Post-translational modifications: emerging directors of cell-fate decisions during endoplasmic reticulum stress in Arabidopsis thaliana

Abstract

Homeostasis of the endoplasmic reticulum (ER) is critical for growth, development, and stress responses. Perturbations causing an imbalance in ER proteostasis lead to a potentially lethal condition known as ER stress. In ER stress situations, cell-fate decisions either activate pro-life pathways that reestablish homeostasis or initiate pro-death pathways to prevent further damage to the organism. Understanding the mechanisms underpinning cell-fate decisions in ER stress is critical for crop development and has the potential to enable translation of conserved components to ER stress-related diseases in metazoans. Post-translational modifications (PTMs) of proteins are emerging as key players in cell-fate decisions in situations of imbalanced ER proteostasis. In this review, we address PTMs orchestrating cell-fate decisions in ER stress in plants and provide evidence-based perspectives for where future studies may focus to identify additional PTMs involved in ER stress management.

Keywords: Arabidopsis thaliana; ER stress; UPR; cell-fate; post translational modification; programmed cell death.

Figure 1.. Proteoforms produce extensive functional diversity.

Gene expression encapsulates the progression from DNA to RNA to Protein and co-ordinately expands functional diversity. Any one gene can encode multiple mRNAs with alternative splicing and produce varying levels of each transcript. Each of those unique transcripts can then be translated, at varying levels, into a protein which can undergo extensive modifications to yield proteoforms that perform different functions within the cell. Created with BioRender.com.

Figure 2.. Technological advancements in molecular biology facilitate the capture of functional diversity within the cell.

Sanger sequencing, developed in the 1970s, allowed researchers to sequence genes and build genomes to obtain some of the first genetic material. Illumina and Oxford Nanopore sequencing facilitate high-throughput DNA and RNA sequencing, expanding the detectable molecular diversity. Current, cutting-edge research on single protein sequencing will provide the most comprehensive understanding of functional diversity in the cell allowing the identification and quantification of proteoforms. Created with BioRender.com.

Figure 3.. Post-translational modifications influence ER stress-induced cell-fate decisions.

Proteolysis, phosphorylation, and ubiquitination and ubiquitin-like (UBL) modifications are critical orchestrators of cell-fate decisions in ER stress conditions. Proteolysis can be digestive, via the 26S proteasomal degradation of proteins into their constituent amino acids or limited which is mediated by a protease to yield gain-of- or switch-of function proteoforms. Phosphorylation is the addition of a phosphate from a donor such as ATP or GTP (represented as XTP) via a kinase to a target protein, which can be removed by the enzymatic activity of phosphatases. Ubiquitin and UBL modifications, including SUMOylation and UFMylation, utilize similar mechanisms for the modification of peptides. In an ATP dependent manner, the modifier is loaded onto an E1 activating protein, transferred to an E2 conjugating protein, and then through an E3 ligase, selects the target protein and transfers the modification. All three modifications are reversible through the action of a protease, and ubiquitination has an E4 chain elongation protein that can build long chains of ubiquitin onto the target protein. Created with BioRender.com.

Figure 4.. PTM of transcription factors regulate life or death pathways in ER stress.

A diagram of a plant cell where red arrows depict molecular occurrences in response to ER stress. In conditions of ER stress, proteolytic cleavage releases the TFs bZIP28, BAG7, NTL6, NTL7, and NTL14 from their membrane bound state for transport into the nucleus. BAG7 also undergoes SUMOylation for its activation. NTL6, NTL7, and BAG7 all promote pro-life gene expression, while NTL14 promotes pro-death gene expression. COP1, a ubiquitin E3 ligase, also translocates to the nucleus in response to ER stress where it ubiquitinates HY5 for degradation, removing its suppression of pro-life genes in ER stress for bZIP28 to bind and drive their expression. Additionally, PIR1, also a ubiquitin E3 ligase, in unresolved ER stress indirectly drives UPS degradation of ABI5, a TF that drives pro-life gene expression. Created with BioRender.com.

Figure 5.. PTMs at the ER membrane regulate cytoplasmic responses that direct cell-fate in ER stress.

A diagram of a plant cell where red arrows depict molecular occurrences in response to ER stress. (A) IRE1, an ancestral and major branch of the UPR that orchestrates many downstream pathways in response to ER stress, is activated via autophosphorylation. (B) ERAD, also a pro-life mechanism in ER stress, relies on ubiquitination to regulate its activity and target its cargo (misfolded proteins) for proteasomal degradation. Auto- and trans-ubiquitination subdue ERAD activity in physiological conditions, but in response to ER stress, this ubiquitination is suppressed to increase ERAD throughput. (C) ER-phagy, a pro-life mechanism in ER stress, utilizes UFMylation and its machinery. Created with BioRender.com.

Figure 6.. Conservation across eukaryotes of modifiable residues.

(A) Alignment with Clustal Omega [177,178] of mouse and A. thaliana IRE1 amino acid sequences. Murine IRE1, S724 is conserved in A. thaliana IRE1a and IRE1c. A. thaliana IRE1b has an alanine at the homologous position but a Serine in the adjacent position. (B) AlphaFold predicted structures [179–181] and alignment with Clustal Omega [177,178] of human, C. reinhardtii, and A. thaliana BiP amino acid sequences. Highlighted is the AMPylation site of the metazoan BiP, the phosphorylation site in C. reinhardtii, and the phosphorylation site in A. thaliana BiP1 and BiP2. Red arrows on the AlphaFold structures denote the modified residue. A. thaliana BiP1 and BiP2 sequences are highly homologous so only BiP1 AlphaFold structure is shown. Created with BioRender.com.