The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

The cyclic AMP (cAMP) signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C_1a or C₂ domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C_1a or C₂ domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl₂, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC₅₀ were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.     

Dynein light chain 1 phosphorylation controls macropinocytosis

Recent studies have identified dynein light chain-1 (DLC1), a component of the dynein motor, as a p21-activated kinase 1 (Pak1)-interacting substrate with binding sites mapped to amino acids 61-89 of DLC1 and phosphorylation site at serine 88. Here we investigated the role of DLC1 phosphorylation by Pak1 upon the process of macropinocytosis. We found that Pak1 associates with dynein motor and that Pak1-DLC1 interaction starts at the initiation of pinosome formation and persists in early and late endosomes. Pak1 phosphorylation of DLC1 on Ser-88 controls vesicle formation and trafficking functions, as Ser-88 substitution for alanine prevents macropinocytosis. A peptide spanning the C-terminal 19-amino acid region of DLC1 efficiently blocked Ser-88 phosphorylation and macropinocytosis. These results suggest that the regulation of DLC1 by Pak1 is a novel mechanism by which a signaling kinase might influence macropinocytosis.     

Differential changes in cell morphology,macromolecular composition and membrane protein profiles of neurons and astrocytes in chronic ethanol treated rats

Cellular morphology, macromolecular composition, (DNA, RNA and Protein content) marker enzyme activities for neurons [neuron specific enolase (NSE)] and astrocytes [glutamine synthetase (GS)] and plasma membrane protein profiles in the bulk isolated neurons and astrocytes from control and ethanol treated rats were studied. One month aged Wistar rats were given ethanol as sole drinking fluid for 10 weeks. Scanning electron microscopy revealed a characteristic cell surface smoothening in astrocytes due to ethanol treatment. DNA levels were unaltered, while RNA and Protein contents were decreased in astrocytes and neurons. Further,³H-leucine incorporation into proteins was decreased in neurons and astrocytes derived from ethanol treated rats indicating reduced protein synthesis in neurons and astrocytes. GS activity was affected severely suggesting impairment in astrocytic functions. Plasma membrane protein composition was analyzed by 2-D electrophoresis. The analysis indicated several protein defects in the plasma membranes of neurons and astrocytes, which might be involved in membrane disorder during ethanol challenge.¹²⁵I-Wheat Germ agglutinin binding studies showed three prominent proteins (160, 116 and 97 kDa) in astrocyte membrane fraction suggesting the possible involvement of N-terminal glycoproteins in altered astrocyte morphology during ethanol ingestion. Impairment in the astrocyte cell functions, protein changes in plasma membrane and cellular morphology studies suggest that astrocytes may be more vulnerable than neurons for ethanol effects.     

Ethanol induced alterations in plasma membrane protein phosphorylation of neurons and astrocytes

The endogenous protein phosphorylation patterns in plasma membranes of bulk isolated neurons and astroglia from control and chronic ethanol treated rats have been investigated. Chronic ethanol treatment resulted in increased phosphorylation of specific proteins with molecular weights 116, 63 and 60 kDa in both neurons and astrocytes. These proteins were further resolved by 2-DE and the analysis suggested an increased phosphorylation of 10–15 proteins, of which 116 kDa protein is phosphorylated to a higher extent by ethanol. Further, decreased phosphorylation was noticed in D-95 and D-63 proteins in neurons and D-78 and D-54 proteins in astrocytes. Alkali stability experiments for identifying the phosphoamino acid involved in phosphorylation of 116, 63 and 60 kDa proteins suggested that tyrosine and threonine are not involved and probably serine is the likely site for phosphorylation during chronic ethanol treatment. The phosphorylation of specific membrane proteins during chronic ethanol treatment might contribute to ethanol evoked cellular dysfunction.     

Molecular modeling of Gly80 and Ser80 variants of human group IID phospholipase A2 and their receptor complexes: potential basis for weight loss in chronic obstructive pulmonary disease

Weight loss is a well known systemic manifestation of chronic obstructive pulmonary disease (COPD). A Gly80Ser mutation on human group IID secretory phospholipase A2 (sPLA₂) enhances expression of the cytokines that are responsible for weight loss. In this study, we seek to establish a structural correlation of wild type sPLA₂ and the Gly80Ser mutation with function. sPLA₂ with glycine and serine at the 80th positions and the M-type receptor were modelled. The enzymes were docked to the receptor and molecular dynamics was carried out to 70 ns. Structural analysis revealed the enzymes to comprise three helices (H1–H3), two short helices (SH1 and SH2), and five loops including a calcium binding loop (L1–L5), and to be stabilized by seven disulfide bonds. The overall backbone folds of the two models are very similar, with main chain RMSD of less than 1 Å. The active site within the substrate binding channel shows a catalytic triad of water–His67–Asp112, showing a hydrogen bonded network. Major structural differences between wild type and mutant enzymes were observed locally at the site of the mutation and in their global conformations. These differences include: (1) loop-L3 between H2 and H3, which bears residue Gly80 in the wild type, is in a closed conformation with respect to the channel opening, while in the mutant enzyme it adopts a relatively open conformation; (2) the mutant enzyme is less compact and has higher solvent accessible surface area; and (3) interfacial binding contact surface area is greater, and the quality of interactions with the receptor is better in the mutant enzyme as compared to the wild type. Therefore, the structural differences delineated in this study are potential biophysical factors that could determine the increased potency of the mutant enzyme with macrophage receptor for cytokine secreting function, resulting in exacerbation of cachexia in COPD.     

Metabolic engineering of sugarcane to accumulate energy‐dense triacylglycerols in vegetative biomass

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy‐rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon‐partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co‐expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1‐2 (DGAT1‐2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP‐glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95‐ or 43‐fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5‐ to 9.5‐fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.