Biology subjects

Biswas, T.

Publications and source records attributed to Biswas, T..

29 records · Page 2Linked to original sources

bioRxiv2023

Wnt signaling couples G2 phase control with differentiation during hematopoiesis

During homeostasis, a critical balance is maintained between myeloid-like progenitors and their differentiated progeny, which function to mitigate stress and innate immune challenges. The molecular mechanisms that help achieve this balance are not fully understood. Using genetic dissection in Drosophila, we show that a Wnt6/EGFR-signaling network simultaneously controls progenitor growth, proliferation, and differentiation. Unlike G1-quiescence of stem cells, hematopoietic progenitors are blocked in the G2 phase by a {beta}-catenin-independent Wnt6 pathway that restricts Cdc25 nuclear entry and promotes cell growth. Canonical {beta}-catenin-dependent Wnt6 signaling is spatially confined to mature progenitors through localized activation of the tyrosine-kinases EGFR and Abl, which promote nuclear entry of {beta}-catenin and facilitate exit from G2. This strategy combines transcription-dependent and - independent forms of both Wnt6 and EGFR pathways to create a direct link between cell-cycle control and differentiation. This unique combinatorial strategy employing conserved components may underlie homeostatic balance and stress response in mammalian hematopoiesis.

developmental biology↗

bioRxiv2023

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IkappaBalpha without requiring ATP

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of I{kappa}B by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-{kappa}B activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its I{kappa}B substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of I{kappa}B at S32 or S36, but rather directs phosphorylation of I{kappa}B at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to I{kappa}B in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-{kappa}B activation.

biochemistry↗

bioRxiv2023

3D spheroid culturing of Astyanax mexicanus liver-derived cell lines recapitulates distinct transcriptomic and metabolic states of in vivo tissue environment.

In vitro assays are crucial tools for gaining detailed insights into various biological processes, including metabolism. Cave morphs of the river-dwelling fish species, Astyanax mexicanus, have adapted their metabolism allowing them to thrive in the biodiversity-deprived and nutrient-limited environment of caves. Liver-derived cells from the cave and river morphs of Astyanax mexicanus have proven to be excellent in vitro resources to better understand the unique metabolism of these fish. However, the current 2D cultures have not fully captured the complex metabolic profile of the Astyanax liver. It is known that 3D culturing can modulate the transcriptomic state of cells when compared to its 2D monolayer culture. Therefore, in order to broaden the possibilities of the in vitro system by modeling a wider gamut of metabolic pathways, we cultured the liver-derived Astyanax cells of both surface and cavefish into 3D spheroids. We successfully established 3D cultures at various cell seeding densities for several weeks and characterized the resultant transcriptomic and metabolic variations. We found that the 3D cultured Astyanax cells represent a wider range of metabolic pathways, including cell cycle changes and antioxidant activities, associated with liver functioning as compared to its monolayer culture. Additionally, the spheroids also exhibited surface and cave-specific metabolic signatures, making it a suitable system for evolutionary studies associated with cave adaptation. Taken together, the liver-derived spheroids prove to be a promising in vitro model for widening our understanding of metabolism in Astyanax mexicanus and of vertebrates in general.

evolutionary biology↗

bioRxiv2022

Targeting MALAT1 Augments Sensitivity to PARP Inhibition by Impairing Homologous Recombination in Prostate Cancer.

Poly(ADP-ribose) polymerase inhibitors (PARPi) have emerged as the most promising targeted therapeutic intervention for the treatment of metastatic castrate-resistant prostate cancer (mCRPC). However, the clinical utility of PARPi has been limited to a subset of patients who harbor aberrations in the homologous recombination (HR) pathway. Here, we report that targeting MALAT1, an oncogenic lncRNA, known to be elevated in advanced-stage prostate cancer (PCa) demonstrates contextual synthetic lethality with PARPi. We show that MALAT1 silencing reprograms the HR transcriptome, contriving BRCAness-like phenotype, thus enhancing sensitivity towards PARPi. Moreover, transcriptome profiles of mCRPC patients exhibit convergence between expression of MALAT1, HR pathway, and neuroendocrine markers. Mechanistically, we show that targeting MALAT1 leads to a decrease in EZH2, a member of polycomb repressor complex-2 (PRC2), which in turn upregulates the expression of RE1 Silencing Transcription Factor (REST), a key repressor of neuroendocrine differentiation. Overall, we showed that MALAT1 plays a pivotal role in maintaining genomic integrity, thereby promoting disease progression. Conclusively, our findings suggest that inhibiting MALAT1 confers PARPi sensitization in patients resistant to anti-androgens and conventional chemotherapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/494272v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@80d842org.highwire.dtl.DTLVardef@f60793org.highwire.dtl.DTLVardef@6b81eforg.highwire.dtl.DTLVardef@1fa35d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO The oncogenic lncRNA MALAT1 exhibits functional pleiotropy in PCa and promotes neuroendocrine differentiation. MALAT1 fosters PCa progression by modulating several hallmark oncogenic properties, such as malignant transformation, enhanced migratory capabilities, stemness, and ultimately contributes to drug resistance. MALAT1 enhances the transcriptional regulation of genes associated with homologous recombination thereby having a profound impact on the genome integrity in metastatic prostate cancer. Over the course of disease progression, it also promotes neuroendocrine trans-differentiation by depleting the levels of REST, the key repressor for NE transdifferentiation in prostate cancer. C_FIG

cancer biology↗

bioRxiv2022

Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odors

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus L. ( Ityp) and Dendroctonus ponderosae Hopkins ( Dpon) (Scolytinae), and the pine weevil Hylobius abietis L. ( Habi; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. Results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm non-host plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

neuroscience↗

bioRxiv2021

Discovery of an IKK2 Site that Allosterically Controls Its Activation

I{kappa}B kinase 2/{beta} (IKK2) is a critical regulator of inflammation which is inducibly activated by a host of stimuli. Aberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Efforts to prevent these diseases by small-molecule inhibitors of IKK2 activity have not been successful. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. Here we focused on identifying inhibitors to block IKK2 activity from an allosteric site, not the ATP-binding pocket. Using virtual screening, we first identified several candidate allosteric sites and screened for potential small-molecule binders, and then selected candidates inhibitory to IKK2 activity using cell-based functional assays. Hydrogen deuterium exchange coupled to mass-spectrometry (HDX-MS) and MS-MS assays revealed that a class of benzoyl conjugates of pyrrolidinedione covalently bound to a site located at the interface of the kinase domain (KD) and the helical domain (SDD), and inhibited IKK2 activation allosterically by preventing phosphorylation of its activation loop serines. Additionally, this class of inhibitor partially blocks IKK2s catalytic activity by enhancing dynamics within the ATP binding pocket and likely the general active site. Hydrogen deuterium exchange (HDX) experiments further revealed that while binding of substrate ATP perturbs only the local structure surrounding its binding site, binding to ATP-competitive or allosteric inhibitors induces structural perturbations in an expansive area including the helical domain. We propose that these allosteric sites can act as specific targets for the development of novel potent IKK inhibitors. SIGNIFICANCEAberrant activation of IKK2 is the leading cause of most inflammatory diseases and many associated cancers. Most inhibitors developed for IKK2 are ATP-competitive, and they are toxic in vivo due to their off-target effects. By combination of virtual screening and cell-based functional assays, we identified small-molecule binders of the class of benzoyl conjugates of pyrrolidinedione that block IKK2 activity from an allosteric site through covalent attachment and could be specific only for IKK2. HDX-MS and MS-MS assays identified a binding pocket with a Cys-Cys motif for these inhibitors, and revealed specific differences in IKK2 dynamics upon binding to substrate ATP vs ATP-competitive and allosteric inhibitors. Present work provides a framework for the development of allosteric inhibitors to combat IKK2-induced diseases inhibitors.

biochemistry↗

bioRxiv2021

A study of Mutation in ATP7B gene and its correlation with clinical phenotype and radiological features in Wilson Disease patients

IntroductionWilson Disease (WD) is an autosomal recessive disease caused by mutations in the ATP7B gene. Clinical manifestations of WD are variable. Identification of prevalent mutations in a given population is necessary to provide mutation-based molecular diagnosis. Previous studies have detected common mutations in this part of the world and our study aimed to correlate genotype with clinical and radiological features. MethodsA descriptive cross-sectional observational study was conducted over a period of two years in a tertiary care hospital and neurology referral unit of Kolkata, India. All WD patients within the study period and meeting the inclusion criteria were included. Demographic data collection, clinical examination and relevant laboratory investigations were done. Magnetic resonance imaging of brain and cognitive assessment by Mini Mental Score Exam (MMSE) were also performed. Blood was collected for genetic analyses. PCR-Sanger sequencing of exons 2,4,6,8,10,14,16,18 of ATP7B gene was done based on previous reports of mutation hotspots of ATP7B gene for WD in Eastern India. Genotype phenotype correlation was attempted using two supervised machine learning methods, viz. logistic regression with an elastic-net penalty and the random forest. ResultsOf 52 WD patients were included in the study, 57.7% were males. The mean age at diagnosis was 13.96 years. Majority (61.8%) of the patients had dystonia on presentation, followed by dysarthria (41.2%), tremor (17.6%) and ataxia (11.8%). The mean MMSE and Frontal Assessment Battery score were 23.74 and 10.63 respectively and both were lower than the normal baseline values.Out of the total cohort of 52 patients,15(28.8%) harbored previously reported common mutations from this part of the country. Of the 15, 12 had the same mutation of c.813C>A(p.cys271Ter).The presence of common mutationswas associated with several distinct clinical phenotypes in the mathematical models but larger sample sizes are needed to corroborate the correlation. ConclusionsWD patients in eastern India have significant genotypic and phenotypic diversity. Further studies with larger samples and screening of remaining exons are warranted.

neuroscience↗

bioRxiv2019

Spring-loaded inverted pendulum goes through two contraction-extension cycles during the single stance phase of walking

Despite the overall complexity of legged locomotion, the motion of the center of mass (COM) itself is relatively simple, and can be qualitatively described by simple mechanical models. The spring-loaded inverted pendulum (SLIP) is one such model, and describes both the COM motion and the ground reaction forces (GRFs) during running. Similarly, walking can be modeled by two SLIP-like legs (double SLIP or DSLIP). However, DSLIP has many limitations and is unlikely to serve as a quantitative model for walking. As a first step to obtaining a quantitative model for walking, we explored the ability of SLIP to model the single stance phase of walking across the entire range of walking speeds. We show that SLIP can be employed to quantitatively model the single stance phase except for two exceptions: first, it predicts larger horizontal GRFs than empirically observed. A new model - angular and radial spring-loaded inverted pendulum (ARSLIP) can overcome this deficit. Second, even the single stance phase has active elements, and therefore a quantitative model of locomotion would require active elements. Surprisingly, the leg spring undergoes a contraction-extension-contraction-extension (CECE) during walking; this cycling is partly responsible for the M-shaped GRFs produced during walking. The CECE cycle also lengthens the stance duration allowing the COM to travel passively for a longer time, and decreases the velocity redirection between the beginning and end of a step. A combination of ARSLIP along with active mechanisms during transition from one step to the next is necessary to describe walking.

bioengineering↗

bioRxiv2018

Kinematic and biomechanical analyses in Drosophila suggests that most legged locomotion in insects can be understood within a single framework.

Despite the fact that all insects have six legs, they display considerable differences in their legged locomotion. Are these differences irreconcilable, or simply different domains of the same system? In this study, we investigate walking in Drosophila. We find that despite the fact that flies walk extremely slowly relative to their size, they predominantly employ an alternating tripod gait, a gait typically associated with high-speed locomotion. The kinematics of their center of mass (CoM) is diametrically opposite to the CoM kinematics observed in insect runners such as cockroach. We resolve this tension between similar gaits, and differing kinematics in slow and high-speed locomotion by showing that the mechanics of a tripod gait naturally reduces to a simple biomechanical model which can support a range of kinematic patterns including both fly-like and cockroach-like patterns. These findings suggest that legged locomotion in different insects might represent different domains of the same system.

neuroscience↗

bioRxiv2018

Exonic unpaired elements modulate pre-mRNA structure for splice site recognition

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short ([lsim]70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

biochemistry↗

bioRxiv2018

In vitro assembly of an early spliceosome defining both splice sites

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML - a model pre-mRNA substrate - guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.

biochemistry↗