ProEnd Pipeline 1.0

ProEnd Scripts

This project contains two Bash scripts designed to handle and analyze multiple protein sequences. The scripts streamline the extraction and identification of specific C-terminal protein HbYX-motifs from a given FASTA file. The third script shows how to download the proteomes from UniProt.

If you have just one protein sequence or your file is already one protein sequence per line go to script 2 ## Scripts

1. HbYX-First.bash

This script formats a multi-entry FASTA file into a single line per entry format, preparing it for further analysis. Use this script first if you have multiple sequences, a proteome, or alignments.

Usage

Ensure you have the necessary FASTA file in the same directory or specify the path to the file. Execute the script by running:

./HbYX-First.bash

or

bash HbYX-First.bash

This will output a file named multifasta.ol.fa, containing all the sequences from the original FASTA file, formatted for further processing.

2. HbYX-Second-Script.bash

This script searches for a specific HbYX-motif at the C-terminus of the protein sequences in the multifasta.ol.fa file created by the first script.

Usage

Run the script using:

./HbYX-Second-Script.bash

or

bash HbYX-Second-Script.bash

It will produce a file named 2aa.txt, containing the motifs found along with the corresponding header from the FASTA file, if available.

Testing ProEND with Arabidopsis proteome

Download (Reviewed Swiss-Pro) Arabidopsis proteome from Uniprot

1. Fasta linearization

zcat Arabidopsis_uniprot_proteome/uniprotkb_arabidopsis_AND_reviewed_true_2024_10_08.fasta.gz | head -n 2

## >sp|A0A067YMX8|XTH8_DIOKA Xyloglucan endotransglucosylase protein 8 OS=Diospyros kaki OX=35925 GN=XTH8 PE=1 SV=1
## MAASPYSIFAVQLLLLASWMLSSSSSNFNQDFNIAWGGGRARILNNGELVTLSLDKASGS

bash HbYX-First.awk Arabidopsis_uniprot_proteome/uniprotkb_arabidopsis_AND_reviewed_true_2024_10_08.fasta.gz Arabidopsis_uniprot_proteome/arabidopsis_uniprot_proteome.ol.fa

2. HbYX motif prediction

bash HbYX-Second-Script.awk  Arabidopsis_uniprot_proteome/arabidopsis_uniprot_proteome.ol_clean.fa  Arabidopsis_uniprot_proteome/arabidopsis_HbyX_proteome.txt

Total number of HbYX motif candidates:

grep -c ">" Arabidopsis_uniprot_proteome/arabidopsis_HbyX_proteome.txt 

## 219

3. Exploring conserved HbYX proteasome regulatory protein

grep --no-group-separator -A 1 -i "proteasome"  Arabidopsis_uniprot_proteome/arabidopsis_HbyX_proteome.txt 

## >sp|O04019|PS6AB_ARATH 26S proteasome regulatory subunit 6A homolog B OS=Arabidopsis thaliana OX=3702 GN=RPT5B PE=1 SV=3
## YYA
## >sp|Q9SEI2|PS6AA_ARATH 26S proteasome regulatory subunit 6A homolog A OS=Arabidopsis thaliana OX=3702 GN=RPT5A PE=1 SV=1
## YYA
## >sp|Q9SL67|PRS4B_ARATH 26S proteasome regulatory subunit 4 homolog B OS=Arabidopsis thaliana OX=3702 GN=RPT2B PE=1 SV=1
## LYM
## >sp|Q9SZD4|PRS4A_ARATH 26S proteasome regulatory subunit 4 homolog A OS=Arabidopsis thaliana OX=3702 GN=RPT2A PE=1 SV=1
## LYM
## >sp|Q9SEI4|PRS6B_ARATH 26S proteasome regulatory subunit 6B homolog OS=Arabidopsis thaliana OX=3702 GN=RPT3 PE=1 SV=1
## FYK
## >sp|Q9SSB4|PRS7B_ARATH 26S proteasome regulatory subunit 7 homolog B OS=Arabidopsis thaliana OX=3702 GN=RPT1B PE=1 SV=2
## YYI
## >sp|Q9SSB5|PRS7A_ARATH 26S proteasome regulatory subunit 7 homolog A OS=Arabidopsis thaliana OX=3702 GN=RPT1A PE=1 SV=1
## VYN

4. Exploring novel regulatory candidates

Mapping TAIR Arabidopsis IDs using biomaRt

Getting entrezID and running GO terms analysis

uniprot_ids <- read.csv("Arabidopsis_uniprot_proteome/uniprot_ids.txt", header = F)
ensembl <- useMart("plants_mart", dataset = "athaliana_eg_gene", host = "https://plants.ensembl.org")
mapping <- getBM(
  attributes = c("uniprotswissprot", "entrezgene_id"), # "tair_locus"),
  filters = "uniprotswissprot",
  values = uniprot_ids,
  mart = ensembl
)

all_uniprot_df <- as.data.frame( uniprot_ids)
colnames(all_uniprot_df ) <- c("uniprotswissprot")

uniprot2entrez <- merge(all_uniprot_df, mapping, by = "uniprotswissprot", all.x = TRUE)
print(paste("Uniprot ids:", nrow(uniprot_ids), " Entrez ids:", nrow(mapping), " Non mapped ids:",  nrow(uniprot_ids) -  nrow(mapping), sep = ""))

## [1] "Uniprot ids:219 Entrez ids:205 Non mapped ids:14"

#Non mapped IDs
#uniprot2TAIR[is.na(uniprot2TAIR$tair_locus),]

ego <- enrichGO(gene  = uniprot2entrez[!is.na(uniprot2entrez$entrezgene_id),2], 
                OrgDb         = org.At.tair.db,
                ont           = "BP", #"MF"
                pAdjustMethod = "BH",
                pvalueCutoff  = 0.01,
                qvalueCutoff  = 0.05,
                readable      = TRUE) #library(clusterProfiler)

ego_at <- attributes(ego )
Whole_table <- ego_at$result
write.csv(Whole_table, "Arabidopsis_uniprot_proteome/GO_HbYX_Arabidopsis.csv") 
d <- godata('org.At.tair.db', ont="BP") #library(GOSemSim)

## preparing gene to GO mapping data...

## preparing IC data...

ego2 <- pairwise_termsim(ego, method="Wang", semData = d) #library(enrichplot)

GO terms enrichment of Arabidopsis HbYX containing proteins

5. HbYX protein CDC48A as a potential proteasome regulator

One proposed and controversial candidate for 20S proteasome regulation, significantly enriched across several Gene Ontology categories, is the CDC48 gene family, as demonstrated in:

head(Whole_table[grepl("CDC48",Whole_table$geneID),c(2,3,8)])

##                                                          Description GeneRatio
## 5                                      response to misfolded protein     4/196
## 7  proteasome-mediated ubiquitin-dependent protein catabolic process    10/196
## 11                             proteasomal protein catabolic process    10/196
## 12                           protein exit from endoplasmic reticulum     3/196
## 14                 ER-associated misfolded protein catabolic process     3/196
## 17                            cellular response to misfolded protein     3/196
##                                                                 geneID
## 5                                      ATCDC48/AtCDC48C/AtCDC48B/RPT2a
## 7  RPT5B/ATCDC48/AtCDC48C/AtCDC48B/ATS6A.2/RPT3/ARI9/RPT2b/RPT1A/RPT2a
## 11 RPT5B/ATCDC48/AtCDC48C/AtCDC48B/ATS6A.2/RPT3/ARI9/RPT2b/RPT1A/RPT2a
## 12                                           ATCDC48/AtCDC48C/AtCDC48B
## 14                                           ATCDC48/AtCDC48C/AtCDC48B
## 17                                           ATCDC48/AtCDC48C/AtCDC48B

6. Reconstruction a potential interaction of CDC48 and the 20S proteasome.

Given its potential, we decided to proceed with an in-silico reconstruction of the CDC48 complex in this example. CDC48 contains a well-characterized AAA+ domain, which is typically associated with the formation of a homo-oligomer, most commonly a hexamer in proteins of this type. For this reconstruction, AlphaFold V2 Multimer or AlphaFold V3 can be used, the latter allowing the inclusion of ligands such as ATP.

CDC-48 Hexamer prediction with and without ATP. HbYX motif in orange

As expected, the CDC-48 homohexamer predictions show robust pTM values, with 0.61 in the absence of ATP and 0.55 in the presence of ATP. A pTM score above 0.5 indicates that the predicted overall structure of the complex is likely to resemble the true native structure. Interestingly, the HbYX motif can be observed shifting from the outer structure to the inner portion, a conformational change commonly seen in substrate-processing ATPases that interact with the 20S proteasome upon substrate engagement. This change facilitates interaction with the 20S proteasome to open the gate for substrate entry.

You can proceed with molecular docking of the CDC48 complex with the 20S proteasome using ChimeraX or any preferred molecular docking tool.

Potential CDC48-20S complex formation. HbYX motif in orange. 20S in gray

Upon docking, the HbYX motif of CDC48, in the presence of ATP, is positioned optimally for interaction with the alpha pockets of the 20S proteasome, potentially facilitating gate opening and activation

7. Conclusion and Future Potential.

This example demonstrates how to generate and explore hypotheses using our tool, ProEnd. In this case, we focused on HbYX-containing proteins from Arabidopsis thaliana, one of the most extensively studied model organisms in biology. Using ProEnd, we successfully identified known HbYX-containing proteins, including the 19S-26S regulatory proteins, and discovered enriched candidates (CDC48-p97) with potential for novel interactions with the 20S proteasome, expanding our understanding of proteasome biology and proteostasis.

The formation of the CDC48-20S complex remains a topic of debate. Some researchers argue that the 19S-20S complexes, also referred to as 26S proteasomes, represent the vast majority of proteasomes in the cell. However, various cellular contexts demand alternative proteasome configurations. For instance, there are well-documented cases of 20S complexes interacting with other molecules, such as PA28, PA200, and PI31. CDC48 is particularly relevant in unique cellular environments that require the degradation of tightly folded substrates. These substrates, after folding, may directly engage the 20S proteasome without the need for 19S caps, providing a distinct scenario for CDC48-20S complex formation, potentially in the ER or chloroplast.

The conservation of the HbYX motif in CDC48 across different kingdoms (from archea to eukaryotes) suggests a conserved mechanism for direct degradation through the 20S proteasome without intermediaries, further supporting the idea that CDC48 plays a crucial role in specific proteolytic pathways.

Requirements

• Unix-like environment
• AWK installed

Installation

No installation is required. Simply clone this repository or download the scripts to your local machine.

Data Folder HbYX_data_tables

This folder contains data results files for the ProEnd Scripts project.

Cite

This code can be cited currently as BioRxiv preprint [1]

License

The code is freely available to download and run, but it’s protected and licensed under a Creative Commons Attribution-ShareAlike 4.0 International License, meaning you can use it but citing it’s source.

References

1. Salcedo-Tacuma DM, Howells G, McHose C, Gutierrez-Diaz A, Smith DM. ProEnd: A comprehensive database for identifying HbYX motif-containing proteins across the tree of life. bioRxiv. 2024;2024–06.