A Guide To "Chronic Fatigue
Syndrome A Biological Approach" (Edited by Patrick Englebienne Ph.D., Kenny DeMeirleir M.D, Ph.D., CRC
Press. Washington D.C. 2002) by Cort Johnson
Chapter One: Interferon and the 2-5A Pathway by Lionel astide, Edith Demettre, Camille Martinand-Mari,
and Bernard Lebleu
Chapter Two: Ribonuclease L: Overview of a
Multifaceted Protein. by Patrick Englebienne, C. Vincent Herst,
Simon Roelens, Ann D’Haese, Karim El Bakkouri, Karen De Smet, Marc
Fremont, Lionel Bastide, Edith Demettre and Bernard Lebleu.
OVERVIEW OF THE INTERFERON/2-5A/PKR SYSTEM. This
first section provides gives some background information not available
in the text and an overview of the viral inhibition process. To go
straight to the synopsis click
here)
It has long been
evident that individuals suffering from one viral disease rarely
contract another simultaneously. Cells in laboratory cultures
infected with one virus can only with difficulty be infected with
another. One reason for this is a group of soluble factors - the
interferon’s - that cells produce in response to infection by a virus or
other agents.
Two IFN induction pathways are believed to exist; one specific to
viruses, the other stimulated by other agents. While IFN’s have
mostly been studied with reference to viruses, they can be activated by
a wide variety of other agents including some bacteria, rickettsiae,
protozoa, organic compounds, antibiotics, antigens, etc. The two
components of the immune system we are mainly concerned with, RNase L
and PKR, are products of the IFN response; they are activated, however,
only if double stranded (ds) RNA is present.
It is important to note that the interferon response is the cells first
attempt to stop a viral invasion. IFN’s are a fundamental
component of what is called the ‘innate response’ of the immune system.
If the innate response fails to check the virus then the ‘adaptive
response’ driven mainly by T and B cells will eventually kick in.
Most pathogens are, however, stopped by the innate response.
As a virus enters a
cell and begins to replicate, metabolic changes in the cell cause genes
coding for IFN to produce messenger RNA (mRNA) which travels to the
ribosomes and codes for IFN production. The IFN’s bind to
receptors inside the cell which send a signal down a complex pathway
that stimulates a wide variety of what are called interferon stimulated
genes (ISG’s) in the nucleus. (They also travel outside the cell
and alert other cells.) These genes induce a host of cellular
activities that are designed to stop the spread of the virus. Two
of the most significant by-products of IFN stimulation are the
stimulation of the protein kinase R (PKR) and RNase L systems.
As mentioned earlier
both RNase L and PKR are activated by IFN’s when dsRNA is present.
Viral RNA in contrast to human RNA, occasionally forms loops or double
strands that alert the cell to the presence of the virus. (The
cell is then alerted to the presence of a virus by at least two
substances; metabolites produced by the virus initiate the IFN response;
dsRNA produced by the virus results in IFN induced PKR and 2-5OAS
activation.) Once activated PKR prevents the first part of mRNA
from being translated at the ribosomes. When this happens the
ribosomes are unable to read the mRNA and viral replication is stopped.
RNase L degrades
ribonucleic acids or RNA. Once oligoadenylate synthetase (2-5OAS)
is activated by type I IFN’s, it creates something called 2-5A out of
bits of ATP it has chopped up. When 2-5A is present RNase L binds
with it, becomes activated and begins to degrade RNA. Because both
PKR and RNase L degrade both viral and cellular RNA, protein synthesis
in a cell is virtually stopped when they are upregulated. The IFN’s do
not operate with surgical precision; they are blunt instruments
that stop infections by disrupting major cellular functions such as
protein synthesis and cell growth. If a cell cannot be cleansed of
a virus it is readily sacrificed.
So here we have a two
pronged attack on viruses. PKR stops the translation of their mRNA
and RNase L chops up untranslated viral mRNA. Together they
provide a very potent attack, an attack that is so potent, in fact, that
once interferon activates these pathways, the cell is said to have
acquired an ‘antiviral state’. This book is about what happens when this
attack goes awry.)
THE TEXT
First discovered in the late 1950’s, interferon’s (IFN’s) protect
against a wide variety of viruses in mammalian cells. They do this by
activating genes (interferon stimulated genes or ISG’s) that initiate a
diverse array of biological responses.
Two types of interferons
are produced. Almost all cells, upon contact with a wide array of
pathogens (viruses, bacteria or mycoplasma) or cytokines can produce
Type I IFN’s (IFN a/b). Only immune cells (activated T-cells) on the
other hand, can produce type II IFN’s (IFN-y). More than 100 ISG
(interferon stimulated genes) have been identified.
IFN’s induce the
production of a dsRNA dependent protein kinase (PKR) which signals for
various activities to occur, one of which inhibits viral mRNA
translation. Some of the substances PKR activates are also be activated
in response to stress such as amino acid starvation or heme
deprivation. (This essentially means that cells act somewhat
similarly to some kinds of stress and viral attack. We will see in
Chapter 7 that amino acid depletion is commonly found in CFS. Increased
cellular stress is believed to be a predisposing factor in CFS (Chapter
eight)). Nor surprisingly, many viruses have engineered ways to
counteract PKR’s antiviral activity.
The 2-5A/RNase L pathway
was discovered in the mid 1970’s by researchers attempting to understand
the protein inhibition they observed in IFN treated cells. The
production of dsRNA by viruses stimulates type I IFN (IFN’s A, B)
production. IFN’s essentially prompt cells to respond; they interact
with the receptors on cellular membranes to trigger a wide variety of
effects, some of which inhibit viral replication and cell growth. Once
a type I IFN binds to its receptor on a membrane, it initiates a
signaling cascade that induces the transcription of genes in the nucleus
that code for, among other things, 2-5A synthetase (2-5OAS).
Type I and
II IFN’s induce the transcription of several 2-5OAS synthetase isozymes.
(Transcription involves constructing an RNA molecule in the nucleus
that travels into the protein producing ‘factories’ (the ribosomes) in
the cytoplasm. Proteins are extremely complex substances that do the
work of the cells. Isozymes are enzymes that are chemically distinct
but perform the same function). The isozymes are quite different;
they are synthesized from different genes, they are located in different
parts of the cell, they are activated by different types of dsRNA, and
they synthesize different lengths of 2-5A oligomers.
2-5OAS is produced in response to IFN a/b but is
activated either by ss or ds RNA.
(The requirement for dsRNA
and perhaps for certain types of ssRNA provides a check on the system.
Since RNase L is a blunt instrument, it is hopefully not wielded unless
absolutely necessary.)
Three forms of 2-5OAS
exist; small (p40), medium (p69), and large (p100). The different
isoforms are triggered by different kinds of ss or ds RNA and they
produce different lengths of 2-5A. Upon activation the 2-5A’s ‘oligomerize’
(form repeating units.) 2-5OAS cuts ATP up into pieces and then
binds them together again to form oligomers. Oligomers are simply a
number of units of the same compound; i.e. a dimer contains two units; a
tetramer four. Longer strands of dsRNA induce the p69 isoform to
primarily produce 2-5A trimers (2-5A tripled). Lower sized
strands of dsRNA induce the p100 isoform to mostly create 2-5A dimers
(2-5A doubled).
Unless RNase L is bound by
its inhibitor it will, upon binding to the appropriate 2-5A oligomer,
become activated and then bind to another RNase L molecule (it ‘homodimerizes’),
at which point it is able to degrade the viral (and other) mRNA in the
cell and stop the viral attack (as well as shut down protein
synthesis.)
As mentioned earlier RNase
L needs to bind with 2-5A oligomers to become activated. In order to
better understand the binding and activation sites of RNase L a
three-dimensional model was constructed. The model indicated that when
RNase L binds with a 2-5A trimer an internal clamp is released
causing a dramatic change in the shape of the protein and uncovering
RNase L’s catalytic sites. Only after this occurs is RNase L able to
dimerize and become activated. The 2-5A dimer is able to
bind with RNase L, but according to the model produced by the authors,
it is too short to release RNase L’s internal clamp. This will turn out
to be an important bit of information. By binding with RNase L the 2-5
dimers inhibit RNase L activation by the 2-5A trimers. In a
sense they keep it frozen in its monomeric state, something that we
shall see has some very negative consequences.
An inhibitor
for RNase L (RLI) has recently been identified. Because RNase L
inhibition is dependent upon the ratio of RNase L and RLI in the cell,
any RNase L production quickly outstrips RLI’s capacity to bind and
inactivate it. This system ensures that RNase L is active only when it
is upregulated. Some viruses are able to induce RLI production and
inhibit RNase L activation.
RNase L
IN CHRONIC FATIGUE SYNDROME
An
up regulation of RNase L activity in CFS was first reported in the early
1990’s. Further studies indicated that the upregulation was due to the
presence of low molecular weight (LMW) variants of RNase L in peripheral
blood mononuclear cells (PBMC). PBMC’s are the focus of almost
all the tests in this book. Mononuclear cells include
monocytes/macrophages and T and B lymphocytes. These LMW variants (42, 37-kDa versus
the native 83-kDa RNase L) were found to be capable of binding to the
2-5A oligomers synthesized by 2-5OAS and upon binding they became active
just as the native RNase L does. After much research it was determined
(recently) that the 37-kDa RNase L is produced by the cleavage of
the native 83-kDa RNase L. The 37-kDa protein has recently been verified
as a biomarker for CFS.
Putting
the 83-kDa RNase L into the PBMC’s of controls and CFS patients
indicated that while the native RNase L remained whole in the controls,
most of it was broken into the 37-kDa fragment within 30 minutes in CFS
patients.
In order
to identify the enzyme responsible for fragmenting it, RNase L was
incubated with several enzymes and 2-5OAS. Three enzymes (m-calpain,
human leukocyte elastase (HLE)) and cathepsin G) were capable of
cleaving RNase L into fragments identical to those found in CFS
patients.
M-calpain is a cysteine
protease that is particularly active during apoptosis. Cysteine
proteases are able to cleave proteins at their cysteine sites.
Because CFS patients exhibit increased apoptotic activity, it is not
surprising that enhanced calpain activity has been found in CFS
patients. None of the other apoptotic proteases tested fragmented RNase
L.
Elastase and cathepsin G
are serine proteases found in the azurophilic granules of
polymorphonuclear leukocytes or granulocytes. Granulocytes
are white blood cells with granules that contain enzymes,
antibiotics, etc. that destroy and degrade foreign particles that the
cells have phagocytosed or ingested. Lurking underneath the surface
tissues they man the front lines of the bodies defenses. These
proteins are also, interestingly enough, involved in two processes that
appear to upregulated in CFS; host defense and inflammation.
Very quickly we appear to be very
near the source of the problem. The question was what causes the
fragmentation of RNase L? At least three enzymes that participate
in immune defense are at least capable of doing that. But why
would these enzymes all of sudden start to tear apart RNase L? Are
they more abundant than before? Or is there something the matter
with RNase L? The authors will leave us hanging here a bit until
the end of this section while they backtrack a bit and clear up some
questions about this fragment.
In order to determine if some cells have higher levels of the
RNase L fragments, the PBMC cells were separated according to their CD
classification into T-cells (CD3) or monocytes (CD14). The LMW RNase L
fragments were primarily found in monocytes. Because monocytes
give rise to antigen presenting cells (APC’s), which are responsible for
alerting T-cells that an intracellular invader is present, a
disregulation in them could result in the distorted TH1/TH2 response
seen in CFS.
APC’s digest the
invaders, then display bits of them on their surface for T and B cells
to determine if an immune response should be launched. If the APC’s are
dysfunctional then the ThI arm of the immune system - which responds to
intracellular invaders such as viruses could be down regulated. Down
regulation of the ThI arm and the consequent upregulation of the Th2 arm
of the immune system is what is seen in CFS.
The monomeric and dimeric forms of RNase L were then examined to
determine if either were more susceptible to cleavage. It was believed
that the folding that accompanied dimerization might hide the cleavage
points and render the dimer more resistant to cleavage. Interestingly
enough, only the monomeric RNase L was fragmented in CFS
patients. This is a big step! The mystery is at least partially
solved; RNase does not dimerize and is left in an unprotected state and
upregulated apoptotic and/or inflammatory enzymes chop it up.
CONCLUSIONS AND PROSPECTS
The probable involvement of apoptotic and inflammatory proteases
(m-calpain, HLE, cathepsin-G) in RNase L cleavage illustrates some of
the effects that increased levels of cellular stress and inflammation
may have in CFS. The RNase L abnormalities found in monocytes provides
a mechanism whereby the incapacity of T-cells to activate natural killer
cells via nitric oxide mediation might be explained. In this case the T
helper cells simply do not receive a signal for activation because the
dysfunctional monocytes do not properly evolve into APC’s. These
observations suggest that therapeutic approaches using protease
inhibitors and regulators of calcium homeostasis may be fruitful in CFS.
The finding that the monomeric form of RNase L
is more susceptible to cleavage than the homodimeric form, indicates
that 2-5 oligoadenylate synthetase (2-5OAS) plays a major role in the
syndrome. If improper activation of 2-5OAS resulted in a lack of RNase
L homodimerization, then RNase L would remain in its latent monomeric
form, a form that is susceptible to cleavage by inflammatory and
apoptotic proteases. These observations suggest that immmunomodulators
able to (properly) activate the 2-5OAS system such as bile salts
or retinoic acid (vitamin A) derivatives may be helpful.
This suggests that
‘improper’ 2-5OAS activation is very near the heart of this disorder and
that RNase L is more an unwitting victim than an instigator. What would
cause ‘improper’ 2-5OAS activation? Remember that 2-5A trimers activate
RNase L; 2-5A dimers only bind to it. It appears that short sections of
RNA induce 2-5OAS to produce 2-5A dimers that leave the RNase L enzyme
in an unprotected state just as the cell is being flooded with apoptotic
and/or inflammatory enzymes. Where do these short sections of RNA come
from? The authors give us little clue but do note that short sections
of dsRNA that are often associated with ‘subcellular fractions’
including mitochondria, nucleus and microsomes. Are these small bits of
RNA coming from disrupted mitochondrial activity? Are they fragments
from increased apoptotic activity? There is evidence of mitochondrial
dysfunction in CFS. Whatever is going on, it seems just as we appear to
be getting to the heart of the problem, another layer appears. What is
causing CFS? It appears to be these little strands of RNA. But where
do they come from, and what is causing them to appear???
The 40 and 37-kDa fragments lack the regions
(the protein kinase like and ankyrin repeats) that are needed not only
for dimerization but also for regulation. The loss of the ankyrin
‘clamp’ means that RNase L’s internal regulating mechanism is gone and
that the 37-kDa fragment is, unless it is inhibited by RLI, always
active.
Click
to go to
Chapters III, IV