Biology subjects

Borowicz, P.

Publications and source records attributed to Borowicz, P..

8 recordsLinked to original sources

bioRxiv2026

Intrinsically disordered insert from SH2D2A rewires CD19 CAR signaling via Tyr290

Chimeric antigen receptor (CAR) T cells have transformed cancer immunotherapy, yet their truncated or suboptimal intracellular signaling can limit therapeutic efficacy. To enhance proximal signaling of a CD19-targeted CAR, we systematically inserted short Lck-recruiting motifs derived from Lck-adaptor proteins into the CAR intracellular tail. Six candidate sequences from four adaptor molecules (SH2D2A, SKAP1, LAT, LIME), with a sequence from CD3{varepsilon}, known to affect CAR functionality, as a positive control, were tested for expression and functional impact. Three CAR constructs (containing SH2D2A, LAT and LIME1 sequences respectively) displayed reduced surface expression, but only SH2D2A elicited a pronounced rewiring of CAR T cell phenotype following co-culture with CD19+ tumor lines. SH2D2A CAR T cells showed increased CD27 and CD56 expression and reduced expression of effector-associated mediators including granzyme B, IL-2, TNF, and IFN{gamma}. Through systematic mutagenesis and comparative phenotyping of SH2D2A CAR variants, we identified SH2D2A tyrosine 290 (Tyr290) as the critical residue mediating both the altered signaling phenotype and the low surface expression. Additionally, mutation of Tyr254 in the LIME1 CAR restored surface expression in Jurkat T cells, indicating insert- and context-dependent effects on receptor surface expression. Collectively, these results demonstrate that short, intrinsically disordered adaptor-derived sequences -- and single tyrosine residues within them -- can profoundly reprogram CAR signaling and expression.

immunology↗

bioRxiv2025

Identification of DOK2 and PTPN11 as novel interactors of T cell specific adapter protein TSAd

T cells play a crucial role in the adaptive immune system and depend on tightly regulated intracellular signalling pathways to respond in an appropriate manner. Adapter proteins have flexible and dynamic features, which allow them to regulate T cell signal transduction pathways. As adapter proteins are enzymatically inert and may play multiple roles in parallel, it has been a challenge to fully characterise their functions individually. One such protein in T cells, is T cell specific adapter protein (TSAd), which is upregulated following T cell receptor (TCR) stimulation and is believed to mediate Src family tyrosine kinase signalling. However, the functional role remains elusive, possibly due to limited insight into interactors that potentially bind TSAd. The only structurally well-defined feature within TSAd, is the Src homology 2 (SH2) domain. This conserved domain displays prototypic binding of phosphorylated tyrosines, which suggests that the adapter molecule is implicated in phosphotyrosine signalling pathways. Here, we used an unbiased approach to identify ligands of the TSAd SH2 domain, by using affinity-purification mass spectrometry (AP-MS). Several novel ligands, many of which are known to be implicated in negative regulation of T cell intracellular signalling, were identified. More specifically, we showed that TSAd binds DOK2 and PTPN11 and determined the tyrosines responsible for the TSAd SH2 domain-dependent interaction. Ablation of TSAd and DOK2 by CRISPR/Cas9 in Jurkat T cells resulted in altered tyrosine phosphorylation. Taken together, these findings provide new insight into the possible function of TSAd as a negative signalling node in T cells.

immunology↗

bioRxiv2025

A bioinformatic workflow to facilitate the study of less-understood proteins: the case of SH2D2A

Adaptor proteins are key regulators of immune signalling but are challenging to study due to redundant pathways that obscure clear functional "anchor" phenotypes. Making use of public single-cell RNA sequencing (scRNA-seq) datasets, we theorised that anchors could be identified by working in the reverse direction: starting from the transcriptomic level and working "bottom-up" to identify anchoring phenomena at the cell surface. We have produced a prototype workflow for this method using the case of the lymphocyte-enriched adaptor protein SH2D2A, whose function remains uncertain. Using our bottom-up analysis, we have identified a link between SH2D2A and T cell-T cell (T-T) synapses enriched for ITGB2. The functional implications of this link are currently being explored, but further application of this bioinformatic approach to less-understood proteins may prove useful in understanding the minutiae of immune cell signalling, with implications for immunotherapy.

molecular biology↗

bioRxiv2025

SH2D2A is an indicator of favourable prognosis in bladder cancer and is enriched in Treg cells

Given the expanding availability of RNA-seq and other such high dimensional data it is now possible to consider a complementary approach working in the opposite direction i.e., from transcriptomics up towards protein function. This approach may prove fruitful in producing information on the function of cytosolic-bound proteins such as adapter proteins. To test the validity of this method, we made use of several public datasets to interrogate the cancer-specific role of the adapter protein SH2D2A: a protein enriched in T and NK cells and a known interactor of the kinase LCK, whose function remains uncertain. We found that SH2D2A is a favourable marker for prognosis in urothelial bladder cancer (BLCA). Digging further, we identified a population of SH2D2A+ FOXP3+ IL2RAhi activated Tregs as the main expressors of SH2D2A in BLCA. This suggests that the expression of SH2D2A in these Tregs contributes to a beneficial prognostic effect. Further comprehension of SH2D2As function in these cells holds the potential for advancing treatment in BLCA and diversifying possible targets for immunotherapies.

bioinformatics↗

bioRxiv2023

Unleashing the secrets of plant-fungal interactions using a transformation-free confocal staining technique that supports AI-assisted quantitative analysis

Laser scanning confocal microscopys ability to generate high-contrast 3D images has become essential to studying plant-fungal interactions. Techniques such as visualization of native fluorescence, fluorescent protein tagging of microbes, GFP/RFP-fusion proteins, and fluorescent labelling of plant and fungal proteins have been widely used to aid in these investigations. Use of fluorescent proteins have several pitfalls including variability of expression in planta and the requirement of gene transformation. Here we used the unlabeled pathogens Parastagonospora nodorum, Pyrenophora teres f. teres, and Cercospora beticola infecting wheat, barley, and sugar beet respectively, to show the utility of a staining and imaging technique that uses propidium iodide (PI), which stains RNA and DNA, and wheat germ agglutinin labeled with fluorescein isothiocyanate (WGA-FITC), which stains chitin, to visualize fungal colonization of plants. This method relies on the use of KOH to remove the cutin layer of the leaf, increasing its permeability. This permeability allows the staining solution to penetrate and efficiently bind to its targets, resulting in a consistent visualization of cellular structures. We have also used this staining technique in conjunction with machine learning to analyze fungal volume, which indicates the fitness of the pathogen in planta, as well as quantifying nuclear breakdown, an early indicator of programmed cell death (PCD). This technique is simple to use, robust, consistent across host species, and can be applied to any plant-fungal interaction. Therefore, this technique can be used to characterize model systems as well as non-model interactions where transformation is not routine.

plant biology↗

bioRxiv2021

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll

Parastagonospora nodorum, causal agent of septoria nodorum blotch, is a destructive necrotrophic fungal pathogen of wheat. P. nodorum is known to secrete several necrotrophic effectors that target wheat susceptibility genes that trigger classical biotrophic resistance responses but resulting in susceptibility rather than resistance. SnTox5 targets the wheat susceptibility gene Snn5 to induce necrosis. In this study, we used full genome sequences of 197 P. nodorum isolates collected from the US and their disease phenotyping on the Snn5 differential line LP29, to perform genome wide association study analysis to localize the SnTox5 gene to chromosome 8 of P. nodorum. SnTox5 was validated using gene transformation and CRISPR-Cas9 based gene disruption. SnTox5 encoded a small secreted protein with a 22 and 45 amino acid secretion signal and a pro sequence, respectively. The SnTox5 gene is under purifying selection in the Upper Midwest but under strong diversifying selection in the South/East regions of the US. Comparison of wild type and SnTox5-disrupted strains on wheat lines with and without the susceptibility target Snn5 showed that SnTox5 has two functions, 1) facilitating colonization of the mesophyll layer, and 2) targeting Snn5 to induce programmed cell death to provide cellular nutrient to complete its necrotrophic life cycle.

pathology↗

bioRxiv2020

Localization of hyphal growth associated with mycotoxin production during the malting of Fusarium head blight infected grains

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Objectives of the current study were to localize the growth of Fusarium within FHB infected kernels and to associate it with the production of DON that occurred during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in Fusarium Tri5 DNA and trichothecene mycotoxins following malting, were screened for hyphal localization. The growth of hyphae, both on the surface of kernels and within tissues of grain and malt was, imagined by scanning electron microscopy and confocal laser scanning microscopy assisted with WGA-Alexa Fluor 488 pre-staining, respectively. In barley, hyphae were primarily present on or within husk, vascular bundle, and pericarp cavities. Following malting, large amounts of hyphal growth were observed in not only these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. Interestingly, these grains already had an extensive internal presence of hyphae in unmalted grain, occurring in the pericarp, testa, vascular bundle, nucellar projection, aleurone layer, endosperm, pericarp and endosperm cavities, and embryo. Shotgun sequencing followed by metagenomics analysis verified that Fusarium spp. accounted for above 90% of the fungal hyphae growing in the interior of grains during malting, which coincided with the significant production of mycotoxins.

plant biology↗

bioRxiv2020

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization

In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAcope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Thus, we optimized the RNAscope detection method in plants to visualize the spatial expression and semi-quantification of target RNAs which can be employed in other plants such as the widely utilized model dicot plant Arabidopsis.

plant biology↗