Li Mengdong Nie Qinghe

Infectious and familial diseases of human central nervous system characterized by chronic spongiform degeneration, such as Creutzfeldt-Jakob disease (CJD), Creutzfeldt-Jakob syndrome, GSS pathogen, fatal familial insomnia (FFI) and Kuru disease, are thought to be caused by chronic virus infection, but the virus has never been isolated. In 1982, Prusiner et al. [1] formally proposed that the pathogen of this kind of disease may be an infectious protein particle, and the word PrP represents this factor. Someone once translated it into prion or saw protein, but since it seems inappropriate to call it virus because of lack of nucleic acid, it seems more appropriate to translate it into prion. Although there are many hypotheses about the pathogens of these diseases, the prion theory has been deeply studied and gradually recognized.

Protein is also the pathogen of similar diseases in many animals, such as bovine spongiform encephalopathy (BSE), itching of sheep, cats or other animals, infectious mink encephalopathy, chronic wasting diseases of North American black-tailed deer and bighorn deer. Many scholars believe that eating or adding the meat or viscera of animals suffering from pruritus to feed additives can make animals infected and sick, just like eating the brains of dead people in Papua New Guinea Plateau to express sacrifice and thus infect Kuru disease. Mad cow disease is also called mad cow disease. Since 1986, at least 150000 cattle in Britain have suffered from this disease, and more than 28500 cattle have died of mad cow disease, which has caused great losses to British beef exports and related cattle biological and medical products [2]. It is suspected that mad cow disease may be transmitted to human beings, which has caused great threat and fear to human beings. The British government has to slaughter all cattle over 30 months at a rate of 30 thousand per week.

First, the formation of prion theory

As early as 1950s, Alper and others suggested that the infectious factors of animal pruritus might lack nucleic acid. To 1967, Griffith et al. [3] once again stressed that the replication of this pathogenic factor may not require nucleic acid templates. During this period, many experiments have proved that the infectivity of the brain extract of itchy sheep can be filtered by bacteria, so it is suspected that it is caused by virus. However, the extract is treated by inactivating nucleic acids, such as boiling, ultraviolet rays or ion radiation, and preparations that specifically change or destroy nucleic acids, such as nuclease, psoralen, hydroxylamine or zinc ions, are used to treat brain homogenates, microsome components and purified prion preparation columns of pruritic animals. Unable to change its infectivity, Prusiner et al. [1, 2] set out to conduct in-depth research, made a lot of experiments on Syrian golden voles, and obtained many important data.

Purified PrP from pruritic animals was inoculated into voles. The subcellular components obtained from the brain tissue (Ha) of voles rich in infectious factors proved the existence of a protease-sensitive protein PrPC with a relative molecular weight of 33 000 ~ 35 000, while the relative molecular weight of a protease-resistant protein PrPSC was 27 000 ~ 30 000. The latter is an important part of the pathogenicity specificity of the former. After PrP 27 000 ~ 30 000 was further purified into a single component, it was infected with voles and mice (Mo), and then the mRNA of PrP was isolated from the brains of infected animals to construct a cDNA library. Sequencing of the cDNA expression product showed that the protein encoded by Ha and Mo PrP cDNA was 254 amino acids, while the protein encoded by human PrP cDNA was 253 amino acids, and the signal peptide (SP) was located at the NH2 terminal. There are 22 kinds of amino acids. During the synthesis of rough endoplasmic reticulum, PrPC was modified by Golgi apparatus and then transported to the inner surface of cell membrane, where it combined with glucophosphatidylinositol (GPI) as a supporting point. At this time, 23 amino acids can be deleted from the COOH terminal of PrP, and the signal peptide is cleaved [1, 4]. The in-depth study of prion molecular biology and structure may open up a new field in the mechanism of cell self-regulation and internal environment stability.

Second, the structure and characteristics of prions

It is proved by molecular probe technology that normal human PrP gene is located on the short arm of chromosome 20, and PrPC is a normal component in human and animal cells. Sequence analysis shows that there is no difference between PrPC and PrPSC, they belong to the same protein but have two configurations. Both isomers were purified without denaturation, and their secondary structures have been determined by Fourier transform infrared spectroscopy and circular dichroism spectroscopy. It is found that the α helix of PRPC is as high as 42%, and the β fold is only 3%, while the protein skeleton of PrPSC is completely extended, forming the β fold as high as 43%, and the α helix is only 30% [5].

In cultured cells, the transformation from PrPC to PrPSC occurs inside neurons, and then PrPSC is deposited in lysosomes. Lysosomes filled with PrPSC and accompanying amyloid granules in the brain will suddenly explode and damage cells. When the nerve cells of the host die, many pores, such as sponginess, will be left in the brain tissue, and the released PrPSC will attack other cells, which is an important feature of prions [5,6].

About 15% of patients with human prion diseases are hereditary and autosomal dominant, and the rest are infectious cases including iatrogenic and sporadic cases. It has been proved that there is a mutation of PrP gene in the family of hereditary patients (human named PRNP) [7]. Individuals with PrP gene mutation are particularly sensitive to the transition from α -helix to β -lamella When encountering exogenous pathogenic factors (such as PrPSC), about half of the members will get sick, and the incubation period is closely related to exposure dose and strain. Because PrP lacks nucleic acid, the difference of plant type lies in the difference of configuration. One PrP can fold PrPC into pathogenicity with short incubation period, while the other folding method may be ineffective, but the incubation period is long. Even the same configuration may be deposited in different groups of neurons in the brain, resulting in different clinical manifestations.

Third, the replication of prions.

The pulse tracing experiment of itch infection factor infecting cultured cells showed that the transformation of PrPC was a post-translation reaction [1]. When pruritus protein comes into contact with PrPC molecules, it will change the configuration of PrPC and become pathogenic PrPSC. This reaction is like a waterfall, and the converted molecules can be converted into other normal molecules. In individuals with point mutation or insertion mutation of PrP gene, the mutated PrPC molecule can be spontaneously transformed into PrPSC. Although random reaction may not achieve pathogenic effect at first, once such transformation occurs, autocatalytic events will occur, which will increase PrPSC exponentially. This mechanism can explain that some people hide mutations when they have embryos, but the degeneration of the central nervous system will not happen for decades, because the accumulation of PrPSC in the brain is slow [1, 5]. PrPC has accumulated in the brain, but the mRNA of PrPC remains unchanged, which also shows that this transformation is a post-translational reaction of protein.

The transformation and replication of prions from PrPC or its precursors to PrPSC does not seem to require any chemical modification. PrPC molecules combine with PrPSC molecules to form heterodimers or possible trimers, which will be transformed into two PrPSC molecules and then proliferate exponentially. This dimer is an intermediate product of replication. These results have been confirmed in transgenic mice.

Brown et al. [6] and Goldfarb et al. [7] all showed that codon 129 of human PrP gene was polymorphic. In Caucasians, about 38% are homozygous M (methionine) alleles, 5 1% are heterozygous, and 1 1% are homozygous V (valine). Most sporadic CJD occurs in this homozygous individual, and those individuals heterozygous at codon 129 have the lowest risk of prion diseases [7,8]. Familial CJD and FFI are obviously different subtypes in clinic and pathology, but both of them have a mutation from 178 codon aspartic acid (ASP 178) to asparagine (ASN 178) in PrP gene, and only the polymorphism of 129 codon is different. Patients with 15 FFI in five families are all ASN 178 alleles and M 129 alleles, while patients with subtype 15 CJD in six families are all ASN 178 alleles and V 129 alleles [6]. Other gene mutations in patients with prion diseases are also found in codons 102, 200, 1 17, 198, etc.

Four. Prion transmission and species barrier

Brown et al. [9] collected 1963 ~ 1993 from the National Institutes of Health (NIH) in the United States (mainly in the United States and Europe) and inoculated 300 cases of various neuropathy in primates by brain tissue infusion. 300 cases of spongiform encephalopathy include 278 cases of CJD, including 234 cases of sporadic, 36 cases of familial and 8 cases of iatrogenic (such as corneal transplantation or other operations); There were 65438 08 cases of Kulu disease and 4 cases of GSS syndrome. The results showed that the transmission rate of iatrogenic Creutzfeldt-Jakob disease was the highest (100%), followed by Kulu disease (95%), sporadic Creutzfeldt-Jakob disease was also high (90%), and the transmission rate of most familial diseases was low (68%).

At present, people are generally concerned about whether mad cow disease can be transmitted to people. It is difficult to answer this question directly, because it is impossible to inoculate people with animal brain infusion, and the incubation period of observation is very long before a conclusion can be drawn. There are a lot of research materials about species barriers. The experimental results showed that 16 of the 254 amino acids in mouse PrP gene were different from those in vole gene. Transgenic mice inoculated with mouse PrPSC will produce more mouse PrPSC, and inoculated with vole PrPSC will also produce vole PrPSC. However, it is preferred to interact with PrPC with the same or similar sequence, indicating that the species barrier is not tight [2], so the PrP gene of sheep and cattle is only 7 codons different, so the scrapie of sheep is easy to spread to cattle, while the PrP gene of human and cattle has more than 30 codons different, so some scholars think that it is less likely for cattle to spread to people, but it has been confirmed that human PrPSC can spread to voles [2]. There are 28 codon differences between human and mouse PrP genes. When the brain tissue of patients with Creutzfeldt-Jakob disease or GSS syndrome is inoculated into mice with some human PrP genes, it is found that the disease occurs faster and more frequently than mice with all human PrP genes, indicating that the similarity of core components of PrP molecules is more important in promoting the disease [2].

In the past, it was thought that prion diseases mostly occurred over 60 years old. There are only two young people (16 and 18 years old) [10,1] among the patients with sporadic creutzfeldt-Jakob disease reported in Britain. Recently, Howard et al. [8] reported 1 case of atypical CJD in a 28-year-old female, which was confirmed by frontal lobe biopsy and autopsy. After Will et al. [12] reported 10 cases of variant creutzfeldt-Jakob disease in British youth, Chazot et al. [13] reported/kloc-0 cases of 26-year-old French men suffering from creutzfeldt-Jakob disease. They believe that although these patients have no history of contact with cattle, their contact with beef or eating mad beef may be the cause of the disease. It is considered that even if there is an efficient species barrier between cattle and people, the possibility that a few susceptible people are infected among a large number of people who have been exposed to mad cow disease cannot be ruled out [8]. Recently, the fact that two cattle breeders in Britain who suffered from mad cow disease died of Creutzfeldt-Jakob disease has attracted people's attention [2].

By the end of February, 1997, at least 17 patients with new variant creutzfeldt-Jakob disease (nvCJD) were diagnosed [14].

The research of Collinge et al. [15, 16] not only proved that transgenic mice expressing human PrP can also produce human PrPSC and develop prion diseases, but also found that PRP obtained from various CJD cases can be divided into four types (strains) by western blot detection, and only the electrophoresis band of PrPSC of nvCJD patients (type 4) and naturally transmitted BSE.

In the past, the diagnosis of prion diseases mainly depended on brain biopsy or autopsy. At present, monoclonal antibodies against protein 14-3-3 can be used for immunological screening and preliminary diagnosis of patients' cerebrospinal fluid [17], or the diagnosis can be made by tonsil biopsy [18]. Many important advances have been made in the study of prions. In addition to the traditional pathogenic microorganisms and parasites of infectious diseases, a new pathogenic factor has been added. This research won the 1997 Nobel Prize in Medicine.

Authors: Chongqing 400038, Center of Infectious Diseases, Southwest Hospital of the Third Military Medical University.

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(This article was edited by Xin Li)

(Received:1997-1-14 Modified: 1998-06-30)