CSC

epestfind

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Function

Description

epestfind rapidly and objectively identifies PEST motifs in an input protein sequence. PEST motifs reduce the half-lives of proteins dramatically and hence, that they target proteins for proteolytic degradation. epestfind writes an output file with putative PEST motifs and (optionally) the PEST scores are plotted for the whole sequence length.

The epestfind output file includes poor and potential PEST motifs together with their PEST score, mass percent of DEPST and their hydrophobicity index. 'Valid' PEST motifs below the specified threshold score are considered as 'poor', while PEST scores above the threshold score are of real biological interest. The higher the PEST score, the more likely is degradation of proteins mediated via 'potential' PEST motifs in eukaryotic cells.

Algorithm

The epestfind algorithm defines the last criterion even more stringently in that PEST motifs are required to be flanked by positively charged amino acids. Though this implication greatly facilitates computer scanning, a few PEST sequences might be missed. Especially sequences with a high local concentration of critical amino acids but with a long distance between positively charged amino acids are error prone. Due to their length, these PEST motifs might become diluted, which results in scores apparently lower than initially expected. Another side effect of scanning for positively charged amino acids is that very long PEST motifs are sub-divided into adjacent smaller ones. However, identification of PEST motifs is achieved by an initial scan for positively charged amino acids arginine (R), histidine (H) and lysine (K) within the specified protein sequence. All amino acids between the positively charged flanks are counted and only those motifs are considered further, which contain a number of amino acids equal to or higher than the window-size parameter. Additionally, all 'valid' PEST regions are required to contain at least one proline (P), one aspartate (D) or glutamate (E) and at least one serine (S) or threonine(T). Sequences that do not meet the above criteria are classified as 'invalid' PEST motifs and excluded from further analysis.

The quality of 'valid' PEST motifs is refined by means of a scoring parameter based on the local enrichment of critical amino acids as well as the motif's hydrophobicity. Enrichment of D, E, P, S and T is expressed in mass percent (w/w) and corrected for one equivalent of D or E, one of P and one of S or T. Calculation of hydrophobicity follows in principle the method of J. Kyte and R.F. Doolittle [4]. For simplified calculations, Kyte-Doolittle hydropathy indices, which originally ranged from -4.5 for arginine to +4.5 for isoleucine, were converted to positive integers. This was achieved by the following linear transformation, which yielded values from 0 for arginine to 90 for isoleucine.

The motif's hydrophobicity is calculated as the sum over the products of mole percent and hydrophobicity index for each amino acid species. The desired PEST score is obtained as combination of local enrichment term and hydrophobicity term as expressed by the following equation:

Although, the formula above differs from the publication [1], it is in fact the correct one, which was also implemented in the original BASIC programme (personal communication). In addition, the programme includes a correction for the hydropathy index of tyrosine, introduced by Robert H. Stellwagen from the University of Southern California. However, PEST scores can range from -45 for poly-isoleucine to about +50 for poly-aspartate plus one proline and one serine. 'Valid' PEST motifs below the threshold score (5.0) are considered as 'poor', while PEST scores above the threshold score are of real biological interest. The higher the PEST score, the more likely is degradation of proteins mediated via 'potential' PEST motifs in eukaryotic cells.

Presently, all modified Kyte-Doolittle hydropathy indices are hard-coded into the programme, which might change in future.

The array of linear transformed Kyte-Doolittle hydropathy indices (ltkdhi) is listed in alphabetical order below. (A-M and N-Z as well as N-terminus and C-terminus)

63, 10, 70, 10, 10, 72, 41, 13, 90, 0, 6, 82, 64, 10, 0, 29, 10, 0, 36, 38, 0, 87, 36, 45, 58, 10, 0, 0

The linear transformation was ltkdhi = 10 * kdhi + 45
All values range from Argine R = 0 to Isoleucine I = 90
B=(N|D)=10 since N=10 and D=10
Z=(Q|E)=10 since Q=10 and E=10
X=10*0+45=45

Usage

% epestfind -graph cps -invalid 
Finds PEST motifs as potential proteolytic cleavage sites
Input protein sequence: exu2_drops.embl
Window length [10]: 
Sort order of results
      1 : length
      2 : position
      3 : score
Sort order of results [score]: 
Output file [exu2_drops.epestfind]: 

Created epestfind.ps

Go to the input files for this example
Go to the output files for this example

Command line arguments

Finds PEST motifs as potential proteolytic cleavage sites
Version: EMBOSS:6.4.0.0

   Standard (Mandatory) qualifiers:
  [-sequence]          sequence   Protein sequence USA to be analysed.
   -window             integer    [10] Minimal distance between positively
                                  charged amino acids. (Integer 2 or more)
   -order              selection  [score] Name of the output file which holds
                                  the results of the analysis. Results may be
                                  sorted by length, position and score.
  [-outfile]           outfile    [*.epestfind] Name of file to which results
                                  will be written.
   -graph              xygraph    [$EMBOSS_GRAPHICS value, or x11] Graph type
                                  (ps, hpgl, hp7470, hp7580, meta, cps, x11,
                                  tek, tekt, none, data, xterm, png, gif, pdf,
                                  svg)

   Additional (Optional) qualifiers:
   -mwdata             datafile   [Emolwt.dat] Molecular weights data file
   -threshold          float      [+5.0] Threshold value to discriminate weak
                                  from potential PEST motifs. Valid PEST
                                  motifs are discriminated into 'poor' and
                                  'potential' motifs depending on this
                                  threshold score. By default, the default
                                  value is set to +5.0 based on experimental
                                  data. Alterations are not recommended since
                                  significance is a matter of biology, not
                                  mathematics. (Number from -55.00 to 55.00)

   Advanced (Unprompted) qualifiers:
   -mono               boolean    [N] Use monoisotopic weights
   -[no]potential      boolean    [Y] Decide whether potential PEST motifs
                                  should be printed.
   -[no]poor           boolean    [Y] Decide whether poor PEST motifs should
                                  be printed.
   -invalid            boolean    [N] Decide whether invalid PEST motifs
                                  should be printed.
   -[no]map            boolean    [Y] Decide whether PEST motifs should be
                                  mapped to sequence.

   Associated qualifiers:

   "-sequence" associated qualifiers
   -sbegin1            integer    Start of the sequence to be used
   -send1              integer    End of the sequence to be used
   -sreverse1          boolean    Reverse (if DNA)
   -sask1              boolean    Ask for begin/end/reverse
   -snucleotide1       boolean    Sequence is nucleotide
   -sprotein1          boolean    Sequence is protein
   -slower1            boolean    Make lower case
   -supper1            boolean    Make upper case
   -sformat1           string     Input sequence format
   -sdbname1           string     Database name
   -sid1               string     Entryname
   -ufo1               string     UFO features
   -fformat1           string     Features format
   -fopenfile1         string     Features file name

   "-outfile" associated qualifiers
   -odirectory2        string     Output directory

   "-graph" associated qualifiers
   -gprompt            boolean    Graph prompting
   -gdesc              string     Graph description
   -gtitle             string     Graph title
   -gsubtitle          string     Graph subtitle
   -gxtitle            string     Graph x axis title
   -gytitle            string     Graph y axis title
   -goutfile           string     Output file for non interactive displays
   -gdirectory         string     Output directory

   General qualifiers:
   -auto               boolean    Turn off prompts
   -stdout             boolean    Write first file to standard output
   -filter             boolean    Read first file from standard input, write
                                  first file to standard output
   -options            boolean    Prompt for standard and additional values
   -debug              boolean    Write debug output to program.dbg
   -verbose            boolean    Report some/full command line options
   -help               boolean    Report command line options and exit. More
                                  information on associated and general
                                  qualifiers can be found with -help -verbose
   -warning            boolean    Report warnings
   -error              boolean    Report errors
   -fatal              boolean    Report fatal errors
   -die                boolean    Report dying program messages
   -version            boolean    Report version number and exit

Input file format

The input is a standard EMBOSS sequence query (also known as a 'USA').

Major sequence database sources defined as standard in EMBOSS installations include srs:embl, srs:uniprot and ensembl

Data can also be read from sequence output in any supported format written by an EMBOSS or third-party application.

The input format can be specified by using the command-line qualifier -sformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: gff (gff3), gff2, embl (em), genbank (gb, refseq), ddbj, refseqp, pir (nbrf), swissprot (swiss, sw), dasgff and debug.

See: http://emboss.sf.net/docs/themes/SequenceFormats.html for further information on sequence formats.

Input files for usage example

File: exu2_drops.embl

ID   EXU2_DROPS     STANDARD;      PRT;   477 AA.
AC   Q24617;
DT   01-NOV-1997 (Rel. 35, Created)
DT   01-NOV-1997 (Rel. 35, Last sequence update)
DT   01-NOV-1997 (Rel. 35, Last annotation update)
DE   Maternal exuperantia 2 protein.
GN   EXU2.
OS   Drosophila pseudoobscura (Fruit fly).
OC   Eukaryota; Metazoa; Arthropoda; Tracheata; Hexapoda; Insecta;
OC   Pterygota; Neoptera; Endopterygota; Diptera; Brachycera; Muscomorpha;
OC   Ephydroidea; Drosophilidae; Drosophila.
OX   NCBI_TaxID=7237;
RN   [1]
RP   SEQUENCE FROM N.A.
RX   MEDLINE=94350208; PubMed=8070663;
RA   Luk S.K.-S., Kilpatrick M., Kerr K., Macdonald P.M.;
RT   "Components acting in localization of bicoid mRNA are conserved among
RT   Drosophila species.";
RL   Genetics 137:521-530(1994).
CC   -!- FUNCTION: ENSURES THE PROPER LOCALIZATION OF THE MRNA OF THE
CC       BICOID GENE TO THE ANTERIOR REGIONS OF THE OOCYTE THUS PLAYING
CC       A FUNDAMENTAL ROLE IN THE ESTABLISHMENT OF THE POLARITY OF THE
CC       OOCYTE. MAY BIND THE BCD MRNA (BY SIMILARITY).
CC   --------------------------------------------------------------------------
CC   This SWISS-PROT entry is copyright. It is produced through a collaboration
CC   between  the Swiss Institute of Bioinformatics  and the  EMBL outstation -
CC   the European Bioinformatics Institute.  There are no  restrictions on  its
CC   use  by  non-profit  institutions as long  as its content  is  in  no  way
CC   modified and this statement is not removed.  Usage  by  and for commercial
CC   entities requires a license agreement (See http://www.isb-sib.ch/announce/
CC   or send an email to license@isb-sib.ch).
CC   --------------------------------------------------------------------------
DR   EMBL; L22553; AAA28523.1; -.
DR   FlyBase; FBgn0014783; Dpse\exu2.
KW   Developmental protein; RNA-binding.
FT   DOMAIN      249    271       GLU/SER/PRO/THR-RICH (PEST REGION).
SQ   SEQUENCE   477 AA;  53194 MW;  8B5334A77DB9467B CRC64;
     MVSAISEDSA SATASGQCEV VKEELPAGNY ILVAVEIDTT GRRLIDEIVQ LAGYTSKGNF
     QQYIMPYMNL NQAARQRHQI RVISIGFYRM LKSMQTYKII KSKSEVAALM DFLNWLETLL
     AKQPNKEGIV MLYHDDRKFI PYMILEALKK YSLIDRFNRS VKAFANTCPM AKTFLGKHGI
     KNCGLRKLSM LLAKSKDGNS TKEDEHENPE GNSSITDNSG HKNQKQGAFE GSANVRAKMV
     YEMALQLIES ESTESPESFE SPESSESSEA EVKLLNAVRP FSQLLSSTIL ELKDQNHSLG
     RQNSFRPVFL NYFRTTLNYR VRAVKYRIGL AEHGFTLKSL KAIWSDKRKP GLELVLTAID
     SLKTEETAEL LDLLDSYYDP SKTTIKPRCK RSGNGTRRRN RAKGAASSKN GAIGAGGDNS
     VPDSATKPGG RPRRKRNNIR NNILGPQNTE KGSPKAEMKT STPKSMSIKP PSEFADI
//

Output file format

The output is to the specified graphics device.

The results can be output in one of several formats by using the command-line qualifier -graph xxx, where 'xxx' is replaced by the name of the required device. Support depends on the availability of third-party software packages.

The device names that output to a file are: ps (postscript), cps (colourps), png, gif, pdf, svg, hpgl, hp7470, hp7580, das, data.

The other available device names are: meta, x11 (xwindows), tek (tek4107t), tekt (tektronix), xterm, text.

Output can be turned off by specifying none (null).

See: http://emboss.sf.net/docs/themes/GraphicsDevices.html for further information on supported devices.

Output files for usage example

File: exu2_drops.epestfind

PEST-find: Finds PEST motifs as potential proteolytic cleavage sites.

    14 PEST motifs were identified in EXU2_DROPS
       from positions 1 to 477 and sorted by score.

Potential PEST motif with 34 amino acids between position 238 and 273.
   238 KMVYEMALQLIESESTESPESFESPESSESSEAEVK 273
       DEPST: 54.46 % (w/w)
       Hydrophobicity index: 38.31
       PEST score: 10.80  

Potential PEST motif with 14 amino acids between position 206 and 221.
   206 HENPEGNSSITDNSGH 221
       DEPST: 36.04 % (w/w)
       Hydrophobicity index: 27.16
       PEST score: 6.25  

Poor PEST motif with 18 amino acids between position 363 and 382.
   363 KTEETAELLDLLDSYYDPSK 382
       PEST score: 2.93  

Poor PEST motif with 17 amino acids between position 409 and 427.
   409 KNGAIGAGGDNSVPDSATK 427
       PEST score: -9.71  

Poor PEST motif with 19 amino acids between position 22 and 42.
    22 KEELPAGNYILVAVEIDTTGR 42
       PEST score: -11.01  

Poor PEST motif with 10 amino acids between position 440 and 451.
   440 RNNILGPQNTEK 451
       PEST score: -15.94  

Poor PEST motif with 13 amino acids between position 279 and 293.
   279 RPFSQLLSSTILELK 293
       PEST score: -16.72  

Poor PEST motif with 13 amino acids between position 349 and 363.
   349 KPGLELVLTAIDSLK 363
       PEST score: -19.94  

Invalid PEST motif with 20 amino acids between position 1 and 22.
     1 MVSAISEDSASATASGQCEVVK 22

Invalid PEST motif with 13 amino acids between position 43 and 57.
    43 RLIDEIVQLAGYTSK 57

Invalid PEST motif with 17 amino acids between position 57 and 75.
    57 KGNFQQYIMPYMNLNQAAR 75

Invalid PEST motif with 18 amino acids between position 103 and 122.
   103 KSEVAALMDFLNWLETLLAK 122

Invalid PEST motif with 10 amino acids between position 138 and 149.
   138 KFIPYMILEALK 149

Invalid PEST motif with 10 amino acids between position 225 and 236.
   225 KQGAFEGSANVR 236


       ---------+---------+---------+---------+---------+---------+

     1 MVSAISEDSASATASGQCEVVKEELPAGNYILVAVEIDTTGRRLIDEIVQLAGYTSKGNF 60
        -------------------- OOOOOOOOOOOOOOOOOOO  ------------- ---

    61 QQYIMPYMNLNQAARQRHQIRVISIGFYRMLKSMQTYKIIKSKSEVAALMDFLNWLETLL 120
       --------------                             -----------------

   121 AKQPNKEGIVMLYHDDRKFIPYMILEALKKYSLIDRFNRSVKAFANTCPMAKTFLGKHGI 180
       -                 ----------                                

   181 KNCGLRKLSMLLAKSKDGNSTKEDEHENPEGNSSITDNSGHKNQKQGAFEGSANVRAKMV 240
                                 ++++++++++++++     ----------   ++

   241 YEMALQLIESESTESPESFESPESSESSEAEVKLLNAVRPFSQLLSSTILELKDQNHSLG 300
       ++++++++++++++++++++++++++++++++       OOOOOOOOOOOOO        

   301 RQNSFRPVFLNYFRTTLNYRVRAVKYRIGLAEHGFTLKSLKAIWSDKRKPGLELVLTAID 360
                                                        OOOOOOOOOOO

   361 SLKTEETAELLDLLDSYYDPSKTTIKPRCKRSGNGTRRRNRAKGAASSKNGAIGAGGDNS 420
       OO OOOOOOOOOOOOOOOOOO                            OOOOOOOOOOO

   421 VPDSATKPGGRPRRKRNNIRNNILGPQNTEKGSPKAEMKTSTPKSMSIKPPSEFADI 477
       OOOOOO              OOOOOOOOOO                           

       Symbols   PEST motifs
       +++++++   potential
       OOOOOOO   poor     
       -------   invalid  

Graphics File: epestfind.ps

The output from epestfind is a simple text one. It reports poor and potential PEST motifs together with their PEST score, mass percent of DEPST and their hydrophobicity index.

'Valid' PEST motifs below the threshold score (5.0) are considered as 'poor', while PEST scores above the threshold score are of real biological interest. The higher the PEST score, the more likely is degradation of proteins mediated via 'potential' PEST motifs in eukaryotic cells.

Data files

EMBOSS data files are distributed with the application and stored in the standard EMBOSS data directory, which is defined by the EMBOSS environment variable EMBOSS_DATA.

To see the available EMBOSS data files, run:

% embossdata -showall

To fetch one of the data files (for example 'Exxx.dat') into your current directory for you to inspect or modify, run:

% embossdata -fetch -file Exxx.dat

Users can provide their own data files in their own directories. Project specific files can be put in the current directory, or for tidier directory listings in a subdirectory called ".embossdata". Files for all EMBOSS runs can be put in the user's home directory, or again in a subdirectory called ".embossdata".

The directories are searched in the following order:

• . (your current directory)
• .embossdata (under your current directory)
• ~/ (your home directory)
• ~/.embossdata

Here is the default Eamino.dat file:

# Molecular weights of amino acids
# Also classified as:
#  Tiny, Small, Aliphatic, Aromatic, Non-polar, Polar, Charge, +ve, -ve
#
A        71.0786         1 1 0 0 1 0 0   0 0
B       114.5960         0 1 0 0 0 0 -.5 0 1
C       103.1386         1 1 0 0 1 0 0   0 0
D       115.0884         0 1 0 0 0 1 -1  0 1
E       129.1152         0 0 0 0 0 1 -1  0 1
F       147.1762         0 0 0 1 1 0 0   0 0
G        57.0518         1 1 0 0 1 0 0   0 0
H       137.1408         0 0 0 1 0 1 .5  1 0
I       113.1590         0 0 1 0 1 0 0   0 0
J         0.0            0 0 0 0 0 0 0   0 0
K       128.1736         0 0 0 0 0 1 1   1 0
L       113.1590         0 0 1 0 1 0 0   0 0
# If met gets oxidised to the sulphoxide replace by 147.1926
M       131.1926         0 0 0 0 1 0 0   0 0
N       114.1036         0 1 0 0 0 1 0   0 0
O         0.0            0 0 0 0 0 0 0   0 0
P        97.1164         0 1 0 0 1 0 0   0 0
Q       128.1304         0 0 0 0 0 1 0   0 0
R       156.1870         0 0 0 0 0 1 1   1 0
S        87.0780         1 1 0 0 0 1 0   0 0
T       101.1048         1 1 0 0 0 1 0   0 0
U         0.0            0 0 0 0 0 0 0   0 0
V        99.1322         0 1 1 0 1 0 0   0 0
W       186.2128         0 0 0 1 1 0 0   0 0
X       144.0000         0 0 0 0 0 0 0   0 0
Y       163.1756         0 0 0 1 1 0 0   0 0
Z       128.6228         0 0 0 0 0 1 -.5 0 1

Notes

Briefly, the PEST hypothesis was based on a literature survey that combined both information on protein stability as well as protein primary sequence information. Initially, the study relied on 12 short-lived proteins with well-known properties [1], but was continually extended later [2,3]. The initial group of proteins included E1A, c-myc, p53, c-fos, v-myb, P730 phytochrome, heat shock protein 70 (HSP 70), HMG-CoA reductase, tyrosine aminotransferase (TAT), ornithine decarboxylase (ODC), alpha-Casein and beta-Casein. Although all these proteins exerted various different cellular functions it became apparent that they shared high local concentrations of amino acids proline (P), glutamic acid (E), serine (S), threonine (T) and to a lesser extent aspartic acid (D). From that it was concluded that PEST motifs reduce the half-lives of proteins dramatically and hence, that they target proteins for proteolytic degradation.

PEST means Black Death in German, so that the name of this programme sounds a bit strange, at least in our ears.

References

1. Rogers S.W., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: The PEST hypothesis Science 234, 364-368 (1986)
2. Rechsteiner M., Rogers S., Rote K. Protein structure and intracellular stability Trends Biochem. Sci. 12, 390-394 (1987)
3. Rechsteiner M. and Rogers S.W. PEST sequences and regulation by proteolysis Trends Biochem. Sci. 21, 267-271 (1996)
4. J. Kyte and R.F. Dootlittle A simple method for displaying the hydropathic character of a protein J. Mol. Biol. 157, 105 (1982)

Warnings

Diagnostic Error Messages

Exit status

Known bugs

See also

Author(s)

Please report all bugs to the EMBOSS bug team (emboss-bug © emboss.open-bio.org) not to the original author.

Original program 'epestfind' by Scott Rogers and Martin Rechsteiner (C)1986.

History

Written (March 2002).

Target users

Comments