Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles

Abstract

Ubiquitin-like 3 (UBL3) is a well-conserved ubiquitin-like protein (UBL) in eukaryotes and regulates the ubiquitin cascade, but the significant roles of UBL3 in cellular processes remained unknown. Recently, UBL3 was elucidated to be a post-translational modification factor that promotes protein sorting to small extracellular vesicles (sEVs). Proteins sorted into sEVs have been studied as etiologies of sEV-related diseases. Also, there have been attempts to construct drug delivery systems (DDSs) by loading proteins into sEVs. In this review, we introduce the new concept that UBL3 has a critical role in the protein-sorting system and compare structure conservation between UBL3 and other UBLs from an evolutionary perspective. We conclude with future perspectives for the utility of UBL3 in sEV-related diseases and DDS.Key words: UBL3, small extracellular vesicles, protein sorting, ubiquitin-like protein, post-translational modification.

Keywords: UBL3; post-translational modification; protein sorting; small extracellular vesicles; ubiquitin-like protein.

Fig. 1

UBL3 as a new protein-sorting factor, joining Ub and UBL The new concept of UBL3 that participate in protein sorting to sEVs, ranking UBL3 alongside the prominent protein-sorting systems, Ub-proteasome and autophagy systems. ATG, autophagy-related protein; MVBs, multivesicular bodies; sEVs, small extracellular vesicles; Ub, ubiquitin; UBL, ubiquitin-like protein; UBL3, ubiquitin-like 3.

Fig. 2

The primary and secondary structures of human UBL3 The characteristics of the primary structure of UBL3 are a ubiquitin-like domain from residues 10 to 88 (yellow) and a CAAX sequence (CVIL) at the end of the carboxyl-terminal (red). The secondary structure elements (α-helix: green, β-sheet: blue) are arranged in a ββαβββ order. UBL3, ubiquitin-like 3.

Fig. 3

Comparison of the tertiary structures and amino acid sequences of UBL3, Ub and other UBLs (a) The tertiary structure of the Ub-fold (β-grasp) is shared in ubiquitin and other UBLs, including UBL3. Carboxyl-termini of Ub and the majority of UBLs end in glycine residues, except for UBL3, Rad23a and HUB1. UBL3 is distinguished from other UBLs by a CAAX sequence located at its carboxyl-terminal. As for Rad23a, only the Ub-fold, not the full-length, is described. Three-dimensional diagrams of the Ub/UBLs were drawn using QueMol 2.0 (http://www.cuemol.org/ja/) and the following Protein Data Bank IDs (http://www.rcsb.org/): human UBL3, 2GOW; human Ub, 1C3T: human SUMO-1, 2N1V: human NEDD8, 2KO3: humanATG8, 4ZDV: human ATG12, 4GDL: human UFM1, 1WXS: human Rad23a, 1P98: human HUB1, 1POR. (b) Multiple sequence alignment of UBL3, Ub, and other UBLs. The sequence homology of UBL3 to Ub and to other UBLs is relatively low (13.1–24.7%). Identical amino acids are highlighted in white (≤60%), light blue (61–80%) and mid-blue (81–100%). Clustal colored multiple sequence alignments were performed with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and displayed with Jalview version 2.11.0 (http://www.jalview.org/). ATG, autophagy-related protein; C (in the Ub-fold schema), carboxyl-terminal; C (in the UBL3 schema), cysteine; G, glycine; I, isoleucine; L, leucine; N, amino-terminal; NEDD8, neuronal precursor cell-expressed, developmentally down-regulated 8; SUMO1, small ubiquitin-like modifier 1; Ub, ubiquitin; UBL, ubiquitin-like protein; UBL3, ubiquitin-like 3; UFM1, ubiquitin-fold modifier 1; V, valine.

Fig. 4

Multiple sequence alignments of UBL3 orthologs in various eukaryotes Primary structure homology of UBL3 orthologs in other eukaryotes to HsUBL3 varies widely (18.5–99.2%). Identical amino acids are highlighted in white (≤60%), light blue (61–80%) and mid-blue (81–100%). At-1 and At-3 are orthologs in Arabidopsis thaliana mentioned as AtMUB1 and AtMUB3, respectively by Downes et al. (Downes et al., 2006). Clustal colored multiple sequence alignments were performed with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and displayed with Jalview version 2.11.0 (http://www.jalview.org/). The GenBank^TM accession numbers of the UBL3 orthologs are as follows: Homo sapiens, CAG38489; Mus musculus, BAE22201; Xenopus tropicalis, AAH44683; Danio rerio, NP_998021; Patinopecten yessoensis, XP_021348282; Nematostella vectensis, XP_001626774; Stegodyphus mimosarum, KFM60655; Drosophila melanogaster, NP_001162763; Caenorhabditis elegans, NP_001021222; Ciona intestinalis, XP_009860727; Oryza sativa, XP_015630437; Arabidopsis thaliana, NP_001319438 (At-1), NP_001329044 (At-3); Tetrabaena socialis, TSOC_00702; Chlorella sorokiniana, PRW20945; Monoraphidium neglectum, XP_013901616; Chlamydomonas reinhardtii, XP_001696591; Pyricularia oryzae, XP_003719682; Aspergillus nidulans, XP_662336; Cryptococcus neoformans, XP_567533; Kockovaella imperatae, XP_021868326; Yarrowia lipolytica, AOW02568. An, Aspergillus nidulans; At, Arabidopsis thaliana; C, carboxyl-terminal; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Cn, Cryptococcus neoformans; Cr, Chlamydomonas reinhardtii; Cs, Chlorella sorokiniana; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Ki, Kockovaella imperatae; Mm, Mus musculus; Mn, Monoraphidium neglectum; N, amino-terminal; Nv, Nematostella vectensis; Os, Oryza sativa; Po, Pyricularia oryzae; Py, Patinopecten yessoensis; Sm, Stegodyphus mimosarum; Ts, Tetrabaena socialis; UBL3, ubiquitin-like 3; Xt, Xenopus tropicalis; Yl, Yarrowia lipolytica.

Fig. 5

Phylogenic analysis of UBL3 orthologs in various eukaryotes, focusing on the CAAX sequence at the carboxyl-terminal and nearby cysteine residues The UBL3-ortholog group was separated from the Ub-out-group. Two adjacent cysteine residues are observed in animals, except for Caenorhabditis elegans, and the CAAX sequence (colored red) is evolutionarily conserved in organisms at various stages of evolution. Phylogenic analysis was performed with Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and was drawn using Dendroscope 3 version 3.6.2 (http://ab.inf.uni-tuebingen.de/software/dendroscope/). The GenBank^TM accession numbers of the UBL3 orthologs mutual with Fig. 4 are used, while that of the Ub orthologs and the range of amino acid (AA) sequences used in the analysis are as follows: Homo sapiens, 1C3T_A (full length); Mus musculus, NP_001335156 (1-76AA); Xenopus tropicalis, NP_001005136 (1-74AA); Danio rerio, NP_957031 (1-74AA); Patinopecten yessoensis, XP_021343069 (1-72AA); Nematostella vectensis, XP_001625352 (1-74AA); Stegodyphus mimosarum, KFM61924 (1-72AA); Drosophila melanogaster, AAA28999 (full length); Caenorhabditis elegans, NP_499695 (1-76AA); Ciona intestinalis, XP_009859868 (1-74AA); Oryza sativa, XP_015629795 (1-76AA); Arabidopsis thaliana, NP_565836 (1-76AA); Tetrabaena socialis, PNH01953 (1-76AA); Chlorella sorokiniana, PRW56465 (1-76AA); Monoraphidium neglectum, XP_013899085 (1-76AA); Chlamydomonas reinhardtii, XP_001700313 (1-76AA); Pyricularia oryzae, XP_003711895 (1-76AA); Aspergillus nidulans, FAA00317 (1-76AA); Cryptococcus neoformans, XP_018996320 (1-76AA); Kockovaella imperatae, XP_021870639 (1-76AA); Yarrowia lipolytica, XP_505175 (1-76AA). An, Aspergillus nidulans; At, Arabidopsis thaliana; C, carboxyl-terminal; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Cn, Cryptococcus neoformans; Cr, Chlamydomonas reinhardtii; Cs, Chlorella sorokiniana; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Ki, Kockovaella imperatae; Mm, Mus musculus; Mn, Monoraphidium neglectum; N, amino-terminal; Nv, Nematostella vectensis; Os, Oryza sativa; Po, Pyricularia oryzae; Py, Patinopecten yessoensis; Sm, Stegodyphus mimosarum; Ts, Tetrabaena socialis; Ub, ubiquitin; UBL3, ubiquitin-like 3; Xt, Xenopus tropicalis; Yl, Yarrowia lipolytica.

Fig. 6

Tertiary structures of UBLs harboring the CAAX sequence in unicellular organisms UBLs with a Ub-fold and the unstructured carboxyl-terminal in unicellular algae (Chlorella sorokiniana, Monoraphidium neglectum, Chlamydomonas reinhardtii) and fungi (Cryptococcus neoformans, Kockovaella imperatae, Yarrowia lipolytica) are shown. Three-dimensional diagrams were drawn using SWISS-MODEL (https://swissmodel.expasy.org/) and the amino acid sequences obtained from the following GenBank^TM accession numbers: Chlorella sorokiniana, PRW20945; Monoraphidium neglectum, XP_013901616; Chlamydomonas reinhardtii, XP_001696591; Cryptococcus neoformans, XP_567533; Kockovaella imperatae, XP_021868326; Yarrowia lipolytica, AOW02568. C, carboxyl-terminal; Cn, Cryptococcus neoformans; Cr, Chlamydomonas reinhardtii; Cs, Chlorella sorokiniana; Ki, Kockovaella imperatae; Mn, Monoraphidium neglectum; N, amino-terminal; Ub, ubiquitin; UBL, ubiquitin-like protein; Yl, Yarrowia lipolytica.

Fig. 7

Isoleucine-centered hydrophobic patch conserved in UBL3 and Ub The isoleucine44-centered hydrophobic patch of HsUb corresponds to the isoleucine61-centered hydrophobic patch in HsUBL3, and the associated residues are also well conserved. These hydrophobic patches are the noncovalent binding sites for UBDs. Hydrophobic and basic residues are indicated in magenta and light blue, respectively. Three-dimensional diagrams of the Ub/UBLs were drawn using QueMol 2.0 (http://www.cuemol.org/ja/) and the following Protein Data Bank IDs (http://www.rcsb.org/): human UBL3, 2GOW; human Ub, 1C3T. arg, arginine; gly, glycine; his, histidine; Hs, Homo sapiens; ile, isoleucine; Ub, ubiquitin; UBD, ubiquitin binding domain; UBL3, ubiquitin-like 3; val, valine.

Fig. 8

The proposed UBL3 protein sorting model Post-translational modification of substrate proteins may occur before CAAX processing, although the precise timing of this process is unknown. UBL3 with a geranylgeranylated cysteine residue is anchored to the plasma membrane or MVBs. Substrate proteins are then sorted into intraluminal vesicles together with UBL3. The bonding structure between UBL3 and the substrate protein is unknown. MVBs, multivesicular bodies; UBL3, ubiquitin-like 3.

Fig. 9

Cancer progression-related sEV-proteins that interact with UBL3 Multistep processes of cancer progression, showing sEV-proteins that interact with UBL3. Arrows indicate sEV delivery. BMP, bone morphogenic proteins; EGFRvIII, epidermal growth factor receptor variant III; EMT, epithelial to mesenchymal transition; HSP90, heat shock protein 90; ITG, integrin; MMT, matrix metalloprotease; sEV, small extracellular vesicle; TGF-β, tumor growth factor-β; UBL3, ubiquitin-like 3.

Fig. 10

The prognostic significance of UBL3-mRNA expression on various malignant tumor types The relationship between UBL3-mRNA expression level and patient prognosis depends on the tumor type. High and low UBL3-mRNA expression groups are shown as red and black lines, respectively. Overall survival analysis for UBL3-mRNA expression was performed using datasets in The Cancer Genome Atlas research network (http://cancergenome.nih.gov). Kaplan-Meier plots, hazard ratio, 95% confidence intervals and log-rank P values were generated on Kaplan-Meier Plotter (https://kmplot.com/analysis/). Best cutoff values for discriminating good or poor prognosis groups were auto-selected. Tumor types with significant P values <0.05 are presented. Hazard ratio, HR; UBL3, ubiquitin-like 3.