Introduction

Neddylation is one of the ubiquitin-like post-translational modifications, in which a NEDD8 protein is attached to target lysine residues. Neddylation affects the localization, lifespan, function and structure of a target protein.

Neddylation target proteins are observed to be located predominantly in the nucleus [1]. The most characterized targets of neddylation belong to cullin protein family, whose members act as scaffold proteins of multi-subunit E3 ubiquitin ligases called cullin-RING ligases [2]. Therefore, neddylation mainly acts as a regulator of ubiquitin-protein ligases [1]. In addition, neddylation affects cell-cycle regulation, transcriptional regulation and signal transduction indirectly [3].

Abnormalities in neddylation has been linked with variety of disorders and developmental aberrations, such as Alzheimer’s [4], Parkinson’s [5] and cancer [6]. Therefore, identifying neddylation sites possibly bears a significant role in understanding various disease mechanisms. Moreover, neddylation site identification may help functionalize disease-related genomic variants and understand etiology and pathogenesis of many diseases.

However, experimental approaches are too burdensome and costly for scanning thousands of proteins. In order to overcome this challenge, we have developed a novel neddylation site predictor, named NeddyPreddy, exploiting the sequence characteristics and sequence-derived features of residues located in the vicinity of lysines.


3-D structure of human NEDD8
(PDB ID: 1ndd)

Submission

There are three submission options on NeddyPreddy as shown below. When you open NeddyPreddy, manual input button is selected by default. To make another selection, click one of the options and input area will change accordingly.


To select your FASTA file, please click browse button.

Browse
NeddyPreddy works with FASTA sequences. You can either provide the sequence's itself or Uniprot ID of the protein and NeddyPreddy will fetch the sequence from Uniprot automatically.

Use the text area to enter your FASTA sequence(s) or Uniprot ID(s) of your protein(s).

You can enter one or more sequences or Uniprot IDs for each protein but please use seperate lines for each sequence or Uniprot ID as shown below. Blue arrows indicate the beginning of each sequence or Uniprot ID.


To select your FASTA file, please click browse button.

Browse

You can upload a file for your sequence(s) by clicking the upload button. Then, click and select the file you want to upload.

Before you make a file selection, you will see No file is selected text next to . After file selection, you will see that the text is changed to your file name.

You can also enter extra sequences in the text area in addition to sequences in your file. NeddyPreddy will read both.

Your sequences have to be in FASTA format.


To select your FASTA file, please click browse button.

Browse

If you do not have a sequence of your own or you want to test the server, you can use sample button to generate sample sequences.

Also, sample sequences can be used for demonstration of how you should enter your own sequences.


To select your FASTA file, please click browse button.

Browse

Because NeddyPreddy depends on other servers for certain features, your results may take some time. Even though your results link will be refreshed automatically and will show your results when they are ready, NeddyPreddy also can send an e-mail to you. To recieve e-mails please enter your e-mail address in the field as shown bel before you submit your sequences.


Use the to reset your entries, files and e-mail.


To select your FASTA file, please click browse button.

Browse

To submit your entries and/or file upload, click .


To select your FASTA file, please click browse button.

Browse

In Progress

When you submit your sequence or file, NeddyPreddy starts working on them and keep you informed about the process on in progress page. In this page, you can see how many proteins are submitted, how many proteins are completed and how many of them are queued.

It also creates a unique (as shown below in green) link for your results, which will be active once your results are ready. You can use this link to see your results even if you close in progress window.

The link for your results will be active for 7 days; therefore, you can check your results whenever you want in that period of time.
If you entered your e-mail address on the submission page, results page link will be sent to you when it is active.
NeddyPreddy is working on your request.
If you choose to close this window, you can check the progress at http://neddypreddy.sabanciuniv.edu/jobs/E7IZGLX1XGPYXQM/

Proteins in Submission: 2 (Completed: 0)
Current Status: Queued (Position in Queue: 1, Total Proteins Left: 1)

Results

Results page composed of two sections:

Content

Menu contains 4 sections:

In the dashboard section, a summary of your results will be shown.

Welcome to your neddylation sites!


Proteins

In details page, you will see detailed neddylation prediction results for your proteins in a table composed of 6 columns:

You can make an export of the data in CSV format by clicking . Applied filters will affect data in CSV.

You can sort and/or filter your results by using filter controllers.

To sort your columns, click the header of the column that you want to sort.

Filtering the results is done by threshold levels from Threshold Level dropdown menu and/or by individual column filters that are present under each associated column.

You can clear all filters (including threshold levels) by clicking .

For more information about filtering, please read filtering sections below.

The table is filtered by medium threshold level (notice >=0 on Score column) by default; therefore, only the neddylation sites that were predicted with score 0 or above are shown.

You can filter your results by the ID of your protein. Try H4_HUMAN in the text field under ID column head in the table.

Avoid using | as it will conflict with math sign. For example, when filtering sp|P62805|H4_HUMAN, instead of writing the whole ID, either use P62805 or H4_HUMAN.

You can filter your results by the position of the neddylated site of your protein.

Enter one of the filters in the text field under Position column head in the table.


Single Number 13

Shows neddylated sites at the position 13 and numbers starts with 13 (i.e. 130).


Multiple Numbers 13|17|21|32

Use | to separate numbers when filtering more than one position.


Range 13 - 42

Shows neddylated sites at the position between 13 and 42 (13 and 42 are included).


Greater Than >13 or >=13

>13 shows neddylated sites at the position greater than 13.

>=13 shows neddylated sites at the position 13 and higher.


Less Than <32 or <=32

<32 shows neddylated sites at the position less than 32.

<=32 shows neddylated sites at the position 32 and less than 32.

NeddyPreddy peptides are composed of 21 amino acids with neddylated lysine (K) being in the center.

Neddylated lysine is written in green as shown below:

GRGKGGKGLGKGGAKRHRKVL

If neddylated lysine is near the beginning or the end of the peptide you will see - beyond the first or last amino acid instead of amino acid letter.

For example, -----MSGRGKGGKGLGKGGA

Because neddylated lysine is the 6th amino acid of the protein, only 5 amino acids are present before neddylated lysine and to complete 21 amino acid chain, NeddyPreddy puts - in the absense of amino acid.

The same is applied when neddylated lysine is near the end.

LLPKKTESHHKAKGK------

You can filter the whole peptide or a portion. Enter the one of the filters in the text field under Peptide column head in the table.


Whole Peptide GRGKGGKGLGKGGAKRHRKVL

GRGKGGKGLGKGGAKRHRKVL shows peptides exactly matched with the search.


Portion Peptide GLGKG

GLGKG shows peptides that contain GLGKG amino acid chain.

You can filter your results by the score of the neddylated site prediction.

Enter one of the filters in the text field under Score column head in the table.

- sign indicates sites that were predicted as non-neddylated.

Single Number 0.20

Shows neddylated sites with score 0.20.


Multiple Numbers 0.20|1.13|2.48|-2.85

Use | to separate numbers when filtering more than one sites with different scores.


Range 0.20 - 2.48

Shows neddylated sites with a score between 0.20 and 2.48 (0.20 and 2.48 are included).


Greater Than >0.33 or >=0.33

>0.33 shows neddylated sites with score greater than 0.33.

>=0.33 shows neddylated sites with score 0.33 and higher.


Less Than <1.13 or <=1.13

<1.13 shows neddylated sites with score less than 1.13.

<=1.13 shows neddylated sites with score 1.13 and less.

You can filter your results by the probability of the neddylated site prediction.

Enter one of the filters in the text field under Probability column head in the table.


Single Number 0.17

Shows neddylated sites with probability 0.17.


Multiple Numbers 0.17|0.35|0.64|0.92

Use | to separate numbers when filtering more than one sites with different probabilities.


Range 0.35 - 0.92

Shows neddylated sites with a probability between 0.35 and 0.92 (0.35 and 0.92 are included).


Greater Than >0.17 or >=0.17

>0.17 shows neddylated sites with probability greater than 0.17.

>=0.17 shows neddylated sites with probability 0.17 and higher.


Less Than <0.45 or <=0.45

<0.45 shows neddylated sites with probability less than 0.45.

<=0.45 shows neddylated sites with probability 0.45 and less.


ID Position Peptide Score Probability Extra
sp|P62805|H4_HUMAN 6 -----MSGRGKGGKGLGKGGA 0.52 0.23

More

sp|P62805|H4_HUMAN 9 --MSGRGKGGKGLGKGGAKRH 2.48 0.92

More

sp|P62805|H4_HUMAN 13 GRGKGGKGLGKGGAKRHRKVL 1.13 0.48

More

sp|P62805|H4_HUMAN 17 GGKGLGKGGAKRHRKVLRDNI 1.49 0.64

More

sp|P62805|H4_HUMAN 21 LGKGGAKRHRKVLRDNIQGIT 0.15 0.13

More

sp|P62805|H4_HUMAN 32 VLRDNIQGITKPAIRRLARRG 0.54 0.23

More

sp|P62805|H4_HUMAN 45 IRRLARRGGVKRISGLIYEET -0.86 0.02

More

sp|P62805|H4_HUMAN 60 LIYEETRGVLKVFLENVIRDA -0.97 0.02

More

sp|P62805|H4_HUMAN 78 RDAVTYTEHAKRKTVTAMDVV -2.85 0.00

More

sp|P62805|H4_HUMAN 80 AVTYTEHAKRKTVTAMDVVYA -2.65 0.00

More

sp|P62805|H4_HUMAN 92 VTAMDVVYALKRQGRTLYGFG 0.67 0.28

More

sp|P04908|H2A1B_HUMAN 6 -----MSGRGKQGGKARAKAK 0.21 0.14

More

sp|P04908|H2A1B_HUMAN 10 -MSGRGKQGGKARAKAKTRSS 1.81 0.76

More

sp|P04908|H2A1B_HUMAN 14 RGKQGGKARAKAKTRSSRAGL 0.84 0.35

More

sp|P04908|H2A1B_HUMAN 16 KQGGKARAKAKTRSSRAGLQF -0.76 0.03

More

sp|P04908|H2A1B_HUMAN 37 PVGRVHRLLRKGNYSERVGAG -1.09 0.02

More

sp|P04908|H2A1B_HUMAN 75 ELAGNAARDNKKTRIIPRHLQ -1.30 0.01

More

sp|P04908|H2A1B_HUMAN 76 LAGNAARDNKKTRIIPRHLQL -2.70 0.00

More

sp|P04908|H2A1B_HUMAN 96 LAIRNDEELNKLLGRVTIAQG -2.45 0.00

More

sp|P04908|H2A1B_HUMAN 119 LPNIQAVLLPKKTESHHKAKG 1.08 0.45

More

sp|P04908|H2A1B_HUMAN 120 PNIQAVLLPKKTESHHKAKGK 0.99 0.41

More

sp|P04908|H2A1B_HUMAN 126 LLPKKTESHHKAKGK------ 0.33 0.17

More

sp|P04908|H2A1B_HUMAN 128 PKKTESHHKAKGK-------- 0.59 0.25

More

sp|P04908|H2A1B_HUMAN 130 KTESHHKAKGK---------- 0.20 0.14

More

For each site on table, there is that reveals more information and visualization of the related site.

Average Disorder

In addition to details in the table, there is average disorder column in the more information window table. Intrinsically unstructured/disordered proteins have no single well-defined tertiary structure in their native, functional state.

Range: 0 - 1

Values above 0.5 indicate disorder.

Position on the Protein with Other Predicted and Known Sites

represents the protein.

represents the beginning and end the of the protein.

represents the neddylated site.

represents the neddylated sites with low scores.

represents the neddylated sites with medium scores.

represents the neddylated sites with high scores.

Ace represents experimentally identified lysine Acetylation site.

Gly represents experimentally identified lysine O-linked Glycosylation site.

Met represents experimentally identified lysine Methylation site.

Sum represents experimentally identified Sumoylation site.

Ubi represents experimentally identified Ubiquitylation site.

Compare with Other Sites on the Protein

You can compare the all sites on the protein by using the bar chart. Hover your mouse on the bars of the desired site and see score and probability values. Also, you can turn on/off score or probability bars by clicking buttons below X-axis of the chart.

Other Lysine Modifications Located on Protein

You can view experimentally identified known lysine modifications, their modification sites, and literature and database references in this section. You can click on links to view the original reference or database entry.

ID Position Peptide Average Disorder Score Probability
sp|P04908|H2A1B_HUMAN 6 -----MSGRGKQGGKARAKAK 0.73 0.21 0.14

Position on the Protein with Other Predicted and Known Sites
1 Low Medium High 6 10 14 16 119 120 126 128 130 Ace 96 Ace Ubi Ubi Ubi 130
Compare with Other Sites on the Protein

Other Lysine Modifications Located on Protein (Source: dbPTM)
Sequence ID Uniprot ID Position Modification Reference Source
sp|P04908|H2A1B_HUMAN P04908 10 Acetylation 19608861 Phosphositeplus.1010730
sp|P04908|H2A1B_HUMAN P04908 96 Acetylation 19608861 HPRD 9.0
sp|P04908|H2A1B_HUMAN P04908 96 Ubiquitylation 21890473
21906983
Phosphositeplus.1010730
sp|P04908|H2A1B_HUMAN P04908 119 Ubiquitylation 18781797
20639865
20972266
21906983
15852347
Swiss-Prot 1010711
Phosphositeplus.1010730
SysPTM 1.1
UbiProtDB 1.0
sp|P04908|H2A1B_HUMAN P04908 120 Ubiquitylation 15386022
16359901
16702407
20639865
21906983
Swiss-Prot 1010711
Phosphositeplus.1010730

This page informs you when your result link will be inactive and provides a link to sequence submit page.

just redirects you to main page.

If you do not want to lose current result page, do not forget to save the result link before you go to main page.

Starting Over


You can start over and submit new sequences anytime you want.

These results will be available in this link until 22 August 2015 18:44.

Start Over

I can't see website properly, what should I do?

We have tested the website on Mac, Linux, Windows with Chrome, Safari and Firefox browsers. Please be sure that Javascript is activated on your browser. If you still encounter an issue, please report us.


There are three threshold levels used in your predictor, what do these parameters mean?

Those are pre-defined levels and they filter score value. For more information please visit related documentation section.


I have a large dataset which I cannot run on your server. What should I do?

You can download standalone version of NeddyPreddy from https://bitbucket.org/asyavuz/neddypreddy/.


How can I cite NeddyPreddy?

NeddyPreddy is currently under review for publication. This section will be updated when more information is available.


Can I use NeddyPreddy for commercial applications?

NeddyPreddy is free for all users. However, in order to ensure fair usage, we kindly request commercial users to use standalone version, if they are planning to use NeddyPreddy for intensive applications.


I have a few questions which are not listed above, who should I contact?

You can reach to Prof. Ugur Sezerman via e-mail at ugur.sezerman@acibadem.edu.tr.

[1] J. Herrmann, L. O. Lerman, and A. Lerman, “Ubiquitin and ubiquitin-like proteins in protein regulation.,” Circ. Res., vol. 100, no. 9, pp. 1276–91, May 2007.
[2] J. Wu et al., “Neddylation and deneddylation regulate Cul1 and Cul3 protein accumulation.”, Nat. Cell Biol., vol. 7, pp. 1014–20, 2005.
[3] D. P. Xirodimas, “Novel substrates and functions for the ubiquitin-like molecule NEDD8.,” Biochem. Soc. Trans., vol. 36, no. Pt 5, pp. 802–6, Oct. 2008.
[4] Y. Chen, R. L. Neve, and H. Liu, “Neddylation dysfunction in Alzheimer’s disease.”, J. Cell. Mol. Med., vol. 16, no. 11, pp. 2583–91, Nov. 2012.
[5] Y. S. Choo, G. Vogler, D. Wang, S. Kalvakuri, A. Iliuk, W. A. Tao, R. Bodmer, and Z. Zhang, “Regulation of parkin and PINK1 by neddylation.”, Hum. Mol. Genet., vol. 21, no. 11, pp. 2514–23, Jun. 2012.
[6] W.-T. Yao, J.-F. Wu, G.-Y. Yu, R. Wang, K. Wang, L.-H. Li, P. Chen, Y.-N. Jiang, H. Cheng, H. W. Lee, J. Yu, H. Qi, X.-J. Yu, P. Wang, Y.-W. Chu, M. Yang, Z.-C. Hua, H.-Q. Ying, R. M. Hoffman, L. S. Jeong, and L.-J. Jia, “Suppression of tumor angiogenesis by targeting the protein neddylation pathway.”, Cell Death Dis., vol. 5, p. e1059, Jan. 2014.