The Study of a Novel Paeoniflorin-Converting Enzyme from Cunninghamella blakesleeana.

Paeoniflorin is a glycoside compound found in Paeonia lactiflora Pall that is used in traditional herbal medicine and shows various protective effects on the cardio-cerebral vascular system. It has been reported that the pharmacological effects of paeoniflorin might be generated by its metabolites. However, the bioavailability of paeoniflorin by oral administration is low, which greatly limits its clinical application. In this paper, a paeoniflorin-converting enzyme gene (G6046, GenBank accession numbers: OP856858) from Cunninghamella blakesleeana (AS 3.970) was identified by comparative analysis between MS analysis and transcriptomics. The expression, purification, enzyme activity, and structure of the conversion products produced by this paeoniflorin-converting enzyme were studied. The optimal conditions for the enzymatic activity were found to be pH 9, 45 °C, resulting in a specific enzyme activity of 14.56 U/mg. The products were separated and purified by high-performance counter-current chromatography (HPCCC). Two main components were isolated and identified, 2-amino-2-p-hydroxymethyl-methyl alcohol-benzoate (tirs-benzoate) and 1-benzoyloxy-2,3-propanediol (1-benzoyloxypropane-2,3-diol), via UPLC-Q-TOF-MS and NMR. Additionally, paeoniflorin demonstrated the ability to metabolize into benzoic acid via G6046 enzyme, which might exert antidepressant effects through the blood-brain barrier into the brain.

YihE is a novel binding partner of Rho and regulates Rho-dependent transcription termination in the Cpx stress response.

The conjugative pilus expression (Cpx) stress response system can sense cell envelope stressors, such as misfolded proteins, and upregulate proteases to modify or degrade damaged proteins. YihE is a protein kinase implicated in the Cpx system, and Rho is a transcription termination factor in prokaryotes, but no functional connection between YihE and Rho has been reported. Here, we found that YihE can interact with Rho to form a binary complex with a stoichiometric ratio of 6:1 (Rho:YihE). A low resolution of Rho crystal structure in the presence of YihE was determined. YihE overexpression helped lessen the aberrant effects caused by Rho overexpression, including long cell morphology and other Rho-mediated phenotypes. Overall, YihE is a Rho binding partner that acts as a Rho antagonist in the Cpx stress. YihE binding to Rho would compete RNA from binding to Rho, thereby helping bacteria cope with stress through the regulation of Rho-dependent transcription termination.

PLK1-mediated phosphorylation of PPIL2 regulates HR via CtIP.

Homologous recombination (HR) is an error-free DNA double-strand break (DSB) repair pathway, which safeguards genome integrity and cell viability. Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator of the pathway which initiates the DNA end resection in HR. Ubiquitination modification of CtIP is known in some cases to control DNA resection and promote HR. However, it remains unclear how cells restrain CtIP activity in unstressed cells. We show that the ubiquitin E3 ligase PPIL2 is recruited to DNA damage sites through interactions with an HR-related protein ZNF830, implying PPIL2's involvement in DNA repair. We found that PPIL2 interacts with and ubiquitinates CtIP at the K426 site, representing a hereunto unknown ubiquitination site. Ubiquitination of CtIP by PPIL2 suppresses HR and DNA resection. This inhibition of PPIL2 is also modulated by phosphorylation at multiple sites by PLK1, which reduces PPIL2 ubiquitination of CtIP. Our findings reveal new regulatory complexity in CtIP ubiquitination in DSB repair. We propose that the PPIL2-dependent CtIP ubiquitination prevents CtIP from interacting with DNA, thereby inhibiting HR.

A Novel Porous PDMS-AgNWs-PDMS (PAP)-Sponge-Based Capacitive Pressure Sensor.

The development of capacitive pressure sensors with low cost, high sensitivity and facile fabrication techniques is desirable for flexible electronics and wearable devices. In this project, a highly sensitive and flexible capacitive pressure sensor was fabricated by sandwiching a porous PAP sponge dielectric layer between two copper electrodes. The porous PAP sponge dielectric layer was fabricated by introducing highly conductive silver nanowires (AgNWs) into the PDMS sponge with 100% sucrose as a template and with a layer of polydimethylsiloxane (PDMS) film coating the surface. The sensitivity of the PAP sponge capacitive pressure sensor was optimized by increasing the load amount of AgNWs. Experimental results demonstrated that when the load amount of AgNWs increased to 150 mg in the PAP sponge, the sensitivity of the sensor was the highest in the low-pressure range of 0-1 kPa, reaching 0.62 kPa-1. At this point, the tensile strength and elongation of sponge were 1.425 MPa and 156.38%, respectively. In addition, the specific surface area of PAP sponge reached 2.0 cm2/g in the range of 0-10 nm pore size, and showed excellent waterproof performance with high elasticity, low hysteresis, light weight, and low density. Furthermore, as an application demonstration, ~110 LED lights were shown to light up when pressed onto the optimized sensor. Hence, this novel porous PAP-sponge-based capacitive pressure sensor has a wide range of potential applications in the field of wearable electronics.

YdfD, a Lysis Protein of the Qin Prophage, Is a Specific Inhibitor of the IspG-Catalyzed Step in the MEP Pathway of Escherichia coli.

Bacterial cryptic prophage (defective prophage) genes are known to drastically influence host physiology, such as causing cell growth arrest or lysis, upon expression. Many phages encode lytic proteins to destroy the cell envelope. As natural antibiotics, only a few lysis target proteins were identified. ydfD is a lytic gene from the Qin cryptic prophage that encodes a 63-amino-acid protein, the ectopic expression of which in Escherichia coli can cause nearly complete cell lysis rapidly. The bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for synthesizing the isoprenoids uniquely required for sustaining bacterial growth. In this study, we provide evidence that YdfD can interact with IspG, a key enzyme involved in the MEP pathway, both in vivo and in vitro. We show that intact YdfD is required for the interaction with IspG to perform its lysis function and that the mRNA levels of ydfD increase significantly under certain stress conditions. Crucially, the cell lysis induced by YdfD can be abolished by the overexpression of ispG or the complementation of the IspG enzyme catalysis product methylerythritol 2,4-cyclodiphosphate. We propose that YdfD from the Qin cryptic prophage inhibits IspG to block the MEP pathway, leading to a compromised cell membrane and cell wall biosynthesis and eventual cell lysis.

Structural characterization of the mitochondrial Ca2+ uniporter provides insights into Ca2+ uptake and regulation.

The mitochondrial uniporter is a Ca2+-selective ion-conducting channel in the inner mitochondrial membrane that is involved in various cellular processes. The components of this uniporter, including the pore-forming membrane subunit MCU and the modulatory subunits MCUb, EMRE, MICU1, and MICU2, have been identified in recent years. Previously, extensive studies revealed various aspects of uniporter activities and proposed multiple regulatory models of mitochondrial Ca2+ uptake. Recently, the individual auxiliary components of the uniporter and its holocomplex have been structurally characterized, providing the first insight into the component structures and their spatial relationship within the context of the uniporter. Here, we review recent uniporter structural studies in an attempt to establish an architectural framework, elucidating the mechanism that governs mitochondrial Ca2+ uptake and regulation, and to address some apparent controversies. This information could facilitate further characterization of mitochondrial Ca2+ permeation and a better understanding of uniporter-related disease conditions.

The structure of the MICU1-MICU2 complex unveils the regulation of the mitochondrial calcium uniporter.

The MICU1-MICU2 heterodimer regulates the mitochondrial calcium uniporter (MCU) and mitochondrial calcium uptake. Herein, we present two crystal structures of the MICU1-MICU2 heterodimer, in which Ca2+ -free and Ca2+ -bound EF-hands are observed in both proteins, revealing both electrostatic and hydrophobic interfaces. Furthermore, we show that MICU1 interacts with EMRE, another regulator of MCU, through a Ca2+ -dependent alkaline groove. Ca2+ binding strengthens the MICU1-EMRE interaction, which in turn facilitates Ca2+ uptake. Conversely, the MICU1-MCU interaction is favored in the absence of Ca2+ , thus inhibiting the channel activity. This Ca2+ -dependent switch illuminates how calcium signals are transmitted from regulatory subunits to the calcium channel and the transition between gatekeeping and activation channel functions. Furthermore, competition with an EMRE peptide alters the uniporter threshold in resting conditions and elevates Ca2+ accumulation in stimulated mitochondria, confirming the gatekeeper role of the MICU1-MICU2 heterodimer. Taken together, these structural and functional data provide new insights into the regulation of mitochondrial calcium uptake.

Characterization of kynurenine pathway in patients with diarrhea-predominant irritable bowel syndrome.

Our objectives are to demonstrate whether the kynurenine pathway is activated in diarrhea-type irritable bowel syndrome (IBS-D) patients, and whether the neurotoxic metabolite quinolinic acid (QUIN) is out of balance with the neuroprotective metabolite kynurenic acid (KYNA), and further explore whether this can lead to increase of N-methyl D-aspartate receptor 2B (NMDAR2B) expression in the enteric nervous system and in turn leads to intestinal symptoms and mood disorders. All enrolled healthy controls and patients accepted IBS symptom severity scale (IBS-SSS) score, Self-rating Depression Scale (SDS) and Self-rating Anxiety Scale (SAS) anxiety and depression scores, and also underwent colonoscopy to collect ileum and colonic mucosa specimens. The expression of NMDAR2B in intestinal mucosa was detected by immunofluorescence, and fasting serum was collected to detect the tryptophan (Trp), kynurenine (KYN), KYNA and QUIN by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Our results showed that the kynurenine pathway of IBS-D patients was activated. The production of QUIN and KYNA was imbalanced and resulting in an increased NMDAR2B for patients with IBS-D, which may be involved in intestinal symptoms and mood disorders of IBS-D.

Author Correction: Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Conformational Plasticity of the Active Site Entrance in E. coli Aspartate Transcarbamoylase and Its Implication in Feedback Regulation.

Aspartate transcarbamoylase (ATCase) has been studied for decades and Escherichia coli ATCase is referred as a "textbook example" for both feedback regulation and cooperativity. However, several critical questions about the catalytic and regulatory mechanisms of E. coli ATCase remain unanswered, especially about its remote feedback regulation. Herein, we determined a structure of E. coli ATCase in which a key residue located (Arg167) at the entrance of the active site adopted an uncommon open conformation, representing the first wild-type apo-form E. coli ATCase holoenzyme that features this state. Based on the structure and our results of enzymatic characterization, as well as molecular dynamic simulations, we provide new insights into the feedback regulation of E. coli ATCase. We speculate that the binding of pyrimidines or purines would affect the hydrogen bond network at the interface of the catalytic and regulatory subunit, which would further influence the stability of the open conformation of Arg167 and the enzymatic activity of ATCase. Our results not only revealed the importance of the previously unappreciated open conformation of Arg167 in the active site, but also helped to provide rationalization for the mechanism of the remote feedback regulation of ATCase.

New regulatory mechanism-based inhibitors of aspartate transcarbamoylase for potential anticancer drug development.

Aspartate transcarbamoylase (ATCase) is a key enzyme which regulates and catalyzes the second step of de novo pyrimidine synthesis in all organisms. Escherichia coli ATCase is a prototypic enzyme regulated by both product feedback and substrate cooperativity, whereas human ATCase is a potential anticancer target. Through structural and biochemical analyses, we revealed that R167/130's loop region in ATCase serves as a gatekeeper for the active site, playing a new and unappreciated regulatory role in the catalytic cycle of ATCase. Based on virtual compound screening simultaneously targeting the new regulatory region and active site of human ATCase, two compounds were identified to exhibit strong inhibition of ATCase activity, proliferation of multiple cancer cell lines, and growth of xenograft tumors. Our work has not only revealed a previously unknown regulatory region of ATCase that helps uncover the catalytic and regulatory mechanism of ATCase, but also successfully guided the identification of new ATCase inhibitors for anticancer drug development using a dual-targeting strategy. DATABASE: Structure data are available in Protein Data Bank under the accession numbers: 6KJ7 (G166P ecATCase), 6KJ8 (G166P ecATCase-holo), 6KJ9 (G128/130A ecATCase), and 6KJA (G128/130A ecATCase-holo).

The crystal structure of MICU2 provides insight into Ca2+ binding and MICU1-MICU2 heterodimer formation.

The mitochondrial calcium uniporter (MCU) complex mediates the uptake of Ca2+ into mitochondria. Its activity is regulated by a heterodimer of MICU1 and MICU2, two EF-hand-containing proteins that act as the main gatekeeper of the uniporter. Herein we report the crystal structure of human MICU2 at 1.96 Å resolution. Our structure reveals a dimeric architecture of MICU2, in which each monomer adopts the canonical two-lobe structure with a pair of EF-hands in each lobe. Both Ca2+ -bound and Ca2+ -free EF-hands are observed in our structure. Moreover, we characterize the interaction sites within the MICU2 homodimer, as well as the MICU1-MICU2 heterodimer in both Ca2+ -free and Ca2+ -bound conditions. Glu242 in MICU1 and Arg352 in MICU2 are crucial for apo heterodimer formation, while Phe383 in MICU1 and Glu196 in MICU2 significantly contribute to the interaction in the Ca2+ -bound state. Based on our structural and biochemical analyses, we propose a model for MICU1-MICU2 heterodimer formation and its conformational transition from apo to a more compact Ca2+ -bound state, which expands our understanding of this co-regulatory mechanism critical for MCU's mitochondrial calcium uptake function.

Characterization of metal binding of bifunctional kinase/phosphatase AceK and implication in activity modulation.

A unique bifunctional enzyme, isocitrate dehydrogenase kinase/phosphatase (AceK) regulates isocitrate dehydrogenase (IDH) by phosphorylation and dephosphorylation in response to nutrient availability. Herein we report the crystal structure of AceK in complex with ADP and Mn2+ ions. Although the overall structure is similar to the previously reported structures which contain only one Mg2+ ion, surprisingly, two Mn2+ ions are found in the catalytic center of the AceK-Mn2+ structure. Our enzymatic assays demonstrate that AceK-Mn2+ showed higher phosphatase activity than AceK-Mg2+, whereas the kinase activity was relatively unaffected. We created mutants of AceK for all metal-coordinating residues. The phosphatase activities of these mutants were significantly impaired, suggesting the pivotal role of the binuclear (M1-M2) core in AceK phosphatase catalysis. Moreover, we have studied the interactions of Mn2+ and Mg2+ with wild-type and mutant AceK and found that the number of metal ions bound to AceK is in full agreement with the crystal structures. Combined with the enzymatic results, we demonstrate that AceK exhibits phosphatase activity in the presence of two, but not one, Mn2+ ions, similar to PPM phosphatases. Taken together, we suggest that metal ions help AceK to balance and fine tune its kinase and phosphatase activities.

Syntheses, Structures, and Magnetic Properties of a Series of Heterotri-, Tetra- and Pentanuclear LnIII⁻CoII Compounds.

Three types of Ln(III)⁻Co(II) heterometallic compounds, LnCo2(L1)7(bipy)2 (Ln = Pr-1, Eu-2, Sm-3, Gd-4, Tb-5, Dy-6) (L1 = 4-chlorobenzoate, bipy = 2,2'-bipyridine), Ln2Co2(L2)10(bipy)2 (Ln = Sm-7, Gd-8, Tb-9, Dy-10, Ho-11, Er-12, Yb-13), (L2 = 2,4-dichlorobenzoate, bipy = 2,2'-bipyridine, phen = 1,10-phenanthroline), and Ln2Co3(L1)12(bipy)2 (Ln = Ho-14, Er-15, Yb-16), were synthesized under hydrothermal conditions and characterized by single crystal X-ray diffraction, IR spectroscopy and magnetic measurements. Structural analyses revealed that 14⁻16 take on a unique linear pentanuclear structural motif. Interestingly, the Ho-containing compound 14 exhibits magnetic relaxation behavior.

Separation and purification of lanosterol, dihydrolanosterol, and cholesterol from lanolin by high-performance counter-current chromatography dual-mode elution method.

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high-performance separation methods. In this study, a counter-current chromatography dual-mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head-to-tail mode, followed by the upper phase in the tail-to-head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n-heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high-performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter-current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.

Future Needs for Science-Driven Geospatial and Temporal Extravehicular Activity Planning and Execution.

Future human missions to Mars are expected to emphasize scientific exploration. While recent Mars rover missions have addressed a wide range of science objectives, human extravehicular activities (EVAs), including the Apollo missions, have had limited experience with science operations. Current EVAs are carefully choreographed and guided continuously from Earth with negligible delay in communications between crew and flight controllers. Future crews on Mars will be expected to achieve their science objectives while operating and coordinating with a science team back on Earth under communication latency and bandwidth restrictions. The BASALT (Biologic Analog Science Associated with Lava Terrains) research program conducted Mars analog science on Earth to understand the concept of operations and capabilities needed to support these new kinds of EVAs. A suite of software tools (Minerva) was used for planning and executing all BASALT EVAs, supporting text communication across communication latency, and managing the collection of operational and scientific EVA data. This paper describes the support capabilities provided by Minerva to cope with various geospatial and temporal constraints to support the planning and execution phases of the EVAs performed during the BASALT research program. The results of this work provide insights on software needs for future science-driven planetary EVAs.

Expression and Characterization of MICU2, a Ca2+ Sensor Protein.

MICU2 is a Ca2+ sensor protein of mitochondrial uniporter which is a highly selective Ca2+ channel mediating mitochondrial Ca2+ uptake to regulate cell death, metabolism, and cytoplasmic Ca2+ signaling. Here we describe the procedures for protein preparation of various MICU2 constructs, which have enabled successful in vitro characterizations of MICU2 including interaction with MICU1 using pull-down assays and oligomerization using multi-angle laser light scattering.

AtNPR4 from Arabidopsis thaliana: expression, purification, crystallization and crystallographic analysis.

Salicylic acid (SA) is an important phytohormone that is involved in the regulation of plant defence, growth and development. A large number of proteins have been shown to have the ability to interact with SA, and NPR4 has been demonstrated to be a receptor of SA that plays significant roles in the innate immune response of plants. In this study, Spodoptera frugiperda (Sf9) cells were used to express full-length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N-terminus of the AtNPR4 protein. The recombinant T4L-AtNPR4 protein was expressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The T4L-AtNPR4 crystals have symmetry consistent with space group C2, with unit-cell parameters a = 93.7, b = 85.8, c = 88.2 Å, ß = 90° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75 Å. Structure determination is in progress.

Characterization of interaction and ubiquitination of phosphoenolpyruvate carboxykinase by E3 ligase UBR5.

Phosphoenolpyruvate carboxykinase (PEPCK1) is ubiquitinated by E3 ubiquitin ligase UBR5, which was thought to be facilitated by the acetylation of Lys70, Lys71 and Lys594 in PEPCK1. Here, we made a series of UBR5 HECT domain truncation variants and, through pull-down assay, showed that the N-terminal lobe of the UBR5 HECT domain is largely responsible for interacting with PEPCK1. We mutated all three lysine residues thought to be acetylated in PEPCK1 but were surprised to observe no loss of binding to UBR5 HECT domain. Furthermore, two PEPCK1 truncation variants (74-622 aa and 10-560 aa) lacking these lysine residues were still able to bind with UBR5 and ubiquitinated in HEK293T cells. To discover the ubiquitination site(s) of PEPCK1, which is currently unknown, the Lys residues of PEPCK1 were mutated to Ala and the ubiquitination level of the PEPCK1 mutants was assessed. Results revealed at least two ubiquitination sites (Lys243 and Lys342), which represent the first time that ubiquitination sites of PEPCK1 have been identified. Our pull-down experiments further show that the lack of ubiquitination of PEPCK1 Lys243Ala and Lys342Ala mutants is not due to their binding to UBR5, which remained unchanged. Taken together, our work has provided new insights into UBR5 mediated ubiquitination of PEPCK1.