The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture.

Cholesterol esterase (Che) from Burkholderia stabilis (BsChe) is a homolog of well-characterized and industrially relevant bacterial triacylglycerol lipases (Lips). BsChe is a rare bacterial Lip enzyme that exhibits practical Che activity and is currently used in clinical applications to determine total serum cholesterol levels. To investigate the sterol specificity of BsChe, we determined the X-ray structure of BsChe. We discovered a local structural change in the active-site cleft, which might be related to substrate binding and product release. We also performed molecular docking studies by using the X-ray models of BsChe and cholesterol linoleate (CLL), the most favorable substrate for BsChe. The results showed that the sterol moieties of reasonable CLL docking poses localized to a specific active-site cleft surface formed by Leu266 and Ile287, which are unconserved among Burkholderia Lip homologs. Site-directed mutagenesis identified these residues as essential for the Che activity of BsChe, and Leu or Ile substitution conferred marked Che activity to Burkholderia Lips. In particular, Burkholderia cepacia and Burkholderia ubonensis Lips with the V266L/L287I double mutation exhibited ~50-fold and 500-fold higher Che activities than those of the wild-type enzymes, respectively. These results provide new insights into the substrate-binding mechanisms and selectivities of bacterial Lips.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital and developmental malformation syndrome associated with defective cholesterol biosynthesis. It is characterized by accumulation of 7-dehydrocholesterol (the immediate biosynthetic precursor of cholesterol in the Kandutsch-Russell pathway) and an altered cholesterol to total sterol ratio. Because SLOS is associated with neurological malfunction, exploring the function and trafficking of neuronal receptors and their interaction with membrane lipids under these conditions assume significance. In this work, we generated a cellular model of SLOS in HEK-293 cells stably expressing the human serotonin1A receptor (an important neurotransmitter G-protein coupled receptor) using AY 9944, an inhibitor for the enzyme 3β-hydroxy-steroid-∆7 -reductase (7-DHCR). Using a quantitative flow cytometry based assay, we show that the plasma membrane population of serotonin1A receptors was considerably reduced under these conditions without any change in total cellular expression of the receptor. Interestingly, the receptors were trafficked to sterol-enriched LysoTracker positive compartments, which accumulated under these conditions. To the best of our knowledge, our results constitute one of the first reports demonstrating intracellular accumulation and misregulated traffic of a neurotransmitter GPCR in SLOS-like conditions. We believe these results assume relevance in our overall understanding of the molecular basis underlying the functional relevance of neurotransmitter receptors in SLOS.

Lipases are enzymes able to catalyze the hydrolysis or synthesis of triglycerides, depending on the reaction conditions, whereas sterol esterases show the same ability on sterol esters. Structurally, both kinds of enzymes display an α/β-hydrolase fold, with a substrate-binding pocket formed by a hydrophobic cavity covered by a mobile lid. However, it has been reported that some lipases from the Candida rugosa-like family display wide substrate specificity on both triglycerides and sterol esters. Among them, enzymes with different biotechnological applications, such as the lipase isoenzymes produced by C. rugosa and the sterol esterase from Ophiostoma piceae, have been exhaustively characterized and their crystal structures are available. Differences in substrate affinity among these proteins have been attributed to changes in their hydrophobicity. In this work, we analyzed the full catalytic mechanisms of these proteins using molecular dynamics tools, gaining insight into their mechanistic properties. In addition, we developed an in silico protocol to predict the substrate specificity using C. rugosa and O. piceae lipases as model enzymes and triglycerides and cholesterol esters with different fatty acid chain lengths as model substrates. The protocol was validated by comparing the in silico results with those described in the literature. These results would be useful to perform virtual screening of substrates for enzymes of the C. rugosa-like family with unknown catalytic properties.

Metal- and pesticide-tolerant biocontrol agents are preferred in integrated pest management, as such strains can be applied in combination with different pesticides. The Bacillus velezensis strain SZMC 6161J proved to be sensitive to copper, nickel, zinc, and cadmium, while manganese elevated its growth. At concentrations higher than 1 mmol L-1 , zinc and iron inhibited the chymotrypsin-like activity of this strain. In addition, trypsin-like protease and palmitoyl esterase activities were insensitive to all tested heavy metals in the applied concentration range. We studied the effects of some widely used herbicides and fungicides on the growth of this strain. The presence of sulfonylurea herbicides, like bensulfuron-methyl, cinosulfuron, chlorsulfuron, ethoxysulfuron, triasulfuron, and primisulfuron-methyl strongly inhibited the biomass production of the strain even at the concentration of 6.25 mg L-1 . Glyphosate also inhibited the growth above 30 mg L-1 . Similarly, contact fungicides like captan, maneb, mancozeb, and thiram resulted in total inhibition at the concentration as low as 6.25 mg L-1 . Interestingly, the sterol-biosynthesis-inhibiting fungicides imazalil, fenarimol, penconazole, and tebuconazole also proved to be potent inhibitors. Heavy metal- and fungicide-tolerant strains were isolated from the parental strain and their antagonistic abilities were evaluated. There was no substantial difference between the antagonism capability of wild-type strain and the resistant mutants.

This study aimed to investigate the changes of lipid-related gene and serum metabolites in broiler chickens fed with fermented cottonseed meal (FCSM) diet, through quantitative real-time PCR and metabolomics analysis. Totally, 180 1-day-old Cobb broilers were randomly assigned to two groups with six replicates of 15 birds in each. The two diets consisted of a control diet supplemented with 0% FCSM (CON group) and an experimental diet with 6% FCSM (fermented by Candida tropicalis) replacing the soybean meal (FCSM group). The results showed that both abdominal fat content and subcutaneous fat thickness significantly reduced (p < 0.05) in response to dietary FCSM supplementation at the age of 21 d. Serum concentrations of glucose, triglyceride, and low-density lipoprotein cholesterol decreased (p < 0.05) in FCSM fed broilers compared with CON fed broilers, while the levels of epinephrine and growth hormone in serum, liver and abdominal fat tissue were higher (p < 0.05) in FCSM than in CON fed broilers. The activity of hormone-sensitive esterase and lipoprotein lipase (LPL) in the liver and abdominal fat were higher (p < 0.05) in FCSM than CON group. Additionally, compared with the CON group (p < 0.05), the expression of peroxisome proliferator-activated receptor alpha and LPL genes were upregulated in the livers of FCSM group broilers. Gene expressions of hormone-sensitive lipase and LPL in the abdominal fat tissue were also upregulated (p < 0.05) with the broilers fed with FCSM diets. A total of 20 significantly different metabolites were obtained in the serum of different dietary FCSM supplemented fed broilers. The mainly altered pathways were clustered into organic acid metabolism, fatty acid metabolism, and amino acid metabolism. These results not only provide a better understanding of broilers' lipid metabolism with FCSM but also can be helpful in further improvement of the broilers' healthy production and utilization of FCSM.

Inhibition and aging of neuropathy target esterase (NTE) by exposure to neuropathic organophosphorus compounds (OPs) can result in OP-induced delayed neuropathy (OPIDN). In the present study we aimed to build a model of OPIDN in adult zebrafish. First, inhibition and aging of zebrafish NTE activity were characterized in the brain by using the prototypic neuropathic compounds cresyl saligenin phosphate (CBDP) and diisopropylphosphorofluoridate (DFP). Our results show that, as in other animal models, zebrafish NTE is inhibited and aged by both neuropathic OPs. Then, a neuropathic concentration inhibiting NTE activity by at least 70% for at least 24 h was selected for each compound to analyze changes in phosphatidylcholines (PCs), lysophosphatidylcholines (LPCs) and glycerolphosphocholine (GPC) profiles. In spite to the strong inhibition of the NTE activity found for both compounds, only a mild increase in the LPCs level was found after 48 h of the exposure to DFP, and no effect were observed by CBDP. Moreover, histopathological evaluation and motor function outcome analyses failed to find any neurological abnormalities in the exposed fish. Thus, our results strongly suggest that zebrafish is not a suitable species for the development of an experimental model of human OPIDN.