deconSTRUCT: general purpose protein database search on the substructure level

deconSTRUCT webserver offers an interface to a protein database search engine, usable for a general purpose detection of similar protein (sub)structures. Initially, it deconstructs the query structure into its secondary structure elements (SSEs) and reassembles the match to the target by requiring a (tunable) degree of similarity in the direction and sequential order of SSEs. Hierarchical organization and judicious use of the information about protein structure enables deconSTRUCT to achieve the sensitivity and specificity of the established search engines at orders of magnitude increased speed, without tying up irretrievably the substructure information in the form of a hash. In a post-processing step, a match on the level of the backbone atoms is constructed. The results presented to the user consist of the list of the matched SSEs, the transformation matrix for rigid superposition of the structures and several ways of visualization, both downloadable and implemented as a web-browser plug-in. The server is available at http://epsf.bmad.bii.a-star.edu.sg/struct_server.html

Structure and function of an insect α-carboxylesterase (α Esterase 7) associated with insecticide resistance

Insect carboxylesterases from the αEsterase gene cluster, such as αE7 (also known as E3) from the Australian sheep blowfly Lucilia cuprina (LcαE7), play an important physiological role in lipid metabolism and are implicated in the detoxification of or

CSA: Comprehensive comparison of pairwise protein structure alignments

htmlabstractCSA is a web server for the computation, evaluation and comprehensive comparison of pairwise protein structure alignments. Its exact alignment engine computes either optimal, top-scoring alignments or heuristic alignments with quality guarantee for the inter-residue distance-based scorings of contact map overlap, PAUL, DALI and MATRAS. These and additional, uploaded alignments are compared using a number of quality measures and intuitive visualizations. CSA brings new insight into the structural relationship of the protein pairs under investigation and is a valuable tool for studying structural similarities. It is available at http://csa.project.cwi.nl

Alteration of Splicing Pattern on Angiotensin Converting Enzyme Gene Due To The Insertion of Alu elements

Angiotensin Converting Enzyme (ACE) is a zinc metallopeptidase that has a significant role in blood pressure regulation and the pathophysiology of hypertension. ACE has two protein domains, the N-domain and the C-domain, which each has a single active site that functions independently of each other. There is insertion/deletion by 288 bp Alu elements in the intron 16 of ACE gene. The Alu elements potentially alter splicing process. The effect of the insertion of Alu elements in the splicing pattern of the ACE gene has not been reported. Here, we report on the results of splicing pattern analysis of the ACE gene due to the insertion of Alu elements. Using an in-silico approach, we found the presence of Alu elements insertion in intron 16 of ACE caused alternative splicing and experienced exonization. Further analysis showed that the exonization lead to a premature termination codon (PTC), which is raised protein shortening and lost one of its two protein domains. The loss of one protein domain may affect the catalytic activity of ACE. These findings suggest that the Alu elements I/D polymorphism is related to the differences in the catalytic activity of ACE that may influence blood pressure regulation and hypertension

Structure of the Complete Dimeric Human GDAP1 Core Domain Provides Insights into Ligand Binding and Clustering of Disease Mutations

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders. Despite the common involvement of ganglioside-induced differentiation-associated protein 1 (GDAP1) in CMT, the protein structure and function, as well as the pathogenic mechanisms, remain unclear. We determined the crystal structure of the complete human GDAP1 core domain, which shows a novel mode of dimerization within the glutathione S-transferase (GST) family. The long GDAP1-specific insertion forms an extended helix and a flexible loop. GDAP1 is catalytically inactive toward classical GST substrates. Through metabolite screening, we identified a ligand for GDAP1, the fatty acid hexadecanedioic acid, which is relevant for mitochondrial membrane permeability and Ca2+ homeostasis. The fatty acid binds to a pocket next to a CMT-linked residue cluster, increases protein stability, and induces changes in protein conformation and oligomerization. The closest homologue of GDAP1, GDAP1L1, is monomeric in its full-length form. Our results highlight the uniqueness of GDAP1 within the GST family and point toward allosteric mechanisms in regulating GDAP1 oligomeric state and function.publishedVersio

Challenges in the quantitation of the naturally generated bioactive peptides in processed meats

[EN] Background: The final characteristics of processed meats depend on many factors but one of the most important is the intense proteolysis occurred in muscle proteins due to the action of endogenous enzymes in dry-cured ham, and also microbial peptidases in the case of dry-fermented meats, that not only affects taste and flavour but also the generation of bioactive peptides. Scope and approach: In this review main difficulties in the identification of bioactive peptides in processed meats have been described. This study highlights the novel strategies used during the last years to identify naturally generated peptides, and emphasises the need of robust quantitative methodologies for the adequate characterisation of their bioavailability. In fact, the most common and well established quantitation approaches using proteomics are not adapted for peptidomics analysis, so alternative strategies need to be considered. Key findings and conclusions: The progress in the identification and characterisation of the activity of natural bioactive peptides is highly dependent on modern instruments and bioinformatics tools as well as updated protein databases. In fact, the use of in silico approaches and proteomics can be complementary tools in the identification of peptides from meat protein sources as the empirical experimental design can be simplified by using bioinformatics for computer simulation in most of the steps. Finally, Multiple Reaction Monitoring mass spectrometry methodology previously used in the quantitation of therapeutic peptides and biomarkers arises as a powerful tool for absolute quantitation or semiquantitation of bioactive peptides.The research leading to these results received funding from the European Union 7th Framework Programme (FP7/2007-2013) under Grant Agreement 312090 (BACCHUS). This publication reflects only the author views and the Community is not liable for any use made of the information contained therein. Grant AGL2014-57367-R from MINECO and FEDER funds and the Juan de la Cierva postdoctoral contract to LM are acknowledged. The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, (IPT13/0001 - ISCIII-SGEFI/FEDER).Mora Soler, L.; Gallego-Ibáñez, M.; Reig Riera, MM.; Toldrá Vilardell, F. (2017). Challenges in the quantitation of the naturally generated bioactive peptides in processed meats. Trends in Food Science & Technology. 69:306-314. https://doi.org/10.1016/j.tifs.2017.04.011S3063146

Molecular analysis of olfactory perception in Drosophila melanogaster

Over the last decade the insect olfactory system has emerged as an important model system with which to investigate the biochemical basis of eukaryote signalling processes. It is believed that certain odorant degrading enzymes are required to maintain the ongoing sensitivity of an insect’s olfactory neuronal system by repriming neurons. However, relatively few ODEs have been identified and characterized to date, especially in the model insect Drosophila melanogaster. The study presented here takes biochemical, neurobiological and behavioural approaches to elucidate the role of ODEs in D. melanogaster. After a review of relevant literature in Chapter 1, Chapter 2 decodes the antennal transcriptome of D. melanogaster for the first time. Using high quality genome sequence and transcriptomic data for many other tissues of this species already available, I identified a few antennae-selective esterases, cytochrome P450s (P450s), glutathione S-transferases (GSTs) and UDP-glycosyltransferases (UGTs). Of these, the activity of one esterase JHEdup, against a range of volatile odorants was found to be comparable with other known ODEs from different species, mainly Lepidoptera. I also identified the presence of another esterase, EST6, at high levels in the antennae. It has previously been proposed that EST6 is a catalyst for the transformation of pheromonal and kairomonal esters to the corresponding alcohols and acids, thereby mediating various mating behaviours. I further examined the proposed effect of EST6 by comparing wild type and EST6 null flies at a neurobiological and behavioural level. The findings, presented in Chapter 3, show this enzyme is important for the flies to respond to incoming volatile odorants and affects their subsequent behaviours. Additionally, EST6 has previously been reported to hydrolyse cis-vaccenyl acetate (cVA), the major pheromone known in Drosophila, in vitro and recent electroantennogram (EAG) experiments with EST6 wild type and null flies exposed to cVA suggest that this might also be an in vivo function. I therefore conducted experiments to understand the biochemical activity of EST6 against cVA. I also measured its activity against 84 other bioactive esters. The results categorically show that EST6 has no activity against cVA but has very good activity against a wide range of fruit- and yeast-derived volatiles known to play a role in mediating female reproductive behaviour. These results are presented in Chapter 4, along with a crystal structure of EST6. The final chapter of this thesis then discusses the overall findings of these studies and offers a broader perspective on future directions. The three major conclusions from the work are as follows. Firstly, JHEdup and EST6 are broad range ODEs active against a wide range of food odorants. Secondly, EST6 may also have a role in cVA processing but not actually as an ODE against this substrate. Thirdly, ODEs may be a fruitful system to develop biocontrol systems for pest insects based on disrupting their olfactory system

Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR

The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads b 5.0 Log cfu g−1 . Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy produc