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Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in Sulfolobus islandicus REY15A.

ABSTRACT: DNA damage response (DDR) in eukaryotes is largely regulated by protein phosphorylation. In archaea, many proteins are phosphorylated, however, it is unclear how the cells respond to DNA damage through global protein phosphorylation. We previously found that ?rio1, a Rio1 kinase homolog deletion strain of Sulfolobus islandicus REY15A, was sensitive to UV irradiation. In this study, we showed that ?rio1 grew faster than the wild type. Quantitative phosphoproteomic analysis of the wild type and ?rio1, untreated and irradiated with UV irradiation, revealed 562 phosphorylated sites (with a Ser/Thr/Tyr ratio of 65.3%/23.8%/10.9%) of 333 proteins in total. The phosphorylation levels of 35 sites of 30 proteins changed with >1.3-fold in the wild type strain upon UV irradiation. Interestingly, more than half of the UV-induced changes in the wild type did not occur in the ?rio1 strain, which were mainly associated with proteins synthesis and turnover. In addition, a protein kinase and several transcriptional regulators were differentially phosphorylated after UV treatment, and some of the changes were dependent on Rio1. Finally, many proteins involved in various cellular metabolisms exhibited Riol-related and UV-independent phosphorylation changes. Our results suggest that Rio1 is involved in the regulation of protein recycling and signal transduction in response to UV irradiation, and plays regulatory roles in multiple cellular processes in S. islandicus.

SUBMITTER: Huang Q

PROVIDER: S-EPMC7744417 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in Sulfolobus islandicus REY15A.

Huang Qihong Q Lin Zijia Z Wu Pengju P Ni Jinfeng J Shen Yulong Y

Frontiers in microbiology 20201203

DNA damage response (DDR) in eukaryotes is largely regulated by protein phosphorylation. In archaea, many proteins are phosphorylated, however, it is unclear how the cells respond to DNA damage through global protein phosphorylation. We previously found that Δrio1, a Rio1 kinase homolog deletion strain of Sulfolobus islandicus REY15A, was sensitive to UV irradiation. In this study, we showed that Δrio1 grew faster than the wild type. Quantitative phosphoproteomic analysis of ...[more]

PMID: 33343525

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Project description:Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the ΔslaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which the cell cycle is tightly regulated. Together, these data suggest that the lack of SlaA results in either cell fusion or irregularities in cell division. Our studies show the key physiological and cellular functions of the S-layer in this archaeal cell.IMPORTANCE The S-layer is considered to be the sole component of the cell wall in Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

Project description:We investigated the interaction between Sulfolobus spindle-shaped virus (SSV9) and its native archaeal host Sulfolobus islandicus. We show that upon exposure to SSV9, S. islandicus strain RJW002 has a significant growth delay where the majority of cells are dormant (viable but not growing) for 24 to 48 hours postinfection (hpi) compared to the growth of controls without virus. We demonstrate that in this system, dormancy (i) is induced by both active and inactive virus particles at a low multiplicity of infection (MOI), (ii) is reversible in strains with active CRISPR-Cas immunity that prevents the establishment of productive infections, and (iii) results in dramatic and rapid host death if virus persists in the culture even at low levels. Our results add a new dimension to evolutionary models of virus-host interactions, showing that the mere presence of a virus induces host cell stasis and death independent of infection. This novel, highly sensitive, and risky bet-hedging antiviral response must be integrated into models of virus-host interactions in this system so that the true ecological impact of viruses can be predicted and understood. Viruses of microbes play key roles in microbial ecology; however, our understanding of viral impact on host physiology is based on a few model bacteria that represent a small fraction of the life history strategies employed by hosts or viruses across the three domains that encompass the microbial world. We have demonstrated that rare and even inactive viruses induce dormancy in the model archaeon S. islandicus. Similar virus-induced dormancy strategies in other microbial systems may help to explain several confounding observations in other systems, including the surprising abundance of dormant cell types found in many microbial environments, the difficulty of culturing microorganisms in the laboratory, and the paradoxical virus-to-host abundances that do not match model predictions. A more accurate grasp of virus-host interactions will expand our understanding of the impact of viruses in microbial ecology.

Project description:Frontotemporal lobar degeneration (FTLD) is a progressive neurodegenerative disease characterized by behavioral abnormalities, personality changes, language dysfunction, and can co-occur with the development of motor neuron disease. One major pathological form of FTLD is characterized by intracellular deposition of ubiquitinated and phosphorylated TAR DNA binding protein-43 (TDP-43), suggesting that dysregulation in phosphorylation events may contribute to disease progression. However, to date systematic analysis of the phosphoproteome in FTLD brains has not been reported. In this study, we employed immobilized metal affinity chromatography (IMAC) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify phosphopeptides from FTLD and age-matched control post-mortem human brain tissue. Using this approach, we identified 786 phosphopeptides in frontal cortex (control and FTLD), in which the population of phosphopeptides represented approximately 50% of the total peptides analyzed. Label-free quantification using spectral counts revealed six proteins with significant changes in the FTLD phosphoproteome. N-myc-Downstream regulated gene 2 (NDRG2) and glial fibrillary acidic protein (GFAP) had an increased number of phosphospectra in FTLD, whereas microtubule associated protein 1A (MAP1A), reticulon 4 (RTN4; also referred to as neurite outgrowth inhibitor (Nogo)), protein kinase C gamma (PRKCG), and heat shock protein 90 kDa alpha, class A member 1(HSP90AA1) had significantly fewer phosphospectra compared to control brain. To validate these differences, we examined NDRG2 phosphorylation in FTLD brain by immunoblot analyses, and using a phosphoserine-13 (pSer13) GFAP monoclonal antibody we show an increase in pSer13 GFAP levels by immunoblot concomitant with increased overall GFAP levels in FTLD cases. These data highlight the utility of combining proteomic and phosphoproteomic strategies to characterize post-mortem human brain tissue.

Dataset Information

Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in Sulfolobus islandicus REY15A.

Publications

Phosphoproteomic Analysis Reveals Rio1-Related Protein Phosphorylation Changes in Response to UV Irradiation in <i>Sulfolobus islandicus</i> REY15A.

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