Cellular Machinery Research Articles

ATM Pathway Is Essential for HPV-Positive Human Cervical Cancer-Derived Cell Lines Viability and Proliferation.

Infection with some mucosal human papillomavirus (HPV) types is the etiological cause of cervical cancer and of a significant fraction of vaginal, vulvar, anal, penile, and head and neck carcinomas. DNA repair machinery is essential for both HPV replication and tumor cells survival suggesting that cellular DNA repair machinery may play a dual role in HPV biology and pathogenesis. Here, we silenced genes involved in DNA Repair pathways to identify genes that are essential for the survival of HPV-transformed cells. We identified that inhibition of the ATM/CHK2/BRCA1 axis selectively affects the proliferation of cervical cancer-derived cell lines, without altering normal primary human keratinocytes (PHK) growth. Silencing or chemical inhibition of ATM/CHK2 reduced the clonogenic and proliferative capacity of cervical cancer-derived cells. Using PHK transduced with HPV16 oncogenes we observed that the effect of ATM/CHK2 silencing depends on the expression of the oncogene E6 and on its ability to induce p53 degradation. Our results show that inhibition of components of the ATM/CHK2 signaling axis reduces p53-deficient cells proliferation potential, suggesting the existence of a synthetic lethal association between CHK2 and p53. Altogether, we present evidence that synthetic lethality using ATM/CHK2 inhibitors can be exploited to treat cervical cancer and other HPV-associated tumors.

An Overview on Oxidative Stress in Diabetes Mellitus

Human body is continuously exposed to different types of agents that results in the production of reactive species called as free radicals (ROS/RNS) which by the transfer of their free unpaired electron causes the oxidation of cellular machinery. In order to encounter the deleterious effects of such species, body has got endogenous antioxidant systems or it obtains exogenous antioxidants from diet that neutralizes such species and keeps the homeostasis of body. Any imbalance between the RS and antioxidants leads to produce a condition known as ‘‘oxidative stress’’ that results in the development of pathological condition among which one is diabetes. Most of the studies reveal the inference of oxidative stress in diabetes pathogenesis by the alteration in enzymatic systems, lipid peroxidation, impaired Glutathione metabolism and decreased Vitamin C levels. Lipids, proteins, DNA damage, Glutathione, catalane and superoxide dismutase are various biomarkers of oxidative stress in diabetes mellitus. Oxidative stress induced complications of diabetes may include stroke, neuropathy, retinopathy and nephropathy. The basic aim of this review was to summarize the basics of oxidative stress in diabetes mellitus

SARS-Coronavirus 2, A Metabolic Reprogrammer: A Review in the Context of the Possible Therapeutic Strategies.

Novel coronavirus, SARS-CoV-2, is advancing at a staggering pace to devastate the health care system and foster concerns over public health. In contrast to the past outbreaks, coronaviruses are not clinging themselves as a strict respiratory virus. Rather, becoming a multifaceted virus, it affects multiple organs by interrupting a number of metabolic pathways leading to significant rates of morbidity and mortality. Following infection, they rigorously reprogram multiple metabolic pathways of glucose, lipid, protein, nucleic acid, and their metabolites to extract adequate energy and carbon skeletons required for their existence and further molecular constructions inside a host cell. Although the mechanism of these alterations is yet to be known, the impact of these reprogramming is reflected in the hyperinflammatory responses, so called cytokine storm and the hindrance of the host immune defence system. The metabolic reprogramming during SARSCoV- 2 infection needs to be considered while devising therapeutic strategies to combat the disease and its further complication. The inhibitors of cholesterol and phospholipids synthesis and cell membrane lipid raft of the host cell can, to a great extent, control the viral load and further infection. Depletion of energy sources by inhibiting the activation of glycolytic and hexosamine biosynthetic pathways can also augment antiviral therapy. The cross talk between these pathways also necessitates the inhibition of amino acid catabolism and tryptophan metabolism. A combinatorial strategy that can address the cross talks between the metabolic pathways might be more effective than a single approach, and the infection stage and timing of therapy will also influence the effectiveness of the antiviral approach. We herein focus on the different metabolic alterations during the course of virus infection that help exploit the cellular machinery and devise a therapeutic strategy that promotes resistance to viral infection and can augment body's antivirulence mechanisms. This review may cast light on the possibilities of targeting altered metabolic pathways to defend against virus infection in a new perspective.

Biophysical and Computational Approaches to Unravel pH-Dependent Conformational Change of PspA Assist PspA-PspF Complex Formation in Yersinia enterocolitica.

In enteropathogen, Yersinia enterocolitica, the genes encoding phage shock proteins are organized in an operon (pspA-E), which is activated at the various types of cellular stress (i.e., extracytoplasmic or envelop stress) whereas, PspA negatively regulates PspF, a transcriptional activator of pspA-E and pspG, and is also involved in other cellular machinery maintenance processes. The exact mechanism of association and dissociation of PspA and PspF during the stress response is not entirely clear. In this concern, we address conformational change of PspA in different pH conditions using various in-silico and biophysical methods. At the near-neutral pH, CD and FTIR measurements reveal a ß-like conformational change of PspA; however, AFM measurement indicates the lower oligomeric form at the above-mentioned pH. Additionally, the results of the MD simulation also support the conformational changes which indicate salt-bridge strength takes an intermediate position compared to other pHs. Furthermore, the bio-layer interferometry study confirms the stable complex formation that takes place between PspA and PspF at the near-neutral pH. It, thus, appears that PspA conformational change in adverse pH conditions abandons PspF from having a stable complex with it, and thus, the latter can act as a trans-activator. Taken together, it seems that PspA alone can transduce adverse signals by changing its conformation.

223-OR: ADA Presidents' Select Abstract: Novel Ligand and Kinase-Independent Signaling by the Insulin Receptor

Insulin acts through the insulin receptor (IR) tyrosine kinase and two major canonical post-receptor signaling pathways (the IRS-1/PI3K/Akt pathway and the Shc/Ras/MAP kinase pathway) to exert its classic metabolic and mitogenic actions. To create a detailed map of how different domains of the receptor might be linked to different insulin actions, we created cells in which endogenous IR and IGF1R had been genetically inactivated, after which the cells were reconstituted with either the wild-type IR, a kinase-dead mutant of IR (K1030R-IR) , a truncated IR lacking just the C-terminus of IR (∆CT-IR) and a truncated IR with the juxtamembrane domain only inside the cell (JMO-IR) . Using these cells and a multi-omics approach, we have uncovered a second novel IR signaling pathway that is dependent on the intracellular domain of IR but is ligand and tyrosinekinase-independent (LYK-I for short) . We find that the LYK-I actions of the IR are mediated by changes in a phosphorylation of a network of proteins involved in cell cycle/mitosis, ATM signaling, extracellular matrix organization, and cellular senescence. These actions result in upregulation of expression of multiple extracellular matrix-related genes and proteins, and down-regulation of immune/interferon-related and cellular senescence related genes and proteins. Functionally, LYK-I signaling results in increased cell proliferation and increased sensitivity to apoptosis. Thus, in addition to classical ligand and tyrosine kinase-dependent signaling, the IR regulates a second, novel ligand and tyrosine kinase-independent (LYK-I) pathway which regulates the cellular machinery involved in cellular senescence, extracellular matrix interaction and apoptotic sensitivity. Disclosure H.Nagao: None. W.Cai: None. H.Pan: n/a. T.M.Batista: None. B.Brandao: n/a. C.Kahn: Advisory Panel; Kaleido, Consultant; Cellarity, Flagship Pioneering, Sana Biotechnology Inc.

Fibroblasts Drive Metabolic Reprogramming in Pacemaker Cardiomyocytes.

The sino atrial node (SAN) is characterized by the microenvironment of pacemaker cardiomyocytes (PCs) encased with fibroblasts. An altered microenvironment leads to rhythm failure. Operable cell or tissue models are either generally lacking or difficult to handle. The biological process behind the milieu of SANs to evoke pacemaker rhythm is unknown. We explored how fibroblasts interact with PCs and regulate metabolic reprogramming and rhythmic activity in the SAN. Tbx18 (T-box transcription factor 18)-induced PCs and fibroblasts were used for cocultures and engineered tissues, which were used as the in vitro models to explore how fibroblasts regulate the functional integrity of SANs. RNA-sequencing, metabolomics, and cellular and molecular techniques were applied to characterize the molecular signals underlying metabolic reprogramming and identify its critical regulators. These pathways were further validated in vivo in rodents and induced human pluripotent stem cell-derived cardiomyocytes. We observed that rhythmicity in Tbx18-induced PCs was regulated by aerobic glycolysis. Fibroblasts critically activated metabolic reprogramming and aerobic glycolysis within PCs, and, therefore, regulated pacemaker activity in PCs. The metabolic reprogramming was attributed to the exclusive induction of Aldoc (aldolase c) within PCs after fibroblast-PC integration. Fibroblasts activated the integrin-dependent mitogen-activated protein kinase-E2F1 signal through cell-cell contact and turned on Aldoc expression in PCs. Interruption of fibroblast-PC interaction or Aldoc knockdown nullified electrical activity. Engineered Tbx18-PC tissue sheets were generated to recapitulate the microenvironment within SANs. Aldoc-driven rhythmic machinery could be replicated within tissue sheets. Similar machinery was faithfully validated in de novo PCs of adult mice and rats, and in human PCs derived from induced pluripotent stem cells. Fibroblasts drive Aldoc-mediated metabolic reprogramming and rhythmic regulation in SANs. This work details the cellular machinery behind the complex milieu of vertebrate SANs and opens a new direction for future therapy.

Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells.

The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.

Cellular and Molecular Machinery of Neuropathic Pain: an Emerging Insight

Purpose of ReviewNeuropathic pain (NP) has been ubiquitously characterized by lesion and its linked somatosensory system either the central nervous system (CNS) or peripheral nervous system (PNS) This PNS episode is the most prevalent site of NP origin and is found to be associated with afferent nerve fibers carrying pain signals from injured/trauma site to the CNS including the brain. Several kinds of pharmacotherapeutic drugs shuch as analgesics, anti-convulsants, and anti-depressants are being employed for the its possible interventions. The NP has been a great interest to follow different pathophysiological mechanisms which are often considered to correlate with the metabolic pathways and its mediated disease. There is paucity of knowledge to make such mechanism via NP.Recent FindingMost notably, recent pandemic outbreak of COVID-19 has also been reported in chronic pain mediated diabetes, inflammatory disorders, and cancers. There is an increasing incidence of NP and its complex mechanism has now led to identify the possible investigations of responsible genes and proteins via bioinformatics tools. The analysis might be more instrumental as collecting the genes from pain genetic database, analyzing the variants through differential gene expression (DEG) and constructing the protein–protein interaction (PPI) networks and thereby determining their upregulating and downregulating pathways.SummaryThis review sheds a bright light towards several mechanisms at both cellular and molecular level, correlation of NP-mediated disease mechanism and possible cell surface biomarkers (receptors), and identified genes could be more promising for their pharmacological targets.

Epstein-Barr virus lytic replication activates and is dependent upon MAPK-interacting kinase 1/2 in a cell-type dependent manner.

Epstein-Barr virus (EBV) is known to manipulate its cellular environment to enhance viral replication, which can lead to dysregulation of cellular machinery, setting the stage for potential future disease. Previous research showed that under rapamycin-mediated inhibition of mTORC1, EBV lytic protein production was altered in a cell-type specific manner, suggesting that EBV differentially activates or utilizes signaling pathways in B versus epithelial cells. Here we correlated activation of the mTORC1, ERK1/2, and p38 pathways in relation to EBV lytic replication and discovered that activation of MAPK-interacting kinase 1/2 (Mnk1/2) was strongly associated with EBV lytic replication. Inhibition studies revealed that Mnk1/2 activated lytic replication in epithelial cells yet acted as an inhibitor of lytic replication in B cells. The ability of lytic epithelial cells to migrate, a potentially important aspect of metastasis, was also demonstrated to be dependent upon Mnk1/2 activity.

Complementing machine learning-based structure predictions with native mass spectrometry.

The advent of machine learning‐based structure prediction algorithms such as AlphaFold2 (AF2) and RoseTTa Fold have moved the generation of accurate structural models for the entire cellular protein machinery into the reach of the scientific community. However, structure predictions of protein complexes are based on user‐provided input and may require experimental validation. Mass spectrometry (MS) is a versatile, time‐effective tool that provides information on post‐translational modifications, ligand interactions, conformational changes, and higher‐order oligomerization. Using three protein systems, we show that native MS experiments can uncover structural features of ligand interactions, homology models, and point mutations that are undetectable by AF2 alone. We conclude that machine learning can be complemented with MS to yield more accurate structural models on a small and large scale.

Multiplex base- and prime-editing with drive-and-process CRISPR arrays

Current base- and prime-editing technologies lack efficient strategies to edit multiple genomic loci simultaneously, limiting their applications in complex genomics and polygenic diseases. Here, we describe drive-and-process (DAP) CRISPR array architectures for multiplex base-editing (MBE) and multiplex prime-editing (MPE) in human cells. We leverage tRNA as the RNA polymerase III promoter to drive the expression of tandemly assembled tRNA-guide RNA (gRNA) arrays, of which the individual gRNAs are released by the cellular endogenous tRNA processing machinery. We engineer a 75-nt human cysteine tRNA (hCtRNA) for the DAP array, achieving up to 31-loci MBE and up to 3-loci MPE. By applying MBE or MPE elements for deliveries via adeno-associated virus (AAV) and lentivirus, we demonstrate simultaneous editing of multiple disease-relevant genomic loci. Our work streamlines the expression and processing of gRNAs on a single array and establishes efficient MBE and MPE strategies for biomedical research and therapeutic applications.

Complete genome sequence of Pseudomonas stutzeri S116 owning bifunctional catalysis provides insights into affecting performance of microbial fuel cells

BackgroundPseudomonas stutzeri S116 is a sulfur-oxidizing bacteria isolated from marine sludge. It exhibited excellent electricity generation as bioanode and biocathode applied in microbial fuel cells (MFCs). Complete genome sequencing of P. stutzeri and cyclic voltammetry method were performed to reveal its mechanism in microbial fuel cells system.ResultsThis study indicated that the MFCs generated a maximum output voltage of 254.2 mV and 226.0 mV, and maximum power density of 765 mW/m2 and 656.6 mW/m2 respectively. Complete genome sequencing of P. stutzeri S116 was performed to indicate that most function genes showed high similarities with P. stutzeri, and its primary annotations were associated with energy production and conversion (6.84%), amino acid transport and metabolism (6.82%) and inorganic ion transport and metabolism (6.77%). Homology of 36 genes involved in oxidative phosphorylation was detected, which suggests the strain S116 possesses an integrated electron transport chain. Additionally, many genes encoding pilus-assembly proteins and redox mediators (riboflavin and phenazine) were detected in the databases. Thiosulfate oxidization and dissimilatory nitrate reduction were annotated in the sulfur metabolism pathway and nitrogen metabolism pathway, respectively. Gene function analysis and cyclic voltammetry indicated that P. stutzeri probably possesses cellular machinery such as cytochrome c and redox mediators and can perform extracellular electron transfer and produce electricity in MFCs.ConclusionThe redox mediators secreted by P. stutzeri S116 were probably responsible for performance of MFCs. The critical genes and metabolic pathways involved in thiosulfate oxide and nitrate reduction were detected, which indicated that the strain can treat wastewater containing sulfide and nitrite efficiently.

Epi-miRNAs: Modern mediators of methylation status in human cancers.

Methylation of the fundamental macromolecules, DNA/RNA, and proteins, is remarkably abundant, evolutionarily conserved, and functionally significant in cellular homeostasis and normal tissue/organism development. Disrupted methylation imprinting is strongly linked to loss of the physiological equilibrium and numerous human pathologies, and most importantly to carcinogenesis, tumor heterogeneity, and cancer progression. Mounting recent evidence has documented the active implication of miRNAs in the orchestration of the multicomponent cellular methylation machineries and the deregulation of methylation profile in the epigenetic, epitranscriptomic, and epiproteomic levels during cancer onset and progression. The elucidation of such regulatory networks between the miRNome and the cellular methylation machineries has led to the emergence of a novel subclass of miRNAs, namely "epi-miRNAs" or "epi-miRs." Herein, we have summarized the existing knowledge on the functional role of epi-miRs in the methylation dynamic landscape of human cancers and their clinical utility in modern cancer diagnostics and tailored therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.

Typhoid toxin sorting and exocytic transport from Salmonella Typhi-infected cells.

Typhoid toxin is an essential virulence factor for Salmonella Typhi, the cause of typhoid fever in humans. This toxin has an unusual biology in that it is produced by Salmonella Typhi only when located within host cells. Once synthesized, the toxin is secreted to the lumen of the Salmonella-containing vacuole from where it is transported to the extracellular space by vesicle carrier intermediates. Here, we report the identification of the typhoid toxin sorting receptor and components of the cellular machinery that packages the toxin into vesicle carriers, and exports it to the extracellular space. We found that the cation-independent mannose-6-phosphate receptor serves as typhoid toxin sorting receptor and that the coat protein COPII and the GTPase Sar1 mediate its packaging into vesicle carriers. Formation of the typhoid toxin carriers requires the specific environment of the Salmonella Typhi-containing vacuole, which is determined by the activities of specific effectors of its type III protein secretion systems. We also found that Rab11B and its interacting protein Rip11 control the intracellular transport of the typhoid toxin carriers, and the SNARE proteins VAMP7, SNAP23, and Syntaxin 4 their fusion to the plasma membrane. Typhoid toxin's cooption of specific cellular machinery for its transport to the extracellular space illustrates the remarkable adaptation of an exotoxin to exert its function in the context of an intracellular pathogen.

Regulation of protein complex partners as a compensatory mechanism in aneuploid tumors.

Aneuploidy, a state of chromosome imbalance, is a hallmark of human tumors, but its role in cancer still remains to be fully elucidated. To understand the consequences of whole-chromosome-level aneuploidies on the proteome, we integrated aneuploidy, transcriptomic, and proteomic data from hundreds of The Cancer Genome Atlas/Clinical Proteomic Tumor Analysis Consortium tumor samples. We found a surprisingly large number of expression changes happened on other, non-aneuploid chromosomes. Moreover, we identified an association between those changes and co-complex members of proteins from aneuploid chromosomes. This co-abundance association is tightly regulated for aggregation-prone aneuploid proteins and those involved in a smaller number of complexes. On the other hand, we observed that complexes of the cellular core machinery are under functional selection to maintain their stoichiometric balance in aneuploid tumors. Ultimately, we provide evidence that those compensatory and functional maintenance mechanisms are established through post-translational control, and that the degree of success of a tumor to deal with aneuploidy-induced stoichiometric imbalance impacts the activation of cellular protein degradation programs and patient survival.

Spatial and temporal crosstalk between the cAMP and Ca2+ signaling systems

The ubiquitous secondary messengers, Ca2+ and cAMP, play a vital role in shaping a diverse array of physiological processes. More significantly, accumulating evidence over the past several decades underpin extensive crosstalk between these two canonical messengers in discrete sub-cellular nanodomains across various cell types. Within such specialized nanodomains, each messenger fine-tunes signaling to maintain homeostasis by manipulating the activities of cellular machinery accountable for the metabolism or activity of the complementary pathway. Interaction between these messengers is ensured by scaffolding proteins which tether components of the signaling machinery in close proximity. Disruption of dynamic communications between Ca2+ and cAMP at these loci consequently is linked to several pathological conditions. This review summarizes recent novel mechanisms underlying effective crosstalk between Ca2+ and cAMP in such nanodomains.

Systems Biology of Virus-Host Protein Interactions: From Hypothesis Generation to Mechanisms of Replication and Pathogenesis.

As obligate intracellular parasites, all viruses must co-opt cellular machinery to facilitate their own replication. Viruses often co-opt these cellular pathways and processes through physical interactions between viral and host proteins. In addition to facilitating fundamental aspects of virus replication cycles, these virus-host protein interactions can also disrupt physiological functions of host proteins, causing disease that can be advantageous to the virus or simply a coincidence. Consequently, unraveling virus-host protein interactions can serve as a window into molecular mechanisms of virus replication and pathogenesis. Identifying virus-host protein interactions using unbiased systems biology approaches provides an avenue for hypothesis generation. This review highlights common systems biology approaches for identification of virus-host protein interactions and the mechanistic insights revealed by these methods. We also review conceptual innovations using comparative and integrative systems biology that can leverage global virus-host protein interaction data sets to more rapidly move from hypothesis generation to mechanism.

Laurel Attenuates Dexamethasone-Induced Skeletal Muscle Atrophy In Vitro and in a Rat Model

Prevention of muscle atrophy contributes to improved quality of life and life expectancy. In this study, we investigated the effects of laurel, selected from 34 spices and herbs, on dexamethasone (DEX)-induced skeletal muscle atrophy and deciphered the underlying mechanisms. Co-treatment of C2C12 myotubes with laurel for 12 h inhibited the DEX-induced expression of intracellular ubiquitin ligases—muscle atrophy F-box (atrogin-1/MAFbx) and muscle RING finger 1 (MuRF1)—and reduction in myotube diameter. Male Wistar rats were supplemented with 2% laurel for 17 days, with DEX-induced skeletal muscle atrophy occurring in the last 3 days. Laurel supplementation inhibited the mRNA expression of MuRF1, regulated DNA damage and development 1 (Redd1), and forkhead box class O 1 (Foxo1) in the muscles of rats. Mechanistically, we evaluated the effects of laurel on the cellular proteolysis machinery—namely, the ubiquitin/proteasome system and autophagy—and the mTOR signaling pathway, which regulates protein synthesis. These data indicated that the amelioration of DEX-induced skeletal muscle atrophy induced by laurel, is mainly mediated by the transcriptional inhibition of downstream factors of the ubiquitin-proteasome system. Thus, laurel may be a potential food ingredient that prevents muscle atrophy.

Stress-induced protein disaggregation in the endoplasmic reticulum catalysed by BiP.

Protein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates’ formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone – BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.

Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells.

In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.