Biology subjects

Bajaj, A.

Publications and source records attributed to Bajaj, A..

8 recordsLinked to original sources

bioRxiv2026

Optical Screening Identifies Chemical Modulators of Intracellular α-synuclein Aggregation

Parkinsons disease (PD) is a movement disorder characterized by the accumulation of alpha-synuclein aggregates leading to dopaminergic neuron loss in the substantia nigra. While PD has been associated with environmental and microbiome changes, our ability to assess the mechanistic impact of these factors on synuclein aggregation in cells has remained limited. Here, we designed and optimized a high-throughput optical screening system to assess the effect of metabolites and small molecules on synuclein aggregation in cell lines expressing a synuclein-fluorescent protein fusion and treated with pre-formed fibrils (PFFs). Using this assay, we identified several compounds that modulate synuclein aggregate accumulation in cells, including harman, a {beta}-carboline that led to reduced synuclein aggregation. We further investigated the transcriptional effect of harman and PFFs and identified changes in peroxiredoxins as a potential mechanism linking harman to aggregate accumulation. Altogether, this work establishes a pipeline to prioritize small molecules that can impact synuclein aggregate formation.

cell biology↗

bioRxiv2026

Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting essential functions and phase separation. {beta}-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/706410v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@d15f63org.highwire.dtl.DTLVardef@1cd6221org.highwire.dtl.DTLVardef@e58126org.highwire.dtl.DTLVardef@181ec67_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYThe RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of {beta}-sheets in both function and pathological transition, we engineered {beta}-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate {beta}-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting {beta}-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTSO_LITargeted proline additions disrupt {beta}-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior C_LIO_LI{beta}-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions C_LIO_LIIn vivo models reveal attenuated toxicity of {beta}-sheet-deficient FUS in Drosophila C_LIO_LI{beta}-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology C_LI

biochemistry↗

bioRxiv2026

Coronin1A regulates tumor microenvironment in colitis-associated colorectal cancer in a SUMO-dependent way

Inflammatory bowel disease (IBD), comprising Crohns disease and Ulcerative colitis, is a group of multifactorial illnesses with persistent gastrointestinal inflammation and a series of undesirable consequences. IBD patients have a three times higher risk of developing colitis-associated colorectal cancer (CAC). A higher mutational burden due to persistent inflammation acts as a driver of dysplasia and even tumorigenesis. While the pathological sequence is known, the molecular mechanisms underlying the transition from chronic inflammation to CAC remain largely elusive. Post-translational modification- SUMOylation plays an integral role in shaping gut inflammation as well as several forms of cancers, including sporadic colorectal cancer. In this study, the contribution of SUMOylation to CAC pathogenesis was characterized. In the AOM-DSS CAC mice model and human IBD patient specimens, SENP5, but not other deSUMOylases, shows an altered expression. Notably, both SENP5 expression dynamics and alterations in the SUMOylome occur in chronically inflamed and neoplastic colon tissues. SENP5 interactome analysis identified Coronin1A (Coro1A), an actin-binding protein predominantly expressed in immune cells. Coro1A also shows a state-specific, distinct expression pattern in the colon. Interestingly, in contrast to wild-type mice, Coro 1A knockout mice were resistant to polyp formation, with reduced cell proliferation, oncogene expression, and epithelial-to-mesenchymal transition (EMT) gene activation, and reduced extracellular matrix (ECM) development and fibrosis, suggesting its integral role in tumorigenesis. WT mice with chronically inflamed colons and polyps have a higher number of M2-like macrophages, with increased abundance of Coro1A, suggesting a role of Coro1A in modulating the tissue microenvironment toward a pro-tumorigenic state. Mechanistically, Coro1A physically interacts with TGF-{beta} RI and regulates TGF-{beta}-TGF-{beta} RI signalling endosome stability, thereby controlling TGF-{beta}-mediated macrophage polarization. Detailed in vitro experiments revealed stabilization of Coro 1A through its interaction with SUMOylated Raftlin protein. Overall, Coro 1A is necessary and sufficient for TGF-{beta} signalling, macrophage polarization, and tumorigenesis in CAC.

cancer biology↗

bioRxiv2024

Alternative splicing modulates chromatin interactome and phase separation of the RIF1 C-terminal domain

How RIF1 (RAP1 interacting factor) fulfills its diverse roles in DNA double-strand break (DSB) repair, DNA replication, and nuclear organization remains elusive. Here we show that alternative splicing (AS) of a cassette exon (Ex32) encoding a Ser/Lys-rich (S/K) cassette in the RIF1 C-terminal domain (CTD) gives rise to RIF1-Long (RIF1-L) and RIF1-Short (RIF1-S) isoforms with different functional characteristics. We demonstrate that RIF1-Ex32 splice-in is mediated by an exonic splicing enhancer that is recognized by the splicing factor SRSF1 and antagonized by splicing inhibitors SRSF3 and SRSF7. Exposure to DNA damage inhibited Ex32 splice-in, potentiated the association of SRSF3 and SRSF7 with RIF1 pre-mRNA, and caused an increase in RIF1-S protein expression, which was also observed across a diverse set of primary cancers. Isoform-specific proteomic analyses revealed RIF1-L preferentially associated with mediator of DNA damage checkpoint 1 (MDC1) and sustained MDC1 focus formation to a greater extent than RIF1-S. We further show that the S/K cassette stabilized a novel phase separation activity of the RIF1 CTD and enhanced RIF1-L chromatin retention, which was reversed by CDK1-dependent phosphorylation of the RIF1 CTD in response to G2 DNA damage checkpoint inhibition. These combined findings suggest DNA damage-dependent RIF1 AS contributes to RIF1 functional diversification in genome protection.

molecular biology↗

bioRxiv2024

Lestaurtinib's antineoplastic activity converges on JAK/STAT signaling to inhibit advanced forms of therapy resistant ovarian cancer

Ovarian cancer is the deadliest gynecological malignancy, owing to its late-stage diagnosis and high rates of recurrence and resistance following standard-of-care treatment, highlighting the need for novel treatment approaches. Through an unbiased drug screen, we identified the kinase inhibitor, lestaurtinib, as a potent antineoplastic agent for chemotherapy- and PARP-inhibitor (PARPi)-sensitive and -resistant ovarian cancer cells and patient derived xenografts (PDXs). RNA-sequencing revealed that lestaurtinib potently suppressed JAK/STAT signaling and lestaurtinib efficacy was shown to be directly related to JAK/STAT pathway activity in cell lines and PDX models. Most ovarian cancer cells exhibited constitutive JAK/STAT pathway activation and genetic loss of STAT1 and STAT3 resulted in growth inhibition. Lestaurtinib also displayed synergy when combined with cisplatin and olaparib, including in a model of PARPi resistance. In contrast, the most well-known JAK/STAT inhibitor, ruxolitinib, lacked antineoplastic activity against all ovarian cancer cell lines and PDX models tested. This divergent behavior was reflected in the ability of lestaurtinib to block both Y701/705 and S727 phosphorylation of STAT1 and STAT3, whereas ruxolitinib failed to block S727. Consistent with these findings, lestaurtinib additionally inhibited JNK and ERK activity, leading to more complete suppression of STAT phosphorylation. Concordantly, combinatorial treatment with ruxolitinib and a JNK or ERK inhibitor resulted in synergistic antineoplastic effects at dose levels where single agents were ineffective. Taken together, these findings indicate that lestaurtinib, and other treatments that converge on JAK/STAT signaling, are worthy of further pre-clinical and clinical exploration for the treatment of highly aggressive and advanced forms of ovarian cancer. Statement of significanceLestaurtinib is a novel inhibitor of ovarian cancer, including chemotherapy- and PARPi-resistant models, that acts through robust inhibition of the JAK/STAT pathway and synergizes with standard-of-care agents at clinically relevant concentrations.

cancer biology↗

bioRxiv2023

Rab7 dependent regulation of goblet cell protein CLCA1 modulates gastrointestinal homeostasis

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier coupled with microbial dysbiosis has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease Chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7 dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

cell biology↗

bioRxiv2022

mTORC2 Drives ZFX-mediated Ganglioside Biosynthesis to Promote Breast Cancer Progression.

Sphingolipid and ganglioside metabolic pathways are crucial components of cell signalling, having established roles in tumor cell proliferation, invasion, and migration. However, regulatory mechanisms controlling sphingolipid and ganglioside biosynthesis in mammalian cells is less known. Here, we show that RICTOR, the regulatory subunit of mTORC2, regulates the synthesis of sphingolipids and gangliosides in luminal breast cancer-specific MCF-7 and BT-474 cells through transcriptional and epigenetic mechanisms. RICTOR regulates glucosylceramide levels by modulating the expression of UDP-Glucose Ceramide Glucosyl transferase (UGCG). We identify Zinc Finger protein X-linked (ZFX) as a RICTOR-responsive transcription factor whose recruitment to the UGCG promoter is regulated by DNA methyltransferases and histone demethylase (KDM5A) that are known AKT substrates. We further demonstrate that RICTOR regulates the synthesis of GD3 gangliosides through ZFX and UGCG, and triggers the activation of EGFR signalling pathway, thereby promoting tumor growth. In line with our findings in cell culture and mice models, we observe an elevated expression of RICTOR, ZFX, and UGCG in Indian luminal breast cancer tissues, and in TCGA and METABRIC datasets. Together, we establish a key regulatory circuit, RICTOR-AKT-ZFX-UGCG-Ganglioside-EGFR-AKT, and elucidate its contribution to breast cancer progression.

cancer biology↗

bioRxiv2020

Screening of tomato seed bacterial endophytes for antifungal activity reveals lipopeptide producing Bacillus siamensis as a potential bio-control agent

The current study investigates the diversity pattern and fungicidal potential of bacterial endophytes isolated from two different organic varieties of tomato plants (V1 and V2). A total of seventy-four bacterial isolates identified by 16S rRNA sequencing revealed a single genus Bacillus with 16 different species. The Shannon diversity H (1.45), Simpsons index of diversity (0.9), Magalef index (2.1), Evenness (0.96), and Species richness (8) indicated the high endophytic bacterial diversity in the V1 variety of the tomato. Bacterial endophytes isolated from both the varieties were screened for their antifungal activity against five economically critical fungal pathogens (viz., Botrytis cinerea, Rhizoctonia solani, Fusarium solani, Verticillium lateritium, and Alternaria solani) of tomato crop through dual culture assay. The data revealed B. siamensis KCTC 13613(T) as the most potent antagonist significantly (p < 0.05), inhibiting the mycelial growth between 75 to 90% against selected fungal pathogens. High bioactivity of lipopeptide extract of B. siamensis was recorded against R. solani with IC50 value of 72 ppm. The UPLC-HDMS analysis of this lipopeptide extract revealed the presence of, Surfactin and Bacillomycin D.

microbiology↗