Protein Misfolding Cyclic Amplification
Encyclopedia
Protein Misfolding Cyclic Amplification (PMCA) is an amplification technique (conceptually like PCR
but not involving nucleotide
s) to multiply misfolded prion
s originally developed by Soto and colleagues. It is a test for spongiform encephalopathies like BSE
.
, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into misfolded prion (termed PrPSc).
with a similar efficiency to the in vivo
process, but with accelerated kinetics. PMCA is conceptually analogous to DNA amplification
by PCR. In both systems a template grows at expenses of a substrate in a cyclic reaction, combining growing and multiplication of the template units.
animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.
ic infectious PrPSc. PMCA enables to generate millions infectious units starting with the equivalent to one PrPSc oligomer, that is way below the infectivity
threshold. This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA and open a great promise for development of a highly sensitive detection of PrPSc and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrPSc in blood of animals experimentally infected with prions during both the symptomatic and pre-symptomatic phases as well as in urine.
s. These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute one of the strongest evidence in favor of the prion hypothesis.
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
but not involving nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s) to multiply misfolded prion
Prion
A prion is an infectious agent composed of protein in a misfolded form. This is in contrast to all other known infectious agents which must contain nucleic acids . The word prion, coined in 1982 by Stanley B. Prusiner, is a portmanteau derived from the words protein and infection...
s originally developed by Soto and colleagues. It is a test for spongiform encephalopathies like BSE
Bovine spongiform encephalopathy
Bovine spongiform encephalopathy , commonly known as mad-cow disease, is a fatal neurodegenerative disease in cattle that causes a spongy degeneration in the brain and spinal cord. BSE has a long incubation period, about 30 months to 8 years, usually affecting adult cattle at a peak age onset of...
.
Technique
The technique initially incubates a small amount of abnormal prion with an excess of normal protein, so that some conversion takes place. The growing chain of misfolded protein is then blasted with ultrasoundUltrasound
Ultrasound is cyclic sound pressure with a frequency greater than the upper limit of human hearing. Ultrasound is thus not separated from "normal" sound based on differences in physical properties, only the fact that humans cannot hear it. Although this limit varies from person to person, it is...
, breaking it down into smaller chains and so rapidly increasing the amount of abnormal protein available to cause conversions. By repeating the cycle, the mass of normal protein is rapidly changed into misfolded prion (termed PrPSc).
Development
PMCA was originally developed to mimic prion replication in vitroIn vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
with a similar efficiency to the in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
process, but with accelerated kinetics. PMCA is conceptually analogous to DNA amplification
DNA replication
DNA replication is a biological process that occurs in all living organisms and copies their DNA; it is the basis for biological inheritance. The process starts with one double-stranded DNA molecule and produces two identical copies of the molecule...
by PCR. In both systems a template grows at expenses of a substrate in a cyclic reaction, combining growing and multiplication of the template units.
Replication
PMCA has been applied to replicate the misfolded protein from diverse species. The newly generated protein exhibits the same biochemical, biological, and structural properties as brain-derived PrPSc and strikingly it is infectious to wild typeWild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
animals, producing a disease with similar characteristics as the illness produced by brain-isolated prions.
Automation
The technology has been automated, leading to a dramatic increase on efficiency of amplification. Indeed, one round of 144 PMCA cycles results in a 6000-fold increase of sensitivity of detection, whereas 2 and 7 rounds of successive PMCA result in 10 million and 3 billion folds amplification.Sensitivity
It has been shown that PMCA is capable to detect as little as a single molecule of oligomerOligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
ic infectious PrPSc. PMCA enables to generate millions infectious units starting with the equivalent to one PrPSc oligomer, that is way below the infectivity
Infectivity
In epidemiology, infectivity refers to the ability of a pathogen to establish an infection. More specifically, infectivity is a pathogen's capacity for horizontal transmission that is, how frequently it spreads among hosts that are not in a parent-child relationship...
threshold. This data demonstrates that PMCA has a similar power of amplification as PCR techniques used to amplify DNA and open a great promise for development of a highly sensitive detection of PrPSc and for understanding the molecular basis of prion replication. Indeed, PMCA has been used by various groups to PrPSc in blood of animals experimentally infected with prions during both the symptomatic and pre-symptomatic phases as well as in urine.
Uses
The PMCA technology has been used by several groups to understand the molecular mechanism of prion replication, the nature of the infectious agent, the phenomenon of prion strains and species barrier, the effect of cellular components, to detect PrPSc in tissues and biological fluids and to screen for inhibitors against prion replication. Recent studies by the groups of Supattapone and Ma were able to produce prion replication in vitro by PMCA using purified PrPC and recombinant PrPC with the sole addition of synthetic polyanions and lipidLipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
s. These studies have shown that infectious prions can be produced in the absence of any other cellular component and constitute one of the strongest evidence in favor of the prion hypothesis.