Biology subjects

Ball, K. L.

Publications and source records attributed to Ball, K. L..

3 recordsLinked to original sources

bioRxiv2025

Spatial dynamics of IFITM1: a core component of the interferon-stimulated gene-resistance signature in glioma

The IFITM1 protein is a key component of the Interferon-Stimulated Gene (ISG) network, which has been linked to treatment-resistant signatures in various cancers, including glioblastoma (GBM). Despite its impact, the mechanisms underlying IFITM1s role in cancer remain poorly understood. Here, we demonstrate that the spatial dynamics of IFITM1 localisation are highly context-dependent, particularly in GBM tissue. In the vasculature, IFITM1 is predominantly localised to the plasma membrane of endothelial cells. However, when present in a subset of cancer stem cells, IFITM1 adopts a distinct perinuclear location, where it co-localises with IFITM3 and is notably absent from the membrane. The spatial dynamics of IFITM1 localisation was investigated in patient-derived glioma stem cells (GSCs), primary endothelial cells and engineered cell lines. Endogenous IFITM1 localised primarily to assemblies in the perinuclear space, however loss of functional IFITM3 led to a shift in the distribution of the protein to a predominantly membrane location. In contrast, IFITM3 localisation was unaffected by the loss of IFITM1. The results were recapitulated by transient expression of IFITM1 or -3 into double knockout (DKO) GSCs and by using an engineered system where IFITM1 expression was IFN-independent. Co-expression studies demonstrate that IFITM3 is sufficient to localise IFITM1 to vesicle structures in the perinuclear space and that mutant forms of IFITM3 lead to retention of IFITM1 primarily at the plasma membrane. Our data highlights dynamic changes in the subcellular localisation of IFITM1 suggesting that distinct functions of this resistance factor may present a specific target for therapeutic intervention.

cancer biology↗

bioRxiv2024

Cell fixation improves performance of in situ crosslinking mass spectrometry while preserving cellular ultrastructure

Crosslinking mass spectrometry (XL-MS) has the potential to map the interactome of the cell with high resolution and depth of coverage. However, current in vivo XL-MS methods are hampered by crosslinkers that demonstrate low cell permeability and require long reaction times. Consequently, interactome sampling is not high and long incubation times can distort the cell, bringing into question the validity any protein interactions identified by the method. We address these issues with a fast formaldehyde-based fixation method applied prior to the introduction of secondary crosslinkers. Using human A549 cells and a range of reagents, we show that 4% formaldehyde fixation with membrane permeabilization preserves cellular ultrastructure and simultaneously improves reaction conditions for in situ XL-MS. Protein labeling yields can be increased even for nominally membrane-permeable reagents, and surprisingly, formaldehyde does not compete with conventional amine-reactive crosslinking reagents. Prefixation with permeabilization uncouples cellular dynamics from crosslinker dynamics, enhancing control over crosslinking yield and permitting the use of any chemical crosslinker.

biochemistry↗

bioRxiv2022

SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression

IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFN{gamma} stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFN{gamma}. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins. SignificanceIFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.

cancer biology↗