18,876 research outputs found
The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases
One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs
Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros
This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites
Molecular architecture of human polycomb repressive complex 2.
Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. Here, we present the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, we have localized all the PRC2 subunits and their functional domains and generated a detailed map of interactions. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity.DOI:http://dx.doi.org/10.7554/eLife.00005.001
Computational modeling to elucidate molecular mechanisms of epigenetic memory
How do mammalian cells that share the same genome exist in notably distinct
phenotypes, exhibiting differences in morphology, gene expression patterns, and
epigenetic chromatin statuses? Furthermore how do cells of different phenotypes
differentiate reproducibly from a single fertilized egg? These are fundamental
problems in developmental biology. Epigenetic histone modifications play an
important role in the maintenance of different cell phenotypes. The exact
molecular mechanism for inheritance of the modification patterns over cell
generations remains elusive. The complexity comes partly from the number of
molecular species and the broad time scales involved. In recent years
mathematical modeling has made significant contributions on elucidating the
molecular mechanisms of DNA methylation and histone covalent modification
inheritance. We will pedagogically introduce the typical procedure and some
technical details of performing a mathematical modeling study, and discuss
future developments.Comment: 36 pages, 4 figures, 2 tables, book chapte
Structure- and context-based analysis of the GxGYxYP family reveals a new putative class of glycoside hydrolase.
BackgroundGut microbiome metagenomics has revealed many protein families and domains found largely or exclusively in that environment. Proteins containing the GxGYxYP domain are over-represented in the gut microbiota, and are found in Polysaccharide Utilization Loci in the gut symbiont Bacteroides thetaiotaomicron, suggesting their involvement in polysaccharide metabolism, but little else is known of the function of this domain.ResultsGenomic context and domain architecture analyses support a role for the GxGYxYP domain in carbohydrate metabolism. Sparse occurrences in eukaryotes are the result of lateral gene transfer. The structure of the GxGYxYP domain-containing protein encoded by the BT2193 locus reveals two structural domains, the first composed of three divergent repeats with no recognisable homology to previously solved structures, the second a more familiar seven-stranded β/α barrel. Structure-based analyses including conservation mapping localise a presumed functional site to a cleft between the two domains of BT2193. Matching to a catalytic site template from a GH9 cellulase and other analyses point to a putative catalytic triad composed of Glu272, Asp331 and Asp333.ConclusionsWe suggest that GxGYxYP-containing proteins constitute a novel glycoside hydrolase family of as yet unknown specificity
Deletion within the Src homology domain 3 of Bruton's tyrosine kinase resulting in X-linked agammaglobulinemia (XLA).
The gene responsible for X-linked agammaglobulinemia (XLA) has been recently identified to code for a cytoplasmic tyrosine kinase (Bruton's agammaglobulinemia tyrosine kinase, BTK), required for normal B cell development. BTK, like many other cytoplasmic tyrosine kinases, contains Src homology domains (SH2 and SH3), and catalytic kinase domain. SH3 domains are important for the targeting of signaling molecules to specific subcellular locations. We have identified a family with XLA whose affected members have a point mutation (g-->a) at the 5' splice site of intron 8, resulting in the skipping of coding exon 8 and loss of 21 amino acids forming the COOH-terminal portion of the BTK SH3 domain. The study of three generations within this kinship, using restriction fragment length polymorphism and DNA analysis, allowed identification of the mutant X chromosome responsible for XLA and the carrier status in this family. BTK mRNA was present in normal amounts in Epstein-Barr virus-induced B lymphoblastoid cell lines established from affected family members. Although the SH3 deletion did not alter BTK protein stability and kinase activity of the truncated BTK protein was normal, the affected patients nevertheless have a severe B cell defect characteristic for XLA. The mutant protein was modeled using the normal BTK SH3 domain. The deletion results in loss of two COOH-terminal beta strands containing several residues critical for the formation of the putative SH3 ligand-binding pocket. We predict that, as a result, one or more crucial SH3 binding proteins fail to interact with BTK, interrupting the cytoplasmic signal transduction process required for B cell differentiation
The role of multiple marks in epigenetic silencing and the emergence of a stable bivalent chromatin state
We introduce and analyze a minimal model of epigenetic silencing in budding
yeast, built upon known biomolecular interactions in the system. Doing so, we
identify the epigenetic marks essential for the bistability of epigenetic
states. The model explicitly incorporates two key chromatin marks, namely H4K16
acetylation and H3K79 methylation, and explores whether the presence of
multiple marks lead to a qualitatively different systems behavior. We find that
having both modifications is important for the robustness of epigenetic
silencing. Besides the silenced and transcriptionally active fate of chromatin,
our model leads to a novel state with bivalent (i.e., both active and
silencing) marks under certain perturbations (knock-out mutations, inhibition
or enhancement of enzymatic activity). The bivalent state appears under several
perturbations and is shown to result in patchy silencing. We also show that the
titration effect, owing to a limited supply of silencing proteins, can result
in counter-intuitive responses. The design principles of the silencing system
is systematically investigated and disparate experimental observations are
assessed within a single theoretical framework. Specifically, we discuss the
behavior of Sir protein recruitment, spreading and stability of silenced
regions in commonly-studied mutants (e.g., sas2, dot1) illuminating the
controversial role of Dot1 in the systems biology of yeast silencing.Comment: Supplementary Material, 14 page
In vivo and in vitro characterization of Staphylococcus aureus and Bacillus subtilis polyglycerolphosphate lipoteichoic acid synthases
Staphylococcus aureus lipoteichoic acid (LTA) consists of a 1,3-linked
polyglycerolphosphate chain retained in the bacterial membrane by a glycolipid
anchor. The LTA backbone is produced by the lipoteichoic acid synthase LtaS, a
membrane protein with five transmembrane helices and a large extracellular
enzymatic domain (eLtaS). Proteomic studies revealed that LtaS is efficiently cleaved,
and here it was demonstrated that the eLtaS domain is released into the culture
supernatant as well as partially retained within the cell wall fraction. However, using
an in vivo LtaS activity assay, it was shown that only the full-length LtaS enzyme is
able to synthesize LTA. Neither expression of a secreted eLtaS variant, created by
replacing the N-terminal membrane domain with a conventional signal sequence, nor
expression of eLtaS fused to a single or multi-transmembrane domains of other
staphylococcal proteins resulted in the production of LTA. These data indicate that the
transmembrane domain of LtaS play an essential, yet unknown, role in LtaS enzyme
function. In addition, the protease responsible for LtaS cleavage was identified. It was
found that a S. aureus strain in which the gene encoding for the essential signal
peptidase SpsB was cloned under inducible expression control showed an
accumulation of the full-length LtaS enzyme in the absence of the inducer. These data
suggest that SpsB is involved in LtaS cleavage.
Four LtaS orthologues, YflE, YfnI, YqgS and YvgJ, are present in Bacillus
subtilis. Using an in vitro enzyme assay and purified protein, it was determined that
all four B. subtilis proteins are Mn2+-dependent metal enzymes that use the lipid
phosphatidylglycerol as substrate. It was shown that YflE, YfnI and YqgS are bonafide
LTA synthases capable of producing polyglycerolphosphate chains, while YvgJ
appears to function as an LTA primase, as indicated by the accumulation of a
glycolipid with the expected chromatographic mobility of GroP-Glc2-DAG. Taken
together, experimental evidence for the enzyme function of all four B. subtilis LtaStype
proteins is provided in this work and it was shown that all four enzymes are
involved in the LTA synthesis process
New Insights Into Sunflower (Helianthus annuus L.) FatA and FatB Thioesterases, Their Regulation, Structure and Distribution
Sunflower seeds (Helianthus annuus L.) accumulate large quantities of triacylglycerols (TAG) between 12 and 28 days after flowering (DAF). This is the period of maximal acyl-acyl carrier protein (acyl-ACP) thioesterase activity in vitro, the enzymes that terminate the process of de novo fatty acid synthesis by catalyzing the hydrolysis of the acyl-ACPs synthesized by fatty acid synthase. Fatty acid thioesterases can be classified into two families with distinct substrate specificities, namely FatA and FatB. Here, some new aspects of these enzymes have been studied, assessing how both enzymes contribute to the acyl composition of sunflower oil, not least through the changes in their expression during the process of seed filling. Moreover, the binding pockets of these enzymes were modeled based on new data from plant thioesterases, revealing important differences in their volume and geometry. Finally, the subcellular location of the two enzymes was evaluated and while both possess an N-terminal plastid transit peptide, only in FatB contains a hydrophobic sequence that could potentially serve as a transmembrane domain. Indeed, using in vivo imaging and organelle fractionation, H. annuus thioesterases, HaFatA and HaFatB, appear to be differentially localized in the plastid stroma and membrane envelope, respectively. The divergent roles fulfilled by HaFatA and HaFatB in oil biosynthesis are discussed in the light of our data.España MINECO y FEDER Projects AGL2014- 53537-R y AGL2017-83449-
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