Biology subjects

Raza, M.

Publications and source records attributed to Raza, M..

12 recordsLinked to original sources

bioRxiv2026

OLT1177 (Dapansutrile) inhibits Gasdermin D-dependent IL-1β Release and Pyroptotic Cell Death in Bone Marrow-derived Macrophages

Gasdermins are a family of pore-forming proteins that regulate the release of pro-inflammatory cytokine, interleukin-1{beta} (IL-1{beta}) from infected or PAMP-stimulated cells. During infection or injury, IL-1{beta} is released by both human and mouse macrophages. IL-1{beta} release from mouse macrophages is associated with cell death, often termed "pyroptosis". Mouse macrophages undergoing pyroptosis assemble an exit channel termed gasdermin D (GSDMD). Both the processing of IL-1{beta} and the formation of the exit channel are caspase-1 dependent. Here, in bacterial endotoxin, lipopolysaccharide (LPS), treated mouse bone marrow-derived macrophages (BMDMs), we studied the pharmacologic inhibition of the intracellular nucleotide-binding domain, leucine-rich-containing family, pyrin domain- containing-3 (NLRP3) inflammasome by OLT1177. BMDMs stimulated with LPS plus the potassium efflux inducer nigericin triggered the formation of the NLRP3 inflammasome. Treatment of these BMDMs with OLT1177 suppressed cell death by 42% and ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain)-speck formation by approximately 60%. In addition, OLT1177 dose-dependently inhibited IL-1{beta}, CCL3, and myeloperoxidase (MPO) secretion and the pore-forming (GSDMD) from LPS-primed BMDMs, suggesting the existence of a vicious cycle controlled by IL-1{beta} release. Overall, our study demonstrates that OLT1177 prevents IL-1{beta} release from BMDMs by inhibiting caspase-1 and the conversion of (GSDMD) into its active N-terminal fragment (GSDMD-N). This study thus supports the concept that orally administered OLT1177 can be used to prevent local as well as systemic inflammation in humans.

immunology↗

bioRxiv2025

DDIAS is a single-stranded DNA-binding effector of the TOPBP1-CIP2A complex in mitosis

DNA double-strand breaks and unresolved DNA replication intermediates are particularly dangerous during mitosis. Paradoxically, cells inactivate canonical DNA repair mechanisms during chromosome segregation in favor of alternative pathways that depend on TOPBP1 and CIP2A, but how they function is still poorly defined. Here, we describe the identification of DDIAS as a mitosis-specific DNA damage response protein. We establish DDIAS as a phosphorylation-dependent component and effector of the TOPBP1-CIP2A complex with single-stranded DNA (ssDNA)-binding activity, and thereby delineate a ssDNA protection mechanism that safeguards chromosome integrity during mitosis, particularly in BRCA-deficient cells. We also identify biallelic inactivating mutations in DDIAS in a patient with a severe neurodevelopmental disorder. These findings highlight for the first time a potential physiological role for the DNA damage response in mitosis.

molecular biology↗

bioRxiv2025

ECD, a novel androgen receptor target promotes prostate cancer tumorigenesis by regulating glycolysis

Androgen receptor (AR)-mediated signaling is essential for PC tumorigenesis. In the TCGA database we observed a positive correlation between ECD and AR expression. Consistently, Dihydrotestosterone (DHT) treatment of PC cell lines increased ECD mRNA and protein levels, and AR knockdown (KD) reduced ECD expression. Bioinformatic analysis predicted three consensus androgen response elements in the ECD promoter, and DHT treatment increased AR occupancy at the ECD promoter, and enhanced ECD promoter activity. Enzalutamide treatment decreased ECD levels, and ECD knockout (KO) in PC cells reduced oncogenic traits, suggesting a functional role of ECD to maintain PC oncogenesis. ECD mRNA and protein are overexpressed in PC patient tissues, and its overexpression predicts shorter survival. Overexpression of ECD in PC cell lines enhanced the oncogenic traits in vitro and developed faster and larger highly proliferative xenograft tumors. RNA-seq analysis of mouse tumors revealed an increase in mRNA levels of several glycolytic genes. ECD associates with mRNA of key glycolytic genes and is required for their stability, consistent with our recent demonstration of ECD is an RNA binding protein. Higher glucose uptake and glycolysis was seen upon ECD overexpression in PC cells. Together, we demonstrate the role of a novel AR target gene ECD in PC tumorigenesis.

cancer biology↗

bioRxiv2025

ECD co-operates with ERBB2 to promote tumorigenesis through upregulation of unfolded protein response and glycolysis

The ecdysoneless (ECD) mRNA and protein are overexpressed in breast cancer (BC), and its overexpression correlates with poor prognosis and short patient survival, particularly in ERBB2/HER2-positive BC. This study investigates the co-operative oncogenic mechanism of ECD and ERBB2 by deriving transgenic mice overexpressing ECD and/or ERBB2 (huHER2) in mammary epithelium under MMTV promoter, as well as human mammary immortal epithelial cell lines (hMECs) overexpressing ECD and/or ERBB2. While huHER2Tg mice developed more homogenous solid nodular carcinomas, double transgenic mice (ECD;huHER2Tg) developed heterogenous and histologically aggressive mammary tumors with basal-like phenotype and epithelial mesenchymal transition (EMT) features, like ECDTg tumors, resembling more to patient tumors. Importantly, transcriptomic profile of ECD;huHER2Tg tumors revealed upregulation of two major oncogenic pathways, unfolded protein response (UPR) and glycolysis. Similarly, hMECs expressing both ECD and ERBB2 as compared to single gene expressing cells showed increase in oncogenic traits, and RNA-seq analysis showed a significant upregulation of glycolysis and UPR pathways. ECD is an RNA binding protein, and directly associates with three key glycolytic enzymes (LDHA, PKM2 and HK2) and mRNA of a major UPR regulated gene GRP78, that results in increased mRNA stability. Lastly, we show an increase in glucose uptake and enhanced glycolytic rate in ECD+ERBB2-overexpressing cells as compared to ECD- or ERBB2-overexpressing hMECs. Taken together, our findings support a co-operative role of ECD and ERBB2 in oncogenesis by enhancing two major oncogenic pathways, UPR and glycolysis. SignificanceThis study provides mechanistic insights that overexpression of ECD in ERBB2+ breast cancer patients correlates with shorter patient survival, by identifying direct ECD binding to mRNAs for UPR and glycolysis pathways.

cancer biology↗

bioRxiv2025

ECD functions as a novel RNA-binding protein to regulate mRNA splicing

Human ecdysoneless protein (ECD) plays an essential role in regulating cell cycle progression and cell survival. ECD has previously been implicated in RNA splicing through its association with spliceosomal proteins. Here, using EMSA, fluorescence polarization assays, and mutational analysis, we demonstrate that ECD directly binds to RNA. Enhanced CLIP-seq analysis identified a broad repertoire of mRNAs bound to ECD in cells. RNA-seq analyses revealed that ECD depletion leads to widespread splicing aberrations and altered gene expression. ECD binding to RNAs was enriched near splice sites, and a substantial fraction of ECD-bound transcripts exhibited splicing defects upon ECD depletion. ECD associates with and stabilizes the U5 snRNP complex specific proteins. While depletion of ECD reduced the levels of key U5-specifc proteins, these proteins exhibited an increased association with the R2TP complex in knockout cells. Notably, we found ECD to directly bind to U5 snRNA, and an RNA binding defective mutant of ECD ({Delta}135-148) failed to rescue the reduced levels of U5-specific proteins or the proliferation defect induced by ECD depletion. Collectively, these findings demonstrate that ECD binds to RNAs, including the U5 snRNA, and that RNA-binding is required for ECD to stabilize the U5 snRNP and for cellular functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/634785v4_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@f5aa4forg.highwire.dtl.DTLVardef@80606borg.highwire.dtl.DTLVardef@3a4c8eorg.highwire.dtl.DTLVardef@1780b0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

An integrated and scalable rodent cage system enabling continuous computer vision-based behavioral analysis and AI-enhanced digital biomarker development

1Home cage monitoring enables continuous observation of animals in familiar environments. It has large utility in preclinical testing, mechanistic studies, animal husbandry, and the general practice of the Replacement, Reduction, Refinement (3R) principles. Despite its acknowledged utility, home cage monitoring has not been broadly adopted. This is mainly due to the complexity of the tasks that must be solved to have a successful system that includes hardware and sensor development, data management, machine vision expertise, behavioral expertise, support, and user training. Here, we describe the Digital In Vivo System (DIV Sys), a modern end-to-end system for video-based rodent home cage monitoring. The DIV Sys consists of a cloud-based study design, monitoring, display, and visualization app (DIV App), local hardware for data acquisition cages (DAX), a machine learning model for tracking mice (mHydraNet) optimized for speed and accuracy, a study display and visualization app, and an advanced behavior quantification workbench (DIV Data). The platform seamlessly manages terabytes of video data in the cloud and is built around enterprise-level security and data standards. Collaborative tools enable teams across geographical locations to work together. As a demonstration of its utility, we used DIV Sys to analyze over a century of mouse videos across multiple geographic locations. We also characterized home cage behavior of 8 mouse strains and carried out customized video analysis. Together, we present a scalable home cage monitoring system for advanced behavior quantification for the rodent research community.

animal behavior and cognition↗

bioRxiv2023

A novel phylogenomics pipeline reveals complex pattern of reticulate evolution in Cucurbitales

High-throughput sequencing data, such as target capture, RNA-Seq, genome skimming, and high-depth whole genome sequencing, are used for phylogenomic analyses. Integrating these mixed data types into a single phylogenomic dataset requires several bioinformatic tools and significant computational resources. Here, we present CAPTUS, a novel pipeline to analyze mixed data efficiently. CAPTUS assembles these data types, searches for loci of interest, and produces paralog-filtered alignments. If reference target loci are not available for the studied taxon, CAPTUS can also be used to discover new putative homologs via sequence clustering. Compared to other software, CAPTUS allows the recovery of a greater number of more complete loci across more species. We apply CAPTUS to assemble a comprehensive dataset, comprising the four types of sequencing data for the angiosperm order Cucurbitales, a clade of about 3,100 species in eight mainly tropical plant families, including begonias (Begoniaceae) and gourds (Cucurbitaceae). Our phylogenomic results support the currently accepted circumscription of Cucurbitales except for the position of the holoparasitic Apodanthaceae, which group with Rafflesiaceae in Malpighiales. A subset of mitochondrial gene regions supports the earlier divergence of Apodanthaceae in Cucurbitales. However, the nuclear regions and majority of mitochondrial regions place Apodanthaceae in Malpighiales. Within Cucurbitaceae, we confirm the monophyly of all currently accepted tribes but also reveal hybridization and incomplete lineage sorting both in Cucurbitales and within Cucurbitaceae. We show that contradicting results among earlier phylogenetic studies in Cucurbitales can be reconciled when accounting for gene tree conflict and demonstrate the efficiency of CAPTUS for complex datasets.

bioinformatics↗

bioRxiv2023

OptoPI3K, genetic code expansion, and click chemistry reveal mechanisms underlying reciprocal regulation between TRPV1 and PI3K

ABSTRACTLigands such as insulin, epidermal growth factor, platelet derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLC{gamma}, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell- impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

physiology↗

bioRxiv2023

Environmental stiffness restores mechanical homeostasis in vimentin-depleted cells

Recent experimental evidence indicates a role for the intermediate filament vimentin in regulating cellular mechanical homeostasis, but its precise contribution remains to be discovered. Mechanical homeostasis requires a balanced bi-directional interplay between the cells microenvironment and the cellular morphological and mechanical state - this balance being regulated via processes of mechanotransduction and mechanoresponse, commonly referred to as mechanoreciprocity. Here, we systematically analyze vimentin-expressing and vimentin-depleted cells in a swatch of in vitro cellular microenvironments varying in stiffness and/or ECM density. We find that vimentin-expressing cells maintain mechanical homeostasis by adapting cellular morphology and mechanics to micromechanical changes in the microenvironment. However, vimentin-depleted cells lose this mechanoresponse ability on short timescales, only to reacquire it on longer time scales. Indeed, we find that the morphology and mechanics of vimentin-depleted cell in stiffened microenvironmental conditions can get restored to the homeostatic levels of vimentin-expressing cells. Additionally, we observed vimentin-depleted cells increasing collagen matrix synthesis and its crosslinking, a phenomenon which is known to increase matrix stiffness, and which we now hypothesize to be a cellular compensation mechanism for the loss of vimentin. Taken together, our findings provide further insight in the regulating role of intermediate filament vimentin in mediating mechanoreciprocity and mechanical homeostasis.

biophysics↗

bioRxiv2022

EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry

With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout of EHD2, together with mouse EHD2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2 and CAV1/2-overexpressing BC.

cancer biology↗

bioRxiv2021

Altered Sub-Genomic RNA Expression in SARS-CoV-2 B.1.1.7 Infections

SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT>CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3 of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. One Sentence SummaryThe recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.

genomics↗

bioRxiv2019

Regulation of nerve growth and patterning by cell surface protein disulphide isomerase

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.

developmental biology↗