The senescent mesothelial matrix accentuates colonization by ovarian cancer cells.

Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

ß-Galactosidase-activated nitroxyl (HNO) donors provide insights into redox cross-talk in senescent cells.

The cross-talk among reductive and oxidative species (redox cross-talk), especially those derived from sulfur, nitrogen and oxygen, influence several physiological processes including aging. One major hallmark of aging is cellular senescence, which is associated with chronic systemic inflammation. Here, we report a chemical tool that generates nitoxyl (HNO) upon activation by ß-galactosidase, an enzyme that is over-expressed in senescent cells. In a radiation-induced senescence model, the HNO donor suppressed reactive oxygen species (ROS) in a hydrogen sulfide (H2S)-dependent manner. Hence, the newly developed tool provides insights into redox cross-talk and establishes the foundation for new interventions that modulate levels of these species to mitigate oxidative stress and inflammation.

Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase.

Early detection of Alzheimer's disease (AD) is important for taking proper measures against AD pathogenesis. Acetylcholinesterase (AChE) is widely reported to be associated with the pathogenicity of AD. Here, employing the "acetylcholine-mimic" approach, we designed and synthesized a new class of naphthalimide (Naph)-based fluorogenic probes for specific detection of AChE and avoiding interference of butyrylcholinesterase (BuChE), the pseudocholinesterase. We investigated the action of the probes on Electrophorus electricus AChE, and the native human brain AChE that we expressed in Escherichia coli and purified in the active form for the first time. The probe Naph-3 exhibited a substantial fluorescence enhancement with AChE and majorly avoided BuChE. Naph-3 successfully crossed the cell membrane of the Neuro-2a cells and fluoresced upon reaction with endogenous AChE. We further established that the probe could be effectively used for screening AChE inhibitors. Our study provides a new avenue for the specific detection of AChE, which can be extended to the diagnosis of AChE-related complications.

Exopolyphosphatases PPX1 and PPX2 from Mycobacterium tuberculosis regulate dormancy response and pathogenesis.

Stress adaptation and virulence of various bacterial pathogens require stringent response pathways involving guanosine pentaphosphate and inorganic polyphosphate (PolyP). In M. tuberculosis, intracellular PolyP levels are maintained by the activities of polyphosphate kinase (PPK-1, PPK-2) and exopolyphosphatases (PPX-1, PPX-2). We demonstrate that these exopolyphosphatases cumulatively contribute to biofilm formation and survival of M. tuberculosis in nutrient limiting, low oxygen growth conditions and in macrophages. Characterization of single (Δppx2) and double knock out strain (dkppx) of M. tuberculosis demonstrated that these exopolyphosphatases are essential for establishing infection in guinea pigs and mice. Transcriptional profiling revealed that relative to the parental strain the expression of genes belonging to DosR regulon were significantly reduced in mid-log phase cultures of dkppx strain. We also show that PolyP inhibited the autophosphorylation activities associated with DosT and DosS sensor kinases. Host RNA-seq analysis revealed that transcripts involved in various antimicrobial pathways such as apoptosis, autophagy, macrophage activation, calcium signalling, innate and T-cell response were differentially expressed in lung tissues of dkppx strain infected mice. Taken together, we demonstrate that enzymes involved in PolyP homeostasis play a critical role in physiology and virulence of M. tuberculosis. These enzymes are attractive targets for developing novel interventions that might be active against drug-sensitive and drug-resistant M. tuberculosis.

Leveraging an enzyme/artificial substrate system to enhance cellular persulfides and mitigate neuroinflammation.

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications.

Identification of COVID-19 prognostic markers and therapeutic targets through meta-analysis and validation of Omics data from nasopharyngeal samples.

BACKGROUND: While our battle with the COVID-19 pandemic continues, a multitude of Omics data have been generated from patient samples in various studies. Translation of these data into clinical interventions against COVID-19 remains to be accomplished. Exploring host response to COVID-19 in the upper respiratory tract can unveil prognostic markers and therapeutic targets. METHODS: We conducted a meta-analysis of published transcriptome and proteome profiles of respiratory samples of COVID-19 patients to shortlist high confidence upregulated host factors. Subsequently, mRNA overexpression of selected genes was validated in nasal swabs from a cohort of COVID-19 positive/negative, symptomatic/asymptomatic individuals. Guided by this analysis, we sought to check for potential drug targets. An FDA-approved drug, Auranofin, was tested against SARS-CoV-2 replication in cell culture and Syrian hamster challenge model. FINDINGS: The meta-analysis and validation in the COVID-19 cohort revealed S100 family genes (S100A6, S100A8, S100A9, and S100P) as prognostic markers of severe COVID-19. Furthermore, Thioredoxin (TXN) was found to be consistently upregulated. Auranofin, which targets Thioredoxin reductase, was found to mitigate SARS-CoV-2 replication in vitro. Furthermore, oral administration of Auranofin in Syrian hamsters in therapeutic as well as prophylactic regimen reduced viral replication, IL-6 production, and inflammation in the lungs. INTERPRETATION: Elevated mRNA level of S100s in the nasal swabs indicate severe COVID-19 disease, and FDA-approved drug Auranofin mitigated SARS-CoV-2 replication in preclinical hamster model. FUNDING: This study was supported by the DBT-IISc partnership program (DBT (IED/4/2020-MED/DBT)), the Infosys Young Investigator award (YI/2019/1106), DBT-BIRAC grant (BT/CS0007/CS/02/20) and the DBT-Wellcome Trust India Alliance Intermediate Fellowship (IA/I/18/1/503613) to ST lab.

Nanomotors Sense Local Physicochemical Heterogeneities in Tumor Microenvironments*.

The invasion of cancer is brought about by continuous interaction of malignant cells with their surrounding tissue microenvironment. Investigating the remodeling of local extracellular matrix (ECM) by invading cells can thus provide fundamental insights into the dynamics of cancer progression. In this paper, we use an active untethered nanomechanical tool, realized as magnetically driven nanomotors, to locally probe a 3D tissue culture environment. We observed that nanomotors preferentially adhere to the cancer-proximal ECM and magnitude of the adhesive force increased with cell lines of higher metastatic ability. We experimentally confirmed that sialic acid linkage specific to cancer-secreted ECM makes it differently charged, which causes this adhesion. In an assay consisting of both cancerous and non-cancerous epithelia, that mimics the in vivo histopathological milieu of a malignant breast tumor, we find that nanomotors preferentially decorate the region around the cancer cells.

Biofilms of the non-tuberculous Mycobacterium chelonae form an extracellular matrix and display distinct expression patterns.

Mycobacterium chelonae is an environmental, non-tuberculous mycobacterial species, capable of causing infections in humans. Biofilm formation is a key strategy used by M. chelonae in colonising niches in the environment and in the host. We studied a water-air interface (pellicle) biofilm of M. chelonae using a wide array of approaches to outline the molecular structure and composition of the biofilm. Scanning electron micrographs showed that M. chelonae biofilms produced an extracellular matrix. Using a combination of biochemical analysis, Raman spectroscopy, and fluorescence microscopy, we showed the matrix to consist of proteins, carbohydrates, lipids and eDNA. Glucose was the predominant sugar present in the biofilm matrix, and its relative abundance decreased in late (established) biofilms. RNA-seq analysis of the biofilms showed upregulation of genes involved in redox metabolism. Additionally, genes involved in mycolic acid, other lipid and glyoxylate metabolism were also upregulated in the early biofilms.

Helical nanobots as mechanical probes of intra- and extracellular environments.

A rheological probe that can measure mechanical properties of biological milieu at well-defined locations with high spatial resolution, on a time scale faster than most biological processes, can further improve our understanding of how living systems operate and behave. Here, we demonstrate nanorobots actively driven in realistic ex vivo biological systems for fast mechanical measurements with high spatial accuracy. In the various demonstrations of magnetic nanobots as mechanical probes, we report the first direct observation of the internalization of probes by a living cell, the accurate measurement of the 'fluid phase' cytoplasmic viscosity of ~200 cP for a HeLa cell, demonstration of intracellular measurements in cells derived from human patients; all of which establish the strength of this novel technique for measurements in both intra- and extracellular environments.

Carbonyl Sulfide (COS) Donor Induced Protein Persulfidation Protects against Oxidative Stress.

The emergence of hydrogen sulfide (H2 S) as an important signalling molecule in redox biology with therapeutic potential has triggered interest in generating this molecule within cells. One strategy that has been proposed is to use carbonyl sulfide (COS) as a surrogate for hydrogen sulfide. Small molecules that generate COS have been shown to produce hydrogen sulfide in the presence of carbonic anhydrase, a widely prevalent enzyme. However, other studies have indicated that COS may have biological effects which are distinct from H2 S. Thus, it would be useful to develop tools to compare (and contrast) effects of COS and H2 S. Here we report enzyme-activated COS donors that are capable of inducing protein persulfidation, which is symptomatic of generation of hydrogen sulfide. The COS donors are also capable of mitigating stress induced by elevated reactive oxygen species. Together, our data suggests that the effects of COS parallel that of hydrogen sulfide, laying the foundation for further development of these donors as possible therapeutic agents.

Heterodimerization of Mu Opioid Receptor Protomer with Dopamine D2 Receptor Modulates Agonist-Induced Internalization of Mu Opioid Receptor.

The interplay between the dopamine (DA) and opioid systems in the brain is known to modulate the additive effects of substances of abuse. On one hand, opioids serve mankind by their analgesic properties, which are mediated via the mu opioid receptor (MOR), a Class A G protein-coupled receptor (GPCR), but on the other hand, they pose a potential threat by causing undesired side effects such as tolerance and dependence, for which the exact molecular mechanism is still unknown. Using human embryonic kidney 293T (HEK 293T) and HeLa cells transfected with MOR and the dopamine D2 receptor (D2R), we demonstrate that these receptors heterodimerize, using an array of biochemical and biophysical techniques such as coimmunoprecipitation (co-IP), bioluminescence resonance energy transfer (BRET1), FÓ§rster resonance energy transfer (FRET), and functional complementation of a split luciferase. Furthermore, live cell imaging revealed that D2LR, when coexpressed with MOR, slowed down internalization of MOR, following activation with the MOR agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO).

Luminescence- and Fluorescence-Based Complementation Assays to Screen for GPCR Oligomerization: Current State of the Art.

G protein-coupled receptors (GPCRs) have the propensity to form homo- and heterodimers. Dysfunction of these dimers has been associated with multiple diseases, e.g., pre-eclampsia, schizophrenia, and depression, among others. Over the past two decades, considerable efforts have been made towards the development of screening assays for studying these GPCR dimer complexes in living cells. As a first step, a robust in vitro assay in an overexpression system is essential to identify and characterize specific GPCR-GPCR interactions, followed by methodologies to demonstrate association at endogenous levels and eventually in vivo. This review focuses on protein complementation assays (PCAs) which have been utilized to study GPCR oligomerization. These approaches are typically fluorescence- and luminescence-based, making identification and localization of protein-protein interactions feasible. The GPCRs of interest are fused to complementary fluorescent or luminescent fragments that, upon GPCR di- or oligomerization, may reconstitute to a functional reporter, of which the activity can be measured. Various protein complementation assays have the disadvantage that the interaction between the reconstituted split fragments is irreversible, which can lead to false positive read-outs. Reversible systems offer several advantages, as they do not only allow to follow the kinetics of GPCR-GPCR interactions, but also allow evaluation of receptor complex modulation by ligands (either agonists or antagonists). Protein complementation assays may be used for high throughput screenings as well, which is highly relevant given the growing interest and effort to identify small molecule drugs that could potentially target disease-relevant dimers. In addition to providing an overview on how PCAs have allowed to gain better insights into GPCR-GPCR interactions, this review also aims at providing practical guidance on how to perform PCA-based assays.

Inorganic polyphosphate accumulation suppresses the dormancy response and virulence in Mycobacterium tuberculosis.

Stringent response pathways involving inorganic polyphosphate (PolyP) play an essential role in bacterial stress adaptation and virulence. The intracellular levels of PolyP are modulated by the activities of polyphosphate kinase-1 (PPK1), polyphosphate kinase-2 (PPK2), and exopolyphosphatases (PPXs). The genome of Mycobacterium tuberculosis encodes two functional PPXs, and simultaneous deletion of ppx1 and ppx2 results in a defect in biofilm formation. We demonstrate here that these PPXs cumulatively contribute to the ability of M. tuberculosis to survive in nutrient-limiting, low-oxygen growth conditions and also in macrophages. Characterization of single (Δppx2) and double knockout (dkppx) strains of M. tuberculosis indicated that PPX-mediated PolyP degradation is essential for establishing bacterial infection in guinea pigs. RNA-Seq-based transcriptional profiling revealed that relative to the parental strain, the expression levels of DosR regulon-regulated dormancy genes were significantly reduced in the dkppx mutant strain. In concordance, we also provide evidence that PolyP inhibits the autophosphorylation activities associated with DosT and DosS sensor kinases. The results in this study uncover that enzymes involved in PolyP homeostasis play a critical role in M. tuberculosis physiology and virulence and are attractive targets for developing more effective therapeutic interventions.

In vitro and in vivo Assessment of Protein Acetylation Status in Mycobacteria.

Protein acetylation is one of the standard post-translational modifications found in proteins across all organisms, along with phosphorylation which regulates diverse cellular processes. Acetylation of proteins can be enzymatically catalyzed through acetyltransferases, acetyl CoA synthetases or non-enzymatically through acyl carrier metabolic intermediates. In this protocol, using response regulator proteins as targets we describe the experimental strategy for probing the occurrence of acetylation using purified recombinant proteins in an in vitro setup. Further using M. smegmatis strains overexpressing the wild type or mutant response regulator protein, we also describe how in vivo acetylation can be validated in Mycobacterial proteins. The described approach can be used for analyzing acetylation of any mycobacterial protein under both in vitro and in vivo conditions.

Activation of Bacterial Histidine Kinases: Insights into the Kinetics of the cis Autophosphorylation Mechanism.

Two-component signaling systems (TCSs) are central to bacterial adaptation. However, the mechanisms underlying the reactions involving TCS proteins and their reaction rates are largely undetermined. Here, we employed a combined experimental and theoretical approach to elucidate the kinetics of autophosphorylation of three histidine kinases (HKs) of Mycobacterium tuberculosis, viz., MtrB, PrrB, and PhoR, all known to play a role in regulating its virulence. Using wild-type and mutant proteins, we performed dimerization assays, thermophoretic-affinity measurements, and competition-based phosphorylation assays to establish that for HK, MtrB autophosphorylation occurs in cis, similar to what has been proposed for the PhoR and PrrB HKs. Next, to determine the kinetics of cis autophosphorylation, we used a quantitative high-throughput assay and identified a two-step mechanism of HK activation, involving (i) the reversible association of HK with ATP, followed by (ii) its phosphorylation. We developed a mathematical model based on this two-step cis mechanism that captured the experimental data. Best-fit parameter values yielded estimates of the extent of HK-ATP association and the rates of HK autophosphorylation, allowing quantification of the propensity of HK autophosphorylation. Our combined experimental and theoretical approach presents a facile, scalable tool to quantify reactions involving bacterial TCS proteins, useful in antibacterial drug development strategies.IMPORTANCE Two-component systems consisting of an input-sensing histidine kinase (HK) and an output-generating response regulator (RR) are one of the key apparatuses utilized by bacteria for adapting to the extracellular milieu. HK autophosphorylation is shown to occur primarily in trans (intermolecular) and more recently shown to occur in cis (intramolecular). Although the catalysis of HK activation remains universal, the reaction scheme for evaluation of the kinetic parameter differs between these designs and cis mode largely remains unexplored. We combined experimental and theoretical approach to unravel two-step mechanism of activation of three cis mode HKs of M. tuberculosis The new mathematical model yields best-fit parameters to estimate the rates of HK-ATP association and HK autophosphorylation.

Spatiotemporal Modulation of ERK Activation by GPCRs.

ERK1/2 (extracellular signal-regulated protein kinases) are the nodal proteins that regulate diverse cellular functions primarily in response to activation from receptor tyrosine kinases (RTKs). Not only is ERK activated through a variety of RTKs, but noncanonical signaling through GPCRs also activates them. Such multimodal activation allows appropriate integration of many inputs to critical cell fate decisions such as proliferation and differentiation that MAP kinases typically regulate. MAP kinases also regulate many polar responses such as apoptosis and proliferation, dedifferentiation-differentiation, and the diversity in the outcomes though the same terminal molecule can be explained based on differences in the activation dynamics and rates. However, two processes have now been established as drivers for most of the diversity recorded in the outcomes of MAP kinase signaling. These parameters are cellular compartmentalization, i.e., spatial confinement of the molecules participating in a pathway and changes in the kinetics of the activation-deactivation, i.e., temporal regulation. While phosphorylation is the key to activating responses, specifically for ERK, the terminal MAP kinase, it is the spatiotemporal dynamics that governs the outcome generated by it. This chapter reviews our present understanding of the spatial and temporal regulation of MAP kinase cascade and the ERK activity, specifically through GPCRs.

ERK Activation Pathways Downstream of GPCRs.

GPCRs, the 7-TM receptors, represent a class of cell surface receptors which modulate a variety of physiological responses. The serpentine structure in addition to contributing the diversity of stimuli these receptors can sense also provides flexibility to the extracellular and intracellular regions where other proteins can interact with and can form functionally active multimeric entities. The range in signaling and physiological responses generated by these receptors can be attributed to a large repertoire of the receptor subtypes as well as their differential coupling to various classes of G-protein subunits and other proteins which facilitate multistate activation. A multistate GPCR can engage diverse signaling molecules, thereby modulating not only the canonical cellular responses but also noncanonical responses typically associated with activation of other cascades such as RTK and MAPK/ERK signaling. Given the crucial involvement of MAP kinase/ERK signaling in cell fate determination specially with respect to regulating cell proliferation, cellular apoptosis, and survival, GPCR-mediated cross-activation of MAPK has been explored in various systems and shown to involve functional integration of multiple pathways. This review describes the present knowledge of the different mechanisms of ERK activation downstream of GPCRs and our present understanding of receptor-dependent and -independent MAPK activation cascades.

Maneuverability of Magnetic Nanomotors Inside Living Cells.

Spatiotemporally controlled active manipulation of external micro-/nanoprobes inside living cells can lead to development of innovative biomedical technologies and inspire fundamental studies of various biophysical phenomena. Examples include gene silencing applications, real-time mechanical mapping of the intracellular environment, studying cellular response to local stress, and many more. Here, for the first time, cellular internalization and subsequent intracellular manipulation of a system of helical nanomotors driven by small rotating magnetic fields with no adverse effect on the cellular viability are demonstrated. This remote method of fuelling and guidance limits the effect of mechanical transduction to cells containing external probes, in contrast to ultrasonically or chemically powered techniques that perturb the entire experimental volume. The investigation comprises three cell types, containing both cancerous and noncancerous types, and is aimed toward analyzing and engineering the motion of helical propellers through the crowded intracellular space. The studies provide evidence for the strong anisotropy, heterogeneity, and spatiotemporal variability of the cellular interior, and confirm the suitability of helical magnetic nanoprobes as a promising tool for future cellular investigations and applications.

Visible-Light-Triggered Uncaging of Carbonyl Sulfide for Hydrogen Sulfide (H2S) Release.

Generation of hydrogen sulfide (H2S) is challenging and few methods are capable of localized delivery of this gas. Here, a boron dipyrromethene-based carbamothioate (BDP-H2S) that is uncaged by visible light of 470 nm to generate carbonyl sulfide (COS), which is rapidly hydrolyzed to H2S in the presence of carbonic anhydrase, a widely prevalent enzyme, is reported.