Design, synthesis, and in vitro gene transfer efficacy of novel ionizable cholesterol derivatives.

ABSTACTThe medicinal properties of genetic drugs are highly dependent on the design of delivery systems. Ionizable cationic lipids are considered core materials in delivery systems. However, there has not yet been a widespread consensus on the relationship between the wide diversity of lipid structure design and gene delivery efficiency. The aims of the research work were to synthesize ionizable cholesterol derivatives (iChol-lipids) and to evaluate their potential applications as gene delivery vector. A series of iChol-lipids with different head groups were synthesized with carbamate bond spacer. The chemical structures were characterized by 1H NMR, MS, melting range, and pKa. The interactions between iChol-lipids and MALAT1-siRNA were studied by molecular dynamics simulations and compared with market available DC-Chol, which revealed that hydrogen bonds, salt-bridge, and electrostatic interaction were probably involved. The self-assemble behaviors of these lipids were intensively investigated and evaluated by dynamic laser scattering in the presence of different helper lipids and PEGylated lipids. Their plasmid binding ability, transfection efficiency, hemolytic toxicity, and cytotoxicity were fully studied. IZ-Chol-LNPs was proved to be highly potential to effectively complex with DNA, and endosome escape mechanisms mediated by proton sponge effect was verified by pH-sensitive fluorescence probe BCFL.

Transition shock, future time perspective, professional self-concept, and professional identity among nursing interns: A cross-sectional study.

Background: Transition shock is prevalent among nursing interns. Future time perspective helps nursing interns learn and work more effectively and improve their problem-solving skills. Professional self-concept and professional identity play an important central and driving role in nursing interns' career choices and career development. However, the mechanism by which future time perspective, professional self-concept and professional identity are linked to transition shock among nursing interns remains unknown. Objectives: We assess the degree of transition shock experienced by nursing interns and investigate the connections among transition shock, future-focused viewpoint, professional identity, and professional self-concept. Design: A descriptive, cross-sectional design was conducted. Setting: Eight hospitals in Hunan Province, China were included in the study. Participants: Nursing interns at the studied hospital participated in the study. Methods: A total of 1090 nursing interns [929 female, 161 male] were recruited from eight hospitals. Data on transition shock, future time perspective, professional self-concept and professional identity among nursing interns were collected using questionnaires from 30 May to June 15, 2022. Results: On a 4-point rating scale, the participants' felt transition shock had a mean overall score of 2.39 (SD = 0.52). The dimension with the highest score was overwhelming workload (mean = 2.74, SD = 0.58), while the dimension with the lowest score was incongruity between work and personal life (mean = 2.16, SD = 0.70).Professional identity was statistically significantly correlated with transition shock (r = -0.198, p < 0.01). The preferred level of nursing (ß = 0.354, p < 00.01), professional self-concept (ß = 0.226, p < 00.01), professional identity (ß = -0.2576, p < 00.01) and future time perspective (ß = 0.119, p < 00.01) were predictors of transition shock. Conclusions: The development of nursing interns' sense of professional identity and future time perspective should be enhanced during both the education phase and clinical placement to help nursing interns overcome the experience of transition shock.

Efficacy and safety of fractional microneedle radiofrequency for atrophic acne scars: A real-world clinical study of 126 patients.

OBJECTIVE: To analyze the clinical efficacy and safety of fractional microneedle radiofrequency (FMR) for facial atrophic acne scars in a real-world setting. METHODS: The clinical data of patients with atrophic acne scars who had received FMR therapy from February 2018 to August 2022 were retrospectively analyzed. The improvement of atrophic acne scars was assessed using the ECCA Grading Scale (échelle d'évaluation clinique des cicatrices d'acné), Global Aesthetic Improvement Scale (GAIS), and modified Manchester Scar Scale (mMSS). Adverse reactions during FMR treatment were also recorded. Univariate and multivariate logistic regression analyses were performed to evaluate the efficacy and safety of FMR for atrophic acne scars. RESULTS: A total of 126 patients with facial atrophic acne scars were included. A total of 590 FMR treatment sessions were accomplished, with each of 82 patients receiving 4 or more treatment sessions, and 1 receiving a maximum of 14 sessions. All patients showed improvement in symptoms after FMR treatment, with moderate to significant improvement (ECCA score reduction of 26%-100%) in 92 (73.0%) patients. As the number of treatment sessions increased, the ECCA score gradually decreased from an average of 85.6 before to 35.0 after FMR. The average scores for distortion, color, and visual analogue scale (VAS) of mMSS all showed certain reductions. The change in GAIS score indicated improvement after treatment, with minimal improvement in 16 patients (12.7%), good improvement in 57 patients (45.2%), significant improvement in 45 patients (35.7%), and optimal improvement in 8 patients (6.4%). The univariate and multivariate logistic regression analyses revealed that the long pulse width and the number of FMR treatment sessions were positively associated with clinical efficacy. Compared to the short pulse-width group (200 ms), the longer pulse-width group (300 ms) (odds ratio [OR] = 8.3, p = 0.003) and the even longer pulse-width group (400-500 ms) (OR = 52.6, p < 0.001) demonstrated stronger efficacies. Patients who received more than three treatment sessions had better outcomes compared to those who received three or fewer treatment sessions (OR = 4.0, p = 0.036). All patients experienced posttreatment transient erythema, but no crusting, infection, or blister. Six cases developed grid-like erythema around 1 month posttreatment and one case experienced hyperpigmentation, both of which resolved within 1-3 months after appropriate management. CONCLUSION: FMR is a safe and effective treatment modality for improving facial atrophic acne scars, and the number of FMR treatment sessions and pulse width are associated with clinical efficacy.

College Students' Employability, Cognition, and Demands for ChatGPT in the AI Era Among Chinese Nursing Students: Web-Based Survey.

BACKGROUND: With the rapid development of artificial intelligence (AI) and the widespread use of ChatGPT, nursing students' artificial intelligence quotient (AIQ), employability, cognition, and demand for ChatGPT are worthy of attention. OBJECTIVE: We aimed to investigate Chinese nursing students' AIQ and employability status as well as their cognition and demand for the latest AI tool-ChatGPT. This study was conducted to guide future initiatives in nursing intelligence education and to improve the employability of nursing students. METHODS: We used a cross-sectional survey to understand nursing college students' AIQ, employability, cognition, and demand for ChatGPT. Using correlation analysis and multiple hierarchical regression analysis, we explored the relevant factors in the employability of nursing college students. RESULTS: In this study, out of 1788 students, 1453 (81.30%) had not used ChatGPT, and 1170 (65.40%) had never heard of ChatGPT before this survey. College students' employability scores were positively correlated with AIQ, self-regulation ability, and their home location and negatively correlated with school level. Additionally, men scored higher on college students' employability compared to women. Furthermore, 76.5% of the variance was explained by the multiple hierarchical regression model for predicting college students' employability scores. CONCLUSIONS: Chinese nursing students have limited familiarity and experience with ChatGPT, while their AIQ remains intermediate. Thus, educators should pay more attention to cultivating nursing students' AIQ and self-regulation ability to enhance their employability. Employability, especially for female students, those from rural backgrounds, and students in key colleges, deserves more attention in future educational efforts.

Anti-interference and non-destructive identification of textile fabrics using front-face excitation-emission matrix fluorescence spectroscopy combined with multi-way chemometrics.

The identification of trace textile fabrics discovered at crime scenes plays a crucial role in the case of forensic investigations. Additionally, in practical situations, fabrics may be contaminated, making identification more challenging. To address the aforementioned issue and promote the application of fabrics identification in forensic analysis, front-face excitation-emission matrix (FF-EEM) fluorescence spectra coupled with multi-way chemometric methods were proposed for the interference-free and non-destructive identification of textile fabrics. Common commercial dyes in the same color range under different materials (cotton, acrylic, and polyester) that cannot be visually distinguished were investigated, and several binary classification models for the identification of dye were established using partial least squares discriminant analysis (PLS-DA). The identification of dyed fabrics in the presence of fluorescent interference was also taken into consideration. In each kind of pattern recognition model mentioned above, the classification accuracy (ACC) of the prediction set was 100%. The alternating trilinear decomposition (ATLD) algorithm was executed to separate mathematically and remove the interference, and the classification model based on the reconstructed spectra attained an accuracy of 100%. These findings indicate that FF-EEM technology combined with multi-way chemometric methods has broad prospects for forensic trace textile fabric identification, especially in the presence of interference.

Development of tLyP-1 functionalized nanoliposomes with tunable internal water phase for glioma targeting.

tLyP-1 peptide is verified to recognize neuropilin (NRP) receptors overexpressed on the surface of both glioma cells and endothelial cells of angiogenic blood vessels. In the present study, tLyP-1 was conjugated with DSPE-PEG2000 to prepare tLyP-1-DSPE-PEG2000, which was further employed to prepare tLyP-1 functionalized nanoliposome (tLyP-1-Lip) to achieve enhancing target of glioblastoma. Process parameters were systematically studied to investigate the feasibility of tuning the internal water phase of nanoliposomes and encapsulating more Temozolomide (TMZ). The particle size, Zeta potential, and encapsulation efficiency of tLyP-1-Lip/TMZ were fully characterized in comparison with conventional nanoliposomes (Lip-TMZ) and PEGylated nanoliposomes (PEG-Lip/TMZ). The release behaviors of TMZ from PEG-Lip/TMZ and tLyP-1-Lip/TMZ are similar and slower than TMZ-Lip in acidic solutions. The tLyP-1-Lip/TMZ demonstrated the strongest cytotoxicity in comparison with TMZ-Lip and PEG-Lip/TMZ in both U87 and HT22 cells, and displayed the highest cellular internalization. The pharmacokinetic studies in rats revealed that tLyP-1-Lip/TMZ showed a 1.4-fold (p < 0.001) increase in AUCINF_obs and a 1.4-fold decrease (p < 0.01) in clearance compared with PEG-Lip/TMZ. We finally confirmed by in vivo imaging that tLyP-1-Lip were able to penetrate the brains and tumors of mice.

Investigations on the influence of the structural flexibility of nanoliposomes on their properties.

In the present study, nanoliposomes with tuneable structure elasticity were prepared by reverse-phase evaporation. Both Fluorescence Polarization and Fluorescence Resonance Energy Transfer was employed to characterize the structural elasticity of resultant nanoliposomes. Temozolomide, a kind of hydrophilic drug as the first-line treatment choice for glioblastoma, was encapsulated into nanoliposomes. The results showed that the flexibility of nanoliposomes gradually increased with sodium cholate, while decreasing with cholesterol, Labrafac CC and Labrafac PG adding. Furthermore, the structural flexibility of nanoliposomes was positively correlated with the encapsulation efficiency and release rate and cellular uptake. Our research reveals the structural flexibility of nanoliposomes could affect in vitro characteristics and thereafter in vivo behaviors of nanoliposomes.

Effects of Herbaspirillum sp. p5-19 assisted with alien soil improvement on the phytoremediation of copper tailings by Vetiveria zizanioides L.

Microbial assisted phytoremediation and reclamation are both potential contaminated soil remediation technologies, but little is known about the combined application of the two technologies on real contaminated soils. This study investigated the potential of Herbaspirillum sp. p5-19 (p5-19) assisted with alien soil improvement on improving stress tolerance and enhancing the accumulation of Mn, Cu, Zn, and Cd by Vetiveria zizanioides L. in copper tailings. Phytoremediation potential was evaluated by plant biomass and the ability of plants to absorb and transfer heavy metals. Results showed that the biomass was increased by 19.64-173.81% in p5-19 inoculation treatments with and without alien soil improvement compared with control. Meanwhile, photosynthetic pigment contents were enhanced in co-inoculation treatment (p5-19 with alien soil improvement). In addition, the malondialdehyde (MDA) content was decreased, and the activities of antioxidant enzymes such as ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were increased in p5-19 treatment, thereby alleviating the oxidative stress. Moreover, co-inoculation significantly (p < 0.05) increased the concentrations of Mn, Cu, Zn, and Cd in the roots and shoots of V. zizanioides. In particular, the highest concentrations of Mn, Zn, and Cd in the shoots (roots) were obtained in covering 10 cm combined with p5-19 inoculation treatment, which were 4.44- (2.71-), 4.73- (3.87-), and 5.93- (4.35-) fold as that of the controls, respectively. These results provided basis for the change of phytoremediation ability of V. zizanioides after inoculation. We concluded that p5-19 assisted with alien soil improvement was a potential strategy for enhancing phytoremediation ability in tailings.

Physiological responses and metal distributions of different organs of Phragmites australis shoots under acid mine drainage stress.

Phragmites australis, which is widely distributed throughout the world, is often used in the phytoremediation of acid mine drainage (AMD) due to its various mechanisms for survival under extremely harsh conditions. To explore the different responses of different aerial organs of P. australis to stress, soil and plant samples were collected from the AMD-polluted area of the Tongling mining area. The contents of manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), arsenic (As), cadmium (Cd), and lead (Pb) in the soil and the leaf blades, leaf sheaths, and stems of P. australis as well as the contents/activities of cysteine synthase (CSase), superoxide dismutase (SOD), peroxidase (POD), glutathione (GSH), malondialdehyde (MDA), and proline (Pro) in the organs were determined. Our results revealed that the leaf sheath had the highest potential to store metals of all the organs. The highest translocation factor (TF) for Fe was observed from the stems to the leaf sheaths. A higher bioconcentration factor (BCF) for Mn was found in the leaf blades and leaf sheaths, while higher BCFs for Cd and Zn were observed in the stems. The content/activity of enzymatic and non-enzymatic stress-resistance substances varied from organ to organ. In general, the leaf sheaths remained almost as or slightly less stress-resistant than the leaf blades. It can be concluded that different plant organs play different roles in stress resistance, and understanding the tolerance mechanism of leaf sheaths to metals is essential for the application of phytoremediation procedures.

Oxidative carboxylation of olefins with CO2: environmentally benign access to five-membered cyclic carbonates.

The purpose of this focus review is to provide a comprehensive overview of the direct synthesis of five-membered cyclic carbonates via oxidative carboxylation of the corresponding olefins and carbon dioxide with particular attention on the mechanistic features of the reactions. The review is divided into two main sections. The first section is a discussion of the single-step reactions, while the second consists of an overview of one-pot, two-step sequential reactions.

Correction: Oxidative carboxylation of olefins with CO2: environmentally benign access to five-membered cyclic carbonates.

[This corrects the article DOI: 10.1039/C9RA10755J.].

Response of chemical properties, microbial community structure and functional genes abundance to seasonal variations and human disturbance in Nanfei River sediments.

The Nanfei River, located in Hefei City, Anhui Province, subjected to increased nutrient loads from point and/or non-point source. Little is known about the indicators indicating heterogeneity of surface sediments. We aimed to identify the suitable indicators that can reflect the sediment heterogeneity by analyzing the sensitivity of sediment physicochemical properties group, microbial communities and diversity indices group and C, N, S-functional genes group to seasonal and regional changes. River sediments from different areas (urban area, urban-rural fringe and rural area) were collected in the level, dry and wet seasons, respectively. The chemical parameters had most significant regional heterogeneity, but no seasonal differences. Seasons had a greater impact on the overall microbial community structure than the areas. Specifically, the relative abundance of Firmicutes and Bacteroidetes were more sensitive to seasonal changes. Overall, seasonal changes showed the greatest impact on the functional genes group, with the S-functional genes (dsrB and aprA) group providing the clearest seasonal variation. Considering the seasonal distribution of functional genes and their sensitivity to environmental factors, we speculated that the sulfate-reducing gene (dsrB), the methanogenic gene (mcrA) and the anammox gene (hzo) could be identified as sensitive indicators to indicate the seasonal heterogeneity of surface sediments in different river sections of the same river in the short term. We also concluded that environmental variables were more conducive to indicating the regional heterogeneity of sediments. This study provided a valuable reference for assessing the heterogeneity or ecological stress of river sediments.

The spatial and seasonal variations of bacterial community structure and influencing factors in river sediments.

Studying the composition and structure of bacterial communities in sediments helps to understand the contribution of bacteria to environmental changes and the role of feedback in response to disturbances. However, seasonal changes in bacterial communities of river sediments with different pollution levels and sources have not been clear yet. In this study, we collected sediment samples during the dry season, wet season and level season from 40 sites with various pollution sources in three inflow rivers (Fengle-Hangbu River, Nanfei River and Zhegao River) of Chaohu Lake. Bacterial community compositions were determined based on high-throughput sequencing. The 'Bioenv' in the R package 'Vegan' and redundancy analysis was used to explore the influence of environmental factors on the bacterial community in the river sediments. Results showed that a significant deviation in bacterial communities was found among seasons and rivers. In addition, seasonal dynamics had a greater impact on shaping bacterial communities than rivers with different pollution sources. A higher diversity was found in the wet season as compared to the other seasons. The bacterial diversity was negatively correlated with nutrients (OM, TN, NH4+, IP, OP and TP) and metals (Cu and Zn). Bacterial communities were more sensitive to heavy metals pressure than nutrients. We also concluded that heavy metals (Cu and Cd) were the key contributing factors in explaining variations in bacterial communities. This study provided a valuable reference for assessing ecological stress.

Trace Elements in Soils and Selected Agricultural Plants in the Tongling Mining Area of China.

The quality and safety of agricultural products from metal mining areas are of wide concern. In order to investigate the contents and health risks of trace elements in fruit vegetables planted in metal mining areas, 440 samples of fruit vegetables from 117 vegetable plots were collected from Tongling mining area. Trace element contents in fruit vegetables and soil were measured. The results indicated that the total concentration of trace elements in some of the soil samples exceeded the Grade II national standard in China. Transfer factor (TF) of Cd was the highest (8.360), followed by Zn, Cu, As, and Pb. Estimated daily intake (EDI) of the trace elements, except Cd, were generally below the maximum tolerable daily intake (MTDI). The target hazard quotient (THQ) of Zn for children was more than 1 in some vegetables, suggesting potential health hazards for child population. Total target hazard quotient (TTHQ) of Cu and Zn were also more than 1 through consumption of all vegetables, indicating significant health risks. For both adults and children, hazard index (HI) was more than 1 for the consumption of vegetables. The findings reveal the health risks associated with the consumption of trace elements through the intake of selected vegetables in the population of Tongling.

A 22-mer segment in the structurally pliable regulatory domain of metazoan CTP: phosphocholine cytidylyltransferase facilitates both silencing and activating functions.

CTP:phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, α-synuclein, which we show shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing, we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus, both silencing and activating functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or α-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus, full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

Crystal structure of a mammalian CTP: phosphocholine cytidylyltransferase catalytic domain reveals novel active site residues within a highly conserved nucleotidyltransferase fold.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in the synthesis of phosphatidylcholine, the most abundant phospholipid in eukaryotic cell membranes. The CCT-catalyzed transfer of a cytidylyl group from CTP to phosphocholine to form CDP-choline is regulated by a membrane lipid-dependent mechanism imparted by its C-terminal membrane binding domain. We present the first analysis of a crystal structure of a eukaryotic CCT. A deletion construct of rat CCTalpha spanning residues 1-236 (CCT236) lacks the regulatory domain and as a result displays constitutive activity. The 2.2-A structure reveals a CCT236 homodimer in complex with the reaction product, CDP-choline. Each chain is composed of a complete catalytic domain with an intimately associated N-terminal extension, which together with the catalytic domain contributes to the dimer interface. Although the CCT236 structure reveals elements involved in binding cytidine that are conserved with other members of the cytidylyltransferase superfamily, it also features nonconserved active site residues, His-168 and Tyr-173, that make key interactions with the beta-phosphate of CDP-choline. Mutagenesis and kinetic analyses confirmed their role in phosphocholine binding and catalysis. These results demonstrate structural and mechanistic differences in a broadly conserved protein fold across the cytidylyltransferase family. Comparison of the CCT236 structure with those of other nucleotidyltransferases provides evidence for substrate-induced active site loop movements and a disorder-to-order transition of a loop element in the catalytic mechanism.

Contribution of each membrane binding domain of the CTP:phosphocholine cytidylyltransferase-alpha dimer to its activation, membrane binding, and membrane cross-bridging.

CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the full-length homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs.

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.

Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.