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Chakraborty, S.

Publications and source records attributed to Chakraborty, S..

At least 163 records · Page 9Linked to original sources
bioRxiv2023

A Cost-effective Purification Process for Erythropoietin Biosimilar through Downstream Process Engineering

Well-characterized and scalable downstream process for purification of biologics is extremely demanding for delivering quality therapeutics to patients at a reasonable price. Erythropoietin (EPO) is a blockbuster biologic with diverse clinical applications but its application is limited to financially well-off societies due to high price. The high price of EPO is associated with the technical difficulties related to the purification challenge to obtain qualified product with a cost-effective defined process. Though there are reports for purification of EPO but there is no report of well-characterized downstream process with critical process parameters (CPPs) that can deliver EPO consistently satisfying the quality target product profile (QTPP), which is a critical regulatory requirement. To advance the field, we applied quality by design (QbD) principle and design of experiment (DoE) protocol to establish an effective process, which is scalable up to 100x batch size satisfying QTPP. We have successfully transformed the process from static mode to dynamic mode and validated. Insignificant variation (p> 0.05) within and between 1x, 10x and 100x batches showed that the process is reproducible and seamlessly scalable. The biochemical analysis along with the biofunctionality data ensures that the products from different-scale batches were indifferent and comparable to a reference product. Our study thereby established a robust and scalable downstream process of EPO biosimilar satisfying QTPP. The technological scheme presented here can speed-up the production of not only EPO but many other life-saving biologics and make them available to mass population at a reduced cost.

bioengineering↗
bioRxiv2023

The neurodevelopmental transcriptome of the Drosophila melanogaster microcephaly gene abnormal spindle reveals a role for temporal transcription factors and the immune system in regulating brain size

The coordination of cellular behaviors during neurodevelopment is critical for determining the form, function, and size of the central nervous system. Mutations in the vertebrate Abnormal Spindle-Like, Microcephaly Associated (ASPM) gene and its Drosophila melanogaster ortholog abnormal spindle (asp) lead to microcephaly, a reduction in overall brain size whose etiology remains poorly defined. Here we provide the neurodevelopmental transcriptional landscape for a Drosophila model for autosomal recessive primary microcephaly (MCPH) and extend our findings into the functional realm in an attempt to identify the key cellular mechanisms responsible for Asp-dependent brain growth and development. We identify multiple transcriptomic signatures, including new patterns of co-expressed genes in the developing CNS. Defects in optic lobe neurogenesis were detected in larval brains through downregulation of temporal transcription factors (tTFs) and Notch signaling targets, which correlated with a significant reduction in brain size and total cell numbers during the neurogenic window of development. We also found inflammation as a hallmark of asp MCPH brains, detectable throughout every stage of CNS development, which also contributes to the brain size phenotype. Finally, we show that apoptosis is not a primary driver of the asp MCPH phenotype, further highlighting an intrinsic Asp-dependent neurogenesis promotion mechanism that is independent of cell death. Collectively, our results suggest that the etiology of asp MCPH is complex and that a comprehensive view of the cellular basis of the disorder requires an understanding of how multiple pathway inputs collectively determine the microcephaly phenotype. AUTHOR SUMMARYAutosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by a reduction in brain size, intellect, and life span. Over 30 genes have been found mutated in human MCPH patients, with Abnormal Spindle-Like, Microcephaly Associated (ASPM) being the most common. Although the clinical aspects of the disorder are well-characterized, the underlying cellular and molecular mechanisms are not. The fruit fly, Drosophila melanogaster, has an ortholog of the ASPM gene named abnormal spindle (asp), and mutations also give rise to fruit flies with small brains. It had previously been suggested that mitotic spindle defects were responsible for the asp MCPH phenotype, preventing neural stem cells from dividing properly to generate the necessary number of neurons and glia in the brain. However, genetic studies in flies showed that this wasnt the case, suggesting that our knowledge of MCPH remains incomplete and must be revised. In this manuscript, we identified new pathways important for asp-dependent brain growth through transcriptional profiling. We found a number of key pathways disrupted in asp mutants, which have not been described previously. Using genetic tools in the fly, we tested a subset of these pathways to identify their contributions to the brain growth defects seen in asp mutants. Our results add to the growing number of pathways necessary for brain growth control, and provide a suitable foundation for follow-up genetic studies to assess MCPH.

developmental biology↗
bioRxiv2023

E3 Ubiquitin Ligase CHIP Drives Monoubiquitination-mediated Nuclear Import of Tumor Suppressor PTEN

Nuclear localization of Phosphatase and Tensin Homolog Deleted on Chromosome TEN (PTEN) is crucial for its tumor suppressive activities. Monoubiquitination has been established as a principal mechanism driving nuclear translocation of PTEN. In this study, we describe a novel mechanism wherein Carboxy terminus of Hsc70 Interacting Protein (CHIP) mediates monoubiquitination of PTEN, leading to the latters nuclear import. Analysis of subcellular fractions of multiple cell lines revealed a rise in both nuclear and total cellular PTEN levels under monoubiquitination-promoting conditions, an effect that was abrogated when CHIP expression was silenced. We established a time-point kinetics of CHIP-mediated nuclear translocation of PTEN using immunocytochemistry. We identified a role of Karyopherin 1 (KPNA1) in facilitating nuclear transport of monoubiquitinated PTEN. We further established a direct interaction between CHIP and PTEN inside the nuclear compartment, where CHIP could participate in either polyubiquitination or monoubiquitination of the latter. CHIP was found to monoubiquitinate PTEN on K13 and K289 residues. Finally, we showed that CHIP-mediated monoubiquitination of PTEN resulted in increased apoptosis, decreased cell viability and proliferation. This enhancement of PTENs tumor suppressive abilities by CHIP involved modulation of transcriptional activities of PTEN. To the best of our knowledge, this is the first report elucidating CHIPs non-canonical roles on PTEN, which is also established here as a nuclear interacting partner of CHIP.

cancer biology↗
bioRxiv2022

Impaired Barrier Integrity of the Skeletal Muscle Vascular Endothelium Drives Progression of Cancer Cachexia

Cancer patients experience cachexia, which is characterized by extensive skeletal muscle wasting that worsens the quality of life and increases mortality. Currently, there are no approved treatments that can effectively counteract cancer cachexia. Vascular endothelial cells (ECs) are essential for maintaining tissue perfusion, nutrient supply, and preventing inappropriate transmigration of immune cells into the tissue. However, little is known about the role of the muscle vasculature in cancer cachexia. We hypothesized that endothelial dysfunction in the skeletal muscle mediates cancer cachexia. Using transgenic pancreatic ductal adenocarcinoma (PDAC) mice and a tissue clearing and high-resolution 3D-tissue imaging approach, we found that the loss of skeletal muscle vascular density precedes the loss of muscle mass. Importantly, we show that cancer cachexia patients exhibit significantly decreased muscle vascular density and severe muscle atrophy when compared to non-cancer patients. Unbiased single cell transcriptomic analyses of the muscle endothelium unveiled a unique EC population present in cachexia muscles. Increased circulating Activin-A suppresses the expression of the transcriptional co-activator PGC1 in the muscle endothelium, thus disrupting junctional integrity in the vasculature and increasing vascular leakage. Conversely, restoration of endothelial-specific PGC1 prevented the decreased vascular density and muscle loss observed in tumor-bearing mice. Our study suggests that EC-PGC1 is essential for maintaining the integrity of the skeletal muscle vascular barrier and that restoring muscle endothelial function could be a valuable therapeutic approach to prevent or reverse cancer cachexia.

cancer biology↗
bioRxiv2022

Equipment-Free Personal Protective Equipment (PPE) Fabrication from Bacterial Cellulose-Derived Biomaterials via Waste-to-Wealth Conversion

The recent COVID-19 crisis necessitated the universal use of Personal Protection Equipment (PPE) kits, generating tons of plastic wastes that inevitably lead to environmental damage. Circumventing the challenges stemming from such undesirable non-degradability on disposal, here we present an eco-friendly, robust, yet inexpensive and equipment-free method of growing biodegradable PPE fabrics by the fermentation of locally-sourced organic feed stocks in a rural livelihood. Using a pre-acclimatized symbiotic culture, we report the production of a high yield (up to 3.2 g fabric/g substrate) of bacterial cellulose, a biopolymer matrix, obtained by bacterial weaving. This membrane has an intricate, self-assembled, nano-porous 3D architecture formed by randomly oriented cellulose fibres. Scanning electron microscopy reveals that the pore size of the membrane turns out to be in the tune of 140 nanometers on the average, indicating that it can filter out viruses effectively. In-vitro results demonstrate assured breathability through the membrane for a filter thickness of approximately 5 microns. When subjected to soil degradation, the fabrics are seen to disintegrate rapidly and fully decompose within 15 days. With a favourable cost proposition of less than 1 US$ per meter square of the developed fabric unit, our approach stands out in providing a unique sustainable, and production-ready alternative to synthetic PPE fabrics, solving community healthcare and environmental crisis, and opening up new avenues sustainable under-served livelihood at the same time. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/514716v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bfa2forg.highwire.dtl.DTLVardef@5649a3org.highwire.dtl.DTLVardef@fc99a4org.highwire.dtl.DTLVardef@1cae297_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗
bioRxiv2022

Equalizing epigenetically imprinted centromeres in early mammalian embryos

The CENP-A histone variant epigenetically defines centromeres, where its levels and locations are precisely maintained through mitotic cell divisions. However, differences in centromere CENP-A propagation in soma versus female/male germline remains poorly understood. Here, we generated CenpamScarlet mice and followed CENP-A dynamics in gametes, zygotes, and embryos. We found that, unlike somatic cells, progenitor female and male germ cells carry high centromeric CENP-A levels that decrease upon terminal differentiation. The reduction in CENP-A is differentially regulated between sexes, resulting in a ten-fold higher level in oocytes compared to sperm. In the zygote, the parent-of-origin CENP-A asymmetry is equalized prior to initial S-phase by redistribution of nuclear CENP-A from maternal to paternal chromosomes. Redistribution of CENP-A requires both CDK1/2 and PLK1 centromeric machinery. These experiments provide direct evidence for resetting of epigenetically imprinted centromeres in early pronuclear stage embryos and imply a mechanism to sense the non-equivalency of parental chromosomes. HighlightsO_LIIncreased CENP-A density at centromeres is a conserved property of germline stem cells while CENP-A reduction is coincident with germ cell differentiation C_LIO_LIPaternal and maternal CENP-A containing nucleosomes are intergenerationally inherited C_LIO_LICENP-A density at centromeres differs between male and female mature gametes C_LIO_LIUpon fertilization, maternal nuclear CENP-A is redistributed to equalize with parental CENP-A C_LIO_LICENP-C and MIS18BP1 are asymmetrically enriched in the parental pronuclei in accordance with CENP-A asymmetry. C_LIO_LILicensing for centromere equalization begins before zygotic DNA replication C_LI

developmental biology↗
bioRxiv2022

Exome-wide analysis reveals role of LRP1 and additional novel loci in cognition

Cognitive functioning is heritable, with metabolic risk factors known to accelerate ageassociated cognitive decline. Identifying genetic underpinnings of cognition is thus crucial. Here, we undertake single-variant and gene-based association analyses upon six neurocognitive phenotypes across six cognition domains in whole-exome sequencing data from 157,160 individuals in the UK Biobank to expound the genetic architecture of human cognition. We further identify genetic variants interacting with APOE, a significant genetic risk factor for cognitive decline, while controlling for lipid and glycemic risks, towards influencing cognition. Additionally, considering lipid and glycemic traits, we conduct bivariate analysis to underscore pleiotropic effects and also highlight suggestive mediation effects of metabolic risks on cognition. We report 18 independent novel loci associated with five cognitive domains while controlling for APOE isoform-carrier status and metabolic risk factors. Our novel variants are mostly in genes which could also impact cognition via their functions on synaptic plasticity and connectivity, oxidative stress, neuroinflammation. Variants in or near these identified loci show genetic links to cognitive functioning in association with APOE, Alzheimers disease and related dementia phenotypes and brain morphology phenotypes, and are also eQTLs significantly controlling expression of their corresponding genes in various regions of the brain. We further report four novel pairwise interactions between exome-wide significant loci and APOE variants influencing episodic memory, and simple processing speed while accounting for serum lipid and serum glycemic traits. We obtain both APOC1 and LRP1 as significantly associated with complex processing speed and visual attention in our gene-based analysis. They also exhibit significant interaction effect with APOE variants in influencing visual attention. We find that variants in APOC1 and LRP1 act as significant eQTLs for regulating their expression in basal ganglia and cerebellar hemispheres, crucial to visual attention. Taken together, our findings suggest that APOC1 and LRP1 have plausible roles along pathways of amyloid-{beta}, lipid and/or glucose metabolism in affecting visual attention and complex processing speed. Interestingly, variants in MTFR1L, PPFIA1, PCDHB16, ATP2A1 show evidence of pleiotropy and mediation effects through serum glucose/HDL levels affecting four different cognition domains. This is the first report from large-scale exome-wide study with evidence underscoring the effect of LRP1 on cognition. Our research highlights a novel set of loci that augments our understanding of the genetic underpinnings of cognition during ageing, considering cooccurring metabolic conditions that can confer genetic risk to cognitive decline in addition to APOE, which can aid in finding causal determinants of cognitive decline.

genetics↗
bioRxiv2022

Cryo-electron tomography reveals enrichment and identifies microtubule lumenal particles in neuronal differentiation

The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) structures and organizations of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons and pluripotent P19 cells. We obtain in-cell 3D maps of several lumenal particles from the respective cells and detect structural features that are common to all cell-types, underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggests a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggest a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.

cell biology↗
bioRxiv2022

Possible Functional Proximity of Various Organisms Based on Taste Receptors Genomics

Taste is one of the essential senses in providing the organism a faithful representation of the external world. Taste perception is responsible for basic food and drink appraisal and bestows the organism with valuable discriminatory power. Umami and sweet are "good" tastes that promote consumption of nutritive food, whereas bitter and sour are "bad" tastes that alert the organism to toxins and low pH, promoting rejection of foods containing harmful substances. Not every animal has the same sense of taste as humans. Variation in the taste receptor genes contributes to inter and intra organism differences of taste (sweet/bitter) sensation and preferences. Therefore a deeper understanding was needed to comprehend taste perception by various vertebrates and accordingly elucidate a possible proximity among them. In this study, a total 20 Type-1 (sweet) and 189 Type-2 (bitter) taste receptor complete-amino acid sequences were taken from the 20 vertebrate organisms (18 mammalian, 1 aves, and 1 amphibian). Among 10 primates, 8 including humans were very close based on genomics of taste receptors and rodent organisms viz. the rat and mouse were away from them. This investigation throws light on the similitude and dissimilitude of perception of sweet and bitter taste among 20 different organisms, steered by quantitative analysis of their genomic data. Furthermore, it enlightened that ligand binding affinity of sweet/bitter taste molecules in the taste receptors of any proximal pair of organisms would be similar.

genomics↗
bioRxiv2022

Decoding the Effects of Spike Receptor Binding Domain Mutations on Antibody Escape Abilities of Omicron Variants

Recent times witnessed an upsurge in the number of COVID cases which is primarily attributed to the emergence of several omicron variants, although there is substantial population vaccination coverage across the globe. Currently, many therapeutic antibodies have been approved for emergency usage. The present study critically evaluates the effect of mutations observed in several omicron variants on the binding affinities of different classes of RBD-specific antibodies using a combined approach of immunoinformatics and binding free energy calculations. Our binding affinity data clearly show that omicron variants achieve antibody escape abilities by incorporating mutations at the immunogenic hotspot residues for each specific class of antibody. K417N and Y505H point mutations are primarily accountable for the loss of class I antibody binding affinities. The K417N/Q493R/Q498R/Y505H combined mutant significantly reduces binding affinities for all the class I antibodies. E484A single mutation, on the other hand, drastically reduces binding affinities for most of the class II antibodies. E484A and E484A/Q493R double mutations cause a 33-38% reduction in binding affinity for the approved therapeutic monoclonal antibodies, Bamlanivimab (LY-CoV555). The Q498R RBD mutation observed across all the omicron variants can reduce ~12% binding affinity for REGN10987, a class III therapeutic antibody, and the L452R/Q498R double mutation causes a ~6% decrease in binding affinities for another class III therapeutic antibody, LY-CoV1404. Our data suggest that achieving the immune evasion abilities appears to be the selection pressure behind the emergence of omicron variants.

bioinformatics↗
bioRxiv2022

KDM6A loss sensitizes human acute myeloid leukemia to PARP and BCL2 inhibition

Acute myeloid leukemia (AML) is a heterogeneous, aggressive malignancy with dismal prognosis and with limited availability of targeted therapies. AML exhibits epigenetic deregulation and transcriptional plasticity that contributes to pathogenesis. KDM6 proteins are histone-3 lysine-27 demethylases that play major context dependent roles in AML evolution and therapy resistance. Here, we demonstrate that KDM6 demethylase function critically regulates DNA damage repair (DDR) gene expression programs in AML. Mechanistically, KDM6 family protein expression is regulated by genotoxic stress, with deficiency of KDM6A (UTX) and KDM6B (JMJD3) impairing DDR transcriptional activation and compromising repair potential. Acquired KDM6A loss-of-function mutations have been implicated in chemoresistance, although a significant percentage of relapsed AML have upregulated KDM6A. Based on these mechanistic findings, olaparib treatment significantly reduced engraftment of patient-derived xenografts. Thus KDM6A-mutant human primary AML samples have increased susceptibility to Poly-(ADP-ribose)-polymerase (PARP) inhibition in vivo. Crucially, a higher KDM6A expression is correlated with venetoclax tolerance. Loss of KDM6A increased mitochondrial activity, BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6A loss was accompanied with a downregulated BCL2A1, which is commonly associated with venetoclax resistance. Corroborating these results, dual targeting of PARP and BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing AML apoptosis, and primary AML cells carrying acquired KDM6A-domain mutations were even more sensitive to the combination. Together, our study illustrates a mechanistic rationale in support for a novel combination targeted therapy for human AML based on subtype heterogeneity, and establishes KDM6A as an important molecular regulator for determining therapeutic efficacy.

cancer biology↗
bioRxiv2022

EHD2 overexpression promotes tumorigenesis and metastasis in triple-negative breast cancer by regulating store-operated calcium entry

With nearly all cancer deaths a result of metastasis, elucidating novel pro-metastatic cellular adaptations could provide new therapeutic targets. Here, we show that overexpression of the EPS15-Homology Domain-containing 2 (EHD2) protein in a large subset of breast cancers (BCs), especially the triple-negative (TNBC) and HER2+ subtypes, correlates with shorter patient survival. The mRNAs for EHD2 and Caveolin-1/2, structural components of caveolae, show co-overexpression across breast tumors, predicting shorter survival in basal-like BC. EHD2 shRNA knockdown and CRISPR-Cas9 knockout of EHD2, together with mouse EHD2 rescue, in TNBC cell line models demonstrate a major positive role of EHD2 in promoting tumorigenesis and metastasis. Mechanistically, we link these roles of EHD2 to store-operated calcium entry (SOCE), with EHD2-dependent stabilization of plasma membrane caveolae ensuring high cell surface expression of the SOCE-linked calcium channel Orai1. The novel EHD2-SOCE oncogenic axis represents a potential therapeutic target in EHD2 and CAV1/2-overexpressing BC.

cancer biology↗
bioRxiv2022

Understanding the genome-wide transcription response to varying cAMP levels using phenomenological models in bacteria

Attempts to understand gene regulation by global transcription factors (TF) have largely been limited to expression studies under binary conditions of presence and absence of the TF. Studies addressing genome-wide transcriptional responses to changing TF concentration at high resolution are lacking. Here, we create a dataset containing the entire E.coli transcriptome as it responds to 10 different cAMP concentrations spanning the biological range. We use the Hills model to accurately summarise individual gene responses into 3 intuitively understandable parameters - k, n and Emax reflecting the midpoint of dynamic range, non-linearity and sensitivity of a gene. cAMP-regulated genes show a small dynamic range with midpoints centred around wild-type cAMP concentrations, with genes activating in a switch-like fashion. Using this approach we show that cAMP-CRP affinity at promoters is well correlated to the sensitivity(Emax) of genes but not to the midpoints of dynamic range(k). Finally, genes belonging to different functional classes are tuned to different k, n and Emax. We show phenomenological models to be a better alternative for studying gene expression trends compared to classical clustering methods with the phenomenological constants providing greater insights into how genes are tuned in a regulatory network.

bioinformatics↗
bioRxiv2022

Assessment of local potato cultivars found in Cis-Himalayan region of West Bengal through morphology and biochemical profiling

Potato is a major global food crop grown for tubers (underground storage stems) that are high in carbohydrates, minerals, and vitamins. The presence of anthocyanins in tuber skin and flesh can have a significant impact on customer preferences. Potatoes are also high in resistant starches, which have a variety of health benefits, including enhanced fullness, cholesterol reduction, and a reduced risk of colon cancer etc. In West Bengal, diabetes is prevalent. Similarly, according to ICMR studies, colon cancer affects 8.9% of all cancer patients in West Bengal cancer which is caused by ill eating habits or the consumption of foods that are low in antioxidants. 5-10% of colon cancers are genetically caused, while the remainder are caused by poor eating habits or the consumption of foods that are low in antioxidants. To address these issues, one strategy is to eat foods with a low glycemic index and high antioxidant content as a staple food. The tuber tissues of the potato (S. tuberosum L.) accumulate various quantities of anthocyanins, which are commonly consumed around the world. Anthocyanins are pigments that range in colour from red to purple and are found throughout the plant kingdom. Anthocyanins are powerful antioxidants that are water soluble. The red skin potato is well-known among the general public. The epidermal layer contains a high quantity of red anthocyanins, which causes the skin to be red. With this background the present study has been undertaken to address the following objectives morphological (quantitative and qualitative traits) and biochemical characterization of local potato cultivars and identification of most stable genotypes based on the anthocyanin content and tuber yield of local potato cultivars. For our recent research 14 local potato cultivar from North Bengal were collected and evaluated for DUS characterization showed wide range of variability with respect to different phenotypic variants of ten characters. Overall predominant DUS characters of local potato cultivars found in North Bengal are medium sized apical length of sprout, short stem height, compact foliage structure, open leaf structure, ovate lanceolate type leaflet shape, purple leaf sprout predominant colour, spherical leaf sprout shape, medium intensity of anthicyanin coloraton at the base, light intensity of anthicyanin coloraton at the tip and weak nature of light sprout pubescence base. The results of the present investigation suggest that local potato cultivars collected from the northern part of West Bengal showed a high level of genetic variation. The differences between genotypes were highly significant at the 1% and 5% alpha level for all characters like tuber yield, length and weight of tuber, length of sprout, chlorophyll content, canopy temperature, and number of tuber per plant, according to the analysis of variance. Three quantitative traits namely tuber yield, tuber breadth, tuber length played major role in the genetic variance. Anthocyanin content had more contribution to diversify the local potato cultures according to biochemical characterization AMMI analysis suggested that Jalpai from CoochBehar is the most stable potato cultivars in respect to anthocyanin content and tuber yield per plant.

plant biology↗
bioRxiv2022

Trimethylamine N-oxide (TMAO) enhances substrate mechanical stability probed by single molecule magnetic tweezers

Trimethylamine N-oxide (TMAO) is a well-known osmolyte to stabilize the folded proteins through a variety of mechanisms. Since mechanical strength of proteins is a critical determinant of its stabilization, TMAO might play a relevant role by favoring its folding dynamics or by enhancing its mechanical stability. To address this question, we have performed the single-molecule magnetic tweezers experiment to explore the TMAO effect on two structurally distinct substrates-protein L and talin. We observed that TMAO increases the mechanical stability of these proteins through increasing their unfolding force. Additionally, we are able to demonstrate that TMAO retards the unfolding kinetics, while accelerating the refolding kinetics under force; which eventually tilts the energy landscape towards the folded state. Interestingly, this TMAO-enhanced protein folding generates mechanical work output upto [~]67 zJ, allowing the protein folding under higher force regime. Overall this TMAO-enhanced mechanical stability provides a significant implication to folding-induced structural stability of proteins.

biophysics↗
bioRxiv2022

Gαq modulates the energy metabolism of osteoclasts

The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are small, their resorptive activity is high and destroys the nasal turbinate bones of pigs. Analysis of the proteome of classical and toxin-derived osteoclasts showed that PMT induces the upregulation of metabolic pathways. This includes strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components are also expressed more efficiently, cells display increased mitochondrial respiration. We found that the heterotrimeric G protein Gq plays a central role in this hypermetabolic cell activation. Gq triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. Overexpression of Gq in Hoxb8 cells mimicked this hypermetabolic phenotype and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. Rheumatoid arthritis patients show an increase in Gnaq expression especially in the synovial fluid, suggesting that Gq is a target of pathophysiological relevance.

immunology↗
bioRxiv2022

Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation

Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.

immunology↗