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Proteome analysis of Arabidopsis Lon1 protease mutant

ABSTRACT: Intracellular selective proteolysis is an important protein quality control process for maintenance of protein homeostasis by removing either the defective polypeptides or deleterious protein aggregates. The ATP-dependent Lon protease is a key component of protein quality control and highly conserved across the kingdoms of living organisms. In Arabidopsis thaliana Lon1 (At5g26860) is dual targeted to mitochondria and chloroplasts. Lon1 mutant plants (lon1-1) exhibit a post-embryonic growth retardation phenotype accompanied with aberrant mitochondria morphology and reduced respiratory capacity. To identify the effect of loss of Lon1 proteolytic activity on the protein level of the plant, the proteome profile of lon1-1 mutant was analyzed by mass spectrometry and compared to the proteome of the wild type plants (Col-0) used as a reference. Total protein extraction was performed under denature conditions of 8 days old seedling, grown vertically on petri dishes at 22 o C in the dark from independent biological replicates. The protein extract was subjected to Ultra High Pressure nanoLC-MS/MS.

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ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Whole Body

SUBMITTER: Martina Samiotaki

LAB HEAD: Stamatis Rigas

PROVIDER: PXD055804 | Pride | 2025-06-10

REPOSITORIES: Pride

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Project description:Arabidopsis Lon1 serves dual roles as a mitochondrial protease and chaperone. Loss of Lon1 function reduced autophagy flux at transcriptional, protein and cellular levels. Meanwhile, changes occurred in the levels of cellular subunit proteins, especially a significant upregulation of mitochondrial proteins overall in lon1 mutants. Lon1 is a key enzyme in maintaining mitochondrial protein homeostasis. In lon1 mutants, other mitochondrial proteases including mitochondrial autophagy receptor proteins showed significant increases in abundance. Mitophagy is an important mechanism for cellular quality control. lon1-2atg5-1 double mutant were constructed to investigate how they cooperatively regulate mitochondrial protein homeostasis. Transcriptome sequencing results revealed that Lon1 inactivation led to widespread unfolded protein responses (UPRs) pathway activation in lon1-2atg5-1, consistent with lon1-2, while loss function of ATG5 inactivation did not affect mitochondrial homeostasis. The double mutant also exhibited independent plant phenotypes from single parents, yet more severe seed number and embryo development issues, suggesting an additive effect of lon1-2 and atg5-1. Protein Storage Vacuole (PSV) and oil body phenotypes also showed more severe developmental damage in the double mutants. Brassinosteroid (BR) acts as a key plant hormone controlling seed and embryo development. GO and KEGG analysis of genes simultaneously downregulated in lon1-2, atg5-1, and lon1-2atg5-1 revealed significant downregulation of genes involved in BR biosynthesis and its homeostasis, indicating that this may be one of the key factors leading to abnormal seed and embryo development in the mutants. In general, the loss function of Lon1 reduces autophagy flux and induces UPRs to regulate protein homeostasis. Meanwhile, the simultaneous absence of Lon1 and autophagy significantly down-regulates the expression of genes related to BR biosynthesis and homeostasis, thereby leading to pronounced blockage in the development of double mutant seeds and embryos.

Project description:We developed a human cerebral organoid model derived from induced pluripotent stem cells (iPSCs) with targeted genome editing to abolish protein expression of the Contactin Associated Protein-like 2 (CNTNAP2) autism spectrum disorder (ASD) risk gene, mimicking loss-of-function mutations seen in patients. CNTNAP2-/- cerebral organoids displayed accelerated cell cycle, ventricular zone disorganisation and increased cortical folding. Proteomic analysis revealed disruptions in glutamatergic/GABAergic synaptic pathways and neurodevelopment, highlighting increased protein expression of corticogenesis and neurodevelopment-related genes such as Forkhead box protein G1 (FOXG1) and Paired box 6 (PAX6). Transcriptomic analysis revealed differentially expressed genes (DEG) belonging to inhibitory neuron-related gene networks. Interestingly, there was a weak correlation between the transcriptomic and proteomic data, suggesting nuanced translational control mechanisms. Along these lines we find upregulated Protein Kinase B (Akt)/mechanistic target of rapamycin (mTOR) signalling in CNTNAP2-/- organoids. Spatial transcriptomics analysis of CNTNAP2-/- ventricular zones demonstrated pervasive changes in gene expression, particularly in PAX6- cells, implicating upregulation of cell cycle regulation pathways, synaptic and glutamatergic/GABAergic pathways. We noted a significant overlap of all omics datasets with idiopathic ASD (macrocephaly) iPSC-derived telencephalic organoids DEG, where FOXG1 was upregulated, along with aberrant expression of Glutamate decarboxylase 1 (GAD1) and T-Box Brain Transcription Factor 1 (TBR1), suggesting altered GABAergic/glutamatergic neuron development. These findings potentially highlight a shared mechanism in the early cortical development of various forms of ASD, further elucidate the role of CNTNAP2 in ASD pathophysiology and cortical development and pave the way for targeted therapies using cerebral organoids as preclinical models.

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Proteome analysis of Arabidopsis Lon1 protease mutant

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