The Functional Human C-Terminome

Abstract

All translated proteins end with a carboxylic acid commonly called the C-terminus. Many short functional sequences (minimotifs) are located on or immediately proximal to the C-terminus. However, information about the function of protein C-termini has not been consolidated into a single source. Here, we built a new "C-terminome" database and web system focused on human proteins. Approximately 3,600 C-termini in the human proteome have a minimotif with an established molecular function. To help evaluate the function of the remaining C-termini in the human proteome, we inferred minimotifs identified by experimentation in rodent cells, predicted minimotifs based upon consensus sequence matches, and predicted novel highly repetitive sequences in C-termini. Predictions can be ranked by enrichment scores or Gene Evolutionary Rate Profiling (GERP) scores, a measurement of evolutionary constraint. By searching for new anchored sequences on the last 10 amino acids of proteins in the human proteome with lengths between 3-10 residues and up to 5 degenerate positions in the consensus sequences, we have identified new consensus sequences that predict instances in the majority of human genes. All of this information is consolidated into a database that can be accessed through a C-terminome web system with search and browse functions for minimotifs and human proteins. A known consensus sequence-based predicted function is assigned to nearly half the proteins in the human proteome. Weblink: http://cterminome.bio-toolkit.com.

Fig 1. Functional landscape of C-terminome.

(A) A pie chart shows the number of experimentally verified C-terminal minimotifs with different functions. The molecular function for each category is shown in the other panels: bind (B), traffic (C), and modify (D). This graph includes both consensus sequences and instances. (B) A pie chart showing the types of domain targets for binding minimotifs. The “Other domains” category includes Transducin-like enhancer proteins, Tetratricopeptide repeat domain, 14-3-3 domain, G protein coupled receptor, COPI and COPII binding proteins, and ubiquitin- binding proteins. The category 'domain not known' indicates that the specific interaction domain of the target protein was not identified. (C) A pie chart showing the different compartments for trafficking motifs. (D) A pie chart showing functional categories for post-translational modifications. The “other PTMs” category (1%) includes methylation, prenylation, glycosylation, crotonylation, amidation, farnesylation, sulphation, de-phosphorylation, o-glcnacation, geranyl-geranylation, glycation, carboxy-methylation, deamination, sumoylation, tri-iodination, malonylation, mevalonation, and palmitoylation.

Fig 2. Predicted C-terminal PTM minimotifs inferred from rodent orthologs.

Pie charts show the number and types of instances of (A) known minimotifs in rodent proteome and (B) predictions using consensus sequences derived from rodent cell experimentation (see keys).

Fig 3. Predicted C-terminal instances from minimotif sequences.

Pie charts showing predicted instances from matches to minimotif sequences for three major minimotif categories (A), binding minimotif types (B), PTM types (C), and trafficking motif types (D). The “Other domains” and the “Other PTMs” are as described for Fig 1. (D) The “Other” category includes trafficking to vesicles, nuclear export, cell surface, rod outer segment and prevents secretion from ER.

Fig 4. de novo consensus sequence matches in the human proteome.

Consensus sequences and instances of length 3–10 residues and up to 5 degenerate positions were generated and used to search the last 10 amino acids of each protein in the human proteome (A). This included alternatively spliced isoforms. The inset shows the total number of sequences searched and number of occurrences identified. For each set of sequences with a given length and degeneracy (e.g. 3–1), the number of sequences searched and occurrences identified are shown in the bar graph.

Fig 5. Fold-enrichment scores of minimotifs and predicted sequences.

Bar graph showing the percentage of occurrences with different proteome-wide (A), and discrete-proteome (B) fold-enrichments. Dark gray bars represent the percentage of C-terminal minimotifs, and light gray bars represent the percentage of predicted consensus sequences and instances from de novo generated sequences.

Fig 6. GERP score analysis of minimotif prediction.

Line graph showing the percentage of positive predictive values and accuracy for average minimotif GERP score (A), and minimum minimotif GERP score (B) thresholds.

Fig 7. C-terminome protein search and browse pages.

(A) The main C-terminome search page can be used to select, search and browse proteins or minimotifs. Question marks open a popup window with acceptable syntax. When a protein is entered into the textbox and a search is initiated, a results page shows the top hits for the protein search term (bottom panel). Presented information includes the RefSeq protein accession number, gene name, and protein sequence with the highlighted C-terminus. (B) A protein can also be selected from the Browse Proteins page. This list can be searched for protein names alphabetically or browsed for C-termini of different proteins. A key at the top indicates the GERP score for each residue at the C-termini. (C) Both Search and Browse Proteins produce a page with the results shown in C. This includes the RefSeq protein accession number, protein name, protein sequence with the C-terminus highlighted, and alternative spliced variants with the RefSeq accession number, and isoform name (top panel); a list of consensi present in the protein including whether the consensus sequence was experimental or predicted, number of instances, and both proteome-wide and discrete proteome fold enrichment (bottom panel).

Fig 8. Browse C-termini minimotifs and predicted minimotifs page.

The Browse Minimotifs page has two tabs for searching different types of data: C-termini minimotifs, and predicted minimotifs. (A) The main display for the minimotif instances and consensus sequences tab has several different motif type and function selectors for identifying minimotif instances. (B) An example of a minimotif result page shows the information provided for an identified minimotif (top panel). If you mouse-click the motif sequence hyperlink, it reveals the set of minimotifs with that consensus sequence (bottom panel). (C) Browse Predicted Minimotifs page for de novo sequences that are highly repetitive in the human proteome. Top panel shows the main selector display page, where the length and degeneracy of the sequence is chosen. A list of minimotifs produced from the search is shown in the bottom panel. Sequences are hyperlinked to more information about the predicted minimotif.

Fig 9. Search minimotifs page.

(A) When a minimotif is entered into the search page textbox (top panel) and a search is initiated, it produces a results page with the top hits for the consensus sequence (bottom panel). Presented information includes information about how the motif was identified and how many times it occurs in the human proteome. The search can be restricted to prediction or minimotif instances. Selecting the question mark open a popup window with acceptable syntax. (B) Once a particular minimotif is selected, a new results page displays more specific information. Selecting one of the sets of instances reveals a list of proteins containing the consensus sequence and the C-termini of these proteins.

Fig 10. Variability of C-terminal minimotifs in the human population.

The pie chart represents breakdown of functional classes of 730 single nucleotide polymorphisms (SNPs) identified in C-terminal minimotifs post-translational modification. The breakdown of the modification function is shown in an extended pie chart. The “other PTMs” category includes methylation, prenylation, crotonylation, glycosylation, lipidation, and sumoylation minimotifs. PDZ domain binding motifs (1%) are not shown.

Fig 11. Selection of C-terminal minimotifs in humans.

Density distribution plots showing the GERP scores for different genomic regions.