Biology subjects

Chaudhuri, D.

Publications and source records attributed to Chaudhuri, D..

At least 19 recordsLinked to original sources

bioRxiv2026

Citrobacter rodentium infection reveals preserved intestinal resilience in aged intestine

Ageing is generally associated with increased susceptibility to enteric infection and is widely considered to reflect progressive deterioration of intestinal function. However, whether increased susceptibility represents complete loss of intestinal resilience or a distinct host response to infection remains unclear. Using Citrobacter rodentium (CR) infection as a model of infectious colitis, we investigated how ageing shapes epithelial responses to enteric bacterial infection. Infection of aged mice with wild type CR (CRWT) resulted greater bacterial colonisation, deeper crypt localisation, impaired fluid-ion homeostasis and more severe colitis than young mice. Conversely, infection of aged mice with CRM12, a CR strain lacking 12 type III secretion system effectors, resulted in reduced epithelial colonisation, preservation of epithelial barrier function and fluid-ion homeostasis, restrained inflammatory responses, and markedly lower disease severity than CRWT infection. Deep quantitative proteomic analysis demonstrated that these divergent outcomes were accompanied by distinct adaptive molecular programmes within the aged intestinal epithelium. Together, our findings demonstrate that although aged mice exhibit increased susceptibility to enteric infection, the aged intestine retains the ability to mount distinct epithelial, inflammatory and molecular responses to pathogens with different virulence repertoires. Thus, increased susceptibility in aged mice should not be interpreted as complete loss of intestinal resilience.

microbiology↗

bioRxiv2026

Paclitaxel sensitizes TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-resistant breast cancer cells towards TRAIL-mediated apoptosis

BackgroundThe ability of TRAIL to specifically induce apoptosis in cancer cells makes it a promising candidate to be an effective chemotherapeutic drug. But resistance to TRAIL treatment is a major obstacle. Finding combinatorial therapies that make resistant tumors more susceptible to TRAIL is an effective preclinical approach. In this work, we investigated the possibility that pre-treatment of paclitaxel may promote apoptosis in TRAIL-resistant breast cancer cells. MethodsIn silico analysis was done to investigate the binding affinity between TRAIL receptors (DR5 and DCR2) and paclitaxel via docking and MD simulation. To check whether any non-lethal dose of paclitaxel can modulate the expression of TRAIL receptors, qPCR was done in paclitaxel treated breast cancer cells. Next, paclitaxel was pre-administered to TRAIL-resistant MCF7 and MDA-MB-453 human breast cancer cells followed by rhTRAIL treatment. Cell viability and survival was evaluated using the MTT assay and colony formation assay, respectively. Immunoblot for caspase-3 was performed to study apoptosis. The expression level changes of DR5 and DCR2 were analyzed post-treatment using qPCR and immunoblot assay. ResultsIn silico analysis showed that paclitaxel can bind with higher stability to DCR2 in comparison to DR5 thereby changing the preference of TRAIL molecules towards DR5. Next, in cell line experiments we observed that administering a non-lethal dose of paclitaxel to MDA-MB-231 and MCF7 breast cancer cells resulted in no significant cell death but led to an increase in DR5 and a decrease in DCR2 expression at both the transcript and protein levels. Furthermore, in TRAIL-resistant MCF7 and MDA-MB-453 cells, pre-treatment with paclitaxel followed by rhTRAIL administration induced significant cell death due to paclitaxel induced increase in DR5 as well as decrease in DCR2 expression at both the transcript and protein levels. Moreover, long term survival of MDA-MB-453 cells was significantly lower when pretreated with paclitaxel and exposed to rhTRAIL compared to control, paclitaxel alone or rhTRAIL alone group. ConclusionThus, our study uncovers a novel therapeutic strategy to overcome TRAIL resistance underscoring the clinical potential of using a non-lethal dose of paclitaxel to modulate TRAIL receptor dynamics. Future research should be aimed at exploring the potentiality of using paclitaxel-based combinatorial approaches in crafting effective TRAIL therapies.

cancer biology↗

bioRxiv2026

Excessive Ca2+-dependent ER-mitochondrial contact stabilization by EFHD1 drives liver injury

Metabolic-associated steatohepatitis (MASH) involves hepatocyte damage that cannot be explained solely by lipid accumulation. Here, to discover injury-specific pathways, we focused on a gene of uncertain function, EF-Hand Domain Family Member D1 (EFHD1), identified in human genome-wide association studies of liver injury but not liver fat. We show that EFHD1, a Ca2+-dependent actin crosslinker, stabilizes endoplasmic reticulum-mitochondria contact sites (ERMCS), detecting spatiotemporal coincidence of inter-organellar proximity and ER Ca2+ release. During MASH, EFHD1 upregulation drives pathological mitochondrial fragmentation via excessive contact persistence. This structural failure promotes mitochondrial double-stranded RNA escape and activation of a maladaptive antiviral PKR-dependent stress response, a causal relationship also supported by Mendelian randomization in humans. Consequently, inhibiting EFHD1 in human and mouse models blunts hepatocyte damage. These findings identify EFHD1 as a Ca2+-dependent ERMCS stabilizer, reveal a hepatocyte-intrinsic injury pathway, and suggest EFHD1 inhibition as a therapeutic strategy.

cell biology↗

bioRxiv2025

GBP1 recruitment to actin-rich pedestals induced by extracellular Gram-negative bacteria promotes pyroptosis.

The IFN{gamma}-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that also induce LPS-and caspase-4-dependent pyroptosis. However, whether GBP1 is involved in this process remains unknown. EPEC and EHEC adhere intimately to intestinal epithelial cells via avid interactions between the bacterial adhesin Intimin and Tir (Translocated intimin receptor), a type 3 secretion system effector protein. Intimin-mediated clustering of Tir triggers actin polymerisation, leading to pedestal-like structures at bacterial attachment sites. Here we show that GBP1 is recruited to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1-dependent caspase-4 trafficking to these sites leads to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals, pointing to LPS-independent mobilisation of GBP1 by actin polymerisation induced by extracellular A/E pathogens. To further dissect the underlying mechanism, we engineered a chimeric receptor (Fc{gamma}R-Tir) by combining the intracellular signalling domain of Tir and the extracellular ligand-binding domain of the Fc{gamma} receptor. Clustering of Fc{gamma}R-Tir with IgG-coated beads produced sterile actin-rich pedestals that were sufficient to recruit GBP1 independently of bacteria. Our findings reveal that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. We establish that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton. 175-word AbstractThe IFN{gamma}-induced GTPase guanylate binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic Gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures called actin-rich pedestals. Here we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC and EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localised to EPEC pedestals. Our novel assay that mimics pathogenic effector activity revealed GBP1 recruitment to sterile actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.

immunology↗

bioRxiv2024

The Novel Chaperone Activity of p47 Modulates Force-Mediated Retrotranslocation from the Endoplasmic Reticulum to the Cytosol

Protein quality control in the endoplasmic reticulum (ER) maintains cellular proteostasis by regulating protein folding, assembly, and degradation. While protein translocation from the cytosol into the ER is well-studied, the reverse process, known as retrotranslocation, which exports proteins from the ER lumen or membrane into the cytosol, is less understood. In this study, we investigate the role of p47, a cofactor of the ATPase p97, in retrotranslocation and its chaperone-like activity on the model substrate talin using single-molecule magnetic tweezers. We find that p47 enhances the mechanical stability of talin, by increasing its folding probabilities by 8 pN, which generate a maximum work output of up to [~]199.5 zJ to facilitate the extraction of the protein from the ER lumen. These findings suggest that p47 interacts with substrates emerging from the ER, generating pulling forces that aid retrotranslocation, which uncovers a novel mechanistic role for p47 in ER-associated degradation, advancing the understanding of retrotranslocation.

biophysics↗

bioRxiv2024

Mechanistic Insights into Periplasmic Chaperones as Regulators of Protein Folding and Translocation in Gram-Negative Bacteria

In Gram-negative bacteria, periplasmic chaperones mediate the transport and folding of outer membrane proteins. While their substrate interaction mechanisms are well studied, their functional cycle during periplasmic translocation remains unclear. Using single-molecule force spectroscopy, we demonstrate that SecYEG-associated chaperones PpiD and DsbC act as foldases, delivering up to 45.5 zJ of mechanical work. This activity facilitates substrate extraction from the SecYEG translocation tunnel, reducing ATP consumption during SecA-mediated translocation. In contrast, chaperones such as Spy and Skp function as holdases, stabilizing unfolded substrates to prevent misfolding during translocation. Our findings reveal a unique mechanism by which chaperones adapt to mechanical constraints, optimizing protein folding and translocation in the periplasm.

biophysics↗

bioRxiv2024

Mechanism of MCUB-dependent inhibition of mitochondrial calcium uptake

Mitochondrial Ca2+ levels are regulated to balance stimulating respiration against the harm of Ca2+ overload. Contributing to this balance, the main channel transporting Ca2+ into the matrix, the mitochondrial Ca2+ uniporter, can incorporate a dominant-negative subunit (MCUB). MCUB is homologous to the pore-forming subunit MCU, but when present in the pore-lining tetramer, inhibits Ca2+ transport. Here, using cell lines deleted of both MCU and MCUB, we identify three factors that contribute to MCUB-dependent inhibition. First, MCUB protein requires MCU to express. The effect is mediated via the N-terminal domain (NTD) of MCUB. Replacement of the MCUB NTD with the MCU NTD recovers autonomous expression but fails to rescue Ca2+ uptake. Surprisingly, mutations to MCUB that affect interactions with accessory subunits or the conduction pore all failed to rescue Ca2+ uptake, suggesting the mechanism of inhibition may involve global rearrangements. Second, using concatemeric tetramers with varying MCU:MCUB ratios, we find that MCUB incorporation does not abolish conduction, but rather inhibits Ca2+ influx proportional to the amount of MCUB present in the channel. Reducing rather than abolishing Ca2+ transport is consistent with MCUB retaining the highly-conserved selectivity filter DIME sequence. Finally, we apply live-cell Forster resonance energy transfer to establish that the endogenous stoichiometry is 2:2 MCU:MCUB. Taken together, our results suggest MCUB preferentially incorporates into nascent uniporters, and the amount of MCUB protein present linearly correlates with the degree of inhibition of Ca2+ transport, creating a precise, tunable mechanism for cells to regulate mitochondrial Ca2+ uptake.

cell biology↗

bioRxiv2024

Direct mitochondrial import of lactate supports resilient carbohydrate oxidation

Lactate is the highest turnover circulating metabolite in mammals. While traditionally viewed as a waste product, lactate is an important energy source for many organs, but first must be oxidized to pyruvate for entry into the tricarboxylic acid cycle (TCA cycle). This reaction is thought to occur in the cytosol, with pyruvate subsequently transported into mitochondria via the mitochondrial pyruvate carrier (MPC). Using 13C stable isotope tracing, we demonstrated that lactate is oxidized in the myocardial tissue of mice even when the MPC is genetically deleted. This MPC-independent lactate import and mitochondrial oxidation is dependent upon the monocarboxylate transporter 1 (MCT1/Slc16a1). Mitochondria isolated from the myocardium without MCT1 exhibit a specific defect in mitochondrial lactate, but not pyruvate, metabolism. The import and subsequent mitochondrial oxidation of lactate by mitochondrial lactate dehydrogenase (LDH) acts as an electron shuttle, generating sufficient NADH to support respiration even when the TCA cycle is disrupted. In response to diverse cardiac insults, animals with hearts lacking MCT1 undergo rapid progression to heart failure with reduced ejection fraction. Thus, the mitochondrial import and oxidation of lactate enables carbohydrate entry into the TCA cycle to sustain cardiac energetics and maintain myocardial structure and function under stress conditions.

biochemistry↗

bioRxiv2024

Enhancing mitochondrial pyruvate metabolism ameliorates myocardial ischemic reperfusion injury.

The established clinical therapy for the treatment of acute myocardial infarction is primary percutaneous coronary intervention (PPCI) to restore blood flow to the ischemic myocardium. PPCI is effective at reperfusing the ischemic myocardium, however the rapid re-introduction of oxygenated blood also can cause ischemia-reperfusion (I/R) injury. Reperfusion injury is the culprit for up to half of the final myocardial damage, but there are no clinical interventions to reduce I/R injury. We previously demonstrated that inhibiting the lactate exporter, monocarboxylate transporter 4 (MCT4), and re-directing pyruvate towards oxidation can blunt isoproterenol-induced hypertrophy. Based on this finding, we hypothesized that the same pathway might be important during I/R. Here, we establish that the pyruvate-lactate metabolic axis plays a critical role in determining myocardial salvage following injury. Post-I/R injury, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium following I/R injury. MPC loss in cardiomyocytes caused more cell death with less myocardial salvage, which was associated with an upregulation of MCT4 in the myocardium at risk of injury. We deployed a pharmacological strategy of MCT4 inhibition with a highly selective compound (VB124) at the time of reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential ({Delta}{psi}), and Ca2+, increased pyruvate entry to TCA cycle, and improved myocardial salvage and functional outcomes following I/R injury. Altogether, our data suggest that normalizing the pyruvate-lactate metabolic axis via MCT4 inhibition is a promising pharmacological strategy to mitigate I/R injury. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/577463v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1a7a9b8org.highwire.dtl.DTLVardef@779e0aorg.highwire.dtl.DTLVardef@128d1f3org.highwire.dtl.DTLVardef@efc1bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2023

Mitochondria possess a large, non-selective ionic current that is enhanced during cardiac injury.

Mitochondrial ion channels are essential for energy production and cell survival. To avoid depleting the electrochemical gradient used for ATP synthesis, channels so far described in the mitochondrial inner membrane open only briefly, are highly ion-selective, have restricted tissue distributions, or have small currents. Here, we identify a mitochondrial inner membrane conductance that has strikingly different behavior from previously described channels. It is expressed ubiquitously, and transports cations non-selectively, producing a large, up to nanoampere-level, current. The channel does not lead to inner membrane uncoupling during normal physiology because it only becomes active at depolarized voltages. It is inhibited by external Ca2+, corresponding to the intermembrane space, as well as amiloride. This large, ubiquitous, non-selective, amiloride-sensitive (LUNA) current appears most active when expression of the mitochondrial calcium uniporter is minimal, such as in the heart. In this organ, we find that LUNA current magnitude increases two- to threefold in multiple mouse models of injury, an effect also seen in cardiac mitochondria from human patients with heart failure with reduced ejection fraction. Taken together, these features lead us to speculate that LUNA current may arise from an essential protein that acts as a transporter under physiological conditions, but becomes a channel under conditions of mitochondrial stress and depolarization.

cell biology↗

bioRxiv2023

A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9

CRISPR-Cas9 is the newest technology available for targeted genome editing. It is very efficient and cheap compared to other genome editing techniques. However, its therapeutic application is limited due to its off-target activity. To have a better understanding of this off-target effect, we concentrated our efforts on its mismatch-prone PAM distal end. Current off-target prediction algorithms use RNA-DNA complementation derived energy as a major factor in predicting off-target effect. RNA-DNA complementation derived energy drives Cas9 conformational change, which in turn drives its functional activity. In the case of lower RNA-DNA complementarity, a partial conformational change occurs resulting in a slower reaction rate and partial activity. However, extensive mismatches are often tolerated despite lower complementation derived energy available from RNA: DNA duplex formation. Thus, the off-target activity of Cas9 depends directly on the nature of mismatches which in turn result in deviation of the active site of the enzyme due to structural instability in the duplex strand. In order to test the hypothesis, we have designed an array of mismatched target sites and performed in vitro and cell line-based experiments to assess the effects of PAM distal mismatches in Cas9 activity. For further mechanistic validation, Molecular dynamics simulation was performed and it revealed that certain mismatch mutations induced pronounced conformational instability within the RNA-DNA duplex, leading to elevated root mean square deviation (RMSD) values. We found that, target sites having mismatches in the 18th to 16th position upstream of the PAM showed no to little activity.

genomics↗

bioRxiv2023

F-box protein FBXB-65 regulates anterograde transport of UNC-104 through modification near the PH domain

Axonal transport is essential for cargo movement between the neuronal cell body and synapses. UNC-104/KIF1A, a Kinesin-3 motor in C. elegans that anterogradely transports precursors of synaptic vesicles (pre-SVs), is known to be degraded at synapses through the ubiquitin pathway. Knockdown of the E1 ubiquitin-activating enzyme, uba-1, leads to increased accumulation of UNC-104 at neuronal ends and at synapses of touch receptor neurons (TRNs). Loss of the F-box protein FBXB-65, a putative E3 ligase, leads to UNC-104 accumulation at distal ends of neurons, alters net anterograde movement of UNC-104, and the intensity of moving UNC-104 puncta likely bound to cargo without changes in synaptic UNC-104 levels. Using a theoretical model, we analyze the steady state distribution of the anterogradely moving UNC-104 motor. A good agreement between the model and the experimental distributions leads to a crucial hypothesis that UNC-104 may exhibit cooperative binding with moving motor puncta likely associated with cargo, which is regulated by fbxb-65. FBXB-65 regulates the modification of UNC-104 motor in a region besides the cargo binding PH-domain. Both fbxb-65 and UNC-104 motor independent of FBXB-65 regulate the extent of cargo transport in the axon and transport behaviour of cargo at branch points. Our study shows that modification of UNC-104 near its cargo-binding domain may regulate number of motors on the cargo surface and this regulation can fine-tune cargo transport to its destination, the synapse.

cell biology↗

bioRxiv2022

Mitochondrial Phosphatidylethanolamine Directly Regulates UCP1 to Promote Brown Adipose Thermogenesis

Thermogenesis by uncoupling protein 1 (UCP1) is one of the primary mechanisms by which brown adipose tissue (BAT) increases energy expenditure. UCP1 resides in the inner mitochondrial membrane (IMM), where it dissipates membrane potential independent of ATP synthase. Here we provide evidence that mitochondrial phosphatidylethanolamine (PE) directly regulates UCP1-dependent proton conductance across IMM to modulate thermogenesis. Mitochondrial lipidomic analyses revealed PE as a signature molecule whose abundance bidirectionally responds to changes in thermogenic burden. Reduction in mitochondrial PE by deletion of phosphatidylserine decarboxylase (PSD) made mice cold intolerant and insensitive to {beta}3 adrenergic receptor agonist-induced increase in whole-body oxygen consumption. High-resolution respirometry and fluorometry of BAT mitochondria showed that loss of mitochondrial PE specifically lowers UCP1-dependent respiration without compromising electron transfer efficiency or ATP synthesis. These findings were confirmed by a reduction in UCP1 proton current in PSD-deficient mitoplasts. Thus, PE performs a previously unknown role as a temperature-responsive rheostat that regulates UCP1-dependent thermogenesis.

biochemistry↗

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

Talin-drug interaction reveals a key molecular determinant for biphasic mechanical effect: studied under single-molecule resolution

Talin as an adhesion protein, exhibits a strong force-dependent structure-function dynamics. Being a mechanosensitive focal adhesion (FA) protein, talin might interact to several FA targeting drugs; however, the molecular mechanism of talin-drug interactions remains elusive. Here we combined magnetic tweezers and molecular dynamics (MD) simulation to explore mechanical stability of talin with three drugs based on their talin specificity. Interestingly, our study revealed that talin displays a bimodal force distribution with a low and high unfolding force population. We observed that talin nonspecific drugs (tamoxifen and letrozole) display biphasic effect: increase talin mechanical stability upto optimum concentration, followed by a decrease in stability with further concentration increase. By contrast, talin-specific cyanidin 3-O-glucoside promotes a steady increase to talin mechanical stability with its concentration. We reconciled our observation from the simulation study: tamoxifen enters into talin hydrophobic core, eventually destabilizing the protein; whereas cyanidin 3-O-glucoside stabilizes the protein core by maintaining the inter-helix distance. Finally, we observed a strong correlation among hydrophobicity and cavity analysis, illustrating a detailed mechanistic analysis of drug effect on the mechanosensitive protein. Overall this study presents a novel perspective for drug designing against mechanosensitive proteins and studying off-target effects of already known drugs.

biophysics↗

bioRxiv2022

Deceiving The Big Eaters: Salmonella Typhimurium SopB subverts host cell Xenophagy through the Akt-TFEB axis in macrophages

Salmonella, a stealthy facultative intracellular pathogen, harbors an array of host immune evasion strategies. This facilitates successful survival and replicative niches establishment in otherwise hostile host innate immune cells such as macrophages. Salmonella survives and utilizes macrophages for effective dissemination throughout the host causing systemic infection. One of the central host defense mechanisms in macrophages is bacterial xenophagy or macro-autophagy. Here we report for the first time that Salmonella pathogenicity island-1 (SPI-1) effector SopB is involved in subverting host autophagy through dual mechanisms. SopB is known to act as a phosphoinositide phosphatase and thereby can alter the phosphoinositide dynamics of the host cell. Here we demonstrate that this activity helps the bacterium escape autophagy by inhibiting terminal fusion of Salmonella containing vacuole (SCV) with both lysosomes and autophagosomes. We also report the second mechanism, wherein SopB downregulates overall lysosomal biogenesis through Akt- transcription factor EB (TFEB) axis. TFEB is a master regulator of lysosomal biogenesis and autophagy, and SopB restricts the nuclear localization of TFEB. This reduces the overall lysosome content inside host macrophages, further facilitating survival in macrophages and systemic dissemination of Salmonella in the host.

microbiology↗

bioRxiv2021

EFHD1 ablation reduces cardiac mitoflash activation and protects cardiomyocytes from ischemia.

Altered levels of intracellular calcium (Ca2+) is a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

cell biology↗

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↗