LPS-Dephosphorylating Cobetia amphilecti Alkaline Phosphatase of PhoA Family Divergent from the Multiple Homologues of Cobetia spp.

A highly active alkaline phosphatase (ALP) of the protein structural family PhoA, from a mussel gut-associated strain of the marine bacterium Cobetia amphilecti KMM 296 (CmAP), was found to effectively dephosphorylate lipopolysaccharides (LPS). Therefore, the aim of this work was to perform a comprehensive bioinformatics analysis of the structure, and to suggest the physiological role of this enzyme in marine bacteria of the genus Cobetia. A scrutiny of the CmAP-like sequences in 36 available Cobetia genomes revealed nine homologues intrinsic to the subspecies C. amphilecti, whereas PhoA of a distant relative Cobetia crustatorum JO1T carried an inactive mutation. However, phylogenetic analysis of all available Cobetia ALP sequences showed that each strain of the genus Cobetia possesses several ALP variants, mostly the genes encoding for PhoD and PhoX families. The C. amphilecti strains have a complete set of four ALP families' genes, namely: PhoA, PafA, PhoX, and two PhoD structures. The Cobetia marina species is distinguished by the presence of only three PhoX and PhoD genes. The Cobetia PhoA proteins are clustered together with the human and squid LPS-detoxifying enzymes. In addition, the predicted PhoA biosynthesis gene cluster suggests its involvement in the control of cellular redox balance, homeostasis, and cell cycle. Apparently, the variety of ALPs in Cobetia spp. indicates significant adaptability to phosphorus-replete and depleted environments and a notable organophosphate destructor in eco-niches from which they once emerged, including Zostera spp. The ALP clusterization and degree of similarity of the genus-specific biosynthetic genes encoding for ectoine and polyketide cluster T1PKS, responsible for sulfated extracellular polysaccharide synthesis, coincide with a new whole genome-based taxonomic classification of the genus Cobetia. The Cobetia strains and their ALPs are suggested to be adaptable for use in agriculture, biotechnology and biomedicine.

PUP-IT2 as an alternative strategy for PUP-IT proximity labeling.

PUP-IT is a proximity labeling method based on the prokaryotic enzyme PafA. PafA mediates the ligation of Pup, a small peptide, to the proximal proteins. It is different from other proximity labeling methods, such as BioID and APEX, in that both the enzyme and the labeling tag are proteins, which allows for potential in vivo applications. All proximity labeling involves the genetic fusion of the proximity labeling enzyme with the bait protein. However, PafA is a 55 kDa enzyme which sometimes interferes with the bait function. In this study, we tested an alternative proximity labeling strategy, PUP-IT2, in which only a small 7 kDa protein is fused to the bait protein. We examined the activity of PUP-IT2 in vitro and in cells. We also compared it with the original PUP-IT. Finally, we applied PUP-IT2 coupled mass spectrometry to map protein-protein interactions. Overall, we established a new way to use PUP-IT2 for proximity labeling, and this method may have a broad application.

A widely distributed phosphate-insensitive phosphatase presents a route for rapid organophosphorus remineralization in the biosphere.

The regeneration of bioavailable phosphate from immobilized organophosphorus represents a key process in the global phosphorus cycle and is facilitated by enzymes known as phosphatases. Most bacteria possess at least one of three phosphatases with broad substrate specificity, known as PhoA, PhoX, and PhoD, whose activity is optimal under alkaline conditions. The production and activity of these phosphatases is repressed by phosphate availability. Therefore, they are only fully functional when bacteria experience phosphorus-limiting growth conditions. Here, we reveal a previously overlooked phosphate-insensitive phosphatase, PafA, prevalent in Bacteroidetes, which is highly abundant in nature and represents a major route for the regeneration of environmental phosphate. Using the enzyme from Flavobacterium johnsoniae, we show that PafA is highly active toward phosphomonoesters, is fully functional in the presence of excess phosphate, and is essential for growth on phosphorylated carbohydrates as a sole carbon source. These distinct properties of PafA may expand the metabolic niche of Bacteroidetes by enabling the utilization of abundant organophosphorus substrates as C and P sources, providing a competitive advantage when inhabiting zones of high microbial activity and nutrient demand. PafA, which is constitutively synthesized by soil and marine flavobacteria, rapidly remineralizes phosphomonoesters releasing bioavailable phosphate that can be acquired by neighboring cells. The pafA gene is highly diverse in plant rhizospheres and is abundant in the global ocean, where it is expressed independently of phosphate availability. PafA therefore represents an important enzyme in the context of global biogeochemical cycling and has potential applications in sustainable agriculture.

PUPIL enables mapping and stamping of transient electrical connectivity in developing nervous systems.

Currently, many genetic methods are available for mapping chemical connectivity, but analogous methods for electrical synapses are lacking. Here, we present pupylation-based interaction labeling (PUPIL), a genetically encoded system for noninvasively mapping and stamping transient electrical synapses in the mouse brain. Upon fusion of connexin 26 (CX26) with the ligase PafA, pupylation yields tag puncta following conjugation of its substrate, a biotin- or fluorescent-protein-tagged PupE, to the neighboring proteins of electrical synapses containing CX26-PafA. Tag puncta are validated to correlate well with functional electrical synapses in immature neurons. Furthermore, puncta are retained in mature neurons when electrical synapses mostly disappear-suggesting successful stamping. We use PUPIL to uncover spatial subcellular localizations of electrical synapses and approach their physiological functions during development. Thus, PUPIL is a powerful tool for probing electrical connectivity patterns in complex nervous systems and has great potential for transient receptors and ion channels as well.

COSMOS next generation - A public knowledge base leveraging chemical and biological data to support the regulatory assessment of chemicals.

The COSMOS Database (DB) was originally established to provide reliable data for cosmetics-related chemicals within the COSMOS Project funded as part of the SEURAT-1 Research Initiative. The database has subsequently been maintained and developed further into COSMOS Next Generation (NG), a combination of database and in silico tools, essential components of a knowledge base. COSMOS DB provided a cosmetics inventory as well as other regulatory inventories, accompanied by assessment results and in vitro and in vivo toxicity data. In addition to data content curation, much effort was dedicated to data governance - data authorisation, characterisation of quality, documentation of meta information, and control of data use. Through this effort, COSMOS DB was able to merge and fuse data of various types from different sources. Building on the previous effort, the COSMOS Minimum Inclusion (MINIS) criteria for a toxicity database were further expanded to quantify the reliability of studies. COSMOS NG features multiple fingerprints for analysing structure similarity, and new tools to calculate molecular properties and screen chemicals with endpoint-related public profilers, such as DNA and protein binders, liver alerts and genotoxic alerts. The publicly available COSMOS NG enables users to compile information and execute analyses such as category formation and read-across. This paper provides a step-by-step guided workflow for a simple read-across case, starting from a target structure and culminating in an estimation of a NOAEL confidence interval. Given its strong technical foundation, inclusion of quality-reviewed data, and provision of tools designed to facilitate communication between users, COSMOS NG is a first step towards building a toxicological knowledge hub leveraging many public data systems for chemical safety evaluation. We continue to monitor the feedback from the user community at support@mn-am.com.

Exploring Protein Space: From Hydrolase to Ligase by Substitution.

The understanding of how proteins evolve to perform novel functions has long been sought by biologists. In this regard, two homologous bacterial enzymes, PafA and Dop, pose an insightful case study, as both rely on similar mechanistic properties, yet catalyze different reactions. PafA conjugates a small protein tag to target proteins, whereas Dop removes the tag by hydrolysis. Given that both enzymes present a similar fold and high sequence similarity, we sought to identify the differences in the amino acid sequence and folding responsible for each distinct activity. We tackled this question using analysis of sequence-function relationships, and identified a set of uniquely conserved residues in each enzyme. Reciprocal mutagenesis of the hydrolase, Dop, completely abolished the native activity, at the same time yielding a catalytically active ligase. Based on the available Dop and PafA crystal structures, this change of activity required a conformational change of a critical loop at the vicinity of the active site. We identified the conserved positions essential for stabilization of the alternative loop conformation, and tracked alternative mutational pathways that lead to a change in activity. Remarkably, all these pathways were combined in the evolution of PafA and Dop, despite their redundant effect on activity. Overall, we identified the residues and structural elements in PafA and Dop responsible for their activity differences. This analysis delineated, in molecular terms, the changes required for the emergence of a new catalytic function from a preexisting one.

Inter- and intramolecular regulation of protein depupylation in Mycobacterium smegmatis.

Whereas intracellular proteolysis is essential for proper cellular function, it is a destructive process, which must be tightly regulated. In some bacteria, a Pup-proteasome system tags target proteins for degradation by a bacterial proteasome. Pup, a small modifier protein, is attached to target proteins by PafA, the sole Pup ligase, in a process termed pupylation. In mycobacteria, including Mycobacterium smegmatis and Mycobacterium tuberculosis, Pup undergoes a deamidation step by the enzyme Dop prior to its PafA-mediated attachment to a target. The catalytic mechanism of Pup deamidation is also used by Dop to perform depupylation, namely the removal of Pup from already tagged proteins. Hence, Dop appears to play contradictory roles: On the one hand, deamidation of Pup promotes pupylation, while on the other hand, depupylation reduces tagged protein levels. To avoid futile pupylation-depupylation cycles, Dop activity must be regulated. An intramolecular regulatory mechanism directs Dop to catalyze deamidation more effectively than depupylation. A complementary intermolecular mechanism results in Dop depletion under conditions where protein pupylation and degradation are favorable. In this work, we studied these regulatory mechanisms and identified a flexible loop in Dop, previously termed the Dop-loop, that acts as an intramolecular regulatory element that allosterically controls substrate preference. To investigate regulation at the intermolecular level, we used the CRISPR interference system to knock down the expression of M. smegmatis ATP-dependent intracellular proteases and found that the ClpCP protease is responsible for Dop depletion under starvation conditions. These findings clarify previous observations and introduce a new level for the regulation of Dop activity. DATABASE: Structural data are available in the PDB database under the accession numbers 4BJR and 4B0S.

The transcription of pafA, encoding the prokaryotic ubiquitin-like protein ligase, is regulated by PafBC.

AIM: Mycobacterium tuberculosis possesses an intracellular tagging and degradation system, which has emerged as a target for development of anti-tuberculosis agents. In this system, PafA is the ligase that marks proteins for degradation by their covalent modification with a protein modifier. Here, we studied pafA transcriptional regulation, which remained elusive despite its importance for M. tuberculosis virulence. MATERIALS & METHODS: Working with Mycobacterium smegmatis, a mycobacterial model organism, we examined the involvement of the global regulators PafB and PafC in pafA regulation. RESULTS: PafBC activated pafA transcription following DNA damage, resulting in efficient cellular recovery. CONCLUSION: The results unraveled the involvement of PafBC in pafA transcription, and revealed the importance of proper PafA regulation in mycobacterial physiology.

Identification of Serine 119 as an Effective Inhibitor Binding Site of M. tuberculosis Ubiquitin-like Protein Ligase PafA Using Purified Proteins and M. smegmatis.

Owing to the spread of multidrug resistance (MDR) and extensive drug resistance (XDR), there is a pressing need to identify potential targets for the development of more-effective anti-M. tuberculosis (Mtb) drugs. PafA, as the sole Prokaryotic Ubiquitin-like Protein ligase in the Pup-proteasome System (PPS) of Mtb, is an attractive drug target. Here, we show that the activity of purified Mtb PafA is significantly inhibited upon the association of AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride) to PafA residue Serine 119 (S119). Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on the activity of purified Mtb PafA. Structural analysis reveals that although S119 is distant from the PafA catalytic site, it is located at a critical position in the groove where PafA binds the C-terminal region of Pup. Phenotypic studies demonstrate that S119 plays critical roles in the function of Mtb PafA when tested in M. smegmatis. Our study suggests that targeting S119 is a promising direction for developing an inhibitor of M. tuberculosis PafA.

Proteasome accessory factor A (PafA) transferase activity makes sense in the light of its homology with glutamine synthetase.

The Pup-proteasome system (PPS) is a prokaryotic tagging and degradation system analogous in function to the ubiquitin-proteasome system (UPS). Like ubiquitin, Pup is conjugated to proteins, tagging them for proteasomal degradation. However, in the PPS, a single Pup-ligase, PafA, conjugates Pup to a wide variety of proteins. PafA couples ATP hydrolysis to formation of an isopeptide bond between Pup and a protein lysine via a mechanism similar to that used by glutamine synthetase (GS) to generate glutamine from ammonia and glutamate. GS can also transfer the glutamyl moiety from glutamine to a hydroxyl amine in an ATP-independent manner. Recently, the ability of PafA to transfer Pup from one protein to another was demonstrated. Here, we report that such PafA activity mechanistically resembles the transferase activity of GS. Both PafA and GS transferase activities are ATP-independent and proceed in two catalytic steps. In the first step catalyzed by PafA, an inorganic phosphate is used by the enzyme to depupylate a Pup donor, while forming an acyl phosphate Pup intermediate. The second step consists of Pup conjugation to the new protein, alongside the release of an inorganic phosphate. Detailed experimental analysis, combined with kinetic modeling of PafA transferase activity, allowed us to correctly predict the kinetics and magnitude of Pup transfer between two targets, and analyze the effects of their affinity to PafA on the efficiency of transfer. By deciphering the mechanism of the PafA transferase reaction in kinetic detail, this work provides in-depth mechanistic understanding of PafA, a key PPS enzyme.

How to control an intracellular proteolytic system: Coordinated regulatory switches in the mycobacterial Pup-proteasome system.

Intracellular proteolysis is critical for the proper functioning of all cells, owing to its involvement in a wide range of processes. Because of the destructive nature of protein degradation, intracellular proteolysis is restricted by control mechanisms at almost every step of the proteolytic process. Understanding the coordination of such mechanisms is a challenging task, especially in systems as complex as the eukaryotic ubiquitin-proteasome system (UPS). In comparison, the bacterial analog of the UPS, the Pup-proteasome system (PPS) is much simpler and, therefore, allows for insight into the control of a proteolytic system. This review integrates available information to present a coherent picture of what is known of PPS regulatory switches and describes how these switches act in concert to enforce regulation at the system level. Finally, open questions regarding PPS regulation are discussed, providing readers with a sense of what lies ahead in the field.

The regulatory significance of tag recycling in the mycobacterial Pup-proteasome system.

Pup, a ubiquitin analog, tags proteins for degradation by the bacterial proteasome. As an intracellular proteolytic system, the Pup-proteasome system (PPS) must be carefully regulated to prevent excessive protein degradation. Currently, those factors underlying PPS regulation remain poorly understood. Here, experimental analysis combined with theoretical modeling of in vivo protein pupylation revealed how the basic PPS design allows stable and controlled protein pupylation. Specifically, the recycling of Pup when targets are degraded allows the PPS to maintain steady-state levels of protein pupylation and degradation at a rate limited by proteasome function, and at a pupylome level limited by Pup concentrations. This design allows the Pup-ligase, a highly promiscuous enzyme, to act in a controlled manner without causing damage, and the PPS to be effectively tuned to control protein degradation. This study thus provides understanding of how the inherent design of an intracellular proteolytic system serves crucial regulatory purposes.

An Extended Loop of the Pup Ligase, PafA, Mediates Interaction with Protein Targets.

Pupylation, the bacterial equivalent of ubiquitylation, involves the conjugation of a prokaryotic ubiquitin-like protein (Pup) to protein targets. In contrast to the ubiquitin system, where many ubiquitin ligases exist, a single bacterial ligase, PafA, catalyzes the conjugation of Pup to a wide array of protein targets. As mediators of target recognition by PafA have not been identified, it would appear that PafA alone determines pupylation target selection. Previous studies indicated that broad specificity and promiscuity are indeed inherent PafA characteristics that probably dictate which proteins are selected for degradation by the Pup-proteasome system. Nonetheless, despite the canonical role played by PafA in the Pup-proteasome system, the molecular mechanism that dictates target binding by PafA remains uncharacterized since the discovery of this enzyme about a decade ago. In this study, we report the identification of PafA residues involved in the binding of protein targets. Initially, docking analysis predicted the residues on PafA with high potential for target binding. Mutational and biochemical approaches subsequently confirmed these predictions and identified a series of additional residues located on an extended loop at the edge of the PafA active site. Mutating residues in this loop rendered PafA defective in the pupylation of a wide variety of protein targets but not in its catalytic mechanism, suggesting an important role for this extended loop in the binding of protein targets. As such, these findings pave the way toward an understanding of the molecular determinants that dictate the broad substrate specificity of PafA.

Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system.

The proper functioning of any biological system depends on the coordinated activity of its components. Regulation at the genetic level is, in many cases, effective in determining the cellular levels of system components. However, in cases where regulation at the genetic level is insufficient for attaining harmonic system function, posttranslational regulatory mechanisms are often used. Here, we uncover posttranslational regulatory mechanisms in the prokaryotic ubiquitin-like protein (Pup)-proteasome system (PPS), the bacterial equivalent of the eukaryotic ubiquitin-proteasome system. Pup, a ubiquitin analog, is conjugated to proteins through the activities of two enzymes, Dop (deamidase of Pup) and PafA (proteasome accessory factor A), the Pup ligase. As Dop also catalyzes depupylation, it was unclear how PPS function could be maintained without Dop and PafA canceling the activity of the other, and how the two activities of Dop are balanced. We report that tight Pup binding and the limited degree of Dop interaction with high-molecular-weight pupylated proteins results in preferred Pup deamidation over protein depupylation by this enzyme. Under starvation conditions, when accelerated protein pupylation is required, this bias is intensified by depletion of free Dop molecules, thereby minimizing the chance of depupylation. We also find that, in contrast to Dop, PafA presents a distinct preference for high-molecular-weight protein substrates. As such, PafA and Dop act in concert, rather than canceling each other's activity, to generate a high-molecular-weight pupylome. This bias in pupylome molecular weight distribution is consistent with the proposed nutritional role of the PPS under starvation conditions.

A kinetic model for the prevalence of mono- over poly-pupylation.

Bacteria belonging to the phyla Actinobacteria and Nitrospira possess proteasome cores homologous to the eukaryotic 20S proteasome particle. In these bacteria, the cytoplasmic signal for proteasomal degradation is a small protein termed Pup (prokaryotic ubiquitin-like protein). PafA, the only known Pup ligase, conjugates Pup to lysine side chains of target proteins. In contrast to the eukaryotic ubiquitin-proteasome system, where poly-ubiquitin chains are the principal tags for proteasomal degradation, mono-Pup moieties are almost exclusively observed in vivo and are sufficient as degradation tags. Although Pup presents lysines, raising the possibility of poly-Pup chain assembly, these do not predominate. At present, the factors promoting the distinct predominance of mono- over poly-pupylation remain poorly understood. To address this issue, we conducted a detailed biochemical analysis characterizing the pupylation of model proteins in vitro. We found that Pup can indeed serve as a pupylation target for PafA either in its free form or when already conjugated to proteins, thus allowing for the formation of poly-Pup chains. However, our results indicate that pupylation of an already pupylated protein is unlikely to occur due to low affinity of PafA for such species. This alone prevents predominance of poly- over mono-pupylation in vitro. This effect is likely to be magnified in vivo by the combination of PafA kinetics with the high abundance of non-pupylated proteins. Overall, this work provides a kinetic explanation for the prevalence of mono- rather than poly-pupylation in vivo, and sheds light on PafA substrate specificity.

Transfer of peanut IgE sensitisation after combined pancreas-kidney transplant.

BACKGROUND: The transfer of peanut allergy has been reported following solid organ transplantation. OBJECTIVE: Present a case of peanut sensitization following combined pancreas-and-kidney transplantation. METHODS: Circulating specific IgE against peanut were measured in serum samples collected from the transplant recipient 1 month before transplantation, and 1, 3 and 6 months after transplantation. Skin tests were performed 1, 3 and 6 months following transplant. RESULTS: The organ recipient's pre-transplant peanut IgE levels were negative. At 1 month post-transplant, the patient had a 6 mm skin test to peanut and had serum IgE to peanut Arah1 of 0.79 kU/L. At 3 months, skin test size and IgE to peanut Arah1 decreased to 4 mm, and 0.69 kU/L respectively. At 6 months, the patient's skin test and IgE to peanuts were negative. At that time, the patient underwent a peanut food challenge without a clinical reaction to suggest allergy. CONCLUSION AND CLINICAL RELEVANCE: We report the development of IgE peanut sensitization in a recipient of a combined pancreas and kidney transplantation. Increasing awareness of this allergen sensitization following transplantation may help prevent serious allergic reactions in transplant recipients.

Efficient and simple generation of unmarked gene deletions in Mycobacterium smegmatis.

Genetic research in molecular laboratories relies heavily on directed mutagenesis and gene deletion techniques. In mycobacteria, however, genetic analysis is often hindered by difficulties in the preparation of deletion mutants. Indeed, in comparison to the allelic exchange systems available for the study of other common model organisms, such as Saccharomyces cerevisiae and Escherichia coli, mycobacterial gene disruption systems suffer from low mutant isolation success rates, mostly due to inefficient homologous recombination and a high degree of non-specific recombination. Here, we present a gene deletion system that combines efficient homologous recombination with advanced screening of mutants. This novel methodology allows for gene disruption in three consecutive steps. The first step relies on the use of phage Che9c recombineering proteins for directed insertion into the chromosome of a linear DNA fragment that encodes GFP and confers hygromycin resistance. In the second step, GFP positive and hygromycin resistant colonies are selected, and in the last step, the gfp-hyg cassette is excised from the chromosome, thus resulting in the formation of an unmarked deletion. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the prcSBA operon of Mycobacterium smegmatis.

Hybrid fusion of linear, non-linear and spectral models for the dynamic modeling of sEMG and skeletal muscle force: an application to upper extremity amputation.

Estimating skeletal muscle (finger) forces using surface Electromyography (sEMG) signals poses many challenges. In general, the sEMG measurements are based on single sensor data. In this paper, two novel hybrid fusion techniques for estimating the skeletal muscle force from the sEMG array sensors are proposed. The sEMG signals are pre-processed using five different filters: Butterworth, Chebychev Type II, Exponential, Half-Gaussian and Wavelet transforms. Dynamic models are extracted from the acquired data using Nonlinear Wiener Hammerstein (NLWH) models and Spectral Analysis Frequency Dependent Resolution (SPAFDR) models based system identification techniques. A detailed comparison is provided for the proposed filters and models using 18 healthy subjects. Wavelet transforms give higher mean correlation of 72.6 ± 1.7 (mean ± SD) and 70.4 ± 1.5 (mean ± SD) for NLWH and SPAFDR models, respectively, when compared to the other filters used in this work. Experimental verification of the fusion based hybrid models with wavelet transform shows a 96% mean correlation and 3.9% mean relative error with a standard deviation of ± 1.3 and ± 0.9 respectively between the overall hybrid fusion algorithm estimated and the actual force for 18 test subjects' k-fold cross validation data.

Data gaps in toxicity testing of chemicals allowed in food in the United States.

In the United States, chemical additives cannot be used in food without an affirmative determination that their use is safe by FDA or additive manufacturer. Feeding toxicology studies designed to estimate the amount of a chemical additive that can be eaten safely provide the most relevant information. We analyze how many chemical additives allowed in human food have feeding toxicology studies in three toxicological information sources including the U.S. Food and Drug Administration's (FDA) database. Less than 38% of FDA-regulated additives have a published feeding study. For chemicals directly added to food, 21.6% have feeding studies necessary to estimate a safe level of exposure and 6.7% have reproductive or developmental toxicity data in FDA's database. A program is needed to fill these significant knowledge gaps by using in vitro and in silico methods complemented with targeted in vivo studies to ensure public health is protected.