Dynamin interacts with members of the sumoylation machinery

Dynamin is a GTP-binding protein whose oligomerization-dependent assembly around the necks of lipid vesicles mediates their scission from parent membranes. Dynamin is thus directly involved in the regulation of endocytosis. Sumoylation is a post-translational protein modification whereby the ubiquitin-like modifier Sumo is covalently attached to lysine residues on target proteins by a process requiring the concerted action of an activating enzyme (ubiquitin-activating enzyme), a conjugating enzyme (ubiquitin carrier protein), and a ligating enzyme (ubiquitin-protein is opeptide ligase). Here, we show that dynamin interacts with Sumo-1, Ubc9, and PIAS-1, all of which are members of the sumoylation machinery. Ubc9 and PIAS-1 are known ubiquitin carrier protein and ubiquitin-protein isopeptide ligase enzymes, respectively, for the process of sumoylation. We have identified the coiled-coil GTPase effector domain (GED) of dynamin as the site on dynamin that interacts with Sumo-1, Ubc9, and PIAS-1. Although we saw no evidence of covalent Sumo-1 attachment to dynamin, Sumo-1 and Ubc9 are shown here to inhibit the lipid-dependent oligomerization of dynamin. Expression of Sumo-1 and Ubc9 in mammalian cells down-regulated the dynamin-mediated endocytosis of transferrin, whereas dynamin-independent fluid-phase uptake was not affected. Furthermore, using high resolution NMR spectroscopy, we have identified amino acid residues on Sumo-1 that directly interact with the GED of dynamin. The results suggest that the GED of dynamin may serve as a scaffold that concentrates the sumoylation machinery in the vicinity of potential acceptor proteins

Ashwagandha Derived Withanone Targets TPX2-Aurora A Complex: Computational and Experimental Evidence to its Anticancer Activity

Cancer is largely marked by genetic instability. Specific inhibition of individual proteins or signalling pathways that regulate genetic stability during cell division thus hold a great potential for cancer therapy. The Aurora A kinase is a Ser/Thr kinase that plays a critical role during mitosis and cytokinesis and is found upregulated in several cancer types. It is functionally regulated by its interactions with TPX2, a candidate oncogene. Aurora A inhibitors have been proposed as anticancer drugs that work by blocking its ATP binding site. This site is common to other kinases and hence these inhibitors lack specificity for Aurora A inhibition in particular, thus advocating the need of some alternative inhibition route. Previously, we identified TPX2 as a cellular target for withanone that selectively kill cancer cells. By computational approach, we found here that withanone binds to TPX2-Aurora A complex. In experiment, withanone treatment to cancer cells indeed resulted in dissociation of TPX2-Aurora A complex and disruption of mitotic spindle apparatus proposing this as a mechanism of the anticancer activity of withanone. From docking analysis, non-formation/disruption of the active TPX2-Aurora A association complex could be discerned. Our MD simulation results suggesting the thermodynamic and structural stability of TPX2-Aurora A in complex with withanone further substantiates the binding. We report a computational rationale of the ability of naturally occurring withanone to alter the kinase signalling pathway in an ATP-independent manner and experimental evidence in which withanone cause inactivation of the TPX2-Aurora A complex. The study demonstrated that TPX2-Aurora A complex is a target of withanone, a potential natural anticancer drug

Nations within a nation: variations in epidemiological transition across the states of India, 1990–2016 in the Global Burden of Disease Study

18% of the world's population lives in India, and many states of India have populations similar to those of large countries. Action to effectively improve population health in India requires availability of reliable and comprehensive state-level estimates of disease burden and risk factors over time. Such comprehensive estimates have not been available so far for all major diseases and risk factors. Thus, we aimed to estimate the disease burden and risk factors in every state of India as part of the Global Burden of Disease (GBD) Study 2016

Deficiency in Cardiolipin Reduces Doxorubicin-Induced Oxidative Stress and Mitochondrial Damage in Human B-Lymphocytes

<div><p>Cardiolipin (CL) is an inner mitochondrial membrane phospholipid which plays an important role in mitochondrial function. Perturbation in CL biosynthesis alters mitochondrial bioenergetics causing a severe genetic disorder commonly known as Barth syndrome. Barth syndrome patients are known to have a reduced concentration and altered composition of CL. Cardiolipin is also known to have a high affinity for the chemotherapeutic agent doxorubicin (Dox), resulting in an extensive mitochondrial accumulation of the drug. Our results indicate that B-lymphocytes from healthy individuals are more sensitive to Dox-induced oxidative stress and cellular toxicity compared to the B-lymphocytes from Barth syndrome as indicated by greater cell death and greater level of cleaved caspase-3 following Dox treatment. Barth lymphocytes, when compared to healthy lymphocytes, showed a greater basal level of mitochondrial reactive oxygen species (mito-ROS), yet exhibited a lower level of induced mito-ROS production in response to Dox. Significantly less ATP content and slightly greater OXPHOS protein levels were detected in healthy cells compared to Barth cells after Dox treatment. Consistent with greater mitochondrial ROS, treatment with Dox induced a higher level of lipid peroxidation and protein carbonylation in healthy lymphocytes compared to Barth lymphocytes. The final remodeling of CL during CL synthesis is catalyzed by the tafazzin protein. Knockdown of <i>tafazzin</i> gene in H9c2 cardiomyocytes using siRNA showed decreased oxidant-induced damage, as observed in Barth lymphocytes. Our findings demonstrate that a deficiency in CL might provide a therapeutic advantage in favor of oxidant-induced anticancer activities.</p></div

Determination of lipid peroxidation and total protein carbonylation in healthy and Barth lymphocytes.

<p>Quantitative determination of lipid peroxidation product in (A) healthy and Barth lymphocytes at basal level and (B) Dox-induced lipid peroxidation in healthy and Barth lymphocytes normalized to control. MDA-TBA adduct was measured at 532 nm and normalized to mg of protein in the cell lysate. To determine the Dox-induced changes in lipid peroxidation between healthy and Barth lymphocytes, basal levels of MDA for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in MDA level were normalized against corresponding control. (C) Total protein carbonylation in healthy and Barth lymphocytes with and without Dox (1μM) treatment was determined by ELISA. *p<0.05; **p<0.001.</p

Dox-induced cell death in Barth and healthy lymphocytes.

<p>(A) Annexin V and propidium iodide staining was used to measure induction of apoptosis by 0.1, 0.5, and 1 μM of Dox following 24 h treatment. VP16 (10 μM) was used as a positive control. (B) Representative western blot showing the level of cleaved caspase-3 in healthy and Barth lymphocytes after 0.5 and 1 μM Dox treatment for 24 h. (C) Quantitative analysis of cleaved caspase-3 from (B) normalized by α-tubulin. Cleaved caspase-3 levels for control (untreated) healthy and Barth lymphocytes were normalized to 100 and Dox-induced changes in cleaved caspase-3 were normalized against corresponding control. (D) Representative western blot showing the level of autophagy induction determined by LC3-II levels in healthy and Barth lymphocytes after 0.5 and 1 μM Dox treatment for 24 h with or without 5 nM bafilomycin A1 for final 2 h of Dox treatment. (E) Quantitative analysis of LC3-II from (D) normalized by α-tubulin. LC3-II levels for control (untreated) healthy and Barth lymphocytes were normalized to 100 and Dox-induced changes in LC3-II level in the presence or absence of bafilomycin A1 were normalized against corresponding control. *p<0.05; **p<0.001; H = healthy B-lymphocytes, B = Barth B-lymphocytes, Baf = bafilomycin A1.</p

<i>Tafazzin</i> knockdown and Dox-sensitivity in H9c2 cardiomyocytes.

<p>(A) knockdown of <i>tafazzin</i> was confirmed in H9c2 cells after transfection with 20 nM <i>tafazzin</i> siRNA for 48 h by western blot analysis. (B) Representative western blot showing the level of cleaved caspase-3. After transfection with <i>tafazzin</i> and NTP siRNA for 24 h, cells were treated with 0.5 and 1 μM Dox for 24 h and cleaved caspase-3 levels were quantified using tubulin as a loading control. (C) Quantitative analysis of cleaved caspase-3 from (B) normalized by α-tubulin. (D) Mitochondrial superoxide levels were measured using MitoSOX Red dye in <i>tafazzin</i> and NTP transfected control H9c2 cells at basal levels and (E) after 0.5 and 1μM Dox treatment for 24 h. The changes in mean fluorescence intensity of mitochondrial superoxide sensitive dye MitoSOX Red in healthy and Barth lymphocytes was normalized by the corresponding control sample to compare the Dox-induced effect in both cell types. Mitochondrial complex III inhibitor Antimycin A (20 μM) was used as a positive control. (F) Measurement of mitochondrial membrane potential using JC-1 dye for NTP and Taz siRNA transfected H9c2 after treatment with Dox for 2 h. Y-axis represents the relative fluorescence ratio of 590/540 nm normalized to control without Dox treatment. Basal level of mitochondrial membrane potential in Taz siRNA transfected cells was slightly lower than NTP siRNA transfected cells (1.03 for NTP siRNA transfected cells and 0.9 for Taz siRNA transfected cells). CCCP (10 μM), a commonly used mitochondrial membrane depolarization agent was used as a positive control. To determine the Dox-induced effect between NTP and Taz siRNA transfected H9c2 cells, basal level of ROS and mitochondrial membrane potential of untreated (control) NTP and Taz siRNA transfected H9c2 cells were normalized to 100; and Dox-induced changes in the ROS and mitochondrial membrane potential were normalized against corresponding control. *p<0.05; **p<0.001.</p

Determination of Dox-induced ATP levels in healthy and Barth lymphocytes.

<p>Healthy and Barth lymphocytes were treated with vehicle (control) or 0.5, 1, and 5 μM of Dox for 8 and 24 h and cellular ATP content was determined (A) at basal levels without Dox treatment and (B-C) in control and Dox-treated lymphocytes using a kit from Molecular Probes. Amount of ATP was calculated as pmol of ATP per 1x10⁶ cells. The y-axis shows the amount of ATP per 1x10⁶ cells. To determine the Dox-induced changes in the ATP level between healthy and Barth lymphocytes, basal level of ATP for control (untreated) healthy and Barth lymphocytes were normalized to 100; and Dox-induced changes in ATP level were normalized against corresponding control. **p<0.001.</p