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2004
Shao-En Ong, Gerhard Mittler
& Matthias Mann
Nature
Methods 1, 119 - 126 (2004)
Protein methylation is a stable
post-translational modification (PTM) with important biological functions.
It occurs predominantly on arginine and lysine residues with varying
numbers of methyl groups, such as mono-, di- or trimethyl lysine. Existing
methods for identifying methylation sites are laborious, require large
amounts of sample and cannot be applied to complex mixtures. We have
previously described stable isotope labeling by amino acids in cell culture
(SILAC) for quantitative comparison of proteomes. In heavy methyl SILAC,
cells metabolically convert [13CD3]methionine to the sole biological methyl
donor, [13CD3]S-adenosyl methionine. Heavy methyl groups are fully
incorporated into in vivo methylation sites, directly labeling the PTM.
This provides markedly increased confidence in identification and relative
quantitation of protein methylation by mass spectrometry. Using antibodies
targeted to methylated residues and analysis by liquid
chromatography–tandem mass spectrometry, we identified 59 methylation
sites, including previously unknown sites, considerably extending the
number of in vivo methylation sites described in the literature.
Mouse
embryonic fibroblasts derived from Odin deficient mice display a
hyperproliiferative phenotype.
Kristiansen TZ, Nielsen MM, Blagoev B, Pandey A, Mann M.
DNA Res. 2004 Aug 31;11(4):285-92.
Odin is a recently identified cytosolic
phosphotyrosine binding (PTB) domain containing negative regulatory
protein, that was discovered on the basis of its ability to undergo
tyrosine phosphorylation upon stimulation by epidermal growth factor in
HeLa cells. The protein was originally obtained as a KIAA clone (KIAA 0229)
from the Kazusa DNA Research Institute which maintains the HUGE protein
database--a database devoted to the analysis of long cDNA clones encoding
large proteins (>50 kDa). Odin has been demonstrated to cause
downregulation of c-Fos promoter activity and to inhibit PDGF-induced
mitogenesis in cell lines. To further investigate the role of Odin in
growth factor receptor signaling and to elucidate its biological function
in vivo, we have generated mice deficient in Odin by gene targeting.
Odin-deficient mice do not display any obvious phenotype, and histological
examination of the kidney, lung and liver does not show any major
abnormalities as compared to wild-type controls. However, mouse embryonic
fibroblasts (MEFs) generated from Odin-deficient mice exhibit a
hyperproliferative phenotype compared to wild-type-derived MEFs, consistent
with its role as a negative regulator of growth factor receptor signaling.
Our results confirm that although Odin expression in mice is not essential
for any major developmental pathway, it could play a significant functional
role to negatively regulate growth factor receptor signaling pathways.
Proteomic Analysis of the Arabidopsis Nucleolus Suggests Novel
Nucleolar Functions.
Pendle AF, Clark
GP, Boon R, Lewandowska D, Lam YW, Andersen J, Mann M, Lamond AI, Brown JW,
Shaw PJ.
Mol
Biol Cell. 2004 Oct 20 [Epub ahead of print]
John Innes Centre,
Norwich NR4 7UH, United Kingdom; Scottish Crop Research Institute, Dundee
DD2 5DA, Scotland; University of Dundee, Dundee DD1 5HN, Scotland;
University of Southern Denmark, DK-5230 Odense, Denmark.
Monitoring Editor:
Joseph Gall The eukaryotic nucleolus is involved in ribosome biogenesis and
a wide range of other RNA metabolism and cellular functions. An important
step in the functional analysis of the nucleolus is to determine the
complement of proteins of this nuclear compartment. Here, we describe the
first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which
we have identified 217 proteins. This allows a direct comparison of the
proteomes of an important nuclear structure between two widely divergent
species - human and Arabidopsis. The comparison identified many common
proteins, plant-specific proteins, proteins of unknown function found in
both proteomes and proteins which were nucleolar in plants but nonnucleolar
in human. Seventy two proteins were expressed as GFP fusions and 87% showed
nucleolar or nucleolar-associated localization. In a striking and
unexpected finding, we have identified six components of the postsplicing
exon-junction complex (EJC) involved in mRNA export and nonsense-mediated
decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion
protein localization. These results raise the possibility that in plants,
nucleoli may have additional functions in mRNA export or surveillance.
Proteomics.
de Hoog CL, Mann M.
Annu
Rev Genomics Hum Genet. 2004;5:267-93.
Department of
Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230
Odense M, Denmark. carmen@bmb.sdu.dk
The genome
sequences of important model systems are available and the focus is now
shifting to large-scale experiments enabled by this data. Following in the
footsteps of genomics, we have functional genomics, proteomics, and even
metabolomics, roughly paralleling the biological hierarchy of the
transcription, translation, and production of small molecules. Proteomics
is initially concerned with determining the structure, expression, localization,
biochemical activity, interactions, and cellular roles of as many proteins
as possible. There has been great progress owing to novel instrumentation,
experimental strategies, and bioinformatics methods. The area of
protein-protein interactions has been especially fruitful. First pass
interaction maps of some model organisms exist, and the proteins in many
important organelles are about to be determined. Researchers are also
beginning to integrate large-scale data sets from various "omics"
disciplines in targeted investigations of specific biomedical areas and in
pursuit of a general framework for systems biology.
Schulze WX, Gleixner G, Kaiser K,
Guggenberger G, Mann M, Schulze ED.
Oecologia. 2004 Sep 22 [Epub ahead of
print]
Mass
spectrometry-based proteomics was applied to analyze proteins isolated from
dissolved organic matter (DOM). The focal question was to identify the type
and biological origin of proteins in DOM, and to describe diversity of
protein origin at the level of higher taxonomic units, as well as to detect
extracellular enzymes possibly important in the carbon cycle. Identified
proteins were classified according to their phylogenetic origin and
metabolic function using the National Center for Biotechnology Information
(NCBI) protein and taxonomy database. Seventy-eight percent of the proteins
in DOM from the lake but less than 50% in forest soil DOM originated from
bacteria. In a deciduous forest, the number of identified proteins
decreased from 75 to 28 with increasing soil depth and decreasing total
soil organic carbon content. The number of identified proteins and
taxonomic groups was 50% higher in winter than in summer. In spruce forest,
number of proteins and taxonomic groups decreased by 50% on a plot where
trees had been girdled a year before and carbohydrate transport to roots was
terminated. After girdling, proteins from four taxonomic groups remained as
compared to nine taxonomic groups in healthy forest. Enzymes involved in
degradation of organic matter were not identified in free soil DOM.
However, cellulases and laccases were found among proteins extracted from
soil particles, indicating that degradation of soil organic matter takes
place in biofilms on particle surfaces. These results demonstrate a novel
application of proteomics to obtain a  proteomic fingerprint of presence and activity of organisms in an
ecosystem.
Neuron. 2004 Sep 16;43(6):847-58.
Small conductance Ca(2+)-activated K(+)
channels (SK channels) couple the membrane potential to fluctuations in
intracellular Ca(2+) concentration in many types of cells. SK channels are
gated by Ca(2+) ions via calmodulin that is constitutively bound to the
intracellular C terminus of the channels and serves as the Ca(2+) sensor.
Here we show that, in addition, the cytoplasmic N and C termini of the
channel protein form a polyprotein complex with the catalytic and
regulatory subunits of protein kinase CK2 and protein phosphatase 2A.
Within this complex, CK2 phosphorylates calmodulin at threonine 80,
reducing by 5-fold the apparent Ca(2+) sensitivity and accelerating channel
deactivation. The results show that native SK channels are polyprotein
complexes and demonstrate that the balance between kinase and phosphatase
activities within the protein complex shapes the hyperpolarizing response
mediated by SK channels.
Published online before print September
3, 2004
Proc. Natl. Acad.
Sci. USA, 10.1073/pnas.0405549101
Jesper V. Olsen and Matthias Mann
MS-based proteomics usually involves the
fragmentation of tryptic peptides (tandem MS or MS2) and their
identification by searching protein sequence databases. In ion trap
instruments fragments can be further fragmented and analyzed, a process
termed MS/MS/MS or MS3. Here, we report that efficient ion capture in a
linear ion trap leads to MS3 acquisition times and spectra quality similar
to those for MS2 experiments with conventional 3D ion traps. Fragmentation
of N- or C-terminal ions resulted in informative and low-background
spectra, even at subfemtomol levels of peptide. Typically C-terminal ions
are chosen for further fragmentation, and the MS3 spectrum greatly
constrains the C-terminal amino acids of the peptide sequence. MS3 spectra
allow resolution of ambiguities in identification, a crucial problem in
proteomics. Because of the sensitivity and rapid scan rates of the linear
ion trap, several MS3 spectra per peptide can be obtained even when
sequencing very complex mixtures. We calculate the probability that an
experimental MS3 spectrum originates from fragmentation of a given N- or
C-terminal ion of a peptide under consideration. This MS3 identification
score can be combined with the MS2 scores of the precursor peptide from
existing search engines. When MS3 is performed on the linear ion
trap-Fourier transform mass spectrometer combination, accurate peptide
masses further increase confidence in peptide identification.
-->Hanno Steen
& Matthias Mann
Nature Reviews Molecular Cell Biology 5,
699-711 (2004); doi:10.1038/nrm1468
Proteomics
is an increasingly powerful and indispensable technology in molecular cell
biology. It can be used to identify the components of small protein
complexes and large organelles, to determine post-translational
modifications and in sophisticated functional screens. The key — but
little understood — technology in mass-spectrometry-based proteomics
is peptide sequencing, which we describe and review here in an easily
accessible format.
J Biol Chem. 2004 Aug 16 [Epub ahead of
print]
Dep. Biochemistry
and Molecular Biology, University of Southern Denmark, Odense M 5230.
Signal transduction
by receptor tyrosine kinases is initiated by recruitment of a variety of
signaling proteins to tyrosine phosphorylated motifs in the activated
receptors. Several signaling pathways are thus activated in parallel, the
combination of which decides the cellular response. Here, we present a dual
strategy for extensive mapping of tyrosine phosphorylated proteins and
probing of signal dependent protein interactions of a signaling cascade.
The approach relies on labeling of cells with 'heavy' and 'light' isotopic
forms of Arg to distinguish two cell populations. First, tyrosine
phosphorylated proteins from stimulated ('heavy'-labeled) and control
samples ('normal'-labeled) are isolated and subjected to high sensitivity
Fourier transform ion cyclotron resonance mass spectrometry analysis. Next,
phosphopeptides corresponding to tyrosine phosphorylation sites identified
during the tyrosine phosphoproteomic analysis are used as baits to isolate
phosphospecific protein binding partners, which are subsequently identified
by mass spectrometry. We used this approach to identify 28 components of
the signaling cascade induced by stimulation with the basic fibroblast
growth factor. Insulin receptor substrate-4 was identified as a novel candidate
in fibroblast growth factor receptor signaling, and we defined
phosphorylation dependent interactions with other components, such as
adaptor protein Grb2, of the signaling cascade. Finally, we present
evidence for a complex containing insulin receptor substrate-4 and ShcA in
signaling by the fibroblast growth factor receptor.
Blagoy
Blagoev1, 2, Shao-En Ong1, 2, Irina
Kratchmarova1 & Matthias Mann1
Nature
Biotechnology 22, 1139 - 1145 (2004)
Published online:
15 August 2004; | doi:10.1038/nbt1005
To
study the global dynamics of phosphotyrosine-based signaling events in
early growth factor stimulation, we developed a mass spectrometric method
that converts temporal changes to differences in peptide isotopic
abundance. The proteomes of three cell populations were metabolically
encoded with different stable isotopic forms of arginine. Each population
was stimulated by epidermal growth factor for a different length of time,
and tyrosine-phosphorylated proteins and closely associated binders were
affinity purified. Arginine-containing peptides occurred in three forms,
which were quantified; we then combined two experiments to generate
five-point dynamic profiles. We identified 81 signaling proteins, including
virtually all known epidermal growth factor receptor substrates, 31 novel
effectors and the time course of their activation upon epidermal growth
factor stimulation. Global activation profiles provide an informative
perspective on cell signaling and will be crucial to modeling signaling
networks in a systems biology approach.
A
proteomic study of SUMO-2 target proteins.
Vertegaal AC, Ogg SC, Jaffray E,
Rodriguez MS, Hay RT, Andersen JS, Mann M, Lamond AI
J Biol Chem. 2004 Jun 2 [Epub ahead
of print]
The SUMO family in vertebrates includes
at least three distinct proteins, SUMO-1, 2 3 that are added as
post-translational modifications to target proteins. A considerable number
of SUMO-1 target proteins has been identified, but little is known about
SUMO-2. A stable HeLa cell line expressing 6His-tagged SUMO-2 was
established and used to label and purify novel endogenous SUMO-2 target
proteins. Tagged forms of SUMO-2 were functional and localised
predominantly in the nucleus. 6His-tagged SUMO-2 conjugates were affinity
purified from nuclear fractions and identified by mass spectrometry. Eight
novel potential SUMO-2 target proteins were identified by at least two
peptides. Three of these proteins, SART1, hnRNP M and the U5 snRNP-200kDa
helicase play a role in RNA metabolism. SART1 and hnRNP M were both shown
to be genuine SUMO targets confirming the validity of the approach.
RNA
and RNA Binding Proteins Participate in Early Stages of Cell Spreading
through Spreading Initiation Centers.
De Hoog CL, Foster LJ, Mann M.
Cell. 2004 May 28;117(5):649-62.
Focal adhesions are specialized
attachment and signaling centers that form at sites of cell-matrix
contacts. We employed a quantitative mass spectrometry-based method called
SILAC to identify and quantify proteins interacting in an
attachment-dependent manner with focal adhesion proteins. Subsequent
confocal microscopy revealed a previously undescribed structure, which we
have termed a spreading initiation center (SIC), existing only in early
stages of cell spreading. SICs contain focal adhesion markers, appear to be
surrounded by an actin sheath, and, surprisingly, contain numerous RNA
binding proteins, ribosomal RNA, and perhaps other RNAs. Interfering with
the function of FUS/TLS, hnRNP K, and hnRNP E1 results in increased
spreading. Spreading initiation centers are ribonucleoprotein complexes
distinct from focal adhesions and demonstrate a role for RNA and RNA
binding proteins in the initiation of cell spreading.
Trypsin
cleaves exclusively C-terminal to Arginine and lysine residues.
Olsen JV, Ong SE, Mann M.
Mol Cell Proteomics. 2004 Mar 19 [Epub
ahead of print]
Almost all large-scale projects in mass spectrometry-based proteomics use
trypsin to convert protein mixtures into more readily analyzable peptide
populations. When searching peptide fragmentation spectra against sequence
databases, potentially matching peptide sequences can be required to
conform to tryptic specificity, namely, cleavage exclusively C-terminal to
arginine or lysine. In many published reports, however, significant numbers
of proteins are identified by non-tryptic peptides. Here we use the sub
parts per million mass accuracy of a new ion trap Fourier Transform mass
spectrometer to achieve more than a hundred-fold increased confidence in
peptide identification compared to typical ion trap experiments and show
that trypsin cleaves solely C-terminal to arginine and lysine. We find that
non-tryptic peptides occur only as the C-terminal peptides of proteins and
as breakup products of fully tryptic peptides N-terminal to an internal
proline. Simulating lower mass accuracy led to a large number of proteins
erroneously identified with non-tryptic peptide hits. Our results indicate
that such peptide hits in previous studies should be re-examined and that
peptide identification should be based on strict trypsin specificity.
eIF4A3
is a novel component of the exon junction complex.
Chan CC, Dostie J, Diem MD, Feng W, Mann M, Rappsilber J, Dreyfuss G.
RNA. 2004 Feb;10(2):200-9.
The exon junction complex (EJC) is a
protein complex that assembles near exon-exon junctions of mRNAs as a
result of splicing. EJC proteins play important roles in postsplicing
events including mRNA export, cytoplasmic localization, and
nonsense-mediated decay. Recent evidence suggests that mRNA translation is
also influenced by the splicing history of the transcript. Here we identify
eIF4A3, a DEAD-box RNA helicase and a member of the eIF4A family of
translation initiation factors, as a novel component of the EJC. We show
that eIF4A3 associates preferentially with nuclear complexes containing the
EJC proteins magoh and Y14. Furthermore, eIF4A3, but not the highly related
eIF4A1 or eIF4A2, preferentially associates with spliced mRNA. In vitro
splicing and mapping experiments demonstrate that eIF4A3 binds mRNAs at the
position of the EJC. Using monoclonal antibodies, we show that eIF4A3 is
found in the nucleus whereas eIF4A1 and eIF4A2 are found in the cytoplasm.
Thus, eIF4A3 likely provides a splicing-dependent influence on the
translation of mRNAs.
Cloning
of a novel signaling molecule, AMSH-2, that potentiates transforming growth
factor beta signaling.
Ibarrola N, Kratchmarova I, Nakajima D, Schiemann WP, Moustakas A, Pandey
A, Mann M.
BMC Cell Biol. 2004 Jan 19;5(1):2.
Transforming growth factor-betas (TGF-betas), bone morphogenetic proteins
(BMPs) and activins are important regulators of developmental cell growth
and differentiation. Signaling by these factors is mediated chiefly by the
Smad family of latent transcription factors. RESULTS: There are a large
number of uncharacterized cDNA clones that code for novel proteins with
homology to known signaling molecules. We have identified a novel molecule
from the HUGE database that is related to a previously known molecule, AMSH
(associated molecule with the SH3 domain of STAM), an adapter shown to be
involved in BMP signaling. Both of these molecules contain a coiled-coil
domain located within the amino-terminus region and a JAB (Domain in Jun
kinase activation domain binding protein and proteasomal subunits) domain
at the carboxy-terminus. We show that this novel molecule, which we have
designated AMSH-2, is widely expressed and its overexpression potentiates
activation of TGF-beta-dependent promoters. Coimmunoprecipitation studies
indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2.
We show that overexpression of AMSH-2 decreases the inhibitory effect of
Smad7 on TGF-beta signaling. Finally, we demonstrate that knocking down
AMSH-2 expression by RNA interference decreases the activation of 3TP-lux
reporter in response to TGF-beta. CONCLUSIONS: This report implicates AMSH
and AMSH-2 as a novel family of molecules that positively regulate the TGF-beta
signaling pathway. Our results suggest that this effect could be partially
explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-beta
signaling pathway.
BASP1
is a transcriptional cosuppressor for the Wilms' tumor suppressor protein
WT1.
Carpenter B, Hill KJ, Charalambous M, Wagner KJ, Lahiri D, James DI,
Andersen JS, Schumacher V, Royer-Pokora B, Mann M, Ward A, Roberts SG.
Mol Cell Biol. 2004 Jan;24(2):537-49.
The Wilms' tumor suppressor protein WT1 is a transcriptional regulator that
plays a key role in the development of the kidneys. The transcriptional
activation domain of WT1 is subject to regulation by a suppression region
within the N terminus of WT1. Using a functional assay, we provide direct
evidence that this requires a transcriptional cosuppressor, which we
identify as brain acid soluble protein 1 (BASP1). WT1 and BASP1 associate
within the nuclei of cells that naturally express both proteins. BASP1 can
confer WT1 cosuppressor activity in transfection assays, and elimination of
endogenous BASP1 expression augments transcriptional activation by WT1.
BASP1 is present in the developing nephron structures of the embryonic
kidney and, coincident with that of WT1, its expression is restricted to
the highly specialized podocyte cells of the adult kidney. Taken together,
our results show that BASP1 is a WT1-associated factor that can regulate
WT1 transcriptional activity.
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