scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Epigenetic Regulator ◽  
Novel Target ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

AbstractAging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Progressive Loss ◽  
Epigenetic Regulator ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

Abstract Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

scholarly journals SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

2021 ◽  
Author(s):  
Scott B Biering ◽  
Francielle Tramontini Gomes de Sousa ◽  
Laurentia V. Tjang ◽  
Felix Pahmeier ◽  
Richard Ruan ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Barrier Dysfunction ◽  
Vascular Leak ◽  
Endothelial Barrier Dysfunction ◽  
Mechanistic Insight ◽  
Starting Point ◽  
Signaling Axis ◽  
Insight Into

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

scholarly journals Mechanistic insight into DsyB/DSYB, key enzymes in marine dimethylsulfoniopropionate synthesis

2021 ◽  
Author(s):  
Jonathan Todd ◽  
Chun-Yang Li ◽  
Jason Crack ◽  
Simone Newton-Payne ◽  
Andrew Murphy ◽  
...  
Keyword(s):  
Marine Algae ◽  
Nucleophilic Attack ◽  
Mechanistic Insight ◽  
Signalling Molecule ◽  
Major Nutrient ◽  
Methyltransferase Gene ◽  
Key Enzyme ◽  
Algae And Bacteria ◽  
Insight Into

Abstract Marine algae and bacteria produce eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth’s surface oceans every year. DMSP is an anti-stress compound and, once released into the environment, a major nutrient, signalling molecule and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine-dependent methyltransferase (SAM-MT) 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). dsyB/DSYB, present in most DMSP-producing bacteria and haptophyte and dinoflagellate algae with the highest known DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known DMSP synthesis pathway S-methyltransferase gene. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans, and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a novel mechanism, distinct from any previously reported SAM-MT, in which the DsyB residue Tyr142 activates the sulfur atom of MTHB for nucleophilic attack on the SAM methyl group. Sequence analysis suggests that this mechanism is common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth’s surface oceans.

scholarly journals Microtubule minus-end stability is dictated by the tubulin off-rate

2019 ◽  
Vol 218 (9) ◽  
pp. 2841-2853 ◽  
Author(s):  
Claire Strothman ◽  
Veronica Farmer ◽  
Göker Arpağ ◽  
Nicole Rodgers ◽  
Marija Podolski ◽  
...  
Keyword(s):  
Dynamic Instability ◽  
In Vitro Reconstitution ◽  
Primary Determinant ◽  
Mean Size ◽  
The Mean ◽  
The Difference ◽  
Insight Into ◽  
Dynamic Organization ◽  
In Cells

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. Here, we use an in vitro reconstitution approach to investigate minus-end dynamics. We find that minus-end lifetimes are not defined by the mean size of the protective GTP-tubulin cap. Rather, we conclude that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, our results show that the minus-end–directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. Our results provide novel insight into the mechanisms of minus-end dynamics, essential for our understanding of microtubule minus-end regulation in cells.

scholarly journals Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Suk Min Jang ◽  
Catherine Lachance ◽  
Wenyi Mi ◽  
Jie Lyu ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Memory Impairment ◽  
Target Genes ◽  
Epigenetic Mark ◽  
Mechanistic Insight ◽  
Genomic Analyses ◽  
Insight Into

Abstract Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

scholarly journals PTP4A3, A Novel Target Gene of HIF-1alpha, Participates in Benzene-Induced Cell Proliferation Inhibition and Apoptosis through PI3K/AKT Pathway

2020 ◽  
Vol 17 (3) ◽  
pp. 910
Author(s):  
Yunqiu Pu ◽  
Fengxia Sun ◽  
Rongli Sun ◽  
Zhaodi Man ◽  
Shuangbin Ji ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Target Gene ◽  
Akt Pathway ◽  
Proliferation Inhibition ◽  
Cell Proliferation Inhibition ◽  
A Cell ◽  
Novel Target ◽  
In Cells

Benzene, a commonly used chemical, has been confirmed to specifically affect the hematopoietic system as well as overall human health. PTP4A3 is overexpressed in leukemia cells and is related to cell proliferation. We previously found that HIF-1alpha was involved in benzene toxicity and PTP4A3 may be the target gene of HIF-1alpha via ChIP-seq. The aim of this study is to confirm the relationship between HIF-1alpha and PTP4A3 in benzene toxicity, as well as the function of PTP4A3 on cell toxicity induced by 1,4-benzoquinone (1,4-BQ). Our results indicate that HIF-1alpha could regulate PTP4A3 with in vivo and in vitro experiments. A cell line with suppressed PTP4A3 was established to investigate the function of PTP4A3 in 1,4-BQ toxicity in vitro. The results revealed that cell proliferation inhibition was more aggravated in PTP4A3 low-expression cells than in the control cells after 1,4-BQ treatment. The relative oxygen species (ROS) significantly increased in cells with inhibited PTP4A3, while the rise was inferior to the control cells at the 20 μM 1,4-BQ group. An increase in DNA damage was seen in PTP4A3 down-regulated cells at the 10 μM 1,4-BQ group, whereas the results reversed at the concentration of 20 μM. Moreover, the apoptosis rate increased higher in down-regulated PTP4A3 cells after 1,4-BQ exposure. In addition, PI3K/AKT pathway was significantly restrained in cells with inhibited PTP4A3 after 1,4-BQ treatment. Our results indicate that HIF-1alpha may regulate PTP4A3 to be involved in benzene toxicity. Inhibition of PTP4A3 could aggravate cell proliferation suppression and apoptosis by regulating PI3K/AKT pathway after 1,4-BQ treatment.

scholarly journals Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity

2021 ◽  
Vol 12 ◽  
Author(s):  
Edanur Sen ◽  
Krishna P. Kota ◽  
Rekha G. Panchal ◽  
Sina Bavari ◽  
Erkan Kiris
Keyword(s):  
Small Molecules ◽  
Ubiquitin Proteasome Pathway ◽  
Lead Compounds ◽  
Botulinum Neurotoxins ◽  
Ubiquitin Proteasome ◽  
Proteasome Pathway ◽  
The Stability ◽  
Dose Dependent ◽  
In Cells

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.

scholarly journals A Trypanosoma brucei orphan kinesin employs a convergent microtubule organization strategy to complete cytokinesis

2021 ◽  
Author(s):  
Thomas E Sladewski ◽  
Paul C Campbell ◽  
Neil Billington ◽  
Alexandra D'Ordine ◽  
Christopher L de Graffenried
Keyword(s):  
Trypanosoma Brucei ◽  
Biophysical Properties ◽  
Microtubule Organization ◽  
Division Plane ◽  
Mechanistic Insight ◽  
Organization Strategy ◽  
Cytoskeletal Elements ◽  
Order Structures ◽  
Insight Into

Many single-celled eukaryotes have complex cell morphologies defined by cytoskeletal elements comprising microtubules arranged into higher-order structures. Trypanosoma brucei (T. brucei) cell polarity is mediated by a parallel array of microtubules that underlie the plasma membrane and define the auger-like shape of the parasite. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the division plane and is essential for the completion of cytokinesis. To gain mechanistic insight into how this novel kinesin functions to complete cleavage furrow ingression, we characterized the biophysical properties of the KLIF motor domain in vitro. We found that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked in an antiparallel orientation are translocated relative to one another by KLIF, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we found that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new posterior, which is a unique requirement for the completion of cytokinesis in T. brucei.

scholarly journals Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA

2020 ◽  
Vol 117 (3) ◽  
pp. 1457-1467 ◽  
Author(s):  
Peiyuan Zhang ◽  
Hye-Jin Park ◽  
Jie Zhang ◽  
Eunsung Junn ◽  
Ryan J. Andrews ◽  
...  
Keyword(s):  
Small Molecules ◽  
Small Molecule ◽  
Intrinsically Disordered Proteins ◽  
Lewy Bodies ◽  
Disordered Proteins ◽  
Lewy Neurites ◽  
Protein Levels ◽  
Intrinsically Disordered ◽  
In Cells

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson’s disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5′ untranslated region (5′ UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5′ UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound’s selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

scholarly journals 4040 Investigation of a Series of 1,4-diaryl-pyrazolo-pyridinones as Anti-Leishmanial Agents

2020 ◽  
Vol 4 (s1) ◽  
pp. 10-10
Author(s):  
Hannah Noel Corman ◽  
Douglas A. Shoue ◽  
Bruce J. Melancon ◽  
Mary Ann McDowell
Keyword(s):  
Small Molecules ◽  
High Volume ◽  
Similar Efficacy ◽  
Volume Of Distribution ◽  
Structural Class ◽  
Ic50 Values ◽  
Study Population ◽  
Novel Target

OBJECTIVES/GOALS: This study was conducted in order to identify novel chemical compounds that exhibit anti-leishmanial activity and to further characterize their efficacy and toxicity in in vitro and in vivo systems in the hopes of future chemotherapeutic developments. METHODS/STUDY POPULATION: We developed a novel, target-free fluorometric high-throughput screen (HTS) to identify small molecules with anti-leishmanial activity. Screening of 10,000 small molecules from the ChemBridge DIVERset-EXP library cassette #5 yielded 210 compounds that killed 80% of parasites. One hundred nine (109) molecular scaffolds were represented within the hit compounds, including the 1,4-diaryl-pyrazolo-pyridinone (1,4-DAPP). A total of 27 novel 1,4-DAPP compounds were synthesized and anti-leishmanial efficacy and host cell toxicity was determined using L. donovani mCherry expressing amastigotes and THP-1 macrophages. Additional pharmacokinetic analyses of a potent 1,4-DAPP compound were conducted. RESULTS/ANTICIPATED RESULTS: Four experimental compounds had IC50 values less than 5μM, providing similar anti-leishmanial activity to miltefosine. Compound 9279817 had a clearance almost twice the rate of normal hepatic blood flow and had a relatively high volume of distribution, indicating this compound is rapidly cleared and distributes into tissues. in vitro rat liver microsome assays suggest a rapid metabolism of 9279817, and MS/MS results suggest this metabolite is most likely formed via oxidation of the sulfur on the lower aromatic ring. DISCUSSION/SIGNIFICANCE OF IMPACT: This study revealed a novel structural class of compounds that have anti-leishmanial activity. in vitro experiments show compounds with similar efficacy as miltefosine while having significantly less toxicity, suggesting that this class could be further developed as a potential chemotherapeutic.

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