Study Links Dozens of Proteins to Rare Degenerative Muscle
and Nerve Diseases
By Jason Socrates
Bardi
A team of scientists at The Scripps Research Institute (TSRI)
has identified more than 50 previously unknown proteins and
has associated several of them with rare human degenerative
muscle and nerve diseases. The team is publishing their findings
this week in the journal Science.
Led by TSRI Professors Larry Gerace and John R. Yates III,
the team used a technique called subtractive proteomics to
identify 62 new proteins in the inner nuclear membrane of
the human cell. The team demonstrated that 23 of these proteins
are linked with strong probability to 14 rare muscle-wasting
diseases such as congenital muscular dystrophy, Limb-Girdle
muscular dystrophy, and spinal muscular atrophy, and several
forms of the neurodegenerative Charcot-Marie-Tooth disease.
Knowing the proteins that may cause or contribute to these
diseases is a first step in the long process of looking for
ways to detect, prevent, or treat them.
This study has the potential to clarify a significant number
of the more than 300 human dystrophies for which a causative
gene has not been identified.
"To understand how these diseases happen, we need to understand
more about the players--the network of interlinked proteins,"
says Gerace.
Muscular Dystrophies and the Nuclear Membrane
Many rare but devastating diseases have been linked to the
inner nuclear membrane, which lines the nuclear envelope compartmentalizing
the cell's genetic material or DNA. On the inner surface of
the membrane is a structure referred to as the lamina. The
lamina is important for maintaining the shape and size of
the nucleus. It also contributes to the specialized functions
of different human cells, for example, enabling muscle cells
to perform their particular functions and brain cells to perform
theirs.
The lamina is largely composed of proteins called lamins,
which are like bricks that form a scaffold-like structure
for the nucleus. The lamina also contains membrane proteins
that dock at the lamins.
"There have been a number of human muscular and neuronal
dystrophies that have been linked to [these] proteins," says
Gerace. "When certain lamins and inner membrane proteins are
mutated, they cause disease."
Because of this link between lamina proteins and disease,
scientists would like to know the identity of all the proteins
in the lamina, and previous studies have identified about
20 lamina proteins.
In their current study, Gerace and Yates used a technique
called subtractive proteomics to identify 62 more candidate
human nuclear membrane proteins.
In the study, TSRI Postdoctoral Fellow Eric Schirmer demonstrates
that the genes encoding 23 of these candidate human nuclear
membrane proteins are in regions of the genome that have already
been implicated in 14 muscle- and neuro-degenerative diseases.
However, many of these regions have hundreds of genes in
them; so the identification of these disease gene candidates
should greatly focus identification of the culprits.
"It's highly likely that some of these diseases will be
due to [the newly identified] nuclear envelope proteins,"
says Gerace. "This is a pretty big step forward."
The Power of Subtractive Proteomics
Where "genomics" maps the DNA sequence and seeks to identify
all the genes in an organism, "proteomics" takes a step further
by asking where and when those genes are actually expressed
as proteins.
One of the most important techniques emerging for proteomics
studies is humbly referred to as MudPIT—Multidimensional
Protein Identification Technology—which Yates has pioneered
in the last few years. Using this technique, scientists like
Yates are able to analyze and identify an enormous number
of proteins in a complex mixture.
MudPIT basically combines liquid chromatography (which is
like a molecular "sieve" that separates a complex mixture
into its component parts) with tandem mass spectrometry (which
identifies the components based on their masses). The instrument
detects these masses and uses sophisticated software to identify
thousands of separate proteins.
But MudPIT alone was not enough in this case, because the
inner nuclear membrane is in contact with other structures
of the cell and cannot be isolated without contaminating material.
Identifying which proteins are from the inner nuclear membrane
and which are contaminants presented a huge problem.
So the team used a simple subtractive technique to deal
with this. They analyzed the nuclear membrane components with
contaminants, containing 2,071 different proteins, and subtracted
out the separately isolated contaminants, which accounted
for more than 40 percent of the membrane proteins. From this
list they were able to apply computational methods to limit
the final list of new human nuclear membrane proteins to 62.
The scientists then took eight of these proteins at random
and demonstrated that they all indeed targeted to the nuclear
membrane.
The new nuclear membrane proteins identified in this study
map to chromosomal regions where the following dystrophies
have been localized:
Congenital Ptosis, hereditary type 1
Charcot-Marie-Tooth Disease 2A
Congenital Muscular Dystrophy 1B
Limb-Girdle Muscular Dystrophy 2B
Charcot-Marie-Tooth Disease 2A
Facioscapulohumeral Muscular Dystrophy (FSH)
Spinal Muscular Atrophy, Types 1, 2, and 3
Limb-Girdle Muscular Dystrophy 1A/1B
Arthogryposis: neurogenic, mild
Blepharophimosis 2
Charcot-Marie-Tooth Disease 2A
Distal Arthrogryposis, type 2B
Congenital Fibrosis of Extraocular Muscles 1
Distal Myopathy
The article, "Nuclear Membrane Proteins With Potential Disease
Links Found By Subtractive Proteomics" was authored by Eric
C. Schirmer, Laurence Florens, Tinglu Guan, John R. Yates
III, and Larry Gerace and will appear in the September 5,
2003 issue of the journal Science.
This work was supported by the National Institutes of Health.
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