Complexes {RuCp(PPh
)
-μ-dmoPTA-1κ
:2κ
-
,
'-CuCl}
-μ-Cl-μ-OCH
(CF
SO
)
·(CH
OH)
(1) and {RuCp(PPh
)
-μ-dmoPTA-1κ
:2κ
-
,
'-NiCl}
-μ-Cl-μ-OH(CF
SO
)
(2) have been synthesized and characterized. Their ...antiproliferative activities were assessed against six human solid tumours showing nanomolar GI
values. The effects of 1 and 2 on SW1573 cells colony formation, HeLa cells action mechanism and their interaction with the pBR322 DNA plasmid were evaluated.
During cytokinesis, a specialized set of proteins is recruited to the equatorial region between spindle poles by microtubules and actin filaments, enabling furrow assembly and ingression before cell ...division. We investigate the mechanisms underlying regional specialization of the cytoskeleton in HeLa cells undergoing drug-synchronized monopolar cytokinesis. After forced mitotic exit, the cytoskeleton of monopolar mitotic cells is initially radially symmetric but undergoes a symmetry-breaking reaction that simultaneously polarizes microtubules and the cell cortex, with a concentration of cortical furrow markers into a cap at one side of the cell. Polarization requires microtubules, F-actin, RhoA, Myosin II activity, and Aurora B kinase activity. Aurora B localizes to actin cables in a gap between the monopolar midzone and the furrow-like cortex, suggesting a communication between them. We propose that feedback loops between cortical furrow components and microtubules promote symmetry breaking during monopolar cytokinesis and regional specialization of the cytoskeleton during normal bipolar cytokinesis.
The thioredoxin system, composed of thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH, is ubiquitous in all cells and involved in many redox-dependent signaling pathways. Curcumin, a ...naturally occurring pigment that gives a specific yellow color in curry food, is consumed in normal diet up to 100mg per day. This molecule has also been used in traditional medicine for the treatment of a variety of diseases. Curcumin has numerous biological functions, and many of these functions are related to induction of oxidative stress. However, how curcumin elicits oxidative stress in cells is unclear. Our previous work has demonstrated the way by which curcumin interacts with recombinant TrxR1 and alters the antioxidant enzyme into a reactive oxygen species (ROS) generator in vitro. Herein we reported that curcumin can target the cytosolic/nuclear thioredoxin system to eventually elevate oxidative stress in HeLa cells. Curcumin-modified TrxR1 dose-dependently and quantitatively transfers electrons from NADPH to oxygen with the production of ROS. Also, curcumin can drastically down-regulate Trx1 protein level as well as its enzyme activity in HeLa cells, which in turn remarkably decreases intracellular free thiols, shifting the intracellular redox balance to a more oxidative state, and subsequently induces DNA oxidative damage. Furthermore, curcumin-pretreated HeLa cells are more sensitive to oxidative stress. Knockdown of TrxR1 sensitizes HeLa cells to curcumin cytotoxicity, highlighting the physiological significance of targeting TrxR1 by curcumin. Taken together, our data disclose a previously unrecognized prooxidant mechanism of curcumin in cells, and provide a deep insight in understanding how curcumin works in vivo.
► Curcumin induces oxidative stress by targeting the thioredoxin system. ► Curcumin-modified TrxR quantitatively oxidizes NADPH to generate ROS. ► Knockdown of TrxR1 augments curcumin's cytotoxicity in HeLa cells. ► Curcumin sensitizes HeLa cells to oxidative stress.
Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that ...assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.
Widespread antisense long noncoding RNA (lncRNA) overlap with many protein-coding genes in mammals and emanate from gene promoter, enhancer, and termination regions. However, their origin and ...biological purpose remain unclear. We show that these antisense lncRNA can be generated by R-loops that form when nascent transcript invades the DNA duplex behind elongating RNA polymerase II (Pol II). Biochemically, R-loops act as intrinsic Pol II promoters to induce de novo RNA synthesis. Furthermore, their removal across the human genome by RNase H1 overexpression causes the selective reduction of antisense transcription. Consequently, we predict that R-loops act to facilitate the synthesis of many gene proximal antisense lncRNA. Not only are R-loops widely associated with DNA damage and repair, but we now show that they have the capacity to promote de novo transcript synthesis that may have aided the evolution of gene regulation.
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•R-loops formed within plasmids promote antisense transcription in nuclear extracts•TSS of lncRNA and eRNA are often near R-loop structures and sensitive to RNase H1•Preinitiation complexes associated with lncRNA synthesis are R-loop dependent•Many mammalian lncRNA derive from R-loop promoter activity
Tan-Wong et al. demonstrate that R-loop structures, often formed at the promoter, terminator, and enhancer regions of human protein-coding genes, act as promoters to generate antisense lncRNA. In effect, R-loop promoter activity may account for the existence of many lncRNA that are detected across mammalian genomes.
Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to ...mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.
Recent findings have significantly expanded our understanding of the regulation of actin-depolymerizing factor (ADF)/cofilin proteins and the profound multifaceted impact that these well-established ...regulators of actin dynamics have on cell biology. In this review we discuss new aspects of previously documented regulation, such as phosphorylation, but also cover novel recently established modes of regulation and functions of ADF (also known as destrin)/cofilin. We now understand that their activity responds to a vast array of inputs far greater than previously appreciated and that these proteins not only feed back to the crucially important dynamics of actin, but also to apoptosis cascades, phospholipid metabolism, and gene expression. We argue that this ability to respond to physiological changes by modulating those same changes makes the ADF/cofilin protein family a homeostatic regulator or ‘functional node’ in cell biology.
Transcription factors (TFs) represent a major class of therapeutic targets for the treatment of human diseases including cancer. Although the biological functions and even crystal structures of many ...TFs have been clearly elucidated, there is still no viable approach to target the majority of TFs, thus rendering them undruggable for decades. PROTACs (proteolysis targeting chimeras) emerge as a powerful class of therapeutic modalities, which rely on induced protein–protein interactions between the proteins of interest (POIs) and E3 ubiquitin ligases to aid the degradation of POIs by the ubiquitin-proteasome system (UPS). Here, we report the development of a platform termed TF-PROTAC, which links an DNA oligonucleotide to an E3 ligase ligand via a click reaction, to selectively degrade the TF of interest. The selectivity of these TF-PROTACs depends on the DNA oligonucleotides utilized that can be specific to the TFs of interest. We have developed two series of VHL-based TF-PROTACs, NF-κB-PROTAC (dNF-κB) and E2F-PROTAC (dE2F), which effectively degrade endogenous p65 and E2F1 proteins in cells, respectively, and subsequently display superior antiproliferative effects in cells. Collectively, our results suggest that TF-PROTACs provide a generalizable platform to achieve selective degradation of TFs and a universal strategy for targeting most “undruggable” TFs.
N
-methyladenosine (m
A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and tissue development. It regulates ...multiple steps throughout the RNA life cycle including RNA processing, translation, and decay, via the recognition by selective binding proteins. In the cytoplasm, m
A binding protein YTHDF1 facilitates translation of m
A-modified mRNAs, and YTHDF2 accelerates the decay of m
A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m
A binder of the YTH (YT521-B homology) domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated through YTHDF2. Cells deficient in all three YTHDF proteins experience the most dramatic accumulation of m
A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m
A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m
A RNA methylation.
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