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| Figure 1: Endoplasmic reticulum action and stress response. In
the endoplasmic reticulum, secreted proteins and membrane proteins
are folded, and sugar molecules are added (green arrow). When
altered proteins that are improperly folded or in the process of
being folded accumulate, the endoplasmic reticulum loses its
functions, causing endoplasmic reticulum stress. This stress leads
to three stress responses: (1) Translation of mRNA is repressed,
and the number of proteins carried to endoplasmic reticulum is
reduced. (2) Gene transcription of molecular chaperones, which
assist in the folding of proteins, is induced to promote folding.
(3) Altered proteins are removed from endoplasmic reticulum and are
decomposed. |
| Copyright : RIKEN 2009 |
Takao Iwawaki
Initiative Research Scientist
Iwawaki Initiative Research Unit
RIKEN Advanced Science Institute
The endoplasmic reticulum is a cell organelle that acts as a
processing factory for secreted proteins and membrane proteins. The
accumulation of abnormal proteins in endoplasmic reticulum causes a
functional disorder called ‘endoplasmic reticulum
stress’, which if not arrested leads to cell death. When and
where does endoplasmic reticulum stress occur? How do cells avoid
this stress? Trying to solve these mysteries, Initiative Research
Scientist Takao Iwawaki has developed the world’s first
endoplasmic reticulum stress visualization system, and is using the
system to advance his research. Endoplasmic reticulum stress has
recently been found to play a role in the development of various
diseases including Alzheimer's disease, diabetes and cancer.
Research into endoplasmic reticulum stress is expected to lead to
the development of therapeutic agents for these diseases.
The endoplasmic reticulum — a protein processing
factory
The endoplasmic reticulum stress visualization system developed by
Iwawaki has been dubbed ‘ERAI’, short for the
endoplasmic reticulum stress activated indicator. Erai also means
‘great’ in Japanese. “Please call it
‘e-ra-i’,” says Iwawaki. “I think names are
very important, because people are attracted by names that have a
strong impact.”
What kind of a system is ERAI? Is it really as great as it
seems?
At the microscopic level, a cell in a living organism contains many
multi-layered sack-like cell organelles called endoplasmic
reticulum. The functions of the endoplasmic reticulum can be
divided into three groups: (1) synthesis, modification, transport
and quality control of secreted proteins and membrane proteins; (2)
synthesis of lipids and maintenance of homeostatic properties; and
(3) storage of calcium ions. Iwawaki focuses on the first category
of functions.
The formation of proteins in a cell starts with the DNA in the cell
nucleus. The DNA contains genes, which encode information on when,
where and what kinds of proteins are to be produced. In the process
of making a protein, the four base sequences adenine, thymine,
guanine and cytosine in part of the gene are transcribed into RNA.
Non-coding regions, called introns, are then removed to form
messenger RNA (mRNA), which is carried away from the nucleus. The
mRNA base sequences are, in turn, translated into amino acids in
cell organelles called ribosomes, leading finally to the formation
of proteins. The newly produced ‘nascent’ proteins,
however, are merely long strings of amino acids, and still do not
fulfill their intended function. Secreted and membrane proteins,
such as digestive enzymes, hormones and antibodies, are carried to
endoplasmic reticulum as they are, and there the nascent proteins
are folded and combined with sugar molecules. Properly folded and
functional proteins then emerge from the endoplasmic reticulum to
be carried to the areas where they are needed.
“The endoplasmic reticulum serves as a protein processing
factory in which proteins, at this stage merely long strings of
amino acids or ‘raw materials’, are
‘manufactured’ into properly folded proteins,”
says Iwawaki. “As you know, no factory can avoid producing
defective products. The accumulation of improperly folded proteins
causes the endoplasmic reticulum to lose its functions. This
situation is called ‘endoplasmic reticulum stress’. I
want to elucidate the mechanisms of occurrence and avoidance of
endoplasmic reticulum stress.”
Persistent endoplasmic reticulum stress leads to the death of
cells. Thus, cells are equipped with a stress response mechanism
for avoiding endoplasmic reticulum stress. “In a factory,
when the rate of defective products reaches a certain level, the
production line will be stopped. This is also the case with the
endoplasmic reticulum. A signal is issued to repress the
translation of mRNAs to prevent further new materials from being
carried to it. This is the first stress response. Additionally, the
number of helpers that serve to fold proteins is increased as an
effective measure to process the accumulated proteins. This is the
second stress response, called the unfolding protein response
(UPR), and it is this action that is the focus of my research. We
can also observe a stress response in which defective products are
removed from endoplasmic reticulum.”
The world’s first visualization of endoplasmic reticulum
stress
Iwawaki had a question when he started to study the endoplasmic
reticulum in graduate school. The mammalian UPR has three pathways,
involving either PERK, ATF6 or IRE1 molecules, which are present in
the membrane of the endoplasmic reticulum. These molecules can
sense the accumulation of altered proteins, and serve to induce the
transcription of molecular chaperone genes that help fold proteins.
Yeast cells, however, have only the IRE1 pathway, and it can
develop under ordinary conditions even in the absence of IRE1
molecules. Iwawaki’s question: “Is an endoplasmic
reticulum stress response essential for living organisms to
survive?”
This question was answered in 2000, when it was found that mice
have two types of IRE1, IRE1α and IRE1β, and that fetal
mice die before birth if IRE1α is absent. This showed that an
endoplasmic reticulum stress response is essential for the survival
of mammals. At about the same time, a paper was published reporting
a relationship between endoplasmic reticulum stress and
Alzheimer’s disease. It has since been found that endoplasmic
reticulum stress may play a role in diseases such as
Parkinson’s disease, bipolar disorder, diabetes,
arteriosclerosis, rheumatism, viral infections and cancer.
“These diseases do not appear in yeast or cultured cells,
which made me think that I should use animals to study endoplasmic
reticulum stress at the level of the whole organism.”
“When and where does endoplasmic reticulum stress occur? This
was the first question I wanted to answer,” says Iwawaki. The
common method at the time to investigate the occurrence of
endoplasmic reticulum stress was to measure molecular chaperone
expression. However, such measurements are performed after grinding
the cells and, therefore, it is not possible to tell where the
stress occurred. Iwawaki then began the development of a system for
visualizing endoplasmic reticulum stress. Fortuitously, as a result
of an academic meeting, he happened to become aware that when IRE1
senses stress, the introns of the XBP1 gene are cut off, and the
rest of the gene then acts as a factor to induce molecular
chaperone transcription. “I immediately thought I could use
this mechanism. First, we bring XBP1 genes and genes for green
fluorescent protein, or GFP, together to create ERAI genes. Then,
when endoplasmic reticulum stress occurs in animal cells bearing
the introduced ERAI gene, IRE1 acts to cut off the introns of the
XBP1 gene, which results in the formation of proteins in which XBP1
proteins and GFP are connected, causing light to be emitted. In the
absence of endoplasmic reticulum stress, IRE1 remains inactive, and
XBP1 also remains inactive without emitting light because IRE1 is
not active. This is the basis of the ERAI system.”
The ERAI process is shown in Figure 2, and the results of an
investigation of endoplasmic reticulum stress in the kidney of an
ERAI gene-bearing mouse are shown in Figure 3. “The kidney
did not emit light when a normal saline solution was administered,
but emitted green light when tunicamycin, a medical agent, was
administered. Thus we succeeded in visualizing endoplasmic
reticulum stress for the first time in the world.”
Iwawaki also made another great discovery: the pancreas of the ERAI
gene-bearing mouse glowed even when the mouse was given a normal
saline solution. “The pancreas produces many secreted
proteins, such as insulin. Thus, it was thought that the pancreas
is under endoplasmic reticulum stress because it is constantly in
an overloaded state. ERAI clearly demonstrated that the pancreas is
constantly under endoplasmic reticulum stress. The light from the
pancreas became intense from about 18 days after birth. An average
baby mouse starts weaning at that time, and starts eating solids.
This may change the way insulin or digestive enzymes are secreted,
or how stress occurs in the pancreas.”
When these findings were published in Nature Medicine in December
2003, Iwawaki received many requests for ERAI genes and ERAI mice.
For researchers who want to study the relationship between
endoplasmic reticulum stress and diseases, ERAI is an effective
research tool. However, Iwawaki felt the necessity to tackle new
challenges in ERAI development. “To me, the development of
ERAI at that time was not a complete success.”
Endoplasmic reticulum stress observed even in fetuses
Fetuses cannot survive in the absence of IRE1α. This means
that some organs in fetuses are constantly under endoplasmic
reticulum stress. IRE1α acts even during the fetal
development of a normal mouse, thus providing a means of relieving
this stress. Iwawaki was hoping that he would be able to use the
ERAI system to visualizing endoplasmic reticulum stress in fetuses.
The fetus, however, failed to emit light. “I thought that
this was due to a lack of sensitivity. So I developed the ERAI-LUC
system in which luciferase, a luminescent enzyme used in place of
GFP genes, and XBP1 genes were brought together.”
GFP, used in the original ERAI scheme, fluoresces when exposed to
light. Thus, light is required to investigate internal organs using
GFP genes. Luciferase, on the other hand, emits light by oxidizing
photosubstrates called luciferins, thus eliminating the need to use
light. Using a dedicated system and administering luciferins at the
last minute allows observation of the entire organ without causing
damage.
Figure 4 (click link to article to see figures 4 and 5) shows the
results obtained from investigating endoplasmic reticulum stress in
the fetus of an ERAI-LUC mouse. “A widely accepted theory
says that endoplasmic reticulum stress occurs in the liver of the
fetus, but I found this to be incorrect. My investigation proved
that the stress occurs in the placenta. This result was a surprise,
but also showed that an endoplasmic reticulum stress response
occurs through a new and different pathway.”
It is thought that endoplasmic reticulum stress occurs, in a strict
sense, in a portion of the placenta called the placental labyrinth,
a network of maternal and fetal blood vessels where nutrients,
waste, oxygen and carbon dioxide are exchanged.
IRE1α-deficient mice are known to have fewer blood vessels in
this area, and to have difficulty in exchanging these necessities.
Endoplasmic reticulum stress in the placenta, Iwawaki believes, is
due to a shortage of sugar molecules as a result of the
interruption of placental exchange. Proteins folded in the
endoplasmic reticulum are only completed when combined with sugar
molecules. “When sugar molecules are deficient, endoplasmic
reticulum stress cannot be relieved, no matter how active the
expression of molecular chaperones. New blood vessels need to be
created and sugar molecules supplied. The conventionally known
endoplasmic reticulum stress response completes within a single
cell. However, we found that there is a pathway through which the
endoplasmic reticulum stress can be relieved by exerting an effect
on the extracellular environment beyond the bounds of the cells.
This result was obtained by investigating the organism as a
whole.”
As his next target, Iwawaki aims to develop effective treatments
for diseases in which endoplasmic reticulum stress may play a role.
“Mating an ERAI-LUC mouse and a disease-model mouse will
allow us to discover when and where endoplasmic reticulum stress
occurs as part of the disease. This knowledge will provide an
important clue on how to treat the disease. If the mechanism of the
endoplasmic reticulum stress response can be clarified, we will be
able to adjust the level of the stress response.”
The ERAI-LUC system has been further improved, and now allows for
the expression of ERAI genes only at specific sites. “The
ERAI-LUC system has now been perfected,” Iwawaki says with
confidence. However, the system cannot be used on humans because
gene recombination is required. “We need to develop a better
technique for visualizing human endoplasmic reticulum stress. This
will also serve to help develop therapeutic agents for
disease.”
Inspired by Tonegawa’s Nobel Prize in Physiology or
Medicine
“I was an arts student in high school,” says Iwawaki.
He belonged to an athletics club, and devoted all his time to
training because he wanted to become a physical education teacher.
However, he experienced a turning point in his life when he was in
his third year in 1987. “Dr Susumu Tonegawa was awarded the
Nobel Prize in Physiology or Medicine in 1987, and our high school
biology teacher told us passionately about Dr Tonegawa’s
research results.” Tonegawa is now director of the RIKEN
Brain Science Institute. “Physical and chemical laws can
serve as tools to elucidate ambiguous processes like life
phenomena. I was deeply affected by this fresh and interesting
idea, and it made me think that I would like to be involved in
those kinds of research activities in the future.” However,
at the time of the announcement, in November, it had become too
late to shift his direction from humanities to science before the
university entrance exams. He took the entrance examination for his
originally intended university, and was admitted into the
department of economics. Dissatisfied, he abandoned that course in
his second year and reenrolled in biology. He now works as an
initiative research scientist at RIKEN.
This year is the fifth and final year of the Iwawaki Initiative
Research Unit. “We have achieved some of our initial targets,
but at the same time, new questions have been raised. Research
activities do not have clear goals.”
Takao Iwawaki
Takao Iwawaki was born in Osaka, Japan, in 1969. He graduated from
the Faculty of Education, Nara University of Education, in 1995,
and obtained his PhD in 2001 from the Nara Institute of Science and
Technology (NAIST). He then worked as a special postdoctoral
researcher at the RIKEN Brain Science Institute for two years, and
as a JST PRESTO Researcher at NAIST for another two years. In 2005,
he returned to RIKEN as an initiative research scientist leading
his own research group. His research presently focuses on
functional analysis of endoplasmic reticulum stress responsive
molecules using transgenic and gene targeting mice and on
development of imaging methods for monitoring physiological and
pathological endoplasmic reticulum stress in vivo. |