Nucleic Acids Research Advance Access published online on November 5, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp971
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
MEROPS: the peptidase database
Neil D. Rawlings*,
Alan J. Barrett and
Alex Bateman
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK
*To whom correspondence should be addressed. Tel: +44 1223 494983; Fax: +44 1223 494919; Email: ndr{at}sanger.ac.uk
Received September 11, 2009. Revised October 12, 2009. Accepted October 14, 2009.
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ABSTRACT
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Peptidases, their substrates and inhibitors are of great relevance
to biology, medicine and biotechnology. The
MEROPS database
(
http://merops.sanger.ac.uk) aims to fulfil the need for an
integrated source of information about these. The database has
a hierarchical classification in which homologous sets of peptidases
and protein inhibitors are grouped into protein species, which
are grouped into families, which are in turn grouped into clans.
The classification framework is used for attaching information
at each level. An important focus of the database has become
distinguishing one peptidase from another through identifying
the specificity of the peptidase in terms of where it will cleave
substrates and with which inhibitors it will interact. We have
collected over 39 000 known cleavage sites in proteins, peptides
and synthetic substrates. These allow us to display peptidase
specificity and alignments of protein substrates to give an
indication of how well a cleavage site is conserved, and thus
its probable physiological relevance. While the number of new
peptidase families and clans has only grown slowly the number
of complete genomes has greatly increased. This has allowed
us to add an analysis tool to the relevant species pages to
show significant gains and losses of peptidase genes relative
to related species.
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INTRODUCTION
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The
MEROPS database is a manually curated information resource
for peptidases (also known as proteases, proteinases or proteolytic
enzymes), their inhibitors and substrates. The database has
been in existence since 1996 and can be found at
http://merops.sanger.ac.uk.
The organizational principle of the database is a hierarchical classification in which homologous sets of peptidases and protein inhibitors are grouped into protein species, which are in turn grouped into families, which are grouped into clans. A family contains proteins that can be shown to be related by sequence comparison alone, whereas a clan contains proteins where the sequences are so distantly related that similarity can only be seen by comparing structures. Sequence analysis is restricted to that portion of the protein directly responsible for peptidase or inhibitor activity which is termed the ‘peptidase unit’ or the ‘inhibitor unit’, respectively. A peptidase or inhibitor unit will normally correspond to a structural domain, and some proteins will contain more than one peptidase or inhibitor domain. Examples are potato virus Y polyprotein which contains three peptidase units, each in a different family, and turkey ovomucoid, which contains three inhibitor units all in the same family. At every level in the database a well-characterized type example is chosen, to which all other members of the family or clan must be shown to be related in a statistically significant manner. The type example at the peptidase or inhibitor level is termed the ‘holotype’ (1,2).
The MEROPS database is released quarterly and users can now keep up to date with the latest MEROPS information by subscribing to the MEROPS database Blog at http://meropsdb.wordpress.com. Statistics from release 8.5 (August 2009) of MEROPS are shown in Table 1 and compared with release 7.8 from April 2007. The number of peptidase sequences has more than doubled, whereas the numbers of protein species, families and clans has increased only slightly. The number of inhibitor sequences has tripled, with the majority of increases in three families (I1, I4 and I63) due to large numbers of homologues being present in some eukaryote genomes. These increases reflect the considerable effort being put into sequencing new genomes. It also demonstrates the power of the peptidase classification to make sense of the data deluge.
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Table 1. Counts of identifiers, families and clans for peptidase and protein inhibitor homologues in the MEROPS database
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In 2007 we published criteria for distinguishing one peptidase
from another (
3), and in the last two years much of our effort
has been focussed on implementing these criteria in the
MEROPS database. We have applied these criteria to hypothetical peptidase
homologues identified by analysing completely sequenced genomes
(
4), allowing us to assign a
MEROPS identifier where appropriate.
Two of the important distinguishing criteria are the different
peptidase specificities and the overall arrangement of all the
domains within the proteins. The new displays discussed below
make use of these criteria and enable us to identify novel peptidases.
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GENOME ANALYSES
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The number of completely sequenced genomes from cellular organisms
now exceeds 1300. Because the genomes from several strains of
the same organism have been sequenced, this represents the genomes
of 780 different species. We have recently introduced a feature
in the organism species pages of
MEROPS for a summary analysis
of the peptidase homologues. We highlight instances where the
genome contains members of a peptidase family not found in 90%
or more of other closely related species (an unexpected presence),
or where a peptidase family is missing but present in 90% or
more of other closely related species (an unexpected absence),
or when the organism in question contains more or less members
of a peptidase family than any other closely related species.
This page is a product of a CGI program which progresses up
the organism classification starting from the family level towards
superkingdom, one taxon at a time, collecting the number of
species with completely sequence genomes. When that number exceeds
five, then the analysis is performed and the results are presented
at the foot of the species page. An example analysis is shown
in
Figure 1.
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Figure 1. A summary analysis for the peptidase homologues from the completely sequence genome of the archaean Cenarchium symbiosum. The figure is taken from the species page in the MEROPS website. A list of peptidase homologues arranged alphabetically by MEROPS identifier is shown in the top panel and the genome analysis is shown at the bottom of the page. The peptidase portion of the proteome of C. symbiosum (12) has been compared with those of 17 other species from the class Thermoprotei. There are unexpected absences of members of peptidase families C26, C44, M38, M48, S9 and U62, and an unexpected presence of a homologue from peptidase family M3. Of the species compared, C. symbiosum had the fewest number of peptidase family M20 homologues, but the most for peptidase family S8. The large number of absent peptidase families may indicates that this endosymbiont genome is degenerate.
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DOMAIN ARCHITECTURES
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The images showing domain architectures have been overhauled.
Because only the peptidase and inhibitor units are classified
in the
MEROPS database, it can be useful to compare the architectures
of different proteins within the same peptidase or inhibitor
family. This can now be done for all the holotypes from a family
by clicking on the ‘architecture’ button on the
family page. An example of a family architecture is shown in
Figure 2.
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Figure 2. The domain architectures for holotypes in peptidase subfamily M12B. The figure is taken from the domain architecture page for peptidase subfamily M12B (the adamalysins) from the MEROPS website. The arrangement of regions and domains are shown for a selection of holotype proteins. The structures are arranged from the top of the page in order of MEROPS identifier. The name of the peptidase is given on the left-hand side. All the structures are drawn to the same scale. The sequence length is denoted by the pale blue line. Regions and domains as determined by MEROPS, the Pfam database and Swiss-Prot entries in the UniProt database (7), are shown as coloured rectangles on this bar. The domains that are classified within the MEROPS database are shown as slightly larger boxes, in green for a peptidase unit and grey for an inhibitor unit (not shown). The MEROPS identifier is displayed in the centre in black text. Domains derived from the Pfam database (13) are shown as smaller rectangles in crimson, with the domain name in white text. On clicking on the box, the user will be taken to the relevant Pfam entry. Regions from Swiss-Prot include signal peptides and transmembrane regions (shown as even smaller boxes in black) and propeptides (in dark grey). Active site residues (red ‘lollipops’) and metal ligands (blue ‘lollipops’) are shown along the bottom edge. Carbohydrate-binding residues (orange ‘lollipops’) and disulphide bridges (black lines connecting the cysteines) are shown along the top edge. Mouse-over text gives details of the feature displayed in all cases.
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SUBSTRATES AND SPECIFICITY DISPLAYS
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One of the most important distinguishing features of a peptidase
is its specificity: where it will cleave a substrate protein
or peptide. The
MEROPS substrate cleavage collection began in
1998 with the publication of the CD version of the
Handbook of Proteolytic Enzymes (
5) and has now grown from 1919 cleavages
in release 7.8 (April 2007) to include over 34 000 known cleavages
in proteins and peptides (physiological and non-physiological)
and over 2700 cleavages in synthetic substrates. Protein and
peptide substrates are mapped to a UniProt identifier where
possible, and the P1 residue for each cleavage [the residue
on the amino side of the scissile bond (
6)] mapped to a residue
number within the UniProt database entry. The peptidase responsible
for the cleavage is mapped to the
MEROPS identifier. We have
recently added cleavages to this collection that result in removal
of targeting signals from proteins, including initiating methionines
from cytoplasmic proteins by methionyl aminopeptidases, the
signal peptides from proteins that enter the secretory pathway
by signal peptidases, and removal of targeting peptides for
proteins that are imported into chloroplasts, mitochondria and
peroxisomes. Only those cleavages that have been experimentally
verified, usually by N-terminal sequencing of the mature protein,
have been included.
We have introduced ‘flags’ on the substrate pages to indicate the method used to identify the cleavage position. The flags are as follows: NT shows that the cleavage position was determined by N-Terminal sequencing, MS shows that the peptide composition was determined by mass-spectroscopy (MS) and the cleavage position computed, MU shows that the cleavage position was determined by site-directed MUtagenesis, CS indicates that the cleavage position was postulated from a consensus motif (CS) within the protein sequence. Because the substrates as used by researchers are usually mature proteins and peptides, the substrates page also includes an extra column in the table to show the residue range of the protein or peptide used in each study.
A tool has been assembled to allow the dynamic alignment of substrate protein sequences. On the assumption that a physiologically relevant cleavage will be conserved in orthologous protein sequences from closely related organisms, cleavage sites are highlighted in the alignment to show conservation or lack of it. Cleavage sites with little conservation are probably fortuitous and of no physiological significance (though in a minority of cases they may be pathological). For each substrate where cleavages are known, the corresponding UniRef50 entry (7) is found and all the UniProt protein sequences included within that entry are aligned with MUSCLE (8).
It is assumed that most cleavages in native proteins occur within surface loops and interdomain linkers. Where the tertiary structure has been solved, the secondary structural elements are indicated on the substrate alignment. An example protein substrate alignment with secondary structure indicated is shown in Figure 3.
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Figure 3. An example of a substrate protein sequence alignment. The figure is taken from the MEROPS website and shows a protein sequence alignment of human C–X–C motif chemokine 11 and its close homologues, showing conservation around the matrix metallopeptidase 8 (MMP8, M10.002) cleavage site at residue 84 (14). The sequence of the protein in which the cleavage was discovered is highlighted in green. Residues are numbered according to this sequence. The MEROPS identifiers of the peptidases known to cleave this substrate are shown below the residue numbers on the left. The arrows next to each MEROPS identifier show the residue range of the peptide fragment used in the experiment, which in most cases is the mature protein without the signal peptide (the signal peptidase cleavage at residue 22 is shown). A question mark instead of an angled bracket would indicate that the terminus has not been determined. The scissile bond symbol ( ) shows where cleavage occurs. Each symbol can be clicked, and the alignment will be highlighted to show conservation around that cleavage site. Four residues either side of each cleavage site (P4–P4') (6) are highlighted. Completely conserved residues are highlighted in orange. Although not shown in this example, a residue highlighted in pink would not be conserved, but the amino acid would have been observed in the same position in another MMP8 substrate. Ile84 in the sequence from the European ferret (Mustela putorius fero), labelled UniProt A8DBL7, is shown with a black background because isoleucine is unknown in this position for any MMP8 substrate. The last line shows the secondary structure: an alpha helix is shown as a series of ‘a’s highlighted in red, and a strand as a series of ‘b’s highlighted in green. This example shows that MMP8 is capable of cleaving this protein substrate within an alpha helix.
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The display showing cleavages in a selected protein depends
on the user choosing the correct species from which the substrate
was derived. If no cleavages are known for the user-selected
protein but are known for the same protein from a different
species, then an option is automatically presented to display
the sequence alignment with those cleavages highlighted.
We use the MEROPS substrate cleavage collection to indicate the specificity of a peptidase. This is shown as a WebLogo (9) and a frequency matrix for the residues accepted in binding pockets P4 to P4', provided we know of 10 or more substrates. There are over 300 peptidases for which 10 or more substrates are known. These displays are shown on the relevant peptidase summary page. However, this does not allow easy comparison of one peptidase with another. So in addition to the displays on a peptidase summary, MEROPS now includes displays to compare preferences in binding pockets S4 to S4'. These show preference in terms of all amino acids, amino acid properties and individual amino acids. The first of these shows, for each peptidase, an amino acid if it occurs in the same binding pocket in 40% or more of the substrates. So no more than two amino acids are shown for any one binding pocket. The amino acid is shown with a green background, and brighter the green, the greater the percentage of substrates with the amino acid in that binding pocket. The second display is similar but instead of showing individual amino acids, these are collected into ‘aliphatic’, ‘aromatic’, ‘acidic, ‘basic’ or ‘small’ groups. In the third option the user is prompted to select an amino acid from a pull-down menu and the display shows the percentage of substrates with the selected amino acid in each binding pocket for each peptidase. Where an amino acid has not been observed in a binding pocket, this is highlighted in black. In all three displays where no amino acid is possible (for example P4, P3 and P2 for an aminopeptidase, of P2', P3' or P4' for a carboxypeptidase) the binding pocket is highlighted in grey. Figure 4 shows a portion of one of these new displays.
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Figure 4. Comparison of peptidase specificity. The figure shows a portion of a page from the MEROPS website. Peptidase preference for the amino acid proline is shown. The MEROPS identifiers and names of the peptidases are shown on the left, along with the number of substrate cleavages in the MEROPS collection. Where proline occurs in the same position in 40% or more of substrates, the cell is highlighted in green and the percentage of substrates with proline in this position is shown. Cells are only highlighted if 10 or more substrates are known for the peptidase. Where there can be no binding pocket to accommodate a substrate residue, for example in position P4, P3 and P2 for an aminopeptidase or P2', P3' and P4' for a carboxypeptidase, these cells are highlighted in grey.
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ALIGNMENTS AND TREES
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We have been aware that as more data are collected some of our
alignments are becoming very large. Not only will there be hundreds
(even thousands) of sequences, but the consequences of aligning
so many diverse sequences means that more gap characters are
inserted and the alignments become wider. These are difficult
to view on a computer screen, and on scrolling the screen, the
residue numbers or sequence identifiers disappear off screen.
To help to alleviate these problems, we have made our dendrograms
more interactive. The nodes of the tree are now active links
and on clicking on the node an alignment of all the sequences
derived from that node will be displayed. This alignment also
includes the family type example and the sequence numbering
derived from the type example sequence. The alignment displayed
is not dynamic, but is derived from the full alignment by removing
any insert characters common to all the sequences. In order
to make this happen, we are now including the aligned peptidase
or inhibitor unit sequences and the dendrograms (in New Hampshire
format) in the MySQL database.
The sequence page of the peptidase (or inhibitor) summary now includes an ALIGN VARIANTS button. Many peptidases and inhibitors are sequenced many times and variants exist, either strain-specific or the result of alternative initiation, alternative splicing of exons, allelic variation or single nucleotide polymorphisms (SNPs). Clicking on the ALIGN VARIANTS button will generate a dynamic alignment of all the variants we have collected from the primary sequence databases. Residues that differ from the sequence we have selected for inclusion in our protein sequence collection are highlighted as white text on a black background.
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NEW INDEXES
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Indexes are important tools to allow users to find the data
they want. We have added a number of new indexes to
MEROPS.
A new index of gene names has been added to the main index page (the left-hand menu). You can now search for any peptidase or protein inhibitor homologue knowing the name of its gene or its gene locus.
A new substrate menu has been added, which includes an index of substrate names to make it easier for the user to find a substrate for which we have cleavages in our collection. Substrates are arranged alphabetically by name. Names are usually taken from the UniProt description, but where the substrate is a fragment of a larger protein, the common name of the peptide is favoured over the protein name. For example, a user will find ‘substance P’ in the index as well as the source protein, ‘protachykinin-1’. The index also includes the names of synthetic substrates. The substrate menu also provides access to the pages that compare peptidase specificity.
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LITERATURE
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The
MEROPS database includes an extensive collection of bibliographic
references (over 37 000). Each of these references is tagged
with the
MEROPS identifier for the relevant peptidase, inhibitor,
family or clan, and a list of references is given for each peptidase,
inhibitor, family or clan. We have marked some of the publications
that are relevant to particularly important topics by use of
coloured ‘flags’. The full list of flags is shown
in
Table 2.
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Table 2. Flags used to mark publications that are relevant to particularly important topics and their explanation
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DATABASE CROSS-REFERENCES
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A new item has been added to the Searches menu. The
MEROPS database
includes many cross-references to other databases and bioinformatics
resources. To make it easier for others to map their database
entries to
MEROPS there is a new CGI that presents the cross-references
between
MEROPS and any database selected from a pull-down menu.
There are a considerable number of cross-references between
MEROPS and primary sequence databases, so these are returned
in batches of 50 000.
A distributed annotation system (DAS) server (10) has been set-up for MEROPS. This allows others to extract data directly from the MEROPS MySQL database for inclusion in their own Internet service. The user enters an accession as a parameter in the URL (usually this will be a UniProt accession, but an EMBL/GenBank ProtID will work for MEROPS) and data relating to the sequence stored in our collection will be returned. For a peptidase or protein inhibitor, this will include the MEROPS identifier, family and clan, the extent of the peptidase or inhibitor unit, active site residues (and metal ligands for metallopeptidases), the amino acid sequence and a link to a page in MEROPS for each feature. For a protein substrate, positions of known cleavages and the MEROPS identifiers of the peptidases responsible are returned. Example URL’s are:
http://das.sanger.ac.uk/das/merops/features?segment=P07858 (features for human cathepsin B)
http://das.sanger.ac.uk/das/merops/sequence?segment=P07858 (sequence for human cathepsin B)
http://das.sanger.ac.uk/das/merops/features?segment=P05067 (known cleavages for human amyloid beta A4 protein precursor)
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ENHANCEMENTS TO EXISTING FEATURES
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For eukaryotes with completely sequenced genomes, the chromosomal
location (in megabases) of the peptidase or protein inhibitor
homologue gene is now shown on the organism page. These locations
are derived from the EnSEMBL database (
11) by searching for
entries with a cross-reference to the UniProt protein sequence
database, therefore a location will not be shown for a gene
from any genome where the copy number is low. However, the locations
for all homologues from human and mouse are shown. For human
and mouse these locations are also shown in the Genetics table
of the peptidase or protein inhibitor summary. Here the locations
are linked to the contig view in EnSEMBL, which shows the exon
and intron structure of the gene. The name of the chromosome
(or genomic scaffold) precedes the location and the strand is
indicated by a plus or minus sign in parentheses after the location.
The displays of peptidase or inhibitor distribution among organisms have been enhanced. There is now mouse-over text at every node which gives the name of the taxon.
MEROPS identifiers have been added to the tables of peptidase-inhibitor interactions, and it is now possible to order the tables according to the identifier or the protein name.
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COMMUNITY ANNOTATION
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Facilities have been set-up for our users to contribute to annotation
in
MEROPS via a ‘Submissions’ button. At present
there are only two submission items, both for advising us of
any known protein cleavage sites that we are unaware of. The
first of these is a form for the submission of a single cleavage,
and the second allows the user to upload a file of known cleavage
sites. The latter has been designed with proteomics experiments
in mind. The information provided will allow us to map the cleavage
to an entry in the UniProt database. Users are also welcome
to send comments on any aspect of the
MEROPS website to the
following E-mail address:
merops{at}sanger.ac.uk.
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FUNDING
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Wellcome Trust [grant number WT077044/Z/05/Z]. Funding for open
access charge: Wellcome Trust.
Conflict of interest statement. None declared.
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ACKNOWLEDGEMENTS
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We would like to thank Pfam and Rfam colleagues for helpful
discussions, and Paul Bevan, Jody Clements and Matthew Waller
from the Sanger Institute web team for all their help in maintaining
this resource. We would also like to thank those users who have
pointed out errors and omissions, or who have suggested changes
and improvements.
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REFERENCES
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- Rawlings ND, Tolle DP, Barrett AJ. Evolutionary families of peptidase inhibitors. Biochem. J. (2004) 378:705–716.[CrossRef][Web of Science][Medline]
- Barrett AJ, Rawlings ND. Species of peptidases. Biol. Chem. (2007) 388:1151–1157.[CrossRef][Web of Science][Medline]
- Rawlings ND, Morton FR. The MEROPS batch BLAST: a tool to detect peptidases and their non-peptidase homologues in a genome. Biochimie (2008) 90:243–259.[CrossRef][Web of Science][Medline]
- Barrett AJ, Rawlings ND, Woessner JF, eds. Handbook of Proteolytic Enzymes (1998) London: Academic Press.
- Schechter I, Berger A. On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem. Biophys. Res. Commun. (1968) 32:898–902.[CrossRef][Web of Science][Medline]
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- Dowell RD, Jokerst RM, Day A, Eddy SR, Stein L. The distributed annotation system. BMC Bioinformatics (2001) 2:7.[CrossRef][Medline]
- Hubbard TJ, Aken BL, Ayling S, Ballester B, Beal K, Bragin E, Brent S, Chen Y, Clapham P, Clarke L, et al. Ensembl 2009. Nucleic Acids Res. (2009) 37:D690–D697.[Abstract/Free Full Text]
- Hallam SJ, Konstantinidis KT, Putnam N, Schleper C, Watanabe Y, Sugahara J, Preston C, de la Torre J, Richardson PM, DeLong EF. Genomic analysis of the uncultivated marine crenarchaeote Cenarchaeum symbiosum. Proc. Natl Acad. Sci. USA (2006) 103:18296–18301.[Abstract/Free Full Text]
- Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL, et al. The Pfam protein families database. Nucleic Acids Res. (2008) 36:D281–D288.[Abstract/Free Full Text]
- Cox JH, Dean RA, Roberts CR, Overall CM. Matrix metalloproteinase processing of CXCL11/I-TAC results in loss of chemoattractant activity and altered glycosaminoglycan binding. J. Biol. Chem. (2008) 283:19389–19399.[Abstract/Free Full Text]
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