Biology subjects

Kiianitsa, K.

Publications and source records attributed to Kiianitsa, K..

5 recordsLinked to original sources

bioRxiv2020

Novel TREM2 splicing isoform that lacks the V-set immunoglobulin domain is abundant in the human brain

TREM2 is an immunoglobulin-like receptor expressed by certain myeloid cells, such as macrophages, dendritic cells, osteoclasts and microglia. In the brain, TREM2 plays an important role in the immune function of microglia, and its dysfunction is linked to various neurodegenerative conditions in humans. Ablation of TREM2 or its adaptor protein TYROBP causes Polycystic Lipo-Membranous Osteodysplasia with Sclerosing Leukoencephalopathy (also known as Nasu-Hakola disorder) with early onset of dementia, while some missense variants in TREM2 are associated with an increased risk of late-onset Alzheimers disease. Human TREM2 gene is a subject to alternative splicing, and its major, full-length canonical transcript encompasses 5 exons. Herein, we report a novel alternatively spliced TREM2 isoform without exon 2 ({Delta}e2), which constitutes a sizable fraction of TREM2 transcripts and has highly variable inter-individual expression in the human brain (average frequency 10%; range 3.7-35%). The protein encoded by {Delta}e2 lacks a V-set immunoglobulin domain from its extracellular part but retains its transmembrane and cytoplasmic domains. We demonstrated {Delta}e2 protein expression in TREM2-positive THP-1 cells, in which the expression of full-length transcript was precluded by CRISPR/Cas9 disruption of the exon 2 coding frame. In "add-back" experiments, overexpression of full-length, but not {Delta}e2 TREM2, restored phagocytic capacity and promoted interferon type I response in the knockout cells. Our findings suggest that expression of a {Delta}e2 splice isoform may modify the dosage of full-length transcript potentially weakening some TREM2 receptor functions in the human brain.

immunology↗

bioRxiv2020

Rapid, direct detection of bacterial Topoisomerase 1 - DNA adducts by RADAR/ELISA

Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches.

microbiology↗

bioRxiv2019

The "adductome": a limited repertoire of adducted proteins in human cells

Proteins form adducts with nucleic acids in a variety of contexts, and these adducts may be cytotoxic if not repaired. Here we apply a proteomic approach to identification of proteins adducted to DNA or RNA in normally proliferating cells. This approach combines RADAR fractionation of proteins covalently bound to nucleic acids with quantitative mass spectrometry (MS). We demonstrate that "RADAR-MS" can quantify induction of TOP1- or TOP2-DNA adducts in cells treated with topotecan or etoposide, respectively, and also identify intermediates in physiological adduct repair. We validate RADAR-MS for discovery of previously unknown adducts by determining the repertoires of adducted proteins in two different normally proliferating human cell lines, CCRF-CEM T cells and GM639 fibroblasts. These repertoires are significantly similar with one another and exhibit robust correlations in their quantitative profiles (Spearman r=0.52). A very similar repertoire is identified by the classical approach of CsCl buoyant density gradient centrifugation. We find that in normally proliferating human cells, the repertoire of adducted proteins -- the "adductome" -- is comprised of a limited number of proteins belonging to specific functional groups, and that it is greatly enriched for histones, HMG proteins and proteins involved in RNA splicing. Treatment with low concentrations of formaldehyde caused little change in the composition of the repertoire of adducted proteins, suggesting that reactive aldehydes generated by ongoing metabolic processes may contribute to protein adduction in normally proliferating cells. The identification of an endogenous adductome highlights the importance of adduct repair in maintaining genomic structure and the potential for deficiencies in adduct repair to contribute to cancer.

biochemistry↗

bioRxiv2019

Treatment with 5-aza-dC Induces Apoptotic PARP1-DNA Adducts

The nucleoside analog 5-aza-2-deoxycytidine (5-aza-dC) is used to treat some hematopoietic malignancies. The mechanism of cell killing depends upon DNMT1, but is otherwise not clearly defined. Here we show that PARP1 forms covalent DNA adducts in human lymphoblast or fibroblasts treated with 5-aza-dC. Some adducts recovered from 5-aza-dC-treated cells have undergone cleavage by apoptotic caspases 3/7. Mapping of PARP1-DNA adducts, by a new method, "Adduct-Seq", demonstrates adduct enrichment at CpG-dense genomic locations that are targets of maintenance methylation by DNMT1. Covalent protein-DNA adducts can arrest replication and induce apoptosis, and these results raise the possibility that induction of PARP1-DNA adducts may contribute to cell killing in response to treatment with 5-aza-dC.

biochemistry↗

bioRxiv2018

TREM2 R47H exacerbates immune response in Alzheimer’s disease brain

The R47H variant in the microglial TREM2 receptor is a strong risk factor for Alzheimers disease (AD). To characterize processes affected by R47H we performed integrative network analysis of genes expressed in brains of AD patients with R47H, sporadic AD without the variant and patients with polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), a systemic disease with early onset dementia caused by loss-of function mutations in TREM2 or its adaptor TYROBP. While sporadic AD had few perturbed microglial and immune genes, TREM2 R47H AD demonstrated upregulation of interferon type I response and pro-inflammatory cytokines accompanied by induction of NKG2D stress ligands. In contrast, PLOSL had distinct sets of highly perturbed immune and microglial genes that included inflammatory mediators, immune signaling, cell adhesion and phagocytosis. TREM2 knock-out in THP1, a human myeloid cell line that constitutively expresses the TREM2-TYROBP receptor, inhibited response to the viral RNA mimetic poly(I:C), and overexpression of ectopic TREM2 restored the response. Compared to wild type protein, R47H TREM2 had higher stimulatory effect on the interferon type I response signature. Our findings point to a role of the TREM2 receptor in the control of the interferon type I response in myeloid cells and provide insight regarding the contribution of R47H TREM2 to AD pathology.

genetics↗