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1. Profiling thimet oligopeptidase‐mediated proteolysis in Arabidopsis thaliana

   https://doi.org/10.1111/tpj.15165  

   Iannetta, Anthony A.; Rogers, Holden T.; Al‐Mohanna, Thualfeqar; O’Brien, Juliana N.; Wommack, Andrew J.; Popescu, Sorina C.; Hicks, Leslie M. (February 2021, The Plant Journal)

   SUMMARY Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown inArabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label‐free peptidomics via liquid chromatography‐tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col‐0 wild type andtop1top2knock‐out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated usingin vitroenzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non‐polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized. 

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2. Metal-induced oxidative stress and human plasma protein oxidation after SARS-CoV-2 infection

   https://doi.org/10.1038/s41598-023-29119-5  

   Aryal, Baikuntha; Tillotson, Joseph; Ok, Kiwon; Stoltzfus, Andrew T.; Michel, Sarah L. J.; Rao, V. Ashutosh (February 2023, Scientific Reports)

   Abstract Pathogenesis of COVID-19 by SARS-CoV-2 resulted in a global pandemic and public health emergency in 2020. Viral infection can induce oxidative stress through reactive oxygen species (ROS). Inflammation and environmental stress are major sources of oxidative stress after infection. Micronutrients such as iron, copper, zinc, and manganese play various roles in human tissues and their imbalance in blood can impact immune responses against pathogens including SARS CoV-2. We hypothesized that alteration of free metal ions during infection and metal-catalyzed oxidation plays a critical role towards pathogenesis after infection. We analyzed convalescent and hospitalized COVID-19 patient plasma using orthogonal analytical techniques to determine redox active metal concentrations, overall protein oxidation, oxidative modifications, and protein levels via proteomics to understand the consequences of metal-induced oxidative stress in COVID-19 plasma proteins. Metal analysis using ICP-MS showed significantly greater concentrations of copper in COVID-19 plasma compared to healthy controls. We demonstrate significantly greater total protein carbonylation, other oxidative modifications, and deamidation of plasma proteins in COVID-19 plasma compared to healthy controls. Proteomics analysis showed that levels of redox active proteins including hemoglobulin were elevated in COVID-19 plasma. Molecular modeling concurred with potential interactions between iron binding proteins and SARS CoV-2 surface proteins. Overall, increased levels of redox active metals and protein oxidation indicate that oxidative stress-induced protein oxidation in COVID-19 may be a consequence of the interactions of SARS-CoV-2 proteins with host cell metal binding proteins resulting in altered cellular homeostasis. 

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3. Proteome-wide 4-hydroxy-2-nonenal signature of oxidative stress in the marine invasive tunicate Botryllus schlosseri

   https://doi.org/10.1101/2024.07.19.604351  

   Kültz, Dietmar; Gardell, Alison M; DeTomaso, Anthony; Stoney, Greg; Rinkevich, Baruch; Qarri, Andy; Hamar, Jens (July 2024, bioRxiv)

   The colonial ascidianBoytryllus schlosseriis an invasive marine chordate that thrives under conditions of anthropogenic climate change. We show that theB. schlosseriexpressed proteome contains unusually high levels of proteins that are adducted with 4-hydroxy-2-nonenal (HNE). HNE represents a prominent posttranslational modification resulting from oxidative stress. Although numerous studies have assessed oxidative stress in marine organisms HNE protein modification has not previously been determined in any marine species. LC/MS proteomics was used to identify 1052 HNE adducted proteins inB. schlosserifield and laboratory populations. Adducted amino acid residues were ascertained for 1849 modified sites, of which 1195 had a maximum amino acid localization score. Most HNE modifications were at less reactive lysines (rather than more reactive cysteines). HNE prevelance on most sites was high. These observations suggest thatB. schlosseriexperiences and tolerates high intracellular reactive oxygen species levels, resulting in substantial lipid peroxidation. HNE adducted B. schlosseri proteins show enrichment in mitochondrial, proteostasis, and cytoskeletal functions. Based on these results we propose that redox signaling contributes to regulating energy metabolism, the blastogenic cycle, oxidative burst defenses, and cytoskeleton dynamics duringB. schlosseridevelopment and physiology. A DIA assay library was constructed to quantify HNE adduction at 72 sites across 60 proteins that represent a holistic network of functionally discernable oxidative stress bioindicators. We conclude that the vast amount of HNE protein adduction in this circumpolar tunicate is indicative of high oxidative stress tolerance contributing to its range expansion into diverse environments. 

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4. Detection and editing of the updated Arabidopsis plastid- and mitochondrial-encoded proteomes through PeptideAtlas

   https://doi.org/10.1093/plphys/kiad572  

   van_Wijk, Klaas_J; Bentolila, Stephane; Leppert, Tami; Sun, Qi; Sun, Zhi; Mendoza, Luis; Li, Margaret; Deutsch, Eric_W (October 2023, Plant Physiology)

   Abstract Arabidopsis (Arabidopsis thaliana) ecotype Col-0 has plastid and mitochondrial genomes encoding over 100 proteins. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of RNA editing at the protein level is largely unexplored due to the complexities of detection. Here, we assembled the nonredundant set of identifiers, their correct protein sequences, and 452 predicted nonsynonymous editing sites of which 56 are edited at lower frequency. We then determined accumulation of edited and/or unedited proteoforms by searching ∼259 million raw tandem MS spectra from ProteomeXchange, which is part of PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). We identified all mitochondrial proteins and all except 3 plastid-encoded proteins (NdhG/Ndh6, PsbM, and Rps16), but no proteins predicted from the 4 ORFs were identified. We suggest that Rps16 and 3 of the ORFs are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical detection challenges. We detected 167 predicted edit sites at the proteome level. Minor frequency sites were edited at low frequency at the protein level except for cytochrome C biogenesis 382 at residue 124 (Ccb382-124). Major frequency sites (>50% editing of RNA) only accumulated in edited form (>98% to 100% edited) at the protein level, with the exception of Rpl5-22. We conclude that RNA editing for major editing sites is required for stable protein accumulation. 

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5. Abscisic acid‐controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gβ protein

   https://doi.org/10.1111/nph.18348  

   Smythers, Amanda L.; Bhatnagar, Nikita; Ha, Chien; Majumdar, Parinita; McConnell, Evan W.; Mohanasundaram, Boominathan; Hicks, Leslie M.; Pandey, Sona (July 2022, New Phytologist)

   Summary The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G‐proteins are key mediators of ABA responses. Both ABA and G‐proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G‐protein signaling remains uncharacterized.To probe the role of reversible protein oxidation in plant stress response and its dependence on G‐proteins, we determined the ABA‐dependent reversible redoxome of wild‐type and Gβ‐protein null mutantagb1of Arabidopsis.We quantified 6891 uniquely oxidized cysteine‐containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G‐proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA‐ and G‐protein‐dependent redox changes, many of which occurred on proteins not previously linked to them.We report the most comprehensive ABA‐dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G‐proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G‐proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses. 

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