Autophagy in malignant transformation and cancer progression

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

Influence of indole-3-butyric acid and propagation method on growth and development of in vitro- and ex vitro-derived lowbush blueberry plants

The effects of four indole-3-butyric acid (IBA) concentrations and two propagation methods were studied in a lowbush blueberry (Vaccinium angustifolium Ait.) clone collected from natural stands in Newfoundland and Labrador, Canada. Lowbush blueberry cultures were established in vitro from nodal explants on a modified cranberry (V. macrocarpon Ait.) tissue culture medium containing zeatin (2 μM). Blueberry plants propagated by in vitro shoot proliferation (TC) and by conventional softwood cuttings (SC) were evaluated for growth and morphology. Significant interactions for morphological characteristics were observed among the treatments. The IBA concentration had an effect on morphology of propagated plants, increasing the concentration of IBA increased stem length and leaves per stem across propagation methods. Stems per plant increased with IBA concentration up to 20 μM in SC plants, but not in TC plants. Plant vigor was affected by neither IBA concentration nor propagation method. The TC plants produced longer and more stems with more leaves per stem than the conventional cuttings. In vitro culture on zeatin-containing nutrient medium apparently induces the juvenile branching characteristics that favored enhanced vegetative growth with more stems and leaf production. It is suggested that IBA may serve as a physiologically active form of auxin in contributing to increased stem and leaf production in lowbush blueberry SC plants but not in TC plants.     

Managing Spatial and Temporal Switchgrass Biomass Yield Variability

Biorefineries that plan to use switchgrass exclusively will encounter year-to-year variability in feedstock production. The economic success of the biorefinery will depend in part on the ability of the management team to strategically identify land for conversion from current use to the production of switchgrass enabling a flow of feedstock for the life of the biorefinery. The objective of this research is to determine the optimal quality, quantity, and location of land to lease while considering the spatial and temporal variability of switchgrass biomass yield. A calibrated biophysical simulation model was used to simulate switchgrass biomass yields for 50 years based on historical weather data from 1962 to 2011, for three land capability classes for each of 30 counties. Mathematical programming models were constructed and solved to determine the optimal leasing scheme for each of three strategies for a biorefinery that requires 2,000 Mg/day. As expected, a model based on the assumption that the average yield would be obtained in each year finds that production from land identified for leasing would be insufficient to fulfill the biorefinery’s needs in half of the years. In the absence of other sources of biomass, the feedstock shortage would require forced idling of the biorefinery for an average of 29.5 days during these years. Results of a strategy of leasing sufficient land to cover feedstock needs in the worst year from among 50 years for which data are available are compared to that of a strategy enabling year-to-year storage.     

Phylogenetic analysis of alkaline proteinase producing fluorescent pseudomonads associated with green gram (<Emphasis Type="Italic">Vigna radiata</Emphasis> L.) rhizosphere

Fifty fluorescent pseudomonads were isolated from rhizospheric soil of green gram from nearby area of Kaziranga, Assam, India and assayed for their extracellular proteinase production. Out of these isolates, 20 were found to be prominent in proteinase production. Genetic diversity of the 20 isolates were analyzed through BOX-PCR fingerprinting and 16S rDNA-RFLP along with three reference strains, viz., Pseudomonas fluorescens (NCIM2099^T), Pseudomonas aureofaciens (NCIM2026^T), and Pseudomonas aeruginosa (MTCC2582^T). BOX-PCR produced two distinct clusters at 56% similarity coefficient and seven distinct BOX profiles. 16S rDNA-RFLP with three tetra-cutters restriction enzymes (HaeIII, AluI, and MspI) revealed two major clusters A and B; cluster A contained only single isolate FPS9 while the rest of 22 isolates belonged to the cluster B. Based on phenotypic characters and 16S rDNA sequence similarity, all the eight highly proteinase-producing strains were affiliated with P. aeruginosa. The proteinase was extracted from two most prominent strains (KFP1 and KFP2), purified by a three-step process involving (NH₄)₂SO₄ precipitation, gel filtration, and ion exchange chromatography. The enzyme had an optimal pH of 8.0 and exhibit highest activity at 60°C and 37°C by KFP1 and KFP2 respectively. The specific activities were recorded as 75,050 (for KFP1) and 81,320 U/mg (for KFP2). The purified enzyme was migrated as a single band on native and SDS-PAGE with a molecular mass of 32 kDa. Zn²⁺, Cu²⁺, and Ni²⁺ ion inhibited the enzyme activity. Enzyme activity was also inhibited by EDTA established as their metallo-proteinase nature.     

A cell-penetrating helical peptide as a potential HIV-1 inhibitor

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer α-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 Å. Using this structural information, we have utilized a structure-based rational design approach to stabilize the α-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced α-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.     

Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid

The human immunodeficiency virus type 1 (HIV-1) capsid protein plays a critical role in virus core particle assembly and is an important target for novel therapeutic strategies. In a previous study, we characterized the binding affinity of a hydrocarbon stapled helical peptide, NYAD-1, for the capsid protein (K(d) approximately 1 mum) and demonstrated its ability to penetrate the cell membrane (Zhang, H., Zhao, Q., Bhattacharya, S., Waheed, A. A., Tong, X., Hong, A., Heck, S., Goger, M., Cowburn, D., Freed, E. O., and Debnath, A. K. (2008) J. Mol. Biol. 378, 565-580). In cell-based assays, NYAD-1 colocalized with the Gag polyprotein during traffic to the plasma membrane and disrupted the formation of mature and immature virus particles in vitro systems. Here, we complement the cellular and biochemical data with structural characterization of the interactions between the capsid and a soluble peptide analogue, NYAD-13. Solution NMR methods were used to determine a high resolution structure of the complex between the inhibitor and a monomeric form of the C-terminal domain of the capsid protein (mCA-CTD). The intermolecular interactions are mediated by the packing of hydrophobic side chains at the buried interface and unperturbed by the presence of the olefinic chain on the solvent-exposed surface of the peptide. The results of the structural analysis provide valuable insight into the determinants for high affinity and selective inhibitors for HIV-1 particle assembly.