Science & Insights

@article{Hoffmann2024,

title = {Discovery of Carbonic Anhydrase 9 as a Novel CLEC2 Ligand in a Cellular Interactome Screen},

author = {Sebastian Hoffmann and Benedict-Tilman Berger and Liane Rosalie Lucas and Felix Schiele and John Edward Park},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.3390/cells13242083},

issn = {2073-4409},

year  = {2024},

date = {2024-12-00},

urldate = {2024-12-00},

journal = {Cells},

volume = {13},

number = {24},

publisher = {MDPI AG},

abstract = {Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin’s lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.},

keywords = {CLEC2},

pubstate = {published},

tppubtype = {article}

}

@article{Lant2024,

title = {Poxin-deficient poxviruses are sensed by cGAS prior to genome replication},

author = {Sian Lant and Alasdair J. M. Hood and Joe A. Holley and Ailish Ellis and Lucy Eke and Rebecca P. Sumner and David O. Ulaeto and Carlos Maluquer de Motes},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1099/jgv.0.002036},

issn = {1465-2099},

year  = {2024},

date = {2024-10-21},

urldate = {2024-10-21},

journal = {General Virology},

volume = {105},

number = {10},

publisher = {Microbiology Society},

abstract = {Poxviruses are dsDNA viruses infecting a wide range of cell types, where they need to contend with multiple host antiviral pathways, including DNA and RNA sensing. Accordingly, poxviruses encode a variety of immune antagonists, most of which are expressed early during infection from within virus cores before uncoating and genome release take place. Amongst these antagonists, the poxvirus immune nuclease (poxin) counteracts the cyclic 2′3′-GMP-AMP (2′3′-cGAMP) synthase (cGAS)/stimulator of interferon genes DNA sensing pathway by degrading the immunomodulatory cyclic dinucleotide 2′3′-cGAMP, the product of activated cGAS. Here, we use poxviruses engineered to lack poxin to investigate how virus infection triggers the activation of STING and its downstream transcription factor interferon-responsive factor 3 (IRF3). Our results demonstrate that poxin-deficient vaccinia virus (VACV) and ectromelia virus (ECTV) induce IRF3 activation in primary fibroblasts and differentiated macrophages, although to a lower extent in VACV compared to ECTV. In fibroblasts, IRF3 activation was detectable at 10 h post-infection (hpi) and was abolished by the DNA replication inhibitor cytosine arabinoside (AraC), indicating that the sensing was mediated by replicated genomes. In macrophages, IRF3 activation was detectable at 4 hpi, and this was not affected by AraC, suggesting that the sensing in this cell type was induced by genomes released from incoming virions. In agreement with this, macrophages expressing short hairpin RNA (shRNA) against the virus uncoating factor D5 showed reduced IRF3 activation upon infection. Collectively, our data show that the viral genome is sensed by cGAS prior to and during genome replication, but immune activation downstream of it is effectively suppressed by poxin. Our data also support the model where virus uncoating acts as an immune evasion strategy to simultaneously cloak the viral genome and allow the expression of early immune antagonists.},

keywords = {IRF3},

pubstate = {published},

tppubtype = {article}

}

@article{Tsaalbi-Shtylik2024,

title = {DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines},

author = {Anastasia Tsaalbi-Shtylik and Cécile Mingard and Michael Räz and Rurika Oka and Freek Manders and Ruben Van Boxtel and Niels De Wind and Shana J. Sturla},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service},

doi = {10.1016/j.dnarep.2024.103755},

issn = {1568-7864},

year  = {2024},

date = {2024-10-00},

urldate = {2024-10-00},

journal = {DNA Repair},

volume = {142},

publisher = {Elsevier BV},

abstract = {By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.},

keywords = {TLS},

pubstate = {published},

tppubtype = {article}

}

@article{nokey,

title = {Certification of the amount-of-substance fraction of haemoglobin A2 versus total haemoglobin in blood haemolysate: ERM®-DA485/IFCC and ERM®-DA486/IFCC},

author = {Guy Auclair and Amalia Muñoz-Piñeiro and Jean Charoud-Got and Renata Paleari and Patricia Kaiser and Cristian-Gabriel Arsene and Stefanie Trapmann and Andrea Mosca and Liesbet Deprez},

url = {https://trenzyme.com/protein-production-services/custom-protein-expression-service/, ➥Custom Protein Expression Service

https://shop.trenzyme.com/products/hemoglobin-hba2-%CE%B12%CE%B42, ➥Mentioned protein in trenzyme webshop (SKU P2020-193)

},

doi = {10.2760/877530},

isbn = {978-92-68-14954-6},

year  = {2024},

date = {2024-09-06},

urldate = {2024-09-06},

journal = {ERM®-DA485/IFCC and ERM®-DA486/IFCC, Publications Office of the European Union, 2024},

abstract = {ERM®-DA485/IFCC and ERM®-DA486/IFCC are blood haemolysate materials certified for the amount-of-substance fraction of haemoglobin A2 versus total haemoglobin, produced within the scope of ISO 17034 accreditation [1]. The CRMs are available in glass vials containing at least 2 mL of blood haemolysate, lyophilised into a dry powder. The vials were sealed under an atmosphere of nitrogen. Between-unit homogeneity was quantified and stability during transport and storage was assessed in accordance with ISO 33405:2024 [2]. The minimum volume of reconstituted material to be used for one measurement is 5 μL. This minimum sampling volume gave acceptable repeatability using this sample volume, the within-unit inhomogeneity did not contribute to variation of measurement. The materials were characterised by a reference method [3] through an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025:2017 [4]. All data sets obtained were found technically valid. Uncertainties of the certified values were calculated in accordance with ISO 17034:2016 [1] and include uncertainties related to possible inhomogeneity, instability and characterisation. The materials are intended for the calibration of various analytical methods used for the quantification of the amount-of-substance fraction of haemoglobin A2 versus total haemoglobin in human blood. Before release of the CRMs, the certification project was subjected to peer-review involving both internal and external experts.},

keywords = {HbA2, P2020-193},

pubstate = {published},

tppubtype = {article}

}