In Vitro Glycosylation of the Membrane Protein γ-Sarcoglycan in Nanodiscs.

Membrane glycoproteins are proteins that reside in the membranes of cells and are post-translationally modified to have sugars attached to their amino acid side chains. Studies of this subset of proteins in their native states are becoming more important since they have been linked to numerous human diseases. However, these proteins are difficult to study due to their hydrophobic nature and their propensity to aggregate. Using membrane mimetics allows us to solubilize these proteins, which, in turn, allows us to perform glycosylation in vitro to study the effects of the modification on protein structure, dynamics, and interactions. Here, the membrane glycoprotein γ-sarcoglycan was incorporated into nanodiscs composed of long-chain lipids and membrane scaffold proteins to perform N-linked glycosylation in which an enzyme attaches a sugar to the asparagine side chain within the glycosylation site. We previously performed glycosylation of membrane proteins in vitro when the protein had been solubilized using different detergents and short-chain lipids. This work demonstrates successful glycosylation of a full-length membrane protein in nanodiscs providing a more biologically relevant sample to study the effects of the modification.

Prevalence of operator fatigue in winter maintenance operations.

Similar to commercial motor vehicle drivers, winter maintenance operators are likely to be at an increased risk of becoming fatigued while driving due to long, inconsistent shifts, environmental stressors, and limited opportunities for sleep. Despite this risk, there is little research concerning the prevalence of winter maintenance operator fatigue during winter emergencies. The purpose of this research was to investigate the prevalence, sources, and countermeasures of fatigue in winter maintenance operations. Questionnaires from 1043 winter maintenance operators and 453 managers were received from 29 Clear Road member states. Results confirmed that fatigue was prevalent in winter maintenance operations. Over 70% of the operators and managers believed that fatigue has a moderate to significant impact on winter maintenance operations. Approximately 75% of winter maintenance operators reported to at least sometimes drive while fatigued, and 96% of managers believed their winter maintenance operators drove while fatigued at least some of the time. Furthermore, winter maintenance operators and managers identified fatigue countermeasures and sources of fatigue related to winter maintenance equipment. However, the countermeasures believed to be the most effective at reducing fatigue during winter emergencies (i.e., naps) were underutilized. For example, winter maintenance operators reported to never use naps to eliminate fatigue. These results indicated winter maintenance operations are impacted by operator fatigue. These results support the increased need for research and effective countermeasures targeting winter maintenance operator fatigue.

The Arabidopsis 3-ketoacyl-CoA thiolase-2 (kat2-1) mutant exhibits increased flowering but reduced reproductive success.

The enzyme 3-ketoacyl-CoA thiolase (KAT) (EC 2.3.1.16) catalyses a key step in fatty acid beta-oxidation. In Arabidopsis thaliana, expression of the KAT2 gene is known to be required for the efficient mobilization of triacylglycerol during germination and seedling establishment. Here, data from the Arabidopsis kat2-1 mutant are presented, showing that perturbation of beta-oxidation also affects vegetative growth and reproductive success. In the wild type, the KAT2 protein was detected in all organs tested. In the kat2-1 mutant, rosette leaf area and dry weight, but not leaf number, were greatly increased relative to wild type. Global proliferative arrest of flowering was delayed, resulting in increased silique production in kat2-1 plants. However, total silique dry weight was not increased. kat2-1 siliques were smaller and had a reduced seed number caused by increased ovule abortion. In kat2-1 ovules, carbon flow into sugars via gluconeogeneis and respiration were both reduced in comparison to the wild type. In conclusion, these data indicate that a functional beta-oxidation pathway is required to maintain the balance between silique development and the continued initiation of floral meristems.

The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation.

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure.

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.

PM19, a barley (Hordeum vulgare L.) gene encoding a putative plasma membrane protein, is expressed during embryo development and dormancy.

A barley (Hordeum vulgare L.) cDNA, PM19, encoding a putative plasma membrane protein was isolated through differential screening of a dormant wild oat embryo library. PM19 is expressed in barley embryos from mid-embryogenesis up to maturity. PM19 mRNA levels decline upon germination, whereas dormant embryos retained high levels of message for up to 72 h of imbibition. PM19 mRNA levels also remained high or were reinduced in non-dormant embryos by treatments that prevented germination (250 mm NaCl, 10% sorbitol, or 50 microm ABA). The PM19 protein sequence is highly conserved in monocotyledonous and dicotyledonous plants.