Monte Carlo (MC) methods have become very popular in signal processing during the past decades. The adaptive rejection sampling (ARS) algorithms are well-known MC techniques which draw efficiently ...independent samples from univariate target densities. The ARS schemes yield a sequence of proposal functions that converge towards the target, so that the probability of accepting a sample approaches one. However, sampling from the proposal pdf becomes more computationally demanding each time it is updated. The parsimonious ARS method, where an efficient trade-off between acceptance rate and proposal complexity is obtained, is proposed. Thus, the resulting algorithm is faster than the standard ARS approach.
Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one‐carbon ...units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine‐tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo‐enzyme structure, showing that PLP binding triggers a disorder‐to‐order transition accompanied by a large rigid‐body movement of the two cofactor‐binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer‐to‐tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo.
Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR‐like ...bacterial transcription factors (MocR‐TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA‐binding proteins and pyridoxal 5′‐phosphate (PLP)‐dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino‐compounds; it is the modulation of such binding properties to confer to MocR‐TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR‐TFs control different metabolic processes involving vitamin B6 and amino acids, which are canonical ligands of PLP‐dependent enzymes. However, MocR‐TFs are also implicated in the metabolism of compounds that are not substrates of PLP‐dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR‐TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR‐TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR‐TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor‐binding sites of MocR‐based regulatory systems.
Bacterial MocR‐like transcription factors are chimeric proteins formed by the joining of a DNA binding domain and the protein scaffold of pyridoxal 5′‐phosphate‐dependent enzymes. They control several different metabolic processes. This review reports and analyses the available information concerning their functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor‐binding sites of MocR‐based regulatory systems.
Pyridoxal 5′‐phosphate (PLP), the well‐known active form of vitamin B₆, is an essential enzyme cofactor involved in a large number of metabolic processes. PLP levels need to be finely tuned in ...response to cell requirements; however, little is known about the regulation of PLP biosynthesis and recycling pathways. The transcriptional regulator PdxR activates transcription of the pdxST genes encoding PLP synthase. It is characterized by an N‐terminal helix‐turn‐helix motif that binds DNA and an effector‐binding C‐terminal domain homologous to PLP‐dependent enzymes. Although it is known that PLP acts as an anti‐activator, the mechanism of action of PdxR is unknown. In the present study, we analyzed the biochemical and DNA‐binding properties of PdxR from the probiotic Bacillus clausii. Spectroscopic measurements showed that PLP is the only B₆ vitamer that acts as an effector molecule of PdxR. Binding of PLP to PdxR determines a protein conformational change, as detected by gel filtration chromatography and limited proteolysis experiments. We showed that two direct repeats and one inverted repeat are present in the DNA promoter region and PdxR is able to bind DNA fragments containing any combination of two of them. However, when PLP binds to PdxR, it modifies the DNA‐binding properties of the protein, making it selective for inverted repeats. A molecular mechanism is proposed in which the two different DNA binding modalities of PdxR determined by the presence or absence of PLP are responsible for the control of pdxST transcription.
Monte Carlo methods represent the
de facto
standard for approximating complicated integrals involving multidimensional target distributions. In order to generate random realizations from the target ...distribution, Monte Carlo techniques use simpler proposal probability densities to draw candidate samples. The performance of any such method is strictly related to the specification of the proposal distribution, such that unfortunate choices easily wreak havoc on the resulting estimators. In this work, we introduce a
layered
(i.e., hierarchical) procedure to generate samples employed within a Monte Carlo scheme. This approach ensures that an appropriate equivalent proposal density is always obtained automatically (thus eliminating the risk of a catastrophic performance), although at the expense of a moderate increase in the complexity. Furthermore, we provide a general unified importance sampling (IS) framework, where multiple proposal densities are employed and several IS schemes are introduced by applying the so-called deterministic mixture approach. Finally, given these schemes, we also propose a novel class of adaptive importance samplers using a population of proposals, where the adaptation is driven by independent parallel or interacting Markov chain Monte Carlo (MCMC) chains. The resulting algorithms efficiently combine the benefits of both IS and MCMC methods.
The vitamin B6 salvage pathway, involving pyridoxine 5′‐phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal ...5′‐phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR‐like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR‐like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA‐binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA‐binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor‐binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup.
PtsJ from Salmonella typhimurium, a novel MocR‐like transcriptional regulator, represses expression of the pdxK gene, encoding vitamin B6 salvage pathway enzyme pyridoxal kinase. PtsJ binds to four repeated nucleotide sequences on the ptsJ‐pdxK intergenic region. Binding to DNA is promoted by pyridoxal phosphate (PLP) that shifts binding stoichiometry from a 2 : 1 to a 4 : 1 protein subunits/DNA molar ratio.
Bacillus subtilis is able to use γ‐aminobutyric acid (GABA) found in the soil as carbon and nitrogen source, through the action of GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase ...(GabD). GABA acts as molecular effector in the transcriptional activation of the gabTD operon by GabR. GabR is the most studied member of the MocR family of prokaryotic pyridoxal 5′‐phosphate (PLP)‐dependent transcriptional regulators, yet crucial aspects of its mechanism of action are unknown. GabR binds to the gabTD promoter, but transcription is activated only when GABA is present. Here, we demonstrated, in contrast with what had been previously proposed, that three repeated nucleotide sequences in the promoter region, two direct repeats and one inverted repeat, are specifically recognized by GabR. We carried out in vitro and in vivo experiments using mutant forms of the gabTD promoter. Our results showed that GABA activates transcription by changing the modality of interaction between GabR and the recognized sequence repeats. A hypothetical model is proposed in which GabR exists in two alternative conformations that, respectively, prevent or promote transcription. According to this model, in the absence of GABA, GabR binds to DNA interacting with all three sequence repeats, overlapping the RNA polymerase binding site and therefore preventing transcription activation. On the other hand, when GABA binds to GabR, a conformational change of the protein leads to the release of the interaction with the inverted repeat, allowing transcription initiation by RNA polymerase.
Bacillus subtilis uses ‐aminobutyrate (GABA) found in the soil as nitrogen source, through GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). Transcription of the gabTD operon is activated by the transcriptional factor GabR upon binding of GABA. Our investigation demonstrates that, depending on whether GABA is absent or present, GabR differently interacts with two direct and one inverted repeats in the gabTDpromoter, respectively, preventing or promoting transcription activation.
Value-at-risk is one of the most popular risk management tools in the financial industry. Over the past 20 years, several attempts to include VaR in the portfolio selection process have been ...proposed. However, using VaR as a risk measure in portfolio optimization models leads to problems that are computationally hard to solve. In view of this, few practical applications of VaR in portfolio selection have appeared in the literature up to now. In this paper, we propose to add the VaR criterion to the classical Mean-Variance approach in order to better address the typical regulatory constraints of the financial industry. We thus obtain a portfolio selection model characterized by three criteria: expected return, variance, and VaR at a specified confidence level. The resulting optimization problem consists in minimizing variance with parametric constraints on the levels of expected return and VaR. This model can be formulated as a mixed-integer quadratic programming (MIQP) problem. An extensive empirical analysis on seven real-world datasets demonstrates the practical applicability of the proposed approach. Furthermore, the out-of-sample performance of the more binding optimal Mean-Variance-VaR portfolios seems to be generally better than that of the Equally Weighted and of the Mean-Variance-CVaR portfolios.
Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the ...brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to β-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.
l‐Threonine aldolases (l‐TAs) represent a family of homologous pyridoxal 5′‐phosphate‐dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several ...l‐3‐hydroxy‐α‐amino acids. l‐TAs have great biotechnological potential, as they catalyse the formation of carbon–carbon bonds, and therefore may be exploited for the bioorganic synthesis of l‐3‐hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many l‐TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, l‐TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low‐specificity l‐TA from Escherichia coli at 2.2‐Å resolution, in the unliganded form and cocrystallized with l‐serine and l‐threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro‐aldol cleavage reaction. Interestingly, the very large active site opening of E. coli l‐TA suggests that much larger molecules than l‐threonine isomers may be easily accommodated, and l‐TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues.
Structured digital
eTA and eTA bind by x-ray crystallography (1, 2).
Database
RCSB PDB (www.pdb.org): structural data are available in the Protein Data Bank/BioMagResBank databases under the accession numbers 4LNJ, 4LNM and 4LNL for the unliganded, eTA‐Thr and eTA‐Ser structures.
l‐Threonine aldolases are enzymes whose biological function is unclear but are biotechnologically relevant for bioorganic synthesis of l‐3‐hydroxyamino acids. Our structural and functional characterization of the Escherichia coli enzyme suggests that stereospecificity is determined by the overall microenvironment of the active site. A structural water molecule has been proposed to act as catalytic base in the retro‐aldol cleavage reaction.
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