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Article ; Online: A Novel Method to Isolate RNase MRP Using RNA Streptavidin Aptamer Tags.

Charteau, Violette / Derksen, Merel / Pruijn, Ger J M

2023 Volume 13, Issue 4

Abstract: Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for ... ...

Abstract	Interactions between RNA-binding proteins and RNA molecules are at the center of multiple biological processes. Therefore, accurate characterization of the composition of ribonucleoprotein complexes (RNPs) is crucial. Ribonuclease (RNase) for mitochondrial RNA processing (MRP) and RNase P are highly similar RNPs that play distinct roles at the cellular level; as a consequence, the specific isolation of either of these complexes is essential to study their biochemical function. Since their protein components are nearly identical, purification of these endoribonucleases using protein-centric methods is not feasible. Here, we describe a procedure employing an optimized high-affinity streptavidin-binding RNA aptamer, termed S1m, to purify RNase MRP free of RNase P. This report details all steps from the RNA tagging to the characterization of the purified material. We show that using the S1m tag allows efficient isolation of active RNase MRP.
Language	English
Publishing date	2023-02-20
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325 ; 2331-8325
ISSN (online)	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.4615
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Article ; Online: Citrulline is not a major determinant of autoantibody reactivity to neutrophil extracellular traps.

de Bont, Cynthia / Pruijn, Ger J M

Philosophical transactions of the Royal Society of London. Series B, Biological sciences

2023 Volume 378, Issue 1890, Page(s) 20220249

Abstract: One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays ... ...

Abstract	One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays an important role during NET formation and a substantial fraction of NET-associated proteins appeared to be citrullinated. The release of citrullinated intracellular proteins from netting neutrophils led to the hypothesis that the production of anti-citrullinated protein autoantibodies by autoimmune patients, in particular patients with rheumatoid arthritis, might be initiated when citrullinated NET components are not properly cleared and are exposed to the immune system. Here, we discuss the processes that lead to NET formation, including the role of peptidylarginine deiminase activation and our current knowledge on citrullinated NET-associated proteins. Citrulline-dependent epitopes do not appear to play a major role in the recognition of NETs by autoantibodies from rheumatoid arthritis and systemic lupus erythematosus patients, even though anti-NET autoantibodies are frequently observed in sera from these patients. The neutrophil proteases associated with NETs have a major impact on the integrity of NET-associated proteins when NET formation is induced by activating isolated human neutrophils. Cleavage/degradation of these proteins also resulted in a strong reduction of the reactivity with autoantibodies. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.
MeSH term(s)	Humans ; Arthritis, Rheumatoid/metabolism ; Autoantibodies/metabolism ; Citrulline/metabolism ; Extracellular Traps/metabolism ; Neutrophils
Chemical Substances	Autoantibodies ; Citrulline (29VT07BGDA)
Language	English
Publishing date	2023-10-02
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	208382-6
ISSN	1471-2970 ; 0080-4622 ; 0264-3839 ; 0962-8436
ISSN (online)	1471-2970
ISSN	0080-4622 ; 0264-3839 ; 0962-8436
DOI	10.1098/rstb.2022.0249
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Article ; Online: Circulating tRNA-derived fragments are decreased in patients with rheumatoid arthritis and increased in patients with psoriatic arthritis.

Dunaeva, Marina / Blom, Jan / Thurlings, Rogier / van Weijsten, Margot / van de Loo, Fons A J / Pruijn, Ger J M

Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals

2024 Volume 29, Issue 2, Page(s) 90–99

Abstract: Introduction: tRNA-derived fragments (tRFs) play an important role in immune responses. To clarify the role of tRFs in autoimmunity we studied circulating tRF-levels in patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA), and in a ... ...

Abstract	Introduction: tRNA-derived fragments (tRFs) play an important role in immune responses. To clarify the role of tRFs in autoimmunity we studied circulating tRF-levels in patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA), and in a murine model for arthritis. Material and methods: Circulating tRF-levels were quantified by miR-Q RT-qPCR. tRNA processing and modification enzyme expression was analysed by RT-qPCR and public transcriptomics data. Results: Significant reduction (up to 3-fold on average) of tRF-levels derived from tRNA-Gly-GCC,CCC, tRNA-Glu-CTC and tRNA-Val-CAC,AAC was observed in RA patients, whereas tRNA-Glu-CTC and tRNA-Val-CAC,AAC tRFs were found at significantly higher levels (up to 3-fold on average) in PsA patients, compared to healthy controls. Also in arthritic IL1Ra-KO mice reduced levels of tRNA-Glu-CTC fragments were seen. The expression of NSUN2, a methyltransferase catalysing tRNA methylation, was increased in RA-peripheral blood mononuclear cells (PBMCs) compared to PsA, but this is not consistently supported by public transcriptomics data. Discussion: The observed changes of specific tRF-levels may be involved in the immune responses in RA and PsA and may be applicable as new biomarkers. Conclusion: Circulating tRF-levels are decreased in RA and increased in PsA and this may, at least in part, be mediated by methylation changes.
MeSH term(s)	Humans ; Animals ; Mice ; Arthritis, Psoriatic/genetics ; Leukocytes, Mononuclear/metabolism ; Arthritis, Rheumatoid ; RNA, Transfer/genetics ; Biomarkers/metabolism
Chemical Substances	RNA, Transfer (9014-25-9) ; Biomarkers
Language	English
Publishing date	2024-02-27
Publishing country	England
Document type	Journal Article
ZDB-ID	1324372-x
ISSN	1366-5804 ; 1354-750X
ISSN (online)	1366-5804
ISSN	1354-750X
DOI	10.1080/1354750X.2024.2319297
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Article ; Online: Global Characterization of Circulating Nucleic Acids.

Dunaeva, Marina / Pruijn, Ger J M

Methods in molecular biology (Clifton, N.J.)

2019 Volume 2063, Page(s) 257–268

Abstract: Circulating nucleic acids (CNAs) include genomic and mitochondrial DNA fragments, small RNAs, and bacterial and viral DNA/RNA. Different mechanisms such as cell apoptosis, necrosis, and active CNA release from cells have been proposed to result in ... ...

Abstract	Circulating nucleic acids (CNAs) include genomic and mitochondrial DNA fragments, small RNAs, and bacterial and viral DNA/RNA. Different mechanisms such as cell apoptosis, necrosis, and active CNA release from cells have been proposed to result in nucleic acids in the circulation. Application of next generation sequencing technology demonstrated that CNAs contain specific mutations, indels, microsatellite alterations, and epigenetic changes (DNA methylation) associated with various diseases. Their clinical implications have been demonstrated for diseases such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. Thus, CNAs in blood represent an attractive family of molecules that can serve as biomarkers and the analysis of CNAs can be alternative for immunohistochemical analyses of conventional biopsies. The methods described in this chapter provides details for circulating DNA and small RNA isolation, CNA(-derived cDNA) library preparation, and sequencing data analysis.
MeSH term(s)	Base Sequence ; Cell-Free Nucleic Acids/genetics ; Cell-Free Nucleic Acids/isolation & purification ; Computational Biology/methods ; DNA Methylation/genetics ; DNA, Mitochondrial/blood ; DNA, Mitochondrial/genetics ; DNA, Neoplasm/blood ; DNA, Neoplasm/genetics ; Female ; Genetic Markers/genetics ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Pregnancy ; RNA, Small Untranslated/blood ; RNA, Small Untranslated/genetics ; RNA, Small Untranslated/isolation & purification ; Sequence Alignment
Chemical Substances	Cell-Free Nucleic Acids ; DNA, Mitochondrial ; DNA, Neoplasm ; Genetic Markers ; RNA, Small Untranslated
Language	English
Publishing date	2019-10-30
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-0138-9_18
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Article ; Online: IL-6 and TNF are Potential Inflammatory Biomarkers in Facioscapulohumeral Muscular Dystrophy.

Greco, Anna / Mul, Karlien / Jaeger, Martin H / Dos Santos, Jéssica C / Koenen, Hans / de Jong, Leon / Mann, Ritse / Fütterer, Jurgen / Netea, Mihai G / Pruijn, Ger J M / van Engelen, Baziel G M / Joosten, Leo A B

Journal of neuromuscular diseases

2024 Volume 11, Issue 2, Page(s) 327–347

Abstract: Background: FSHD is a highly prevalent inherited myopathy with a still poorly understood pathology.: Objective: To investigate whether proinflammatory cytokines are associated with FSHD and which specific innate immune cells are involved in its ... ...

Abstract	Background: FSHD is a highly prevalent inherited myopathy with a still poorly understood pathology. Objective: To investigate whether proinflammatory cytokines are associated with FSHD and which specific innate immune cells are involved in its pathology. Methods: First, we measured circulating cytokines in serum samples: IL-6 (FSHD, n = 150; HC, n = 98); TNF (FSHD, n = 150; HC, n = 59); IL-1α (FSHD, n = 150; HC, n = 66); IL-1β (FSHD, n = 150; HC, n = 98); MCP-1 (FSHD, n = 14; HC, n = 14); VEGF-A (FSHD, n = 14; HC, n = 14). Second, we tested trained immunity in monocytes (FSHD, n = 15; HC, n = 15) and NK cells (FSHD, n = 11; HC, n = 11). Next, we explored the cytokine production capacity of NK cells in response to different stimuli (FSHD, n = 39; HC, n = 22). Lastly, we evaluated the cytokine production of ex vivo stimulated MRI guided inflamed (TIRM+) and paired MRI guided non inflamed (TIRM-) muscle biopsies of 21 patients and of 8 HC muscle biopsies. Results: We included a total of 190 FSHD patients (N = 190, 48±14 years, 49% men) and of 135 HC (N = 135, 44±15 years, 47% men). We found that FSHD patients had higher concentrations of IL-6 and TNF measured (a) in the circulation, (b) after ex-vivo stimulation of NK cells, and (c) in muscle specimens. Besides, IL-6 circulating concentrations, as well as its production by NK cells and IL-6 content of FSHD muscle specimens, showed a mild correlation with disease duration, disease severity, and muscle weakness. Conclusion: These results show that IL-6 and TNF may contribute to FSHD pathology and suggest novel therapeutic targets. Additionally, the activation of NK cells in FSHD may be a novel pathway contributing to FSHD pathology.
MeSH term(s)	Female ; Humans ; Male ; Biomarkers ; Biopsy ; Interleukin-6 ; Muscle Weakness ; Muscular Dystrophy, Facioscapulohumeral/pathology
Chemical Substances	Biomarkers ; Interleukin-6 ; IL6 protein, human
Language	English
Publishing date	2024-01-04
Publishing country	Netherlands
Document type	Journal Article
ISSN	2214-3602
ISSN (online)	2214-3602
DOI	10.3233/JND-230063
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Article: Citrullination and carbamylation in the pathophysiology of rheumatoid arthritis.

Pruijn, Ger J M

Frontiers in immunology

2015 Volume 6, Page(s) 192

Abstract: The discovery that citrullination was crucial for the recognition of antigens by the most disease-specific class of autoantibodies in rheumatoid arthritis (RA) had a huge impact on studies aimed at understanding autoimmunity in this disease. In addition ... ...

Abstract	The discovery that citrullination was crucial for the recognition of antigens by the most disease-specific class of autoantibodies in rheumatoid arthritis (RA) had a huge impact on studies aimed at understanding autoimmunity in this disease. In addition to the detailed characterization of anti-citrullinated protein antibodies, various studies have addressed the identity of citrullinated antigens. These investigations were facilitated by new methods to characterize these proteins, the analysis of protein citrullination by peptidylarginine deiminases, the generation of a catalog of citrullinated proteins present in the inflamed joints of patients and the finding that the formation of extracellular traps is dependent on the activity of peptidylarginine deiminase activity. Recently, it was found that in addition to citrullination also carbamylation, which results in chemically highly related modified proteins, yields antigens that are targeted by rheumatoid arthritis patient sera. Here, all of these aspects will be discussed, culminating in current ideas about the involvement of citrullination and carbamylation in pathophysiological processes in autoimmunity, especially RA.
Language	English
Publishing date	2015
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2606827-8
ISSN	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2015.00192
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Article ; Online: Low molecular weight silicones induce cell death in cultured cells.

Onnekink, Carla / Kappel, Rita M / Boelens, Wilbert C / Pruijn, Ger J M

Scientific reports

2020 Volume 10, Issue 1, Page(s) 9558

Abstract: Women with silicone gel-filled breast implants are exposed to organosilicon compounds, in particular methylsiloxanes, as a result of 'gel bleed' and implant rupture. Although these silicones were originally considered to be inert, increasing evidence ... ...

Abstract	Women with silicone gel-filled breast implants are exposed to organosilicon compounds, in particular methylsiloxanes, as a result of 'gel bleed' and implant rupture. Although these silicones were originally considered to be inert, increasing evidence indicates that they can cause serious health problems. Here, we have analyzed the effects of microdroplets of the methylcyclosiloxanes, in particular D4, on the viability of cultured human cells. The exposure of Jurkat suspension and HeLa monolayer cells to D4 resulted in morphological changes of the cells. The analysis of molecular markers for apoptotic and necrotic processes not only demonstrated that caspases were activated and DNA was fragmented in Jurkat cells exposed to D4, but that also the permeability of the plasma membrane was altered. The induction of apoptotic pathways by D4 was substantiated by the inhibition of caspase activation in cells overexpressing Bcl-2. Cleavage of the caspase-3 substrate U1-70K appeared to be dependent on the D4 content and the efficiency of cleavage decreased with increasing size of the methylcyclosiloxanes (D4, D5 and D6). In addition to Jurkat cells, D4-induced U1-70K cleavage was also observed in HeLa cells, but not in HEp-2 cells. Taken together, these results indicate that D4 and, to a lesser extent, D5 can activate cell-death-related pathways in a cell type-specific fashion and suggest that this phenomenon may contribute to the development of Breast Implant Illness.
MeSH term(s)	Apoptosis/drug effects ; Breast/drug effects ; Breast/metabolism ; Breast Implants/adverse effects ; Caspases/metabolism ; Cell Death/drug effects ; Cell Line, Tumor ; Female ; HeLa Cells ; Humans ; Jurkat Cells ; Molecular Weight ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Signal Transduction/drug effects ; Silicones/adverse effects
Chemical Substances	Proto-Oncogene Proteins c-bcl-2 ; Silicones ; Caspases (EC 3.4.22.-)
Language	English
Publishing date	2020-06-12
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-020-66666-7
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Article ; Online: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Korchynskyi, Olexandr / Yoshida, Ken / Korchynska, Nataliia / Czarnik-Kwaśniak, Justyna / Tak, Paul P / Pruijn, Ger J M / Isozaki, Takeo / Ruth, Jeffrey H / Campbell, Phillip L / Amin, M Asif / Koch, Alisa E

International journal of molecular sciences

2023 Volume 24, Issue 3

Abstract: Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid ... ...

Abstract	Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.
MeSH term(s)	Animals ; Humans ; Chemokine CCL2/metabolism ; Glycosylation ; Endothelial Cells/metabolism ; Arthritis, Rheumatoid/metabolism ; Proteins/metabolism ; Mammals/metabolism ; Citrulline/metabolism
Chemical Substances	Chemokine CCL2 ; Proteins ; Citrulline (29VT07BGDA) ; CCL2 protein, human
Language	English
Publishing date	2023-01-18
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms24031862
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Article ; Online: A novel experimental approach for the selective isolation and characterization of human RNase MRP

Derksen, Merel / Mertens, Vicky / Visser, Eline A. / Arts, Janine / Vree Egberts, Wilma / Pruijn, Ger J. M.

RNA Biology. 2022 Dec. 31, v. 19, no. 1 p.305-312

2022

Abstract: RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by ... ...

Abstract	RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.
Keywords	RNA ; cartilage ; catalytic activity ; humans ; mutants ; oligonucleotides ; protein subunits ; ribonucleases ; ribonucleoproteins ; single nucleotide polymorphism ; RNase MRP ; RNase P ; ribonucleoprotein ; RNA aptamer
Language	English
Dates of publication	2022-1231
Size	p. 305-312.
Publishing place	Taylor & Francis
Document type	Article ; Online
ZDB-ID	2159587-2
ISSN	1555-8584
ISSN	1555-8584
DOI	10.1080/15476286.2022.2027659
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Article ; Online: LINE-1 Hypermethylation in Serum Cell-Free DNA of Relapsing Remitting Multiple Sclerosis Patients.

Dunaeva, Marina / Derksen, Merel / Pruijn, Ger J M

Molecular neurobiology

2017 Volume 55, Issue 6, Page(s) 4681–4688

Abstract: Concentrations of cell-free DNA (cfDNA) circulating in blood and its epigenetic variation, such as DNA methylation, may provide useful diagnostic or prognostic information. Long interspersed nuclear element-1 (LINE-1) constitutes approximately 20% of the ...

Abstract	Concentrations of cell-free DNA (cfDNA) circulating in blood and its epigenetic variation, such as DNA methylation, may provide useful diagnostic or prognostic information. Long interspersed nuclear element-1 (LINE-1) constitutes approximately 20% of the human genome and its 5'UTR region is CpG rich. Due to its wide distribution, the methylation level of the 5'UTR of LINE-1 can serve as a surrogate marker of global genomic DNA methylation. The aim of the current study was to investigate whether the methylation status of LINE-1 elements in serum cell-free DNA differs between relapsing remitting multiple sclerosis (RRMS) patients and healthy control subjects (CTR). Serum DNA samples of 6 patients and 6 controls were subjected to bisulfite sequencing. The results showed that the methylation level varies among distinct CpG sites in the 5'UTR of LINE-1 repeats and revealed differences in the methylation state of specific sites in this element between patients and controls. The latter differences were largely due to CpG sites in the L1PA2 subfamily, which were more frequently methylated in the RRMS patients than in the CTR group, whereas such differences were not observed in the L1HS subfamily. These data were verified by quantitative PCR using material from 18 patients and 18 control subjects. The results confirmed that the methylation level of a subset of the CpG sites within the LINE-1 promoter is elevated in DNA from RRMS patients in comparison with CTR. The present data suggest that the methylation status of CpG sites of LINE repeats could be a basis for development of diagnostic or prognostic tests.
MeSH term(s)	Base Sequence ; Case-Control Studies ; Cell-Free Nucleic Acids/blood ; CpG Islands/genetics ; DNA Methylation/genetics ; Female ; Humans ; Long Interspersed Nucleotide Elements/genetics ; Male ; Middle Aged ; Multiple Sclerosis, Relapsing-Remitting/blood ; Multiple Sclerosis, Relapsing-Remitting/genetics ; Promoter Regions, Genetic
Chemical Substances	Cell-Free Nucleic Acids
Language	English
Publishing date	2017-07-13
Publishing country	United States
Document type	Journal Article
ZDB-ID	645020-9
ISSN	1559-1182 ; 0893-7648
ISSN (online)	1559-1182
ISSN	0893-7648
DOI	10.1007/s12035-017-0679-z
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