TRAIN (Transcription of Repeats Activates INterferon) in response to chromatin destabilization induced by small molecules in mammalian cells

Cellular responses to the loss of genomic stability are well-established, while how mammalian cells respond to chromatin destabilization is largely unknown. We previously found that DNA demethylation on p53-deficient background leads to transcription of repetitive heterochromatin elements, followed by an interferon response, a phenomenon we named TRAIN (Transcription of Repeats Activates INterferon). Here, we report that curaxin, an anticancer small molecule, destabilizing nucleosomes via disruption of histone/DNA interactions, also induces TRAIN. Furthermore, curaxin inhibits oncogene-induced transformation and tumor growth in mice in an interferon-dependent manner, suggesting that anticancer activity of curaxin, previously attributed to p53-activation and NF-kappaB-inhibition, may also involve induction of interferon response to epigenetic derepression of the cellular ‘repeatome’. Moreover, we observed that another type of drugs decondensing chromatin, HDAC inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of mammalian cells via elimination of cells with desilenced chromatin.

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Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

10.1101/2020.06.11.147199 ◽

2020 ◽

Cited By ~ 16

Author(s):

Christiane Iserman ◽

Christine Roden ◽

Mark Boerneke ◽

Rachel Sealfon ◽

Grace McLaughlin ◽

...

Keyword(s):

Small Molecules ◽

Mammalian Cells ◽

Upper Airway ◽

Dependent Manner ◽

Rna Sequences ◽

Concentration Dependent Manner ◽

Health Crisis ◽

Viral Packaging ◽

N Protein ◽

Human Body Temperature

AbstractA mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

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Transcription of Repeats Activates INterferon (TRAIN) in response to chromatin destabilization induced with anti-cancer small molecule

10.1101/142471 ◽

2017 ◽

Author(s):

Katerina Leonova ◽

Alfiya Safina ◽

Elimelech Nesher ◽

Poorva Sandlesh ◽

Rachel Pratt ◽

...

Keyword(s):

Small Molecule ◽

Interferon Response ◽

Drug Candidate ◽

Type I ◽

Dependent Manner ◽

Dna Hypomethylation ◽

Deacetylase Inhibitor ◽

Dna Elements ◽

A Cell ◽

Nf Kappab

AbstractThe anticancer activity of genotoxic agents has been intensively studied, while the mechanisms of action of drugs destabilizing the epigenome are far less understood. We previously found that DNA hypomethylation in the absence of p53 leads to transcriptional desilencing of repetitive DNA elements, such as pericentromeric repeats and endogenous retroelements, which is associated with an interferon type I response, a phenomenon we named TRAIN (Transcription ofRepeatsActivatesINterferon). Here, we report that curaxin, a small molecule anticancer drug candidate, which destabilizes nucleosomes via disruption of histone/DNA interactions, can induce TRAIN independently of the p53 status of a cell. Furthermore, curaxin inhibits oncogene-induced transformation in an interferon-dependent manner, suggesting that cancer prevention by curaxin, previously attributed to its p53-activating and NF-kappaB-inhibiting activities, may also involve the induction of the interferon response to epigenetic derepression of the cellular “repeatome.” Moreover, we observed that another type of drugs decondensing chromatin, histone deacetylase inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of cells via elimination of cells with desilenced chromatin.

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Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

10.26434/chemrxiv.6447683.v1 ◽

2018 ◽

Author(s):

Todd D. Gruber ◽

Chithra Krishnamurthy ◽

Jonathan B. Grimm ◽

Michael R. Tadross ◽

Laura M. Wysocki ◽

...

Keyword(s):

Small Molecules ◽

Mammalian Cells ◽

Target Cells ◽

Specific Chemical ◽

E Coli ◽

Enzyme Substrate ◽

Cell Specificity ◽

General Chemical ◽

Increased Activity ◽

Enzyme Variant

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2‑nitro-N-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

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Faculty Opinions recommendation of Induction of an interferon response by RNAi vectors in mammalian cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1014289.194712 ◽

2003 ◽

Author(s):

Jenny Ting

Keyword(s):

Mammalian Cells ◽

Interferon Response

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Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

Scientific Reports ◽

10.1038/s41598-021-92991-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benjamin N. Nelson ◽

Savannah G. Beakley ◽

Sierra Posey ◽

Brittney Conn ◽

Emma Maritz ◽

...

Keyword(s):

Antifungal Activity ◽

Cryptococcus Neoformans ◽

Mammalian Cells ◽

Cysteine Proteases ◽

Dependent Manner ◽

Life Threatening ◽

Opportunistic Fungal Pathogen ◽

Dose Dependent ◽

Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

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Epigenetic Modulation of TLR4 Expression by Sulforaphane Increases Anti-Inflammatory Capacity in Porcine Monocyte-Derived Dendritic Cells

Biology ◽

10.3390/biology10060490 ◽

2021 ◽

Vol 10 (6) ◽

pp. 490

Author(s):

Xueqi Qu ◽

Christiane Neuhoff ◽

Mehmet Ulas Cinar ◽

Maren Pröll ◽

Ernst Tholen ◽

...

Keyword(s):

Dna Methylation ◽

Dendritic Cells ◽

Epigenetic Modifications ◽

Dna Demethylation ◽

Dependent Manner ◽

Experimental Conditions ◽

Hdac Activity ◽

Protein Levels ◽

Anti Inflammatory ◽

Epigenetic Modulation

Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines’ expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs.

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The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01228-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Alyssa N. Coyne ◽

Jeffrey D. Rothstein

Keyword(s):

Nuclear Pore Complex ◽

Mammalian Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Dependent Manner ◽

Structured Illumination Microscopy ◽

Neuronal Nuclei ◽

Complex Injury ◽

Sporadic Als ◽

Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

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Regulation of the tumour suppressor Fbw7α by PKC-dependent phosphorylation and cancer-associated mutations

Biochemical Journal ◽

10.1042/bj20100799 ◽

2010 ◽

Vol 432 (1) ◽

pp. 77-87 ◽

Cited By ~ 12

Author(s):

Joanne Durgan ◽

Peter J. Parker

Keyword(s):

Mammalian Cells ◽

Tumour Suppressor ◽

Dependent Manner ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Pkc Protein Kinase C ◽

Specific Regulation ◽

The Relationship ◽

Substrate Binding Domain

Fbw7 (F-box WD40 protein 7) is a major tumour suppressor, which mediates the degradation of several potent oncogenes. PKC (protein kinase C) comprises a serine/threonine kinase family that can promote transformation when dysregulated. In the present study, we investigated the relationship between Fbw7 and PKC. Multiple members of the PKC superfamily interact with the substrate-binding domain of Fbw7. However, we find no evidence for Fbw7-mediated degradation of PKC. Instead, we demonstrate that Fbw7 is a novel substrate for PKC. Two residues within the isoform-specific N-terminus of Fbw7α are phosphorylated in a PKC-dependent manner, both in vitro and in mammalian cells (Ser10 and Ser18). Mutational analyses reveal that phosphorylation of Fbw7α at Ser10 can regulate its nuclear localization. Cancer-associated mutations in nearby residues (K11R and the addition of a proline residue at position 16) influence Fbw7α localization in a comparable manner, suggesting that mislocalization of this protein may be of pathological significance. Together these results provide evidence for both physical and functional interactions between the PKC and Fbw7 families, and yield insights into the isoform-specific regulation of Fbw7α.

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Phosphorylation: The Molecular Switch of Double-Strand Break Repair

International Journal of Proteomics ◽

10.1155/2011/373816 ◽

2011 ◽

Vol 2011 ◽

pp. 1-8 ◽

Cited By ~ 24

Author(s):

K. C. Summers ◽

F. Shen ◽

E. A. Sierra Potchanant ◽

E. A. Phipps ◽

R. J. Hickey ◽

...

Keyword(s):

Mammalian Cells ◽

Genomic Stability ◽

Molecular Switches ◽

Strand Break ◽

Nonhomologous End Joining ◽

End Joining ◽

Brca1 And Brca2 ◽

Damage Response ◽

Repair Mechanisms ◽

Repair Pathways

Repair of double-stranded breaks (DSBs) is vital to maintaining genomic stability. In mammalian cells, DSBs are resolved in one of the following complex repair pathways: nonhomologous end-joining (NHEJ), homologous recombination (HR), or the inclusive DNA damage response (DDR). These repair pathways rely on factors that utilize reversible phosphorylation of proteins as molecular switches to regulate DNA repair. Many of these molecular switches overlap and play key roles in multiple pathways. For example, the NHEJ pathway and the DDR both utilize DNA-PK phosphorylation, whereas the HR pathway mediates repair with phosphorylation of RPA2, BRCA1, and BRCA2. Also, the DDR pathway utilizes the kinases ATM and ATR, as well as the phosphorylation of H2AX and MDC1. Together, these molecular switches regulate repair of DSBs by aiding in DSB recognition, pathway initiation, recruitment of repair factors, and the maintenance of repair mechanisms.

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