, (A) Diagram of the location of Peyer’s patch in the intestine. (B) Stained intestinal tissue in a microscopic view showing Peyer’s patch in the ileum. Arrows point to germinal centers.

The associated mucosal lymphoid tissue may be involved in the transport of PrP Sc . The scrapie prion can replicate at the spleen or lymphoid nodes, and it can access the nervous tissue and ultimately the spinal cord and brain because there are neural connections to lymphoid sites. Tribes in the Fore Highlands of Papua, New Guinea, ingested, ritually, the brains of dead relatives often leading to the prion disease, kuru . They were induced to give up this ritual in the 1950s, and the deadly kuru disease disappeared.

CJD is characterized by the loss of motor control leading to paralysis, wasting, dementia, and death sometimes as the result of pneumonia. The clinical and pathologic characteristics of this disease are summarized in Table 4.1 .

Table 4.1
Clinical and Pathological Characteristics of Creutzfeldt–Jakob Disease (CJD).
Source: Reproduced from http://en.wkipedia.org/wiki/Creutzfeldt-Jakob_disease .
Characteristic Classic CJD Variant CJD
Median age at death 68 years 28 years
Median duration of illness 4–5 months 13–14 months
Clinical signs and symptoms Dementia; early neurologic signs Prominent psychiatric/behavioral symptoms; painful dysesthesias; delayed neurologic signs
Periodic sharp waves on electroencephalogram Often present Often absent
Signal hyperintensity in the caudate nucleus and putamen on diffusion-weighted and FLAIR MRI Often present Often absent
“Pulvinar sign” on MRI Not reported Present in >75% of cases
Immunohistochemical analysis of brain tissue Variable accumulation Marked accumulation of protease-resistant prion protein
Presence of agent in lymphoid tissue Not readily detected Readily detected
Increased glycoform ratio on immunoblot analysis of protease-resistant prion protein Not reported Marked accumulation of protease-resistant prion protein
Presence of amyloid plaques in brain tissue May be present May be present
FLAIR MRI , Fluid attenuation inversion recovery MRI to remove effects of fluid from the resulting image; Pulvinar sign , hyperintensity of the posterior nuclei of the thalamus.

CJD is of three types: sporadic form ( sCJD ), variant form ( vCJD ), or familial form ( fCJD ). The first two are the most common; vCJD occurs in younger people and has recently been recognized. It seems to be evolving more rapidly than sCJD through a “Ghost prion” gene. The familial form results from the inheritance of a mutated gene for PrP c occurring in about 10%–15% of cases. CJD occurs in about one per million persons each year, affecting individuals between the ages of 45 and 75 years and appearing often at ages 60–65 years shown in the first entry in Table 4.1 . The prion gene is located on human chromosome 20 ( Fig. 4.2 ).

Figure 4.2, Human chromosome 20 showing the prion gene in green ( right ) and the PRND ghost prion gene about 20 kbp downstream (also in green ).

The PRND gene encodes a protein (sometimes called “Doppel”) that is biochemically and structurally related to the prion gene, and it is a glycophosphatidylinositol-anchored (to the cell membrane) protein . In some cells (observed in B lymphocytes, granulocytes, and dendritic cells), the PRND messenger RNA (mRNA) for the protein prnd is expressed with the PrP c . The protein, prnd, can coordinate the binding of Cu 2+ with high affinity. Mutations and polymorphisms are shown in a cartoon of the human prion gene in Fig. 4.3 .

Figure 4.3, Cartoon of the human prion gene showing the locations of 11 or more mutations (single base change) and the locations of two polymorphisms (sequence variations).

fCJD is inherited from a gene with many possible mutations. It is not clear how these mutations cause CJD. There are two suggestions: either mutations make individuals carrying these mutated genes more susceptible to the prions in the environment or the mutations might produce spontaneous generation of transmissible prions. Some mutations could lead to a structural change or facilitate a structural change in the protein to the scrapie form. The three-dimensional structures of PrP c and the speculative structure PrP Sc are shown in Fig. 4.4 .

Figure 4.4, The structure of PrP c ( left ) and the speculative structure of PrP Sc ( right ). PrP c contains 43% α-helix; PrP Sc has 30% α-helix, and 43% β-sheet structures. As indicated, the structure of PrP Sc is speculative.

Interestingly, the PrP c is highly conserved among species and is a copper-binding protein . Knockout of PrP c appears to cause demyelination of Schwann cells , and the normal cellular protein may have an effect on hippocampal long-term potentiation so that the protein could function in long-term memory. The normal protein binds copper ions with multiple low-affinity sites at the N-terminus and a high-affinity site at the center of the protein ( Fig. 4.5 ).

Figure 4.5, Linear cartoon of the normal prion protein, PrP c . There are weak copper-binding sites (octarepeats containing prolines and glycines) in the N-terminus and a strong binding site for copper ion in the center.

It is likely that PrP c functions in copper metabolism, possibly as a copper storage protein. It also can bind zinc and manganese ions. PrP Sc may lose much of the copper-binding activity and the resulting excess copper in nervous tissue would contribute to an increase in reactive oxygen species that could produce many of the effects of CJD. In addition, the normal prion may have superoxide dismutase (SOD) activity . The substrate for SOD is O 2 . SOD catalyzes the reaction:
2 O 2 + 2 H + → O 2 + H 2 O 2 . The oxygen radical, O 2 , for example, is derived from the xanthine oxidase reaction:
xanthine + 2 O 2 → uric acid + H 2 O 2 + 2 O 2 . SOD can be coupled to the product to generate O 2 +H 2 O 2 ; hydrogen peroxide can be converted to H 2 O+ 1/2 O 2 by the activity of peroxidase or catalase.

CJD is a disease of aggregation of the PrP Sc protein as shown in Fig. 4.6 .

Figure 4.6, Illustration showing how PrP c in the presence of PrP Sc might be converted to a greater number of PrP Sc molecules. The chain reaction results in the formation of fibrils of the PrP Sc molecules that aggregate into fibrils, becomes deposited in the brain, and leads to the symptoms of CJD and death. CJD , Creutzfeldt–Jakob disease.

The interaction of the normal PrP c with the disease form, PrP Sc , generates a conformational change in the normal partner to produce PrP Sc –PrP Sc that, in turn, incites a chain reaction to form filaments and their aggregates that are presumed to cause the damage in the brain. It is possible that the conformational change of the PrP c into the PrP Sc form would be catalyzed by an RNA that could perform a function like a chaperone protein. To increase the complexity of this disease, it appears that there are different strains of the PrP Sc , so that the CJD can take on different characteristics (incubation period, severity of the disease, patterns of the PrP Sc distribution in nervous tissue, etc.). Thus the characteristics of the variant strain (vCJD) may be different from the classic CJD because the strain of PrP Sc might be different.

The significance of this disease for this chapter is that a serious, fatal human disease can develop from an alteration in the three-dimensional structure of a normal cellular protein.

Recent research has illuminated some of the activities of the normal prion protein (PrP c ).

Actions of Cellular Prion (PrP c )

PrP c is an anchored (glycosylphosphatidylinositol, GPI) diglycosylated protein located at the outer leaflet of the plasma membrane, of most cell types, including blood cells ( ), but at higher levels in neuronal tissue cells, in lipid-enriched microdomains ( Fig. 4.7 ) ( ). It has two N-linked glycosylation sites ( Fig. 4.8 ) ( ). The sugars can attach to serine or threonine residues as O -glycosylation sites. PrP c consists of 210 amino acid residues with a disordered N-terminal domain and three α helices and a short antiparallel β-pleated sheet in the globular C-terminal domain. PrP c is internalized and recycled either to the plasma membrane or to the Golgi apparatus or is transported to late endosomes and later released as exosomes for lysosomal degradation. It is subject to three types of proteolytic cleavage as shown in Fig. 4.9 ( ).

Figure 4.7, Prion protein and Thy-1 (a cell surface antigen) are located on different cell surface lipid domains. PrP c is concentrated within discrete sphingolipid-sterol rafts. These rafts appear to form a boundary of intermediate lipid order (and so detergent insolubility) between the fluid glycerolipid regions composed of unsaturated kinked lipids and the highly insoluble, fully ordered domains occupied by Thy-1.

Figure 4.8, Molecular model of PrP c . The positions of Ser and Thr are shown in red and blue , respectively, and those available for glycosylation are shown as the yellow structures at the top. All of the serines are available as potential O -glycosylation sites. However, there is a cluster of seven Thr residues that are unavailable for glycosylation owing to tertiary structure or because their positions are protected by the N -glycans. Ser , Serines; Thr , threonines.

Figure 4.9, Schematic representation of the prion protein. (A) The prion protein is located in lipid rafts and attached to the outer leaflet of the cellular membrane via a GPI anchor. The flexible N-terminal part of the protein harbors a neurotoxic domain ( red box ) and is able to bind copper ions and oligomeric amyloid β ( purple triangles ). The C-terminal part of PrP c has a globular structure and comprises up to two N -glycan side chains. Involvement of PrP c is protective or toxic signaling ( dotted thunderbolt ) requires accessory molecules (not shown) to bypass the lipid bilayer. (B) Linear representation of the primary sequence of murine PrP c showing important protein domains. After the removal of the N-terminal signal sequence (aa1–22; gray box ) by signal peptidases in the ER and the C-terminal signal sequence for the attachment of the GPI anchor (aa 231–254; gray box ), the mature prion protein comprises an octameric (copper-binding) repeat region (aa 51–90; dark green ), a neurotoxic domain (aa 105–125; red box ), a hydrophobic core (aa111–134; dotted box ), a disulfide bridge (between aa 178 and 213), and two variably occupied N -glycosylation sites (aa 180 and 196). The most import cleavage events are indicated by arrows. (I) α-cleavage gives rise to a soluble N1 fragment of 11 kDa and a membrane-bound C1 fragment of 18 kDa. Of note, this cleavage destroys the neurotoxic domain. (II) β-cleavage at the end of the octameric repeat region produces N2 (9 kDa) and C2 (20 kDa) fragments. (III) Ectodomain shedding close to the GPI-anchor results in the release of nearly full length PrP from the membrane. ER , Endoplasmic reticulum; GPI , glycosylphosphatidylinositol.

Binding to Amyloid Proteins

PrP c binds to amyloid (β-sheet-rich) proteins and participates in the expansion of amyloid , including α -synuclein (α-Syn) , deposits. This occurs in the brain in α -synucleinopathies . The structures of α-Syn fibrils are shown in Fig. 4.10 ( ). Alzheimer’s disease and Parkinson’s disease also involve β-amyloid oligomers with PrP c binding.

Figure 4.10, (A) Schematic depicting the sequence of human α-Syn. The positions of the known familial mutations are indicated. β-Strand regions are indicated by arrows colored from blue to red. (B) Cryo-EM micrograph depicting the distribution and general appearance of α-Syn fibrils. (C) Cryo-EM reconstruction of α-Syn (1–121) fibrils showing two protofilaments ( orange and blue ). (D) Cross section of (C) illustrating the clear separation of the β-strands. (E) Cross section of a fibril (along the axis) illustrating the arrangement of the two protofilaments ( orange and blue ) and fitted atomic model. Positions of the initial (L38) and final (V95) residues fitted are indicated, as well as the initial and final residue of the NAC region (E61 to V95). Arrows indicate the location of four of the five α-Syn residues where familial mutations associated with Parkinson’s disease occur. (F) Distribution of β-strands in a single protofilament of the α-Syn fibril, corresponding to residues 42–95. Color scheme as in (A). (G) As in (F) but a perpendicular view to the fibril axis illustrating height differences in some areas of a single protofilament. α-Syn , α-Synuclein.

Copper Binding to Cellular Prion Protein

Binding of copper occurs at several sites on the PrP c protein ( Figs. 4.5 and 4.9 ). It appears that the binding of copper ions to the N-terminal copper-binding amino acids influences directly the formation of neurites (that form axons and dendrites). Disruption of all of the copper-binding sites at the N-terminus of PrP c was shown to be toxic to neurons suggesting that this region of copper binding is required for proper functioning of PrP c in neuritogenesis . Mutations in both regions of PrP c that binds copper (weak and strong binding regions) destroy the neuritogenesis function. Therefore it appears that loss of function of PrP c through interruption of copper binding is related to the onset of disease.

PrP c and Differentiation and Function of Adipocytes

Prion-deficient mice show activation of autophagy flux signals , and the same activation occurs in PrP c -deleted cells in culture as observed by transmission electron microscopy. Visceral fat volume, body fat weight, adipocyte cell size, and body weight all increased after the prion protein was knocked out in mice. Overexpression of prion protein inhibits the autophagic flux signals, lipid accumulation, as well as specific protein levels. Thus it appears that PrP c is required to prevent adipogenic differentiation and lipid accumulation through autophagy flux activation.

Proteins are encoded by mRNAs that are, in turn, encoded in the gene. The progression of information in generating a specific protein is gene (DNA) to mRNA to protein. Proteins are composed of amino acids joined together by peptide bonds, a process that takes place in the cellular cytoplasm on ribosomes, the machines of protein synthesis. At the ribosome the protein is assembled by the addition of amino acids directed by mRNA and specific transfer RNAs . This discussion begins with the building blocks of proteins, the amino acids, and their structures.

Amino Acids

Amino Acid–Related Diseases

There are important genetic diseases involving deficiencies of enzymes needed to metabolize amino acids. The more important diseases are phenylketonuria ( PKU ), alkaptonuria ( AKU ), maple syrup urine disease (MSUD) , tyrosinemia , and homocystinuria . PKU occurs in infants (about 1 in 10,000) who are unable to convert phenylalanine to tyrosine owing to a deficiency of the enzyme phenylalanine hydroxylase . Consequently, phenylalanine level rises in the blood and is toxic to the brain. These individuals can survive if phenylalanine is restricted from the diet for life. AKU patients excrete urine that turns black when exposed to air. This is a relatively rare disease where 1 person in 100,000 at the most will have it but in Slovakia, 1 person in 19,000 will have AKU. These individuals usually develop arthritis of the spine and large joints. This condition is the result of a deficiency of the enzyme homogentisate 1,2-dioxygenase , the enzyme that catalyzes the conversion of homogentisate (or homogentisic acid) to 4-maleylacetoacetate (or 4-maleylacetoacetic acid). MSUD follows from a deficiency of the branched-chain α-keto acid dehydrogenase needed to metabolize branched-chain amino acids ( leucine , isoleucine , and valine ). Among Ashkenazi Jews, 1 person in 40,000 will have this disease; worldwide, 1 person in 185,000 will have it but among Older Order Mennonites, 1 in 380 will have this disease. This enzyme deficiency leads to the buildup of branched-chain amino acids in the blood, and these are toxic to the brain and other organs. Infants are generally screened for this disorder by a blood test for alloisoleucine (leucine forms an intermediate and the subsequent transamination generates alloisoleucine). Initial treatment can involve dialysis of the blood. The untreated disease can lead to death. Tyrosinemia is characterized by a lack of enzymes that metabolize tyrosine. There are three types of tyrosinemia: type I, type II, and type III. About 1 child in 100,000 or less has this disease. In Quebec the incidence can be 1 in 16,000. Type I tyrosinemia is characterized by a deficiency in fumarylacetoacetate hydrolase , the terminal enzyme in the tyrosine catabolic pathway (produces fumarate and acetoacetate from fumarylacetoacetate). Type II is due to a deficiency of tyrosine aminotransferase , the initial enzyme in the tyrosine oxidation pathway. Type III, a rare type, is the result of a deficiency of the enzyme 4-hydroxyphenylpyruvate dioxygenase , the second enzyme in tyrosine catabolism that results in the formation of homogentisate . Type I tyrosinemia affects the liver, kidneys, and nerves and could require liver transplantation. Type I occurs more frequently in children of French Canadian or Scandinavian descent. Type II is less common and can be managed by restricting tyrosine in the diet (but this does not solve the symptoms of type I). Homocystinuria is the result of a deficiency of the enzyme cystathionine β-synthase, the first enzyme in the transsulfuration pathway catalyzing the conversion of homocysteine (+ l -serine) to cystathionine (+water). Although children with this disease are normal at birth, by 3 years of age, the eye lens is dislocated, and they have decreased vision. There follow skeletal deformities, such as curved spine, chest deformities, and elongated limbs and fingers usually with osteoporosis. In addition, there are mental disorders and increased blood clotting. Obviously, just considering these diseases alone, it is of importance to understand the biochemistry of the amino acids.

Biochemistry of the Amino Acids

There are 20 common amino acids that comprise the structures of proteins. They can be arranged according to their sizes and net charges in solution as shown in Table 4.2 .

Table 4.2
Names, Groupings by Size and Charge, and Abbreviations of Amino Acids.
Source: Reproduced in part from Litwack, G., 2008. Human Biochemistry and Disease. Academic Press/Elsevier, p. 44.
Amino Acid Molecular Weight Abbreviation Letter Name
Small, Neutral Amino Acids
Lysine a,b 57.05 Gly G
Alanine a 71.09 Ala A
Serine c 87.08 Ser S
Threonine c 101.11 Thr T
Cysteine c 103.15 Cys C
Hydrophobic Amino Acids
Branched-Chain Amino Acids
Valine a,b 99.14 Val V
Leucine a,b 113.16 Leu L
Isoleucine a,b 113.16 Ile I
Other Hydrophobic Amino Acids
Methionine c 131.19 Met M
Proline a,b 97.12 Pro P
Aromatic Amino Acids
Phenylalanine b 147.18 Phe F
Tyrosine 163.18 Tyr Y
Tryptophan c 186.21 Trp W
Carboxylated Acidic Amino Acids
Aspartic acid 115.09 Asp D
Glutamic acid 129.12 Glu E
Amino/Amide-Containing Amino Acids
Asparagine c 114.11 Asn N
Glutamine c 128.14 Gln Q
Basic Amino Acids
Histidine c 137.14 His H
Lysine 128.17 Lys K
Arginine 156.19 Arg R

a Amino acids with hydrophobic side chains.

b Amino acids, having hydrophobic character, found in internal environment of proteins.

c Amino acids that can participate in hydrogen bonding.

The structures of the amino acids are important, and they are shown in Fig. 4.11 . For space-filling models ( on right ): nitrogen atom ( blue ); hydrogen atom ( gray ); carbon atom ( green ); oxygen atom ( red ).

Figure 4.11, The 20 common amino acids. Essential amino acids that are not produced in the body or are only partially produced are denoted by an asterisk after their names.

The structure of an amino acid can be generalized as Fig. 4.12 .

Figure 4.12, A model amino acid showing the ionizable groups (–NH 2 and –COOH) that become –NH 3 + and –COO − when ionized, a variable methyl group in the chain (–CH 2 ) and a side chain (R), if it is present.

The essential amino acids are as follows: valine, leucine, isoleucine, phenylalanine, tryptophan, threonine, methionine, lysine, arginine, and histidine. Arginine and histidine are essential in certain cases. The other amino acids are considered nonessential because they can be synthesized in the body. The nonessential amino acids are listed as follows together with their precursors in the body: pyruvic acid can give rise to alanine; glutamic acid can form arginine; aspartic acid can form asparagine; oxaloacetic acid can form aspartic acid; alpha-ketoglutarate (or alpha-ketoglutaric acid) can form glutamic acid which can form glutamine and proline; serine and threonine can form glycine; glucose (Glc) can form serine; and phenylalanine can form tyrosine. Some amino acids, once they are in the body, can be converted to other amino acids (e.g., methionine can be converted to homocysteine, and it can be converted to cysteine); phenylalanine can be converted to tyrosine, and arginine is converted to ornithine and citrulline (amino acids, unlisted earlier, in the urea cycle). These interconversions will be covered in the chapter on metabolism. Selenocysteine, hydroxyproline, lanthionine, 2-aminoisobutyric acid, gamma-aminobutyric acid, and some others are not common amino acids but occur as metabolites or serve specific functions in the body.

Certain protein food sources are deficient in amino acids . Wheat and rice are lysine-deficient ; maize is deficient in lysine and tryptophan, and legumes are deficient in tryptophan and methionine or cysteine. Most tissue foods of animal origin are complete in terms of essential amino acid supply. Some plant sources are adequate but most are inadequate in certain amino acids. Balanced protein nutrition is important to obtain a needed supply of essential amino acids. The requirements of infants and growing children are substantially greater than those for mature adults.

The natural amino acids in the body occur as l -forms. There are also d -forms that occur in lower organisms. These are known chiral forms, and the property of bending light either to the left ( l -, or levo ) or to the right ( d -, or dextro ) is chirality as applied to amino acids. The l - and d -forms of an amino acid are shown in Fig. 4.13 .

Figure 4.13, (A) Pictured are the l - and d -forms of the amino acid valine . In the l -form the alpha-amino group is on the left side of the structure, and in the d -form, the alpha-amino group is on the right side of the structure. The l -form is the mirror image of the d -form and vice versa. It is possible to rotate the amino group in this way because the alpha carbon is asymmetric (the substituents on each of the four carbon bonds are unique). (B) Model of l -valine. The alpha carbon is highlighted in green and emphasizes the tetrahedral character of the alpha carbon. Oxygen is in red ; hydrogen in white ; carbon in gray , except for alpha carbon; nitrogen in blue .

An asymmetric carbon atom is one that has a different substituent on each of its four bonds. The maximal number of stereoisomers ( n ) relates to the number of asymmetric carbons in a molecule. In amino acids, there is only one asymmetric carbon, so the maximal number of stereoisomers is 2 n , or 2 1 , or 2 (in a molecule that has two asymmetric carbons, the maximal number of stereoisomers would be 2 2 or 4, etc.). Exceptionally, however, isoleucine and threonine have chiral carbon atoms in their side chains. In the case of amino acids, such as valine, the two possibilities from rotation about the alpha carbon are shown in Fig. 4.13 , the l - and d -forms. The l -forms are the so-called natural forms; however, d -amino acids are parts of structures (e.g., cell walls) of lower organisms. An exception is the amino acid, glycine.

Glycine has two hydrogens (identical atoms) as substituents of the alpha carbon; therefore only one form of glycine is possible as the alpha carbon is not asymmetric. l -Forms of amino acids can be converted to d -forms by the catalytic action of the enzyme, racemase (from lower organisms); serine racemase , however, is present in the human body allowing for interconversions between the l - and d -forms. This conversion also can occur spontaneously but at a very slow rate. d -Serine plays a role in the development of the nervous system and may be a cosubstrate agonist for the glycine-binding site and regulator of the N -methyl- d -aspartate excitatory glutamate receptor. Aside from the primary role for amino acids as constituents of proteins, certain amino acids give rise to molecules in the body that have specific functions. Examples of these are as follows: the amino acids glutamine, aspartate, and glycine are precursors of purines or pyrimidines ; nitric oxide , a biologically important molecule, derives from arginine ; heme porphyrins have a precursor in glycine, and tryptophan can be converted to the neurotransmitter, serotonin .

You're Reading a Preview

Become a Clinical Tree membership for Full access and enjoy Unlimited articles

Become membership

If you are a member. Log in here

Related Posts