Biology subjects

Jana, S.

Publications and source records attributed to Jana, S..

30 records · Page 2Linked to original sources

bioRxiv2024

Phenotypic screening of covalent compound libraries identifies chloromethyl ketone antibiotics and MiaA as a new target

The emerging antibiotic resistance requires the development of new antibiotics working on novel bacterial targets. Here, we reported an antibiotic discovery workflow by combining the cysteine-reactive compound library phenotypic screening with activity-based protein profiling, which enables the rapid identification of lead compounds as well as new druggable targets in pathogens. Compounds featuring chloromethyl ketone scaffolds exhibited a notably high hit rate against both gram-negative and gram-positive bacterial strains, but not the more commonly used warheads such as acrylamide or chloroacetamide. Target identification of the lead compound, 10-F05, revealed that its primary targets in S. flexneri are FabH Cys112 and MiaA Cys273. We validated the target relevance through biochemical and genetic interactions. Mechanistic studies revealed modification of MiaA by 10-F05 impair substrate tRNA binding, leading to decreased bacterial stress resistance and virulence. Our findings underscore chloromethyl ketone as a novel antibacterial warhead in covalent antibiotic design. The study showcases that combining covalent compound library phenotypic screening with chemoproteomics is an efficient way to identify new drug targets as well as lead compounds, with the potential to open new research directions in drug discovery and chemical biology. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/576576v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@8d6420org.highwire.dtl.DTLVardef@a89d44org.highwire.dtl.DTLVardef@1ed0ae2org.highwire.dtl.DTLVardef@1659d3e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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

Isolation and characterization of a novel hormone receptor positive mammary adenocarcinoma MCa-P1362 with stromal drivers of tumor growth, metastasis, and drug resistance

Preclinical models that display spontaneous metastasis are necessary to improve therapeutic options for hormone receptor positive breast cancers. In this study, we conducted a detailed cellular and molecular characterization of MCa-P1362, a novel syngeneic Balb/c mouse model of metastatic breast cancer. MCa-P1362 cancer cells expressed estrogen receptors (ER), progesterone receptors (PR), and HER-2 receptors. MCa-P1362 cells proliferate in vitro and in vivo in response to estrogen, yet do not depend on steroid hormones for tumor progression. Further characterization of MCa-P1362 tumor explants shows that they contain a mixture of epithelial cancer cells and stromal cells. Based on transcriptomic and functional analyses of cancer and stromal cells, stem cells are present in both populations. Functional studies demonstrate that crosstalk between cancer and stromal cells promotes tumor growth, metastasis, and drug resistance. MCa-P1362 may serve as a useful preclinical model to investigate the cellular and molecular basis of hormone receptor positive tumor progression and therapeutic resistance.

cancer biology↗

bioRxiv2023

Combinatorial genetic strategy accelerates the discovery of cancer genotype-phenotype associations

Available genetically-defined cancer models are limited in genotypic and phenotypic complexity and underrepresent the heterogeneity of human cancer. Herein, we describe a combinatorial genetic strategy applied to an organoid transformation assay to rapidly generate diverse, clinically relevant bladder and prostate cancer models. Importantly, the clonal architecture of the resultant tumors can be resolved using single-cell or spatially resolved next-generation sequencing to uncover polygenic drivers of cancer phenotypes.

cancer biology↗

bioRxiv2023

PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR (p53 Inactivating TRIM28 associated RNA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to Temozolomide. Thus, we establish an alternate way of p53 inactivation by PITAR, which maintains low p53 levels in normal cells and attenuates the DNA damage response by p53. Finally, we propose PITAR as a potential GBM therapeutic target.

cancer biology↗

bioRxiv2023

Ultra-Fast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

Studying protein structures and dynamics directly in the cellular environments in which they function is essential to fully understand the molecular mechanisms underlying cellular processes. Site-directed spin-labeling (SDSL)--in combination with double electron-electron resonance (DEER) spectroscopy--has emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. In-cell DEER spectroscopy on proteins in mammalian cells has thus far not been possible due to the notable challenges of spin-labeling in live cells. In-cell SDSL requires exquisite biorthogonality, high labeling reaction rates and low background signal from unreacted residual spin label. While the bioorthogonal reaction must be highly specific and proceed under physiological conditions, many spin labels display time-dependent instability in the reducing cellular environment. Additionally, high concentrations of spin label can be toxic. Thus, an exceptionally fast bioorthogonal reaction is required that can allow for complete labeling with low concentrations of spin-label prior to loss of signal. Here we utilized genetic code expansion to site-specifically encode a novel family of small, tetrazine-bearing non-canonical amino acids (Tet-v4.0) at multiple sites in green fluorescent protein (GFP) and maltose binding protein (MBP) expressed both in E. coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxides--including a gem-diethyl-substituted nitroxide with enhanced stability in cells--with rate constants that can exceed 106 M-1 s-1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live HEK293T cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide added directly to the culture medium. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and successfully discerned the conformational state of MBP within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/525763v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1251523org.highwire.dtl.DTLVardef@18ee6ddorg.highwire.dtl.DTLVardef@d6d4b0org.highwire.dtl.DTLVardef@259a53_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Bridging the neural and algorithmic correlates of action-stopping

Rapid stopping of actions is crucial for survival. The algorithm underlying action-stopping is often described using the independent race model, where the race between independent go- and stop-processes determines whether one is able to stop. However, it is unclear whether humans have neural correlates of these processes. Here, using scalp potentials from three studies, we show that the mu (8-13 Hz) desynchronization over the contralateral motor cortex follows the predictions of a go-process: the activity builds up till an action is made. We also show that the low beta (13-20 Hz) activity over the frontal areas follows the predictions of a stop-process: the activity builds up after the stop signal and prior to the stopping latency. Using these neural correlates, we observed that: 1) The failure to stop mostly results from a rapid go-process, 2) The go-process seems to be influenced by the stop-process during the late stages of action-stopping, 3) Intrinsic prefrontal beta activity has a retardive effect on movement, 4) Violations of independence assumption in the failed stop trials may result from the activity of the stop-process. Future studies may use these neural correlates to resolve the mimicry between models of action-stopping.

neuroscience↗

bioRxiv2022

Probing the role of macromolecular crowding in cell volume regulation using fluorescence anisotropy

Cytoplasmic macromolecular crowding (MMC) influences multiple cellular functions. Using fluorescence anisotropy of EGFP, we explore the homeostasis of MMC in the cytoplasm and its role in cell volume regulation. Individual cells of different lineages maintain a distinctly unique average cytoplasmic MMC for considerably long durations. Despite diffusion, actin cytoskeleton facilitates spatially heterogeneous MMC in the cytoplasm. While hypertonic-stress triggers regulatory volume increase (RVI), other methods of increasing cytoplasmic MMC fail to start RVI, suggesting that cells lack capabilities for MMC homeostasis. During spreading or microtubule-depolymerized state, cells neglect changes in cytoplasmic MMC to undergo volume regulation. Inhibition of TNFR1 increases the membrane tension and deprives cells of their ability to undergo volume regulation. Current understanding contemplates that cytoplasmic MMC, the mechanical state of the plasma membrane or the actin cytoskeleton, could be sensors for cell volume regulation. Our observations establish the irrelevance of cytoplasmic MMC and the significance of plasma membrane tension in setting off the cell volume regulation machinery.

cell biology↗

bioRxiv2021

Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

The ability to site-specifically modify proteins at multiple sites in vivo will enable the study of protein function in its native environment with unprecedented levels of detail. Here, we present a versatile two-step strategy to meet this goal involving site-specific encoding of two distinct noncanonical amino acids bearing bioorthogonal handles into proteins in vivo followed by mutually orthogonal labeling. This general approach, that we call dual encoding and labeling (DEAL), allowed us to efficiently encoded tetrazine- and azide-bearing amino acids into a protein and demonstrate for the first time that the bioorthogonal labeling reactions with strained alkene and alkyne labels can function simultaneously and intracellularly with high yields when site-specifically encoded in a single protein. Using our DEAL system, we were able to perform topologically-defined protein-protein crosslinking, intramolecular stapling, and site-specific installation of fluorophores all inside living Escherichia coli cells, as well as study the DNA-binding properties of yeast Replication Protein A in vitro. By enabling the efficient dual modification of proteins in vivo, this DEAL approach provides a tool for the characterization and engineering of proteins in vivo.

synthetic biology↗

bioRxiv2021

An unusually long 5'UTR of ATP dependent cold shock DEAD-box RNA helicase gene csdA negatively regulates its own expression in Escherichia coli

Cold-shock DEAD-box protein A (CsdA) is an ATP dependant cold shock DEAD-box RNA helicase. It is a major cold shock protein needed for the cold adaptation in Escherichia coli. Although the CsdA has been studied at the protein level, further studies are necessary to understand its mechanisms of gene regulations. In this regard, we have constructed a promoter less vector with the ORF of a GFP reporter and found that the promoter of the csdA gene resides far upstream (more than 800 bases) of its coding region. Furthermore, our in vivo deletion experiment has confirmed the existence of this extraordinarily long 5UTR. Our results show that it represses its own expression. In addition, the short peptide encoding (26 aa) yrbN gene resides within this 5UTR as an operon with 8 overlapping nucleotides with the csdA coding region. Besides, we observed that the csdA gene expression may also occur along with immediate upstream (180 nucleotides) nlpI gene both at 37{degrees}C and 15{degrees}C and from the pnp gene (1173 nucleotides upstream) only during cold. In conclusion, csdA gene has operon feature like prokaryotes, in contrast, it also contains an extraordinarily long 5UTR, found in eukaryotes.

molecular biology↗

bioRxiv2020

A velocity plan with internal feedback control best explains modulation of saccade kinematics during eye-hand coordination.

Fast movements like saccadic eye movements that occur in the absence of sensory feedback are often thought to be under internal feedback control. In this framework, a desired input in the form of desired displacement signal is widely believed to be encoded in a spatial map of the superior colliculus (SC). This is then converted into a dynamic velocity signal that drives the oculomotor neurons. However, recent evidence has shown the presence of a dynamic signal within SC neurons, which correlates with saccade velocity. Hence, we used models based on optimal control theory to test whether saccadic execution could be achieved by a velocity based internal feedback controller. We compared the ability of a trajectory control model based on velocity to that of an endpoint control model based on final displacement to capture saccade behavior of modulation of peak saccade velocity by the hand movement, independent of the saccade amplitude. The trajectory control model tracking the desired velocity in optimal feedback control framework predicted this saccade velocity modulation better than an endpoint control model. These results suggest that the saccadic system has the flexibility to incorporate a velocity plan based internal feedback control that is imposed by task context. NEW & NOTEWORTHYWe show that the saccade generation system may use an explicit velocity tracking controller when demand arises. Modulation of peak saccade velocity due to modulation of the velocity of the accompanying hand movement was better captured using a velocity tracking stochastic optimal control model compared to an endpoint model of saccade control. This is the first evidence of trajectory planning and control for the saccadic system based on optimal control theory.

neuroscience↗

bioRxiv2019

Temporal cascade of frontal, motor and muscle processes underlying human action-stopping

Action-stopping is a canonical executive function thought to involve top-down control over the motor system. Here we aimed to validate this stopping system using high temporal resolution methods in humans. We show that, following the requirement to stop, there was an increase of right frontal beta ([~]13 to 30 Hz) at [~]120 ms, likely a proxy of right inferior frontal gyrus; then, at 140 ms, there was a broad skeletomotor suppression, likely reflecting the impact of the subthalamic nucleus on basal ganglia output; then, at [~]160 ms, suppression was detected in the muscle, and, finally, the behavioral time of stopping was [~]220 ms. This temporal cascade confirms a detailed model of action-stopping, and partitions it into subprocesses that are isolable to different nodes and are more precise than the behavioral speed of stopping. Variation in these subprocesses, including at the single-trial level, could better explain individual differences in impulse control.

neuroscience↗