Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca²⁺/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.     

Plant proteomics in India and Nepal: current status and challenges ahead

Plant proteomics has made tremendous contributions in understanding the complex processes of plant biology. Here, its current status in India and Nepal is discussed. Gel-based proteomics is predominantly utilized on crops and non-crops to analyze majorly abiotic (49 %) and biotic (18 %) stress, development (11 %) and post-translational modifications (7 %). Rice is the most explored system (36 %) with major focus on abiotic mainly dehydration (36 %) stress. In spite of expensive proteomics setup and scarcity of trained workforce, output in form of publications is encouraging. To boost plant proteomics in India and Nepal, researchers have discussed ground level issues among themselves and with the International Plant Proteomics Organization (INPPO) to act in priority on concerns like food security. Active collaboration may help in translating this knowledge to fruitful applications.     

An efficient algorithm for protein structure comparison using elastic shape analysis

Background

Protein structure comparison play important role in in silico functional prediction of a new protein. It is also used for understanding the evolutionary relationships among proteins. A variety of methods have been proposed in literature for comparing protein structures but they have their own limitations in terms of accuracy and complexity with respect to computational time and space. There is a need to improve the computational complexity in comparison/alignment of proteins through incorporation of important biological and structural properties in the existing techniques.

Results

An efficient algorithm has been developed for comparing protein structures using elastic shape analysis in which the sequence of 3D coordinates atoms of protein structures supplemented by additional auxiliary information from side-chain properties are incorporated. The protein structure is represented by a special function called square-root velocity function. Furthermore, singular value decomposition and dynamic programming have been employed for optimal rotation and optimal matching of the proteins, respectively. Also, geodesic distance has been calculated and used as the dissimilarity score between two protein structures. The performance of the developed algorithm is tested and found to be more efficient, i.e., running time reduced by 80–90 % without compromising accuracy of comparison when compared with the existing methods. Source codes for different functions have been developed in R. Also, user friendly web-based application called ProtSComp has been developed using above algorithm for comparing protein 3D structures and is accessible free.

Conclusions

The methodology and algorithm developed in this study is taking considerably less computational time without loss of accuracy (Table 2). The proposed algorithm is considering different criteria of representing protein structures using 3D coordinates of atoms and inclusion of residue wise molecular properties as auxiliary information.

     

Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin

Context: Surface-modified pH-sensitive liposomal system may be useful for intracellular delivery of chemotherapeutics.

Objective: Achieving site-specific targeting with over-expressed hyaluronic acid (HA) receptors along with using pH sensitive liposome carrier for intracellular drug delivery was the aim of this study.

Materials and methods: Stealth HA-targeted pH-sensitive liposomes (SL-pH-HA) were developed and evaluated to achieve effective intracellular delivery of doxorubicin (DOX) vis–a-vis enhanced antitumor activity.

Results: The in vitro release studies demonstrated that the release of DOX from SL-pH-HA was pH-dependent, i.e. faster at mildly acidic pH ～5, compared to physiological pH ～7.4. SLpH-HA was evaluated for their cytotoxicity potential on CD44 receptor expressing MCF-7 cells. The half maximal inhibitory concentration (IC50) of SL-pH-HA and SL-HA were about 1.9 and 2.5?μM, respectively, after 48?h of incubation. The quantitative uptake study revealed higher localization of targeted liposomes in the receptor positive cells, which was further confirmed by fluorescent microscopy. The antitumor efficacy of the DOX-loaded HA-targeted pH-sensitive liposomes was also verified in a tumor xenograft mouse model.

Discussion: DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction. The major side-effect of conventional DOX formulation, i.e. cardiotoxicity was also estimated by measuring serum enzyme levels of LDH and CPK and found to be minimized with developed formulation. Overall, HA targeted pH-sensitive liposomes were significantly more potent than the non-targeted liposomes in cells expressing high levels of CD44.

Conclusion: Results strongly implies the promise of such liposomal system as an intracellular drug delivery carrier developed for potential anticancer treatment.     

Acetyltransferase p300 inhibitor reverses hypertension‐induced cardiac fibrosis

Epigenetic dysregulation plays a crucial role in cardiovascular diseases. Previously, we reported that acetyltransferase p300 (ATp300) inhibitor L002 prevents hypertension‐induced cardiac hypertrophy and fibrosis in a murine model. In this short communication, we show that treatment of hypertensive mice with ATp300‐specific small molecule inhibitor L002 or C646 reverses hypertension‐induced left ventricular hypertrophy, cardiac fibrosis and diastolic dysfunction, without reducing elevated blood pressures. Biochemically, treatment with L002 and C646 also reverse hypertension‐induced histone acetylation and myofibroblast differentiation in murine ventricles. Our results confirm and extend the role of ATp300, a major epigenetic regulator, in the pathobiology of cardiac hypertrophy and fibrosis. Most importantly, we identify the efficacies of ATp300 inhibitors C646 and L002 in reversing hypertension‐induced cardiac hypertrophy and fibrosis, and discover new anti‐hypertrophic and anti‐fibrotic candidates.     

Cold chain and virus‐free oral polio booster vaccine made in lettuce chloroplasts confers protection against all three poliovirus serotypes

To prevent vaccine‐associated paralytic poliomyelitis, WHO recommended withdrawal of Oral Polio Vaccine (Serotype‐2) and a single dose of Inactivated Poliovirus Vaccine (IPV). IPV however is expensive, requires cold chain, injections and offers limited intestinal mucosal immunity, essential to prevent polio reinfection in countries with open sewer system. To date, there is no virus‐free and cold chain‐free polio vaccine capable of inducing robust mucosal immunity. We report here a novel low‐cost, cold chain/poliovirus‐free, booster vaccine using poliovirus capsid protein (VP1, conserved in all serotypes) fused with cholera non‐toxic B subunit (CTB) expressed in lettuce chloroplasts. PCR using unique primer sets confirmed site‐specific integration of CTB‐VP1 transgene cassettes. Absence of the native chloroplast genome in Southern blots confirmed homoplasmy. Codon optimization of the VP1 coding sequence enhanced its expression 9–15‐fold in chloroplasts. GM1‐ganglioside receptor‐binding ELISA confirmed pentamer assembly of CTB‐VP1 fusion protein, fulfilling a key requirement for oral antigen delivery through gut epithelium. Transmission Electron Microscope images and hydrodynamic radius analysis confirmed VP1‐VLPs of 22.3 nm size. Mice primed with IPV and boosted three times with lyophilized plant cells expressing CTB‐VP1co, formulated with plant‐derived oral adjuvants, enhanced VP1‐specific IgG1, VP1‐IgA titres and neutralization (80%–100% seropositivity of Sabin‐1, 2, 3). In contrast, IPV single dose resulted in <50% VP1‐IgG1 and negligible VP1‐IgA titres, poor neutralization and seropositivity (<20%, <40% Sabin 1,2). Mice orally boosted with CTB‐VP1co, without IPV priming, failed to produce any protective neutralizing antibody. Because global population is receiving IPV single dose, booster vaccine free of poliovirus or cold chain offers a timely low‐cost solution to eradicate polio.     

Human MICAL1: Activation by the small GTPase Rab8 and small‐angle X‐ray scattering studies on the oligomerization state of MICAL1 and its complex with Rab8

Human MICAL1 is a member of a recently discovered family of multidomain proteins that couple a FAD‐containing monooxygenase‐like domain to typical protein interaction domains. Growing evidence implicates the NADPH oxidase reaction catalyzed by the flavoprotein domain in generation of hydrogen peroxide as a second messenger in an increasing number of cell types and as a specific modulator of actin filaments stability. Several proteins of the Rab families of small GTPases are emerging as regulators of MICAL activity by binding to its C‐terminal helical domain presumably shifting the equilibrium from the free – auto‐inhibited – conformation to the active one. We here extend the characterization of the MICAL1–Rab8 interaction and show that indeed Rab8, in the active GTP‐bound state, stabilizes the active MICAL1 conformation causing a specific four‐fold increase of k_cat of the NADPH oxidase reaction. Kinetic data and small‐angle X‐ray scattering (SAXS) measurements support the formation of a 1:1 complex between full‐length MICAL1 and Rab8 with an apparent dissociation constant of approximately 8 μM. This finding supports the hypothesis that Rab8 is a physiological regulator of MICAL1 activity and shows how the protein region preceding the C‐terminal Rab‐binding domain may mask one of the Rab‐binding sites detected with the isolated C‐terminal fragment. SAXS‐based modeling allowed us to propose the first model of the free full‐length MICAL1, which is consistent with an auto‐inhibited conformation in which the C‐terminal region prevents catalysis by interfering with the conformational changes that are predicted to occur during the catalytic cycle.