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â€‹The Detection of Corrupt Prions

Posted on: Fri 18-12-2015

This research is the leading and pioneering work in the discovery and development of an ultra-sensitive technique for the detection and diagnosis of prion infection using recombinant prion proteins. The significance of an early detection and diagnosis of prion disease, a fatal infection that has no known cure, lies not only in potentially prolonging the lives of many patients through early treatment but saves many human lives by the prevention of its spread. It also can potentially save billions of naira in the food and agricultural industries through the early detection and destruction of infected livestock to prevent the contamination of the healthy ones. The sequential publication of this work in two issues of Nature Methods and the continued multiplication of its numerous citations due to the interest of the global Science Community daily reflected on the Internet proves its popularity among researchers in the fields of infectious diseases and agriculture. The prestige it has brought to the University of Nigeria is best reflected in the acknowledgement and appreciation letter from the editor-in-chief of Nature Methods.

The transmissible spongiform encephalopathies or prion diseases are infectious neuro-degenerative diseases of mammals that include Kreutz-Jakob (CJD) and Kuru diseases in humans, bovine spongiform encephalopathies or Mad Cow Disease in cattle, Scrapie in sheep and Chronic Wasting Disease in Elk. They have also been observed in mink, cats, rodents, exotic engulates and other mammals but not in dogs, rabbits, horses or bird. In humans it exists as CJD or its variant form vCJD which resulted from humans being infected by infected cattle. Transmissible spongiform encephalopathies have incubation periods of months to years, but after the appearance of clinical signs, they are rapidly progressive, untreatable and invariably fatal (Atarashi et al, 2007). Infections can occur from ingestion, inoculation contact with infected surgical materials or body fluids (Onwubiko, H.A. 2012). Transmissible spongiform encephalopathies have been described as the worst way of dying. There are reports of young women going into menopause once infected despite their age, and infected individuals are unable to sleep and at best go into a state of stupor, where they still retain full consciousness (Max, D.T. 2006).

Transmissible spongiform encephalopathies are also making their impact on the world’s agricultural industries. Over four million cattle had been destroyed in Britain alone as a result of prion infection in order to contain its spread to other uninfected cattle and sheep in Europe. Prion diseases are also affecting international commerce, shipping and immigration rules as various nations heighten vigilance and security at their borders and ports to protect their human and livestock populations. Unfortunately in Nigeria there are no strategic plans at present to prevent livestocks or human life from prion infection.

For many years, eminent researchers had sought to know the nature of the disease causing agent of the transmissible spongiform encephalopathies. After having eliminated bacteria, VIRUSES and other micro-organisms, Prusiner in his outstanding Nobel winning work purified the protein components from infected scrapie fibrils and cloned the prion gene (Prusiner et al, 1989). A comparison of the infectiously derived cloned gene showed that it was identical to the host prion gene. Furthermore, other researchers have also cloned the mice and hamster prion gene (Chesebro et al, 1985; Oesh et al, 1985). These workers also found no difference between the normal prion gene and the diseased prion gene. A most convincing proof came with the cloning of the Knock-out mice (mice which lack the prion gene) and the finding that they did not develop prion disease upon infection, (Chesebro, 1998).

How then does the normal prion protein differ from the diseased or infectious prion protein since they are the same protein from the same host gene? The normal prion protein is highly sensitive to protease digestion and is, therefore, designated as PrP-sen (meaning PrP protease sensitive). It is soluble in detergents and present in diverse tissues and cell types. PrP-sen is a monormeric glycophosphatidylinositol (GPI) linked glycoprotein. Its apparent cellular roles include adhesion, differentiation of cells, neuritogenesis, synaptogenesis as well as cell survival.

Free copper is known to be toxic to the central nervous system hence copper levels are mopped up by prion protein which plays a role in their regulation. Also altered sleep patterns and circadian rhythm activity regulated by the penal gland has been observed in mice that lack prions such as the knockout mice (Tobler et al, 1996, 1997). Normal prions (PrP-sen) has also been shown to have super-oxide dismutase activity, thus making it a powerful anti-oxidant for the central nervous system (Brown et al, 1999). Other roles in which normal prion protein (PrP-sen) has been implicated includes signal transduction in neuronal cells which in part may account for the clumsy motor-responses of patients with prion disease (Mouillet-Richard et al, 2000). Normal prion protein has also been implicated in the activation of lymphocytes, a process that may affect patients with prion disease to exhibit an adequate immune response to infections (Li et al 2001). Also evidence from infra red spectroscopy indicate that the normal prion protein (PrP-sen) contains 28% alpha helix and 4% beta pleated sheet.

THIS is reflected by its maximum absorbance in the infrared at 1656cm-1 wavelength while the normal diseased prion protein (PrP-res) has a maximum absorbance at 1628cm-1 due to its higher beta sheet content. In fact infectious prion protein (Pr P-res) contains 43-61% beta sheet content compared to only 4% for the normal prion protein. Thus the infectious and normal prion protein (PrP-res) differ mainly in conformational changes due to the secondary structure of otherwise the same protein, with the same covalent structure and amino acid sequence.

One of the most intriguing problems of molecular biology and biochemistry is the propagation of pathology or to show how normal prion protein (Pr P-sen) is converted by the infectious and abnormal prion protein (PrP-res) into an abnormal newly infectious prion protein (PrP-res).

While conversion mechanistically involved both contact and conformational changes by the normal and pathological prion protein, invitro experiments with radioactive sulfur tagged normal prion protein 35S –PrP-sen incubated with infectious prion (PrP-res) and treated with proteinase K gave the product of 35S – Pr P-res, as an intermediate product not identical to the initial pathological protein which indicated the conversion of normal prion protein (PrP-sen) to its infectious isoform, PrP-res (Kocisko et al, 1994).

Furthermore, continuous amplification of infectivity when crude brain homogenate is used as a source of infectious prions (PrP-res) has also been observed (Saborio et al, 1999; 2001). Also, the structure of the various domains of Pr P-sen upon conversion to PrP-res (infectious) oligomers have been assessed and it can be seen that the octarepeat region which normally binds four copper atoms remain exposed to proteases and easily undergoes alteration. Independent deletion mutagenesis studies have shown that the N-terminus of PrP facilitates prion conversion to its pathological PrPres form (Supattapone et al, 2001; Flechsig et al 2000). However, residues 90 to 231 which include the helix-1 salt bridges are unaltered and stabilized against conversion. This region is also known to form strain dependent beta sheet identified by various infra-red bands to reflect strain specific conformations.

In general, the experience of most researchers about normal prion (PrP-sen) conversion to its pathological form (PrP-res) show that it is stimulated by sulfated glycans, chaperone proteins, partially unfolding detergents and an increase in temperature to 65oC (Wong et al, 2000). It is inhibited by disulfide bond reduction and requires PrP-res (the pathological form) as multimers for seeding the process. Additionally, there appears to be specific bonding of the hosts normal PrP-sen to the diseased prion (PrP-res) polymer (DeBurman et al; 1997). This specificity is thought to be responsible for the strain specificity observed in infected animals. For instance, the pathological form of the prion protein from humans shows greater degree of virulence to humans than to other animals.

Several other factors have been observed to influence the rate of conversion of PrP-sen (normal) to Pr P-res (pathological). (Korth et al, 2000, Saborio et al; 2001). Correlations between PrP sequences show that the closer the sequences between PrPsen and PrPres, the higher the conversion rate. Thus, the more the similarities in structure between species, the higher the rate of conversion and inter-species transmissibilities of prion diseases. It is, therefore, reasonable that the highest rate of conversion is obtainable within the same species (Prusiner et al; 1990).

What is peculiarly striking about the mode of prion infection is that it is characteristically different from all our previous knowledge on how infections occur, with the examples of bacteria, plasmodium in malaria, other micro-organisms and VIRUSES. Our past accumulated knowledge show that infections can only occur through the invasion of an infectious agent into a host cell and the subsequent replication of its nucleic acid. However, prion is a simple inanimate protein without any known nucleic acid for self-replication, yet its abnormal isoform is capable of transforming the normal prion protein to an infectious form.

Based on the capacity of the infected prion protein (PrP-res) to induce the conversion of the normal prion protein to its infectious isoform, we have developed an ultra-sensitive means for the detection and diagnosis of infectious prions and its resulting diseases. In this work, we obtained a quick and steady supply of normal recombinant forms of prion protein (PrP-sen) from bacteria (E. coli) for various species of sheep, human, cattle, hamsters, mouse, etc. Normal prion proteins obtained are labeled as rec – Hu PrP, rec-Ha-PrP, rec-Mo-PrP, rec-Sh-PrP, respectively for human, hamster, mouse, and sheep.

Of course, obvious concerns arise which include the dangers of obtaining test samples of cerebrospinal fluid or brain homogenates from animals in the field without subjecting them to trauma, while applying this technique to test for prion diseases. Many animals which include humans and livestock can go into shock, leading to their death in an attempt to obtain test samples from them. Fortunately, on-going experiments at the Laboratory of Persistent Viral Diseases in Hamilton Montana, a branch of the National Institute for Allergy and Infection Diseases (NIAID) a sub-sector of the United States National Institutes of Health (NIH), has since shown results that nose swaps and saliva or other body fluids could serve as harmless substitutes in the application of our ultrasensitive method for the diagnosis of prion disease, thus putting these results beyond dispute as a significant contribution to the advancement of science in the battle against the present resurgence of infectious diseases.

• Concluded

•Onwubiko is Professor of Biochemistry. This an abridged version of his Inaugural Lecture titled “Ultrasensitive Detection of Corrupt Prions and Diagnosis of Their Neurodegenerative Infectious Diseases: Mad Cow Disease in Cattle, Scrapie In Sheep And CJD in Humans” delivered at the University of Nigeria on Thursday 9th July, 2015

By Henry Onwubiko

Source: Guradian News

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