Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that brief exposure of HEK293T cells to meta-arsenite increases the affinity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis, and to identify the susceptible proteome.

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Quantitative Proteomics Reveals the Defense Response of Wheat against Puccinia striiformis f. sp. tritici

Scientific Reports ◽

10.1038/srep34261 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 12

Author(s):

Yuheng Yang ◽

Yang Yu ◽

Chaowei Bi ◽

Zhensheng Kang

Keyword(s):

Quantitative Proteomics ◽

Rna Binding ◽

Rna Binding Proteins ◽

Puccinia Striiformis ◽

Post Inoculation ◽

Control Groups ◽

Stimulus Response ◽

Wheat Gene ◽

Related Proteins ◽

And Control

Abstract Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is considered one of the most aggressive diseases to wheat production. In this study, we used an iTRAQ-based approach for the quantitative proteomic comparison of the incompatible Pst race CYR23 in infected and non-infected leaves of the wheat cultivar Suwon11. A total of 3,475 unique proteins were identified from three key stages of interaction (12, 24, and 48 h post-inoculation) and control groups. Quantitative analysis showed that 530 proteins were differentially accumulated by Pst infection (fold changes >1.5, p < 0.05). Among these proteins, 10.54% was classified as involved in the immune system process and stimulus response. Intriguingly, bioinformatics analysis revealed that a set of reactive oxygen species metabolism-related proteins, peptidyl–prolyl cis–trans isomerases (PPIases), RNA-binding proteins (RBPs), and chaperonins was involved in the response to Pst infection. Our results were the first to show that PPIases, RBPs, and chaperonins participated in the regulation of the immune response in wheat and even in plants. This study aimed to provide novel routes to reveal wheat gene functionality and better understand the early events in wheat–Pst incompatible interactions.

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Quantitative Proteomics to Identify Nuclear RNA-Binding Proteins of Malat1

International Journal of Molecular Sciences ◽

10.3390/ijms21031166 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1166 ◽

Cited By ~ 2

Author(s):

Marian Scherer ◽

Michal Levin ◽

Falk Butter ◽

Marion Scheibe

Keyword(s):

Quantitative Proteomics ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Full Length ◽

Interacting Proteins ◽

Non Coding Rna ◽

Nuclear Rna ◽

Long Non Coding Rna ◽

Shed Light

The long non-coding RNA Malat1 has been implicated in several human cancers, while the mechanism of action is not completely understood. As RNAs in cells function together with RNA-binding proteins (RBPs), the composition of their RBP complex can shed light on their functionality. We here performed quantitative interactomics of 14 non-overlapping fragments covering the full length of Malat1 to identify possible nuclear interacting proteins. Overall, we identified 35 candidates including 14 already known binders, which are able to interact with Malat1 in the nucleus. Furthermore, the use of fragments along the full-length RNA allowed us to reveal two hotspots for protein binding, one in the 5′-region and one in the 3′-region of Malat1. Our results provide confirmation on previous RNA-protein interaction studies and suggest new candidates for functional investigations.

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Differential urine proteome analysis of a ventilator-induced lung injury rat model by label-free quantitative and parallel reaction monitoring proteomics

Scientific Reports ◽

10.1038/s41598-021-01007-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Weiwei Qin ◽

Xiao Zhang ◽

Lingnan Chen ◽

Qiujie Li ◽

Benwang Zhang ◽

...

Keyword(s):

Lung Injury ◽

Quantitative Proteomics ◽

Urinary Biomarkers ◽

Reaction Monitoring ◽

Label Free ◽

Parallel Reaction ◽

Ventilator Induced Lung Injury ◽

Parallel Reaction Monitoring ◽

Free Data ◽

Differential Proteins

AbstractUrine is a promising resource for biomarker research. Therefore, the purpose of this study was to investigate potential urinary biomarkers to monitor the disease activity of ventilator-induced lung injury (VILI). In the discovery phase, a label-free data-dependent acquisition (DDA) quantitative proteomics method was used to profile the urinary proteomes of VILI rats. For further validation, the differential proteins were verified by parallel reaction monitoring (PRM)-targeted quantitative proteomics. In total, 727 high-confidence proteins were identified with at least 1 unique peptide (FDR ≤ 1%). Compared to the control group, 110 proteins (65 upregulated, 45 downregulated) were significantly changed in the VILI group (1.5-fold change, P < 0.05). The canonical pathways and protein–protein interaction analyses revealed that the differentially expressed proteins were enriched in multiple functions, including oxidative stress and inflammatory responses. Finally, thirteen proteins were identified as candidate biomarkers for VILI by PRM validation. Among these PRM-validated proteins, AMPN, MEP1B, LYSC1, DPP4 and CYC were previously reported as lung-associated disease biomarkers. SLC31, MEP1A, S15A2, NHRF1, XPP2, GGT1, HEXA, and ATPB were newly discovered in this study. Our results suggest that the urinary proteome might reflect the pathophysiological changes associated with VILI. These differential proteins are potential urinary biomarkers for the activity of VILI.

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Development of parallel reaction monitoring (PRM)-based quantitative proteomics applied to HER2-Positive breast cancer

Oncotarget ◽

10.18632/oncotarget.26031 ◽

2018 ◽

Vol 9 (73) ◽

pp. 33762-33777 ◽

Cited By ~ 6

Author(s):

Mathilde Guerin ◽

Anthony Gonçalves ◽

Yves Toiron ◽

Emilie Baudelet ◽

Matthieu Pophillat ◽

...

Keyword(s):

Breast Cancer ◽

Quantitative Proteomics ◽

Reaction Monitoring ◽

Parallel Reaction ◽

Her2 Positive ◽

Her2 Positive Breast Cancer ◽

Parallel Reaction Monitoring ◽

Positive Breast Cancer

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Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates

10.1101/636407 ◽

2019 ◽

Author(s):

Hiroya Yamazaki ◽

Hidetaka Kosako ◽

Shige H. Yoshimura

Keyword(s):

Protein Phosphorylation ◽

Quantitative Proteomics ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Amino Acid Sequences ◽

Mass Spectrometry Analysis ◽

Order Structure ◽

Ontology Term ◽

Related Proteins

AbstractProtein phosphorylation plays a critical role in the regulation and progression of mitosis. More than 10,000 phosphorylated residues and the associated kinases have been identified to date via proteomic analyses. Although some of these phosphosites are associated with regulation of either protein-protein interactions or the catalytic activity of the substrate protein, the roles of most mitotic phosphosites remain unclear. In this study, we examined structural properties of mitotic phosphosites and neighboring residues to understand the role of heavy phosphorylation in non-structured domains. Quantitative mass spectrometry analysis of mitosis-arrested and non-arrested HeLa cells revealed >4,100 and >2,200 residues either significantly phosphorylated or dephosphorylated, respectively, at mitotic entry. The calculated disorder scores of amino acid sequences of neighboring individual phosphosites revealed that >70% of dephosphorylated phosphosites exist in disordered regions, whereas 50% of phosphorylated sites exist in non-structured domains. A clear inverse correlation was observed between probability of phosphorylation in non-structured domain and increment of phosphorylation in mitosis. These results indicate that at entry to mitosis, a significant number of phosphate groups are removed from non-structured domains and transferred to more-structured domains. Gene ontology term analysis revealed that mitosis-related proteins are heavily phosphorylated, whereas RNA-related proteins are both dephosphorylated and phosphorylated, suggesting that heavy phosphorylation/dephosphorylation in non-structured domains of RNA-binding proteins plays a role in dynamic rearrangement of RNA-containing organelles, as well as other intracellular environments.Significance StatementProgression of mitosis is tightly regulated by protein phosphorylation/dephosphorylation. Although proteomic studies have identified tens of thousands of phosphosites in mitotic cells, the roles of them remain to be answered. We approached this question from the viewpoint of the higher-order structure of phosphosites. Quantitative proteomics and bioinformatic analyses revealed that more than 70% of mitotic dephosphorylation events occurred in non-structured regions. Non-structured regions of cellular proteins are attracting considerable attention in terms of their involvement in dynamic rearrangements of intracellular membrane-less organelles and protein assembly/disassembly processes. Our results suggest the possibility that a vast amount of mitosis-associated dephosphorylation/phosphorylation at non-structured regions plays a role in regulating the dynamic assembly/disassembly of intracellular architectures and organelles such as chromosomes and nucleolus.

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The Differential Urine Proteome Analysis of a Ventilator-induced Lung Injury Rat Model by Label-free Quantitative and Parallel Reaction Monitoring Proteomics

10.21203/rs.3.rs-152794/v1 ◽

2021 ◽

Author(s):

Weiwei Qin ◽

Xiao Zhang ◽

Lingnan Chen ◽

Qiujie Li ◽

Benwang Zhang ◽

...

Keyword(s):

Lung Injury ◽

Quantitative Proteomics ◽

Urinary Biomarkers ◽

Reaction Monitoring ◽

Label Free ◽

Parallel Reaction ◽

Ventilator Induced Lung Injury ◽

Parallel Reaction Monitoring ◽

Free Data ◽

Differential Proteins

Abstract Background: Urine is a promising resource for biomarker research. Therefore, the purpose of this study was to investigate potential urinary biomarkers to monitor the disease activity of ventilator-induced lung injury (VILI). Methods: In the discovery phase, a label-free data-dependent acquisition (DDA) quantitative proteomics method was used to profile the urinary proteomes of VILI rats. For further validation, the differential proteins were verified by parallel reaction monitoring (PRM)-targeted quantitative proteomics.Results: In all, 727 high-confidence proteins were identified with at least 1 unique peptide (FDR ≤1%). Compared to the control group, 110 proteins (65 upregulated, 45 downregulated) were significantly changed in the VILI group (1.5-fold change, P<0.05). The canonical pathways and protein-protein interaction analyses revealed that the differentially expressed proteins were enriched in multiple functions, including oxidative stress and inflammatory responses. Finally, thirteen proteins were identified as candidate biomarkers for VILI by PRM validation. Among these PRM-validated proteins, AMPN, MEP1B, LYSC1, DPP4 and CYC were previously reported as lung-associated disease biomarkers. SLC31, MEP1A, S15A2, NHRF1, XPP2, GGT1, HEXA, and ATPB were newly discovered in this study. Conclusions: Our results suggest that the urinary proteome might reflect the pathophysiological changes associated with VILI. These differential proteins are potential urinary biomarkers for the activity of VILI.

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A working model for condensate RNA-binding proteins as matchmakers for protein complex assembly

RNA ◽

10.1261/rna.078995.121 ◽

2021 ◽

pp. rna.078995.121

Author(s):

Xiuzhen Chen ◽

Christine Mayr

Keyword(s):

Protein Complex ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Complex Assembly ◽

Protein Targets ◽

Working Model ◽

Cellular Environment ◽

Protein Complex Assembly

Most cellular processes are carried out by protein complexes, but it is still largely unknown how the subunits of lowly expressed complexes find each other in the crowded cellular environment. Here, we will describe a working model where RNA-binding proteins in cytoplasmic condensates act as matchmakers between their bound proteins (called protein targets) and newly translated proteins of their RNA targets to promote their assembly into complexes. Different RNA-binding proteins act as scaffolds for various cytoplasmic condensates with several of them supporting translation. mRNAs and proteins are recruited into the cytoplasmic condensates through binding to specific domains in the RNA-binding proteins. Scaffold RNA-binding proteins have a high valency. In our model, they use homotypic interactions to assemble condensates and they use heterotypic interactions to recruit protein targets into the condensates. We propose that unoccupied binding sites in the scaffold RNA-binding proteins transiently retain recruited and newly translated proteins in the condensates, thus promoting their assembly into complexes. Taken together, we propose that lowly expressed subunits of protein complexes combine information in their mRNAs and proteins to colocalize in the cytoplasm. The efficiency of protein complex assembly is increased by transient entrapment accomplished by multivalent RNA-binding proteins within cytoplasmic condensates.

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