Biology subjects

Chakraborty, S.

Publications and source records attributed to Chakraborty, S..

At least 199 records · Page 11Linked to original sources

bioRxiv2021

Wnt/β-catenin signaling and p68 conjointly regulate CHIP in colorectal cancer

The differential expression pattern of Carboxy terminus of Hsc70 Interacting Protein (CHIP, alias STIP1 Homology and U-box Containing Protein 1 or STUB1) in cancers is associated with ubiquitination mediated degradation of its client proteins. Emerging evidences suggest its abundant expression of CHIP in colorectal cancer compared to normal tissues, but the mechanistic detail of this augmented expression pattern is unclear. The signature driver of canonical Wnt pathway, {beta}-catenin, and its co-activator RNA helicase p68, are also overexpressed in colorectal cancer. In this study, we describe a novel mechanism of Wnt/{beta}-catenin and p68 mediated transcriptional activation of CHIP gene leading to enhanced proliferation of colorectal cancer cells. Wnt3A treatment and pharmacological activation of canonical Wnt signaling pathway resulted in increased nuclear translocation of {beta}-catenin and elevated expression of CHIP. Likewise, overexpression and knockdown of {beta}-catenin and p68 upregulated and downregulated CHIP expression, respectively, at both mRNA and protein levels. After cloning CHIP promoter, the increased and decreased promoter activities of CHIP induced by overexpression and knockdown of either {beta}-catenin or p68 further confirmed transcriptional regulation of CHIP gene by Wnt/{beta}-catenin signaling cascade. p68 along with {beta}-catenin were found to occupy Transcription Factor 4 (TCF4) binding sites on endogenous CHIP promoter and regulate its transcription. Finally, enhanced cellular propagation and migration of colorectal cancer cells induced by Wnt/{beta}-catenin-p68-CHIP axis established the significance of this pathway in oncogenesis. To the best of our knowledge, this is the first report elucidating the mechanistic details of transcriptional regulation of CHIP (STUB1) gene expression.

cancer biology↗

bioRxiv2021

Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival

Spatiotemporal-heterogeneity of glioblastoma (GBM) originating from the genomic and transcriptional variation in spatially distinct intra-tumor sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. We have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, the unique enrichment of the Mesenchymal subtype (MES) in 5ALA+ cells supports the hypothesis that MES 5ALA+ cells may drive GBM recurrence. Upregulation of the wound response pathway in 5ALA+ cells signifies the possibility of hijacking the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokine CCL2 that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by an IRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular signatures of 5ALA+ cells and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.

cancer biology↗

bioRxiv2021

Divergent early antibody responses define COVID-19 disease trajectories

A damaging inflammatory response is strongly implicated in the pathogenesis of severe COVID-19 but mechanisms contributing to this response are unclear. In two prospective cohorts, early non-neutralizing, afucosylated, anti-SARS-CoV-2 IgG predicted progression from mild, to more severe COVID-19. In contrast to the antibody structures that predicted disease progression, antibodies that were elicited by mRNA SARS-CoV-2 vaccines were low in Fc afucosylation and enriched in sialylation, both modifications that reduce the inflammatory potential of IgG. To study the biology afucosylated IgG immune complexes, we developed an in vivo model which revealed that human IgG-Fc{gamma}R interactions can regulate inflammation in the lung. Afucosylated IgG immune complexes induced inflammatory cytokine production and robust infiltration of the lung by immune cells. By contrast, vaccine elicited IgG did not promote an inflammatory lung response. Here, we show that IgG-Fc{gamma}R interactions can regulate inflammation in the lung and define distinct lung activities associated with the IgG that predict severe COVID-19 and protection against SARS-CoV-2. One Sentence SummaryDivergent early antibody responses predict COVID-19 disease trajectory and mRNA vaccine response and are functionally distinct in vivo.

immunology↗

bioRxiv2021

Real time observation of chaperone-modulated talin mechanics under single molecule resolution

Recent single-molecule studies have recognized talin as a mechanosensitive hub in focal adhesion, where its function is strongly regulated by mechanical force. For instance, at low force (below 5 pN), folded talin binds RIAM for integrin activation; whereas at high force (above 5 pN), it unfolds to activate vinculin binding for focal adhesion stabilization. Being a cytoplasmic protein, talin might interact with several cytosolic chaperones: however, the role of chaperones in talin mechanics is unknown. To address this question, we investigated the force response of a mechanically stable talin domain with a set of well-known holdase (DnaJ, DnaK, Hsp70, and Hsp40) and foldase (DnaKJE, DsbA) chaperones, using single-molecule magnetic tweezers. Our findings demonstrate that chaperone could affect adhesion proteins stability by changing their folding mechanics; while holdase chaperones reduce their unfolding force to [~]6 pN, foldase chaperones shift it up to [~]15 pN. Since talin is mechanically synced within 2 pN force ranges, these changes are significant in cellular condition. Furthermore, we determined the fundamental mechanism of this altered mechanical stability, where chaperones directly reshape their energy landscape: unfoldase chaperone (DnaK) decreases the unfolding barrier height from 26.8 to 21.7 kBT, while foldase chaperone (DsbA) increases it to 33.5 kBT. We reconciled our observations with eukaryotic Hsp70 and Hsp40 chaperones and observed their similar function of decreasing the talin unfolding barrier to 23.1 kBT. The quantitative mapping of this chaperone-induced talin folding landscape directly illustrates that chaperones perturb the adhesion protein stability under physiological force, thereby influencing their force-dependent interactions and adhesion dynamics.

biophysics↗

bioRxiv2021

Spatiotemporal cytokinin signaling imaging reveals IPT3 function in nodule development in Medicago truncatula

Most legumes can establish a symbiotic association with soil rhizobia that triggers the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharide (LCO) signal in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) acts as an essential positive regulator of nodule organogenesis, and specific CK receptors are required for nodule formation. Temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In the present study, using a fluorescence-based CK sensor (TCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the CK responses sequential activation during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYL TRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::tdTOMATO and the CK sensor showed that IPT3 induction in the root stele at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula. One-sentence summaryHigh-resolution spatiotemporal imaging of cytokinin signaling reveals IPT3 function during indeterminate nodule development in Medicago truncatula

plant biology↗

bioRxiv2021

A novel mitragynine analog with low efficacy mu-opioid receptor agonism displays antinociception with attenuated adverse effects

Dried kratom leaves are anecdotally used for the treatment of pain, opioid dependence, and alcohol use disorder. We have previously shown that kratoms natural products (mitragynine) and semi-synthetic analogs (7-hydroxy mitragynine (7OH) and mitragynine pseudoindoxyl) are mu opioid receptor (MOR) agonists that show minimal {beta}-arrestin2 recruitment. To further investigate the structure activity relationships of G-protein potency, efficacy, and {beta}-arrestin2 recruitment, we diversified the mitragynine/7OH templates at the C9, -10 and -12 positions of the aromatic ring of the indole moiety. Three lead C9 analogs, synthesized by swapping the 9-methoxy group with varied substituents, namely phenyl (SC11), methyl (SC12), 3-furanyl (SC13), were further characterized using a panel of in vitro and ex vivo electrophysiology assays. All three compounds were partial agonists with lower efficacy than both DAMGO and morphine in heterologous G-protein assays and synaptic physiology. SC11-13 also showed lower recruitment of both {beta}-arrestin subtypes compared to DAMGO, and in assays with limited MOR receptor reserve, the G-protein efficacy of SC11, SC12 and SC13 was comparable to buprenorphine. In mouse models, at equianalgesic doses SC13 showed MOR-dependent analgesia with potency similar to morphine without respiratory depression, hyperlocomotion, constipation, or place conditioning. Taken together, these results suggest that MOR agonists with a G-protein efficacy profile similar to buprenorphine can be developed into opioids that are effective analgesics with greatly reduced liabilities.

pharmacology and toxicology↗

bioRxiv2021

Exploring the role of glycans in the interaction of SARS-CoV-2 RBD and human receptor ACE2

COVID-19 is a highly infectious respiratory disease caused by the novel coronavirus SARS-CoV-2. It has become a global pandemic and its frequent mutations may pose new challenges for vaccine design. During viral infection, the Spike RBD of SARS-CoV-2 binds the human host cell receptor ACE2, enabling the virus to enter the host cell. Both the Spike and ACE2 are densely glycosylated, and it is unclear how distinctive glycan types may modulate the interaction of RBD and ACE2. Detailed understanding of these determinants is key for the development of novel therapeutic strategies. To this end, we perform extensive all-atom simulations of the (i) RBD-ACE2 complex without glycans, (ii) RBD-ACE2 with oligomannose MAN9 glycans in ACE2, and (iii) RBD-ACE2 with complex FA2 glycans in ACE2. These simulations identify the key residues at the RBD-ACE2 interface that form contacts with higher probabilities, thus providing a quantitative evaluation that complements recent structural studies. Notably, we find that this RBD-ACE2 contact signature is not altered by the presence of different glycoforms, suggesting that RBD-ACE2 interaction is robust. Applying our simulated results, we illustrate how the recently prevalent N501Y mutation may alter specific interactions with host ACE2 that facilitate the virus-host binding. Furthermore, our simulations reveal how the glycan on Asn90 of ACE2 can play a distinct role in the binding and unbinding of RBD. Finally, an energetics analysis shows that MAN9 glycans on ACE2 decrease RBD-ACE2 affinity, while FA2 glycans lead to enhanced binding of the complex. Together, our results provide a more comprehensive picture of the detailed interplay between virus and human receptor, which is much needed for the discovery of effective treatments that aim at modulating the physical-chemical properties of this virus.

biophysics↗

bioRxiv2021

Identification of Blood Based biomarker for Huntington's disease using in-silico gene expression analysis

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder with profound phenotypic characters. HD is at present incurable and there are several trials going on to find a cure. HD is caused when there is a mutation in the Huntingtin gene which is found to be associated with axonal transport. Diagnosis is based on the signs and symptoms of the patients but by that time the psychomotor problems have already reached the level from where reversing the disease is impossible. Blood based biomarkers can be used for the diagnosis of the disease at an early stage. In this study several gene expression study data were analyzed and there were 329 Differentially Expressed Genes (DEGs) in all the three chosen datasets. Protein protein interaction network was created using STRING and CytoHubba plug-in was used to identify top ten hub genes which are CXCL8, PSMC6, UBE2D1, UBE2D1, CD27, UBE2D3, SF3B1, CASP3, EIF4E, BIRC2 and PTEN. Online software Enrichr was used for Gene Ontology and KEGG pathway enrichment analysis to find out the biological process, molecular function, cellular component and the pathways that were enriched in HD. This study finds out that those genes which were present in all the three datasets namely FNDC3A, BCLAF1 and ALCAM were not the hub genes. So further studies are required for identifying a potential biomarker of HD.

neuroscience↗

bioRxiv2021

In-vivo protein nitration and de-nitration facilitate Vibrio cholerae cell survival under anaerobic condition: Consequences of Nitrite induced protein nitration

Protein tyrosine nitration (PTN), a highly selective post translational modification, occurs in both prokaryotic and eukaryotic cells under nitrosative stress1. It is reported that the activities of many proteins are altered due to PTN2. PTN is found to be associated with many pathophysiological conditions like neurodegenerative and cardiac diseases etc.3. However, its physiological function is not yet clear. Like all other gut pathogens Vibrio cholerae also faces nitrosative stress in the gut environment which makes its proteome more vulnerable to PTN. Here, we report for the first time in-vivo PTN in V. cholerae. We show that in-vivo protein nitration is nitrite dependent and nitration-denitration phenomenon actually facilitates V. cholerae cell survival in anaerobic or hypoxic condition. In our study, we found that the extent of in-vivo nitration is negatively correlated with the intracellular nitrite content and maximum nitration occurs during log phase of V. cholerae. Most interestingly, a significant denitration was associated with increase in intracellular nitrate content during anaerobic incubation of aerobically grown late log phase cultures. In-vivo nitration could provide an avenue for toxic nitrite storage and nitrosative stress tolerance mechanism in many gut pathogens, whereas denitration could supply nitrate for cell survival in anaerobic nitrate deficient environment.

microbiology↗

bioRxiv2021

Effect of ligand sensing on flagellar bundle formation in bacteria

E. coli swims in liquid media by rotating long appendages called flagella. The direction of rotation of each flagellum is governed by a transmembrane rotary nanomotor, which receives signals from ligand-specific receptors. Attractants bias the motor to rotate in CCW direction causing flagella to bundle and provide thrust for locomotion. Recent studies have shown that sensing not only leads to increase in CCW bias but also increases the motor rotation speed due to the recruitment of additional stator units bound to the rotor. Despite the detailed studies on bacterial motility, the effect of ligand sensing on the synchronization of flagellar filaments leading to bundle formation and changes in bundle geometry are not clear. In this work, we performed real-time imaging of the flagellar bundle of swimming cells in metabolising (glucose) and non-metabolisable (2-Deoxy-d-glucose) attractants. We characterized bundles during swimming by measuring visible distal length and the spread of filaments at poles. We show that sensing of attractant by receptor leads to the formation of tight bundles when compared to control buffer. Contrary to previous studies, the swimming speeds were proportional to the bundle tightness with the latter dependent not only on the bias but also on the torque exerted by the motor. We further show that the observed wiggles in the swimming trajectory of cells is directly proportional to the spread angles of bundle and is effected by both motor CCW bias and torque. Mutant cells, which were rendered non-motile due to the absence of the PTS (phosphotransferase system) sugar uptake mechanism, exhibited motility when exposed to the non-metabolisable attractant confirming that mere sensing can induce torque in flagellar motor. These results clarify the role of sensing and metabolism on bundle formation and its impact on the motility of cells. Statement of significancePeritrichously flagellated E. coli swims away or towards ligands by biasing the direction of rotation of its flagellar motor. Recently, it has been shown that motor speed is also modulated on merely sensing a ligand. How does this impact flagellar bundle formation and swimming behavior? Using real-time imaging, we show that the bundle geometry changes in response to both metabolisable and non-metabolisable ligand. Mere sensing of a ligand temporarily increases the motor torque and CCW bias that causes tight flagellar bundles and leads to smooth swimming trajectories at high speeds. Our result provides strong evidence of a new signalling pathway that controls the flagellar motor speed to enable the bacteria to respond efficiently to changes in its environment.

biophysics↗

bioRxiv2021

Effects of treatment with three antibiotics, vancomycin, neomycin, and AVNM on gut microbiome in C57BL/6 mice

Higher organisms, especially mammals, harbor diverse microbiota in the gut that plays a major role in maintaining health and physiological homeostasis. Perturbation of gut flora helps identifying their roles. Antibiotics are potent perturbing agents of microbiome. Select antibiotics like vancomycin, neomycin, and AVNM (an antibiotic cocktail containing ampicillin, vancomycin, neomycin, and metronidazole) were used to perturb the gut microbiota of C57BL/6 male mice to understand their roles in host immunity and metabolism. The current study revealed that the resulting gut microbial composition was different, and diversity (at the phylum and genus level) was reduced differentially following each antibiotic treatment. Vancomycin treatment caused a significant increase in Verrucomicrobia and Proteobacteria phyla. The treatment with neomycin yielded an increase in the Bacteroidetes phylum, while the treatment with AVNM led to an increase in Proteobacteria phylum with lowest diversity of microbiome in the gut. The current results also revealed that the different antibiotics treatment caused variation in the cecal index, expression of immune genes (TNF-, IL-10, IFN-{gamma}) in the colon, and short-chain fatty acids (SCFA) level in the blood of mice. A strong correlation was observed for antibiotic-induced differential dysbiosis patterns of gut microbiota and the altered immune and SCFA profile of the host. The outcome of the present study could be clinically important.

immunology↗

bioRxiv2021

Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

bioinformatics↗

bioRxiv2021

Baseline cell proliferation rates and response to UV differ in Lymphoblastoid Cell Lines derived from healthy individuals of extreme constitution types

Differences in human phenotypes and susceptibility to complex diseases are an outcome of genetic and environmental interactions. This is evident in diseases that progress through a common set of intermediate patho-endophenotypes. Precision medicine aims to delineate the molecular players for individualized and early interventions. Functional studies in Lymphoblastoid Cell Line (LCL) model of phenotypically well characterized healthy individuals can help deconvolute and validate these molecular mechanisms. We developed LCLs from eight healthy individuals belonging to three extreme constitution types, deep phenotyped on the basis of Ayurveda. LCLs were characterized by karyotyping and immunophenotyping. Growth characteristics and response to UV was studied in these LCLs. We observed significant differences in cell proliferation rates between the contrasting groups such that one type (Kapha) proliferates significantly slower than the other two (Vata, Pitta). In response to UV, one fast growing group (Vata) shows higher cell death but recovers its numbers due to inherent higher rates of proliferation. The baseline differences in cell proliferation are key to understanding the survival of cells in UV stress. Variability in baseline cellular phenotypes not only explains the cellular basis of different constitutions types but can also help set priors during designing an individualized therapy with DNA damaging agents. This is the first study of its kind that shows variability of intermediate patho-phenotypes amongst healthy individuals that have implications in precision medicine.

cell biology↗

bioRxiv2021

Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

microRNA-122 (miR-122), a liver specific regulatory RNA, plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 is also a proinflammatory microRNA and when exported to tissue resident macrophage induces expression of inflammatory cytokines there. We found intercellular transfer of miR-122 in lipid exposed liver plays a role in liver inflammation. Exploring the mechanism of intercellular miR-122 transfer from hepatic cells, we detected MMP2 on the membrane of extracellular vesicles derived from hepatic cells which proved to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. Matrix metalloprotease 2 or MMP2 is a metalloproteinase that plays a key role in shaping and remodelling the extracellular matrix of human tissue by targeting degradation of matrix proteins. MMP2 was found to increase the movement of the EVs along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries. By targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/425217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@aec3b0org.highwire.dtl.DTLVardef@1b18e87org.highwire.dtl.DTLVardef@47e75org.highwire.dtl.DTLVardef@10a48e0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuman hepatocytes on exposure to high lipid export out miRNAs including proinflammatory miR-122. C_LIO_LIExtracellular miR-122 is taken up by tissue macrophages to get them activated to produce inflammatory cytokines. C_LIO_LIMMP2 present on the surface of the EVs released by hepatocyte is essential for miRNA transfer to macrophage cells C_LIO_LIInhibition of MMP2 prevents miR-122 transfer to macrophage and stops activation of recipient macrophage. C_LI

cancer biology↗

bioRxiv2020

APOE4 affects basal and NMDAR mediated protein synthesis in neurons by perturbing calcium homeostasis

Apolipoprotein E (APOE), one of the primary lipoproteins in the brain has three isoforms in humans - APOE2, APOE3, and APOE4. APOE4 is the most well-established risk factor increasing the pre-disposition for Alzheimers disease. The presence of the APOE4 allele alone is shown to cause synaptic defects in neurons and recent studies have identified multiple pathways directly influenced by APOE4. However, the mechanisms underlying APOE4 induced synaptic dysfunction remain elusive. Here, we report that the acute exposure of primary cortical neurons to APOE4 leads to a significant decrease in global protein synthesis. APOE4 treatment also abrogates the NMDA mediated translation response indicating an impairment of synaptic signaling. Importantly, we demonstrate that both APOE3 and APOE4 generate a distinct translation response which is closely linked to their respective calcium signature. Acute exposure to APOE3 causes a short burst of calcium through NMDARs in neurons leading to an initial decrease in protein synthesis which quickly recovers. Contrarily, APOE4 leads to a sustained increase in calcium levels by activating both NMDARs and L-VGCCs, thereby causing sustained translation inhibition through eEF2 phosphorylation, which in turn disrupts NMDAR response. Thus, we show that APOE4 affects basal and activity mediated protein synthesis response in neurons by affecting calcium homeostasis. We propose this as a possible mechanism to explain the synaptic dysfunction caused by APOE4. Highlights / SummaryO_LIAPOE3 treatment causes a short burst of calcium through NMDARs, leading to an acute increase in eEF2 phosphorylation which eventually recovers to basal levels. C_LIO_LIGlobal translation follows a similar temporal profile of initial inhibition followed by recovery in APOE3 treated neurons, thus unaffecting the NMDA mediated translation response. C_LIO_LIAPOE4 treatment activates both NMDARs and L-VGCCs leading to a marked elevation in calcium levels, thus causing sustained increase in eEF2 phosphorylation as well as global translation inhibition. C_LIO_LIHence, the NMDA mediated response is perturbed, potentially causing a stress-related phenotype in APOE4 treated neurons. C_LIO_LIThus, different calcium signatures and sources lead to distinct temporal profiles of translation. C_LI

neuroscience↗

bioRxiv2020

The Mechanical Roles of Chaperons

Protein folding under force is an integral source of generating mechanical energy in various cellular processes, ranging from protein translation to degradation. Although chaperones are well known to interact with proteins under mechanical force, how they respond to force and control cellular energetics remains unknown. To address this question, we introduce novel real-time magnetic-tweezers technology to mimic physiological force environment on client proteins, keeping the chaperones unperturbed. We studied two structurally distinct client proteins with seven different chaperones, independently and in combination, and proposed novel mechanical activity of chaperones. We found chaperones behave differently, while these client proteins are under force than its previously known functions. For instance, tunnel associated chaperones (DsbA and trigger factor), otherwise working as holdase without force, assist folding under force. This process generates an additional mechanical energy up to [~]147 zJ to facilitate translation or translocation. However, well-known cytoplasmic foldase chaperones (PDI, thioredoxin, or DnaKJE), does not possess the mechanical folding ability under force. Notably, the transferring chaperones (DnaK, DnaJ, SecB), act as unfoldase and slow down folding process, both in the presence and absence of force, to prevent misfolding of the client proteins. This provides an emerging insight of mechanical roles of chaperones: they can generate or consume energy by shifting energy landscape of the client proteins towards folded or unfolded state; suggesting an evolutionary mechanism to minimize the energy consumption in various biological processes.

biophysics↗

bioRxiv2020

BANCOVID, the first D614G variant mRNA-based vaccine candidate against SARS-CoV-2 elicits neutralizing antibody and balanced cellular immune response

Effective vaccine against SARS-CoV-2 is the utmost importance in the current world. More than 1 million deaths are accounted for relevant pandemic disease COVID-19. Recent data showed that D614G genotype of the virus is highly infectious and responsible for almost all infection for 2nd wave. Despite of multiple vaccine development initiatives, there are currently no report that has addressed this critical variant D614G as vaccine candidate. Here we report the development of an mRNA-LNP vaccine considering the D614G variant and characterization of the vaccine in preclinical trial. The surface plasmon resonance (SPR) data with spike protein as probe and competitive neutralization with RBD and S2 domain revealed that immunization generated specific antibody pools against the whole extracellular domain (RBD and S2) of the spike protein. The anti-sera and purified IgGs from immunized mice on day 7 and 14 neutralized SARS-CoV-2 pseudovirus in ACE2-expressing HEK293 cells in a dose dependent manner. Importantly, immunization protected mice lungs from pseudovirus entry and cytopathy. The immunologic responses have been implicated by a balanced and stable population of CD4+ cells with a Th1 bias. The IgG2a to IgG1 and (IgG2a+IgG2b) to (IgG1+IgG3) ratios were found 1{+/-}0.2 and 1.24{+/-}0.1, respectively. These values are comparatively higher than relevant values for other published SARS-CoV-2 vaccine in development,1, 2 and suggesting higher viral clearance capacity for our vaccine. The data suggested great promise for immediate translation of the technology to the clinic.

immunology↗

bioRxiv2020

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively opening these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a kinetic gating mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how opening is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed open undamaged DNA in solution and that such opening can largely occur without one or the other of the {beta}-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound open DNA adopts multiple conformations in solution notwithstanding the DNAs original structure or the {beta}-hairpins. Molecular dynamics simulations reveal compensatory roles of the {beta}-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA opening of undamaged sites and the dynamic nature of open DNA may shed light on how the protein functions within and beyond NER in cells.

biochemistry↗