Biology subjects

Gupta, D.

Publications and source records attributed to Gupta, D..

At least 73 records · Page 4Linked to original sources

bioRxiv2023

Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery

Extracellular vesicles (EVs) are promising tools to transfer macromolecular therapeutic molecules to recipient cells, however, efficient functional intracellular protein delivery by EVs remains challenging. Here, we have developed novel and versatile systems that leverage selected molecular tools to engineer EVs for robust cytosolic protein delivery both in vitro and in vivo. These systems, termed VSV-G plus EV-sorting Domain-Intein-Cargo (VEDIC) and VSV-G-Foldon-Intein-Cargo (VFIC), exploit an engineered mini-intein (intein) protein with self-cleavage activity to link cargo to an EV-sorting domain and release it from the EV membrane inside the EV lumen. In addition, we utilize the fusogenic protein VSV-G to facilitate endosomal escape and cargo release from the endosomal system to the cytosol of recipient cells. Importantly, we demonstrate that the combination of the self-cleavage intein, fusogenic protein and EV-sorting domain are indispensable for efficient functional intracellular delivery of cargo proteins by engineered EVs. As such, nearly 100% recombination and close to 80% genome editing efficiency in reporter cells were observed by EV-transferred Cre recombinase and Cas9/sgRNA RNPs, respectively. Moreover, EV-mediated Cre delivery by VEDIC or VFIC engineered EVs resulted in significant in vivo recombination in Cre-LoxP R26-LSL-tdTomato reporter mice following both local and systemic injections. Finally, we applied these systems for improved treatment of LPS-induced systemic inflammation by delivering a super-repressor of NF-B activity. Altogether, this study describes a platform by which EVs can be utilized as a vehicle for the efficient intracellular delivery of macromolecular therapeutics for treatments of disease. Graphic summary: Development of VEDIC and VFIC systems for high-efficiency intracellular protein delivery in vitro and in vivo.Intein in tripartite fusion protein (EV-sorting Domain-Intein-Cargo) performs C-terminal cleavage during the process of EV-biogenesis, resulting in enriched free cargo proteins inside of vesicles. Together with fusogenic protein, VSV-G, these engineered EVs achieve high-efficiency intracellular delivery of cargo protein (Cre and super repressor of NF-B) or protein complex (Cas9/sgRNA RNPs) both in reporter cells and in mice models. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/535834v4_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@d0da37org.highwire.dtl.DTLVardef@1a6514corg.highwire.dtl.DTLVardef@235c9aorg.highwire.dtl.DTLVardef@194bbfa_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Mobilisation and analyses of publicly available SARS-CoV-2 data for pandemic responses

The COVID-19 pandemic has seen large-scale pathogen genomic sequencing efforts, becoming part of the toolbox for surveillance and epidemic research. This resulted in an unprecedented level of data sharing to open repositories, which has actively supported the identification of SARS-CoV-2 structure, molecular interactions, mutations and variants, and facilitated vaccine development and drug reuse studies and design. The European COVID-19 Data Platform was launched to support this data sharing, and has resulted in the deposition of several million SARS-CoV-2 raw reads. In this paper we describe (1) open data sharing, (2) tools for submission, analysis, visualisation and data claiming (e.g. ORCiD), (3) the systematic analysis of these datasets, at scale via the SARS-CoV-2 Data Hubs as well as (4) lessons learned. As a component of the Platform, the SARS-CoV-2 Data Hubs enabled the extension and set up of infrastructure that we intend to use more widely in the future for pathogen surveillance and pandemic preparedness.

bioinformatics↗

bioRxiv2023

MmcA is an electron conduit that facilitates both intracellular and extracellular electron transport in Methanosarcina acetivorans

Methanogens are a diverse group of Archaea that couple energy conservation to the production of methane gas. While most methanogens have no alternate mode of energy conservation, strains like Methanosarcina acetivorans are known to also conserve energy by dissimilatory metal reduction (DSMR) in the presence of soluble ferric iron or iron-containing minerals. The ecological ramifications of energy conservation decoupled from methane production in methanogens are substantial, yet the molecular details are poorly understood. In this work, we conducted in vitro and in vivo studies with a multiheme c-type cytochrome (MHC), called MmcA, to establish its role during methanogenesis and DSMR in M. acetivorans. MmcA purified from M. acetivorans can donate electrons to methanophenazine, a membrane-bound electron carrier, to facilitate methanogenesis. In addition, MmcA can also reduce Fe(III) and the humic acid analog anthraquinone-2,6-disulfonate (AQDS) during DSMR. Furthermore, mutants lacking mmcA have slower Fe(III) reduction rates. The redox reactivities of MmcA are consistent with the electrochemical data where MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. MmcA is prevalent in members of the Order Methanosarcinales but does not belong to a known family of MHCs linked to extracellular electron transfer, bioinformatically, and instead forms a distinct clade that is closely related to octaheme tetrathionate reductases. Taken together, this study shows that MmcA is widespread in methanogens with cytochromes where it acts as an electron conduit to support a variety of energy conservation strategies that extend beyond methanogenesis.

microbiology↗

bioRxiv2023

Novel endogenous engineering platform for robust loading and delivery of functional mRNA by extracellular vesicles

Messenger RNA (mRNA) has emerged as an attractive therapeutic molecule for a plethora of clinical applications. For in vivo functionality, mRNA therapeutics require encapsulation into effective, stable, and safe delivery systems to protect the cargo from degradation and reduce immunogenicity. Here, a bioengineering platform for efficient mRNA loading and functional delivery using bionormal nanoparticles, Extracellular Vesicles (EVs), is established by expressing a highly specific RNA-binding domain fused to CD63 in EV producer cells stably expressing the target mRNA. The additional combination with a fusogenic endosomal escape moiety, VSVg, enables functional mRNA delivery in vivo at doses substantially lower than currently used clinically with synthetic lipid-based nanoparticles. Importantly, the application of EVs loaded with effective cancer immunotherapy proves highly effective in an aggressive melanoma mouse model. This technology addresses substantial drawbacks currently associated with EV-based nucleic acid delivery systems and is a leap forward to clinical EV applications. Graphical Abstract(Figure created using BioRender) O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/533081v3_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@140058eorg.highwire.dtl.DTLVardef@2ceaccorg.highwire.dtl.DTLVardef@eafd52org.highwire.dtl.DTLVardef@19d4019_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

ORC1 binds to cis-transcribed RNAs for efficient activation of replication origins

Cells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins. One sentence summaryThe human origin recognition complex subunit 1 ORC1, binds to RNAs transcribed from genes with origins of replication at the TSS, which is required for optimal origin activation.

molecular biology↗

bioRxiv2023

Trial-history biases in evidence accumulation can give rise to apparent lapses

Trial history biases and lapses are two of the most common suboptimalities observed during perceptual decision-making. These suboptimalities are routinely assumed to arise from distinct processes. However, several hints in the literature suggest that they covary in their prevalence and that their proposed neural substrates overlap - what could underlie these links? Here we demonstrate that history biases and apparent lapses can both arise from a common cognitive process that is normative under misbeliefs about non-stationarity in the world. This corresponds to an accumulation- to-bound model with history-dependent updates to the initial state of the accumulator. We test our models predictions about the relative prevalence of history biases and lapses, and show that they are robustly borne out in two distinct rat decision-making datasets, including data from a novel reaction time task. Our model improves the ability to precisely predict decision-making dynamics within and across trials, by positing a process through which agents can generate quasi-stochastic choices.

animal behavior and cognition↗

bioRxiv2022

High-throughput microgel biofabrication via air-assisted co-axial jetting for cell encapsulation, 3D bioprinting, and scaffolding applications

Microgels have recently received widespread attention for their applications in a wide array of domains such as tissue engineering, regenerative medicine, and cell and tissue transplantation because of their properties like injectability, modularity, porosity, and the ability to be customized in terms of size, form, and mechanical properties. However, it is still challenging to mass produce microgels with diverse sizes and tunable properties. Herein, we developed an air-assisted co-axial device (ACAD) for continuous production of microgels in a high-throughput manner. To test its robustness, microgels of multiple hydrogels and their combination, including alginate (Alg), gelatin methacrylate (GelMA) and Alg-GelMA, were formed at a maximum production rate of 65,000 microgels per sec while retaining circularity and a size range of 50-500 m based on varying air pressure levels. The ACAD platform allowed single and multiple cell encapsulation with around 75% efficiency. These microgels illustrated appealing rheological properties such as yield stress, viscosity, and shear modulus for bioprinting applications. Specifically, Alg microgels have the potential to be used as a sacrificial support bath while GelMA microgels have potential for direct extrusion both on their own or when loaded in a bulk GelMA hydrogel. Generated microgels showed high cell viability (>90%) and proliferation over 7 days with their increased interactions with cells, particularly for GelMA microgels. The developed strategy provides a facile and rapid approach without any complex or expensive consumables and accessories for scalable high-throughput microgel production for cell therapy, tissue regeneration and 3D bioprinting applications.

bioengineering↗

bioRxiv2022

Characterization of Plasmodium falciparum RAP domain proteins-RAP291 and RAP070, and their association with ribosomal RNAs

Plasmodium genomes encode multiple RAP (RNA-binding domain abundant in Apicomplexan) domain proteins that contain a conserved module of 56 to 73 amino acids. Here, we characterized two of the P. falciparum RAP domain proteins; PfRAP291 & PfRAP070, for their expression and role at asexual blood stages. RNA binding assays and high-throughput CLIP-seq analysis showed that these proteins mainly bind ribosome associated RNAs. Blue-native PAGE and protein-protein interaction studies suggested association of these proteins with MSP-1 complex. Anti-PfRAP291 and anti-PfRAP070 antibodies showed moderate inhibitions in in-vitro merozoite invasion assays. Together, these results suggest multiple roles of these proteins; PfRAP291 and PfRAP070, in merozoite invasion and in ribosome regulation during asexual stages of the parasite.

microbiology↗

bioRxiv2022

The dual role of a multi-heme cytochrome in methanogenesis: MmcA is important for energy conservation and carbon metabolism in Methanosarcina acetivorans

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c-type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex ({Delta}rnf) or just the MmcA subunit ({Delta}mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA are required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin regeneration that arise in each strain. Transcriptomic data suggest that the {Delta}mmcA mutant might regenerate ferredoxin by upregulating the cytosolic Wood-Ljundahl pathway for acetyl-CoA synthesis, whereas the {Delta}rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to regenerate ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of Rnf or MmcA leads to some shared and some unique transcriptional changes in methyltransferase genes and regulatory proteins. Overall, our study provides systems-level insights into the non-overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA. ImportanceMethane is a greenhouse gas that is ten times more potent than carbon dioxide and efforts to curb emissions are crucial to meet climate goals. Methane emissions primarily stem from the metabolic activity of microorganisms called methanogenic archaea (methanogens). The electron transport chain (ETC) in methanogens that belong to the Order Methanosarcinales has been the focus of many in vitro studies to date, but the endogenous functions of the bioenergetic complexes that comprise the ETC have rarely been investigated. In this study, we use genetic techniques to functionally characterize the Rnf bioenergetic complex and the associated multi-heme c-type cytochrome MmcA in the model methanogen, Methanosarcina acetivorans. Our results show that MmcA and Rnf have shared and unique roles in the cell, and that, contrary to current knowledge, M. acetivorans has the capacity to induce at least two alternative pathways for ferredoxin regeneration in the absence of a functional Rnf complex.

microbiology↗

bioRxiv2022

Anti-COVID-19 Activity of FDA Approved Drugs through RNA G-quadruplex Binding

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ, PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ/PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against fast mutating viruses like SARS-CoV-2.

molecular biology↗

bioRxiv2022

The non-catalytic role of DNA polymerase epsilon in replication initiation in human cells

DNA polymerase epsilon in an essential enzyme, responsible for the synthesis of the leading strand during DNA replication. Deficiencies and mutations in DNA polymerase epsilon catalytic subunit (POLE1) cause severe developmental abnormalities and cancers. Paradoxically, the non-catalytic C-terminal domain of yeast polymerase epsilon catalytic subunit (Pol2) is sufficient for cell survival. The non-catalytic essential function of Pol2 in yeast has been associated with its role in the assembly of the replicative helicase CMG. However, the understanding of POLE1 functions in DNA replication initiation in human cells is falling behind. In this study we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in human cells. Surprisingly, in the absence of POLE1, human cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 C-terminal non-catalytic domain was enough to rescue replication initiation and support slow, but processive DNA synthesis, which was dependent on the POLE1-POLE2 interaction. We propose a model where in human cells POLE1/POLE2 are not essential for CMG assembly, but are required during later steps of replication initiation. Our study provides some insights into the role of DNA polymerase epsilon in replication initiation in human cells.

cell biology↗

bioRxiv2022

SARS-CoV-2 Variant Delta Potently Suppresses Innate Immune Response and Evades Interferon-Activated Antiviral Responses

Delta variant of SARS-CoV-2 has caused more severe infections than its previous variants. We studied the host innate immune response to Delta, Alpha and two earlier variants to map the evolution of the recent ones. Our biochemical and transcriptomic studies reveal that Alpha and Delta have progressively evolved over the ancestral variants by silencing innate immune response, thereby limiting cytokine and chemokine production. Though Alpha silenced RLR pathway just as Delta, it failed to persistently silence the innate immune response unlike Delta. Both Alpha and Delta have evolved to resist IFN treatment while they are still susceptible to RLR activation, further highlighting the importance of RLR-mediated, IFN-independent mechanisms in restricting SARS-CoV-2. Our studies reveal that SARS-CoV-2 Delta has integrated multiple mechanisms to silence host innate immune response and evade IFN response. Deltas silent replication and sustained suppression of host innate immune response, possibly resulting in delayed or reduced intervention by the adaptive immune response, could potentially contribute to the severe symptoms and poor recovery index associated with it.

immunology↗

bioRxiv2022

An Archaea-Specific c-type Cytochrome Maturation Machinery is Crucial for Methanogenesis in Methanosarcina acetivorans

C-type cytochromes (cyt c) are proteins that covalently bind heme and are integral to electron transport chains. A growing body of evidence suggests that cyt c play a vital role in both intra- and extra-cellular electron transfer processes in Archaea, especially in members that metabolize methane and other short chain alkanes. Elaborate mechanisms for the biogenesis of cyt c are known in Bacteria and Eukarya but this process remains largely uncharacterized in Archaea. Here, we have used the model methanogenic archaeon Methanosarcina acetivorans to characterize a distinct form of the system I cyt c maturation machinery (referred to as the Ccm machinery henceforth) that is broadly distributed in members of the Archaea. Phenotypic analyses of M. acetivorans mutants deficient in essential components of the Ccm machinery reveal that cyt c are broadly important for growth and methanogenesis, but the magnitude of their impact can vary substantially depending on the growth substrate. Heterologous expression of a synthetic operon with the Ccm machinery (CcmABCEF) from M. acetivorans is both necessary and sufficient for cyt c biogenesis in a non-native host (M. barkeri Fusaro) that is incapable of cyt c biogenesis. Even though components of the Ccm machinery are universally conserved across the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this pathway through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we have demonstrated the convergent evolution of a novel Archaea-specific Ccm machinery for cyt c biogenesis and its role in methane metabolism. Significance StatementMicroorganisms belonging to the domain Archaea play an especially important role in regulating atmospheric methane levels. Specifically, methanogens are the primary source of biogenic methane and anaerobic methanotrophic archaea (ANME) consume a substantial proportion of methane released in marine sediments. Genomic studies have implicated a class of electron-transfer proteins called c-type cytochromes as being crucial in mediating archaeal methane metabolism in the environment. However, neither the biogenesis nor the role of c-type cytochromes in methane metabolism has ever been investigated. Here, we have used a model methanogen, Methanosarcina acetivorans, to characterize a distinct pathway for maturation of c-type cytochromes that seems to be uniformly conserved across the Archaea and have also identified substrate-specific functional roles for c-type cytochromes during methanogenesis.

microbiology↗

bioRxiv2022

Panoramic visual statistics shape retina-wide organization of receptive fields

Statistics of natural scenes are not uniform - their structure varies dramatically from ground to sky. It remains unknown whether these non-uniformities are reflected in the large-scale organization of the early visual system and what benefits such adaptations would confer. Here, by relying on the efficient coding hypothesis, we predict that changes in the structure of receptive fields across visual space increase the efficiency of sensory coding. We show experimentally that, in agreement with our predictions, receptive fields of retinal ganglion cells change their shape along the dorsoventral retinal axis, with a marked surround asymmetry at the visual horizon. Our work demonstrates that, according to principles of efficient coding, the panoramic structure of natural scenes is exploited by the retina across space and cell-types.

neuroscience↗

bioRxiv2021

Limitations of a proposed correction for slow drifts in decision criterion

Trial history biases in decision-making tasks are thought to reflect systematic updates of decision variables, therefore their precise nature informs conclusions about underlying heuristic strategies and learning processes. However, random drifts in decision variables can corrupt this inference by mimicking the signatures of systematic updates. Hence, identifying the trial-by-trial evolution of decision variables requires methods that can robustly account for such drifts. Recent studies (Lak20, Mendonca 20) have made important advances in this direction, by proposing a convenient method to correct for the influence of slow drifts in decision criterion, a key decision variable. Here we apply this correction to a variety of updating scenarios, and evaluate its performance. We show that the correction fails for a wide range of commonly assumed systematic updating strategies, distorting ones inference away from the veridical strategies towards a narrow subset. To address these limitations, we propose a model-based approach for disambiguating systematic updates from random drifts, and demonstrate its success on real and synthetic datasets. We show that this approach accurately recovers the latent trajectory of drifts in decision criterion as well as the generative systematic updates from simulated data. Our results offer recommendations for methods to account for the interactions between history biases and slow drifts, and highlight the advantages of incorporating assumptions about the generative process directly into models of decision-making.

neuroscience↗

bioRxiv2021

Regulation of eIF2α Phosphorylation by MAPKs Influences Polysome Stability and Protein Translation

Regulation of protein translation occurs primarily at the level of initiation and is mediated by multiple signaling pathways, majorly mechanistic target of rapamycin complex 1 (mTORC1), mitogen-activated protein kinases (MAPKs), and the eukaryotic translation initiation factor eIF2. While mTORC1 and eIF2 influence the polysome stability, MAPKs influence the phosphorylation of the cap-binding protein eIF4E and are known to influence translation of only a small set of mRNAs. Here, we demonstrate that p38 MAPK and ERK1/2 regulate translation through integrated stress response (ISR) pathways. Dual inhibition (dual-Mi) of p38 MAPK and ERK1/2 caused substantial phosphorylation of eIF2 in a synergistic manner, resulting in near-absolute collapse of polysomes. This regulation was independent of Mnk1/2, a well-studied mediator of translation regulation by the MAPKs. Dual-Mi-induced polysome dissociation was far more striking than that caused by sodium arsenite, a strong inducer of ISR. Interestingly, induction of ISR caused increased p38 phosphorylation, and its inhibition resulted in stronger polysome dissociation, indicating the importance of p38 in the translation activities. Thus, our studies demonstrate a major, unidentified role for ERK1/2 and more particularly p38 MAPK in the maintenance of homeostasis of polysome association and translation activities.

biochemistry↗

bioRxiv2021

Involvement of translocon complex in hemoglobin import from infected erythrocyte cytoplasm into the Plasmodium parasite

Haemoglobin degradation is crucial for the growth and survival of Plasmodium falciparum in human erythrocytes. Although the process of Hb degradation has been studied in great detail, the mechanisms of Hb uptake remain ambiguous to date. Here, we characterized Heme Detoxification Protein (PfHDP), a protein localized in the parasitophorous vacuole, parasite food vacuole and infected erythrocyte cytosol for its role in Hb uptake. Immunoprecipitation of PfHDP-GFP fusion protein from a transgenic line using anti-GFP antibody and of Plasmodium parasite extract using anti-human Hb antibodies respectively, showed the association of PfHDP/Hb with each other as well as with the members of PTEX translocon complex. Some of these associations such as PfHDP/Hb and PfHDP/Pfexp-2 interactions were confirmed by in vitro protein-protein interaction tools. To know the roles of PfHDP and translocon complex in Hb import into the parasites, we next studied the Hb uptake by the parasite in PfHDP knock-down line using the GlmS ribozyme strategy. PfHDP knock-down significantly reduced the Hb uptake in these parasites in comparison to the wild type parasites. Further, the transient knock-down of one of the members of the translocon complex; PfHSP101 showed considerable reduction in Hb uptake. Morphological analysis of PfHDP-HA-GlmS transgenic parasites in the presence of GlcN showed food vacuole abnormalities and parasite stress, thereby causing a growth defect in the development of these parasites. Together, we implicate the translocon complex in the trafficking of PfHDP/Hb complex in the parasite and suggest a role for PfHDP in the uptake of Hb and parasite development. The study thus reveals new insights into the function of PfHDP, making it an extremely important target for developing new antimalarials.

molecular biology↗

bioRxiv2021

Artemisinin acts by inhibiting Plasmodium falciparum Ddi1, a retropepsin, resulting into the accumulation of ubiquitinated proteins

Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A2 family protease that hydrolyzes ubiquitinated substrates. We further show that treatment with ARTs lead to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of the ubiquitinated substrates by PfDdi1. Besides, whereas the PfDdi1 is predominantly localised in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, Ddi1 knock-out Saccharomyces cerevisiae cells are more suceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act by inducing DNA and protein damage, and impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

cell biology↗