Biology subjects

Mojumdar, A.

Publications and source records attributed to Mojumdar, A..

11 recordsLinked to original sources

bioRxiv2025

Phospotyrosine proteomics reveals novel Zap70 and Itk pathway targets downstream of TCR and CAR in Jurkat T cells

{zeta}-associated protein of 70 kDa (Zap70) and interleukin-2-inducible T cell kinase (Itk) propagate the primary and CD28-integrated phosphotyrosine (pY) signalling, respectively, to achieve full T cell activation. Despite their canonical roles in T cell activation, our understanding of how each kinase controls canonical and noncanonical pY signalling is incomplete. Here, using three T cell activation methods (soluble antibodies, APC- pMHC/TCR, and CD19-CAR/Raji), we evaluated the effects of two novel inhibitors, RDN2150 (RDN, Zap70) and Soquelitinib (Soq, Itk), on T cell activation. We validated the published working concentrations of RDN and Soq on phosphorylation of key T cell signalling proteins and on T cell activation markers, finding that RDN provides more complete inhibition of T cell signalling and activation. We used LC-MS/MS to evaluate how RDN and Soq treatment affected the phosphotyrosine (pY) signalling and proteome of T cells, finding that RDN, as opposed to Soq, completely downregulated the TCR signalling pathway. Finally, we identified new, noncanonical pY sites responsive to RDN and Soq, providing new insights into the pathways regulated by Zap70 and Itk. Together, our work provides a basis for further study on RDN and Soq, as well as a molecular roadmap for the effects of these inhibitors.

immunology↗

bioRxiv2025

Evaluating First-Pass, High Protein Capacity Desalting Techniques For Phosphoproteomics Applications

Many commercial desalting products exist for pre-MS peptide cleanup, although few exist that can handle the high protein input ([≥] 4 mg) required for phosphotyrosine enrichment. For these desalting products, the technical aptitude required for effective and organized desalting is often a barrier to entry for new users. Here, we evaluate four commercially available desalting techniques with varying degrees of automation, operational organization, and chemistries to determine the most cost-effective, user-friendly, and sensitive technique for protein profiling and phosphotyrosine (pY) enrichment. We find that TECAN Narrow Bore Extraction (NBE) products are the most cost effective per sample and least difficult to use, whereas ProtiFis S-Trap are the most expensive per sample and Pierce C18 spin columns have the worst operational organization. ProtiFi S-Trap vastly outperforms other desalting methods for peptide sequencing and protein profiling applications, uniquely identifying 25,654 unique peptide sequences and 375 unique proteins. Consistently, ProtiFi S-Trap samples show the deepest pY sequencing after Src SH2 superbinder enrichment, leading to the highest identification of significantly changing, biologically relevant pY sites in a Jurkat T cell signalling model. Our data show that ProtiFi S-Trap columns provide high peptide recovery, thus increasing meaningful pY site identification. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/657744v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@13b77e2org.highwire.dtl.DTLVardef@13bb4cforg.highwire.dtl.DTLVardef@dba171org.highwire.dtl.DTLVardef@1440f7d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR

The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, LckY394. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.

systems biology↗

bioRxiv2024

CSF1R-CAR T cells induce CSF1R signalling and promote cancer cell growth

Chimeric antigen receptor (CAR) T cells have transformed the landscape of cancer therapy and demonstrate unprecedented success in treating relapsed/refractory blood cancers. The mechanism underlaying the interactions and responses of CAR T cells and their targets remain incompletely understood. Previous studies focus on the activation of CAR T cells and attempt to optimise CAR design to increase efficacy, meanwhile ignoring tumours and their responses to CAR ligation. Here, we evaluate the signalling capacity of a second generation CSF1-tageted CSF1R CAR compared with a scFv-targeted CD19-CAR using a SILAC co- culture approach coupled with phosphotyrosine (pY) enrichment and LC-MS/MS. We show that ligation of CSF1R-expressing THP1 cells with CSF1R-CAR T cells induces CSF1R-like signalling in THP1 cells, whereas no target cell signalling response is observed after CD19- CAR/Raji B cell ligation. Using small molecule inhibitors of Lck, actin polymerisation, and CSF1R, we find that CAR-induced CSF1R signalling in THP1 cells depends exclusively on CSF1R kinase activity with no participation from T cell activation. Consistently, CSF1R- CAR T cells promote THP1 growth at low effector-to-target (E:T) ratios but prevent THP1 growth at high E:T ratios. Our data provide evidence for an unintended consequence of CARs; CAR-induced signalling in cancer cells. These data may have broad implications for the choice of CAR antigen for optimal clinical efficacy. One Sentence Summary: CSF1R-CAR activates intracellular signalling cascades in THP1 cells, which promote THP1 cell growth.

cancer biology↗

bioRxiv2024

Dna2 nuclease resolves RNA:DNA hybrids at double-strand breaks

DNA double-strand breaks (DSBs) are highly detrimental to cells, as improper repair can result in inheritable genetic rearrangements or cell death. The role of RNA:DNA hybrids (RDHs) in DSB repair remains poorly understood, but their transient accumulation and subsequent resolution are crucial for accurate repair. The absence of the end-joining factor Nej1 at DSBs significantly reduced RDH levels, which was linked to increased activity of the Dna2 nuclease. Dna2 limits the accumulation of hybrids at DSBs, with levels rising in the presence of a nuclease-dead Dna2 and RNH201 deletion. Dna2 has a heightened preference for resolving RDHs with 5 RNA overhangs compared to duplex substrates with 5 DNA overhangs. This selective resolution by Dna2 helps restrict hybrid accumulation at DSBs and promotes resection, a function not shared by Exo1. This study underscores the multifunctional roles of canonical repair factors in ensuring efficient homology-directed repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/609401v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1578f07org.highwire.dtl.DTLVardef@16f3614org.highwire.dtl.DTLVardef@4789f1org.highwire.dtl.DTLVardef@b15f9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Loss of Dna2 nuclease activity results in decreased Exo1-mediated resection at DNA double strand breaks.

A DNA double strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination (HR) or non-homologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of HR, is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harbouring nuclease dead dna2-1 compared to dna2{Delta} pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harbouring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater defect in resection compared to cells where DNA2 was deleted. Both the decreased level of resection and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change, however Nej1 increased as did the frequency of alt-EJ/ MMEJ repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/564088v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c8280borg.highwire.dtl.DTLVardef@1bcf245org.highwire.dtl.DTLVardef@1c59f43org.highwire.dtl.DTLVardef@15b18d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

During replicative aging, changes in DNA double-strand break repair correlate with an increased rate of mutation.

A double -strand break (DSB) is one of the most deleterious forms of DNA damage. In eukaryotic cells, two main repair pathways have evolved to repair DSBs, homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is the predominant pathway of repair in the unicellular eukaryotic organism, S. cerevisiae. However, during replicative aging the relative use of HR and NHEJ shifts in favor of end-joining repair. By monitoring repair events in the HO-DSB system, we find that early in replicative aging there is a decrease in the association of long-range resection factors, Dna2-Sgs1 and Exo1 at the break site and a decrease in DNA resection. Subsequently, as aging progressed, the recovery of Ku70 at DSBs decreased and the break site associated with the nuclear pore complex at the nuclear periphery, which is the location where DSB repair occurs through alternative pathways that are more mutagenic. End-bridging remained intact as HR and NHEJ declined, but eventually it too became disrupted in cells at advanced replicative age. In all, our work provides insight into the molecular changes in DSB repair pathway during replicative aging. HR first declined, resulting in a transient increase in the NHEJ. However, with increased cellular divisions, Ku70 recovery at DSBs and NHEJ subsequently declined. In wild type cells of advanced replicative age, there was a high frequency of repair products with genomic deletions and microhomologies at the break junction, events not observed in young cells which repaired primarily by HR. HighlightsO_LIDecreased DNA resection at DSBs is an early event of replicative aging C_LIO_LIEnd-joining repair increases as resection decreases at DSBs in older cells C_LIO_LIIn older cells the products of DSB repair contain deletions and microhomologies C_LIO_LIDSBs associate with the NPC at the nuclear periphery more in older cells C_LIO_LIOld Cell Enrichment method suitable for molecular biology approaches in budding yeast C_LI

molecular biology↗

bioRxiv2022

Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330-338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection. HighlightsO_LINej1 C-terminus is critical for repair pathway choice. C_LIO_LIThe KKRK region of Nej1 is important for interactions with ssDNA and dsDNA. C_LIO_LINej1-F335 and V338 are key residues for end-joining and inhibition of resection at DSB. C_LIO_LINej1-mediated end-bridging is not sufficient for end-joining repair. C_LI

molecular biology↗

bioRxiv2021

Nej1(XLF) inhibits Sae2(CTIP)-Dna2(DNA2) mediated resection at DNA double strand breaks

The two major pathways of DNA double strand break (DSB) repair, non-homologous end-joining (NHEJ) and homologous recombination (HR), are highly conserved from yeast to mammals. The regulation of 5 DNA resection controls repair pathway choice and influences repair outcomes. Nej1 was first identified as a canonical NHEJ factor involved in stimulating the ligation of broken DNA ends and more recently, it was shown to be important for DNA end-bridging and inhibiting 5 resection mediated by Dna2-Sgs1. Nej1 interacts with Sae2 and this impacts DSB repair in three ways. First, Nej1 inhibits MRX-Sae2 interactions and Sae2 localization to a DSB. Second, Nej1 inhibits Sae2-dependent recruitment of Dna2 in the absence of Sgs1. Third, NEJ1 and SAE2 showed an epistatic relationship for DNA end-bridging, an event that restrains the broken ends and reduces the frequency of genomic deletions from developing at the DSB. Deletion of NEJ1 suppressed the synthetic lethality of sae2{Delta} sgs1{Delta} and was dependent on the nuclease activity of Dna2. These Nej1 functions promote end-joining DSB repair, but could also be relevant for controlling resection initiation during HR repair. HighlightsO_LINej1 physically interacts with Sae2 and inhibits end-resection at a DSB. C_LIO_LINej1 inhibits Sae2 interactions with the MRX complex. C_LIO_LINej1 inhibits Sae2-dependent recruitment of Dna2 to a DSB. C_LIO_LINEJ1 and SAE2 are epistatic for DNA end-bridging. C_LIO_LIDeletion of NEJ1 suppresses the synthetic lethality of sae2{Delta} sgs1{Delta}, which is dependent on Dna2 nuclease activity. C_LI

genetics↗

bioRxiv2020

A Fob1 independent role for the Smc5/6 complex in the rDNA that modulates lifespan.

The ribosomal DNA (rDNA) in Saccharomyces cerevisiae is in one tandem repeat array on Chromosome XII. Two spacer regions within each repetitive element, called non-transcribed spacer 1 (NTS1) and NTS2, are important in nucleolar organization. Smc5/6 localizes to both NTS1 and NTS2 and is involved in the regulation of Sir2 and Cohibin binding at NTS1, whereas Fob1 and Sir2 are required for optimal binding of the complex to NTS1 and NTS2, respectively. We demonstrate that Smc5/6 functions in chromatin silencing at NTS1 independently of its role in homologous recombination (HR) when forks pause at the replication fork barrier (RFB). In contrast, when the complex does not localize to the rDNA in nse3-1 mutants, the shortened lifespan correlates with NTS2 homeostasis independently of FOB1 status. Our data identify the importance of Smc5/6 integrity in NTS2 transcriptional silencing and repeat tethering, which in turn underscores rDNA stability and replicative lifespan. HighlightsO_LISmc5/6 is important for transcriptional silencing in the rDNA. C_LIO_LISmc5/6 tethers the rDNA array to the periphery. C_LIO_LITranscriptional silencing of ncRNA at NTS1 and NTS2 is differentially regulated. C_LIO_LISmc5/6 has a role in rDNA maintenance independent of HR processing at the RFB. C_LIO_LIFob1-independent disruption of Smc5/6 at NTS2 leads to lifespan reduction. C_LI

molecular biology↗

bioRxiv2019

Modeling Cancer Genomic Data in Yeast Reveals Selection Against ATM Function During Tumorigenesis

The DNA damage response (DDR) comprises multiple functions that collectively preserve genomic integrity and suppress tumorigenesis. The Mre11 complex and ATM govern a major axis of the DDR and several lines of evidence implicate that axis in tumor suppression. Components of the Mre11 complex are mutated in approximately five percent of human cancers. Inherited mutations of complex members cause severe chromosome instability syndromes, such as Nijmegen Breakage Syndrome, which is associated with strong predisposition to malignancy. And in mice, Mre11 complex mutations are markedly more susceptible to oncogene-induced carcinogenesis. The complex is integral to all modes of double strand break (DSB) repair and is required for the activation of ATM to effect DNA damage signaling. To understand which functions of the Mre11 complex are important for tumor suppression, we undertook mining of cancer genomic data from the clinical sequencing program at Memorial Sloan Kettering Cancer Center, which includes the Mre11 complex among the 468 genes assessed. Twenty five mutations in MRE11 and RAD50 were modeled in S.cerevisiae and in vitro. The mutations were chosen based on recurrence and conservation between human and yeast. We found that a significant fraction of tumor-borne RAD50 and MRE11 mutations exhibited separation of function phenotypes wherein Tel1/ATM activation was defective while DNA repair functions were mildly or not affected. At the molecular level, the gene products of RAD50 mutations exhibited defects in ATP binding and hydrolysis. The data reflect the importance of Rad50 ATPase activity for Tel1/ATM activation and suggest that inactivation of ATM signaling confers an advantage to burgeoning tumor cells.\n\nAuthor SummaryA complex network of functions is required for suppressing tumorigenesis. These include processes that regulate cell growth and differentiation, processes that repair damage to DNA and thereby prevent cancer promoting mutations and signaling pathways that lead to growth arrest and programmed cell death. The Mre11 complex influences both signaling and DNA repair. To understand its role in tumor suppression, we characterized mutations affecting members of the Mre11 complex that were uncovered through cancer genomic analyses. The data reveal that the signaling functions of the Mre11 complex are important for tumor suppression to a greater degree than its role in DNA repair.

genetics↗