Facile Synthesis of Novel Ti2C Nano Bipyramids for Photothermal and Photodynamic Therapy of Breast Cancer.

Photo-responsive synergetic therapeutics achieved significant attraction in cancer theranostic due to the versatile characteristics of nanomaterials. There have been substantial efforts in developing the simplest nano-design with exceptional synergistic properties and multifunctionalities. In this work, biocompatible Ti2C MXene nano bipyramids (MNBPs) were synthesized by hydrothermal method with dual functionalities of photothermal and photodynamic therapies. The MNBPs shape was obtained from two-dimensional (2D) Ti2C nanosheets by controlling the temperature of the reaction mixture. The structure of these Ti2C MNBPs was characterized by a high-resolution transmission electron microscope, scanning electron microscope, atomic force microscope, X-ray photoelectron spectroscopy, and X-ray diffraction. The Ti2C NBPs have shown exceptional photothermal properties with increased temperature to 72.3 °C under 808 nm laser irradiation. The designed nano bipyramids demonstrated excellent cellular uptake and biocompatibility. The Ti2C NBP has established a remarkable photothermal therapy (PTT) effect against 4T1 breast cancer cells. Moreover, Ti2C NBPs showed a profound response to UV light (6 mW/cm2) and produced reactive oxygen species, making them useful for photodynamic therapy (PDT). These in-vitro studies pave a new path to tune the properties of photo-responsive MXene nanosheets, indicating a potential use in biomedical applications.

Ferroptosis and WDFY4 as novel targets for immunotherapy of lung adenocarcinoma.

BACKGROUND: Lung cancer exhibits the world's highest mortality rate among malignant cancers worldwide, thereby presenting a significant global challenge in terms of reducing patient mortality. In the field of oncology, targeted immunotherapy has emerged as a novel therapeutic approach for lung cancer. This study aims to explore potential targets for immunotherapy in lung adenocarcinoma (LUAD) through the analysis of Ferroptosis Index (FPI) and Single Cell RNA-Sequencing (scRNA-seq) data. The findings of this research can potentially offer valuable insights for improving LUAD immunotherapy strategies and informing clinical decision-making. METHODS: Firstly, the relationship between survival and ferroptosis in LUAD patients was analyzed by FPI. Subsequently, the association between ferroptosis and infiltration and regulation of immune cells was explored by immune infiltration analysis and correlation statistics. Lastly, the relationship between major infiltrating immune cell populations and related pathways and prognosis of LUAD patients was analyzed by GSEA and GSVA. To screen out core genes regulating infiltration of immune cell populations, scRNA-seq data of cancer and para-cancerous tissues of LUAD patients were downloaded, followed by cell clustering analysis, cell identification of core subpopulations, pseudotime analysis, single-cell GSVA and pathway enrichment analysis, and identification and functional analysis of core regulatory genes. Moreover, the expression levels of core functional genes in LUAD tissue microarray were detected by immunohistochemistry, and its relationship with the prognosis of LUAD patients was verified. Finally, we used lentivirus with WDFY4 to transfect LUAD A549 cells. CCK-8, flow cytometry apoptosis detection, Scratch wound healing assay, Transwell migration assay, Xenograft nude mice model, immunohistochemical analysis and other experimental methods were used to explore the biological effects of WDFY4 on LUAD in vitro and in vivo. RESULTS: Survival analysis of FPI values in LUAD patients revealed a positive correlation between smaller FPI values and longer overall survival. Immuno-infiltration analysis and its correlation with FPI values revealed that B cells were most strongly associated with ferroptosis. Ferroptosis of cancer cells could promote infiltration and activation of B cell populations, and LUAD patients with more infiltration of B cell populations had longer long-term survival. scRNA-seq data analysis indicated that the B cell population is one of the major cell populations infiltrated by immune cells in LUAD. During the later phases of B cell differentiation in LUAD, there was a decrease in the expression levels of ACAP1, LINC00926, TLR10, MS4A1, WDFY4, and TRIM22 genes, whereas the expression levels of TMEM59, TP53INP1, and METTL7A genes were elevated. The protein-protein interaction (PPI) network analysis indicated that WDFY4 plays a crucial role in regulating B cell differentiation in LUAD. Immunohistochemical analysis of LUAD tissue microarray revealed a significant downregulation of WDFY4 expression, which was closely related to the occurrence sites of LUAD. Moreover, LUAD patients with a low WDFY4 expression exhibited a poorer prognosis. Additionally, experimental findings demonstrated that the overexpression of WDFY4 could inhibit the proliferation and metastasis of A549 cells while promoting apoptosis. It was also confirmed that WDFY4 could inhibit cancer growth in vivo. CONCLUSIONS: The results indicate that promoting infiltration and activation of B cell populations could improve the long-term survival of LUAD patients, thereby offering a potential novel immunotherapeutic approach for LUAD. Besides, the promotion of cancer cell ferroptosis and upregulation of WDFY4 expression have been shown to induce the infiltration and activation of B cell populations. Furthermore, the overexpression of WDFY4 can significantly inhibit the growth of lung adenocarcinoma in vitro and in vivo, highlighting its potential as a target for immunotherapy in LUAD.

A cane toad (Rhinella marina) N-methyltransferase converts primary indolethylamines to tertiary psychedelic amines.

Psychedelic indolethylamines have emerged as potential medicines to treat several psychiatric pathologies. Natural sources of these compounds include 'magic mushrooms' (Psilocybe spp.), plants used to prepare ayahuasca, and toads. The skin and parotid glands of certain toads accumulate a variety of specialized metabolites including toxic guanidine alkaloids, lipophilic alkaloids, poisonous steroids, and hallucinogenic indolethylamines such as DMT, 5-methoxy-DMT, and bufotenin. The occurrence of psychedelics has contributed to the ceremonial use of toads, particularly among Mesoamerican peoples. Yet, the biosynthesis of psychedelic alkaloids has not been elucidated. Herein, we report a novel indolethylamine N-methyltransferase (RmNMT) from cane toad (Rhinella marina). The RmNMT sequence was used to identify a related NMT from the common toad, Bufo bufo. Close homologs from various frog species were inactive, suggesting a role for psychedelic indolethylamine biosynthesis in toads. Enzyme kinetic analyses and comparison with functionally similar enzymes showed that recombinant RmNMT was an effective catalyst and not product inhibited. The substrate promiscuity of RmNMT enabled the bioproduction of a variety of substituted indolethylamines at levels sufficient for purification, pharmacological screening, and metabolic stability assays. Since the therapeutic potential of psychedelics has been linked to activity at serotonergic receptors, we evaluated binding of derivatives at 5-HT1A and 5-HT2A receptors. Primary amines exhibited enhanced affinity at the 5-HT1A receptor compared with tertiary amines. With the exception of 6-substituted derivatives, N,N-dimethylation also protected against catabolism by liver microsomes.

Highly Efficient Light Helicity Detection of Enantiomers by Chiral Metal-Organic Framework Thin Films.

The potential of chiral metal-organic frameworks (MOFs) for circularly polarized (CP) optics has been largely unexplored. Herein, we have successfully deposited monolithic and highly oriented chiral MOF thin films prepared by a layer-by-layer method (referred to as surface-coordinated MOF thin films, SURMOF) to fabricate CP photodetection devices and distinguish enantiomers. The helicity-sensitive absorption induced by a pair of enantiopure oriented SURMOF was found to be excellent, with an anisotropy factor reaching 0.41. Moreover, the chiral SURMOFs exhibited a pronounced difference in the uptake of the l- and d-tryptophan enantiomers. To demonstrate the potential of these novel MOF thin films for chirality analysis, we fabricated a portable sensor device that allows for chiral recognition by monitoring the photocurrent signals. Our findings not only introduce a new concept of using chiral building blocks for realizing direct CP photodetectors but also provide a blueprint for novel devices in chiral optics.

[Discuss Application of Real-world Data from the Boao Lecheng Pilot Zone to Support Premarket Clinical Evaluation of Medical Device].

The real-world data of Hainan Boao Lecheng International Tourism Pilot Zone has the advantage of supporting pre-market clinical evaluation of medical devices. Based on the relevant requirements of clinical evaluation of medical devices and based on the practical experience of pilot devices in the early stage, the application of Boao Lecheng real-world data in the pre-market clinical evaluation path of medical devices from the perspective of review is discussed. At the same time, the elements that should be considered in real-world study design and the way of data quality evaluation are proposed. Expect to provide a reference in order to allow registration applicants to use real world data wisely to help declare device registration for marketing.

A metal-porphyrinic framework film as an efficient optical limiting layer in an electro-optical switchable device.

Herein we report a 2D surface-coordinated porphyrinic metal-organic framework film (SURMOF) CuTCPP prepared by a layer by layer method as an optical limiting layer in a polymer-dispersed liquid crystal (PDLC) device. The results show that the CuTCPP SURMOF/PDLC device has excellent switchable transparency and optical limiting performance, providing a new route to achieve mutilfunctional electro-optical applications.

The Characteristics and Motivations of Taiwanese People toward Advance Care Planning in Outpatient Clinics at a Community Hospital.

Advance care planning (ACP) provides access to complete advance decisions (ADs). Despite the legalization of ACP in Taiwan, it is underutilized in community settings. The objective of this study is to describe the service at a community hospital in Southern Taiwan. We retrospectively analyzed participants who were engaged in ACP consultations from January 2019 to January 2020. The characteristics, motivations, content, and satisfaction of participants are reported. Factors associated with refusing life-sustaining treatments (LST) or artificial nutrition/hydration (ANH) were analyzed using multivariate logistic regression. Of the 178 participants, 123 completed the ACP. The majority were female (64.2%), aged 61 on average and more than 80% had never signed a do-not-resuscitate order. In the ADs, most participants declined LST (97.2%) and ANH (96.6%). Family-related issues (48.9%) were the most prevalent motivations. Rural residence (OR 8.6, p = 0.005), increased age (OR 7.2, p = 0.025), and reluctance to consent to organ donation (OR 5.2, p = 0.042) correlated with refusing LST or ANH. Participants provided a positive feedback regarding overall satisfaction (good, 83%) compared to service charge (fair/poor, 53%). The study demonstrated high AD completion when refusing LST or ANH. These findings may facilitate the development of ACP as a community-based service.

N-Heterocyclic Carbene as a Surface Platform for Assembly of Homochiral Metal-Organic Framework Thin Films in Chiral Sensing.

N-heterocyclic carbenes (NHCs) have attracted increasing attention on surface assembly due to their strong metal binding property, but an NHC-modified metal surface as a new growth platform to assemble other functional materials is still a challenge. Here, we report the preparation and chiral sensing properties of homochiral metal-organic framework thin films on carboxyl-containing NHC self-assembled monolayer-modified gold (Au(NHC)) substrates. By using a liquid-phase epitaxial layer-by-layer method, enantiopure [Cu2(cam)2dabco]n thin films with preferred [110] crystal orientation have been successfully grown on Au(NHC) surfaces. The results of electrochemical cyclic voltammetry and quartz crystal microbalance uptakes of (R)- and (S)-1-phenylethanol show that the chiral porous thin film on the robust Au(NHC) surface has good enantiomeric electrochemical recognition and enantioselective adsorption. The present work is a new step to develop metal-NHCs as surface platforms for the preparation of multifunctional thin films for sensing applications.

Purine Permease-Type Benzylisoquinoline Alkaloid Transporters in Opium Poppy.

Although opiate biosynthesis has been largely elucidated, and cell-to-cell transport has been long postulated, benzylisoquinoline alkaloid (BIA) transporters from opium poppy (Papaver somniferum) have not been reported. Investigation of a purine permease-type sequence within a recently discovered opiate biosynthetic gene cluster led to the discovery of a family of nine homologs designated as BIA uptake permeases (BUPs). Initial expression studies in engineered yeast hosting segments of the opiate pathway showed that six of the nine BUP homologs facilitated dramatic increases in alkaloid yields. Closer examination revealed the ability to uptake a variety of BIAs and certain pathway precursors (e.g. dopamine), with each BUP displaying a unique substrate acceptance profile. Improvements in uptake for yeast expressing specific BUPs versus those devoid of the heterologous transporters were high for early intermediates (300- and 25-fold for dopamine and norcoclaurine, respectively), central pathway metabolites [10-fold for (S)-reticuline], and end products (30-fold for codeine). A coculture of three yeast strains, each harboring a different consecutive segment of the opiate pathway and BUP1, was able to convert exogenous Levodopa to 3 ± 4 mg/L codeine via a 14-step bioconversion process involving over a dozen enzymes. BUP1 is highly expressed in opium poppy latex and is localized to the plasma membrane. The discovery of the BUP transporter family expands the role of purine permease-type transporters in specialized metabolism, and provides key insight into the cellular mechanisms involved in opiate alkaloid biosynthesis in opium poppy.

Liquid-Phase Epitaxial Growth of Azapyrene-Based Chiral Metal-Organic Framework Thin Films for Circularly Polarized Luminescence.

Development of chiral metal-organic frameworks (MOFs) for circularly polarized luminescence (CPL) is a challenging but important task. In this work, we report a first example of azapyrene-based chiral MOF thin films [Zn2Cam2DAP]n grown on functionalized substrates (named SURchirMOF-4) for CPL property. By using a liquid-phase epitaxial layer-by-layer method, the resulted SURchirMOF-4 was constructed from chiral camphoric acid and 2,7-diazapyrene ligand, which has high orientation and homogeneity. The circular dichroism, CPL, and enantioselective adsorption results show that SURchirMOF-4 has strong chirality and CPL property as well as good enantioselective adsorption toward enantiomers of methyl-lactate. The synthesis of azapyrene-based chiral MOF thin films not only represents an ideal model for studying the enantioselective adsorption, but also will be a valuable approach for development of the chiral thin film exhibiting CPL property.

Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy.

Codeinone reductase (COR) catalyzes the reversible NADPH-dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy (Papaver somniferum). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3-O-demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O-demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast (Saccharomyces cerevisiae) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR-B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1-1.4 and COR-B) were used to determine sequence-function relationships that influence product selectivity. Biochemical characterization and site-directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms.

A pathogenesis-related 10 protein catalyzes the final step in thebaine biosynthesis.

The ultimate step in the formation of thebaine, a pentacyclic opiate alkaloid readily converted to the narcotic analgesics codeine and morphine in the opium poppy, has long been presumed to be a spontaneous reaction. We have detected and purified a novel enzyme from opium poppy latex that is capable of the efficient formation of thebaine from (7S)-salutaridinol 7-O-acetate at the expense of labile hydroxylated byproducts, which are preferentially produced by spontaneous allylic elimination. Remarkably, thebaine synthase (THS), a member of the pathogenesis-related 10 protein (PR10) superfamily, is encoded within a novel gene cluster in the opium poppy genome that also includes genes encoding the four biosynthetic enzymes immediately upstream. THS is a missing component that is crucial to the development of fermentation-based opiate production and dramatically improves thebaine yield in engineered yeast.

Cardiolipin synthase A colocalizes with cardiolipin and osmosensing transporter ProP at the poles of Escherichia coli cells.

Osmosensing by transporter ProP is modulated by its cardiolipin (CL)-dependent concentration at the poles of Escherichia coli cells. Other contributors to this phenomenon were sought with the BACterial Two-Hybrid System (BACTH). The BACTH-tagged variants T18-ProP and T25-ProP retained ProP function and localization. Their interaction confirmed the ProP homo-dimerization previously established by protein crosslinking. YdhP, YjbJ and ClsA were prominent among the putative ProP interactors identified by the BACTH system. The functions of YdhP and YjbJ are unknown, although YjbJ is an abundant, osmotically induced, soluble protein. ClsA (CL Synthase A) had been shown to determine ProP localization by mediating CL synthesis. Unlike a deletion of clsA, deletion of ydhP or yjbJ had no effect on ProP localization or function. All three proteins were concentrated at the cell poles, but only ClsA localization was CL-dependent. ClsA was shown to be N-terminally processed and membrane-anchored, with dual, cytoplasmic, catalytic domains. Active site amino acid replacements (H224A plus H404A) inactivated ClsA and compromised ProP localization. YdhP and YjbJ may be ClsA effectors, and interactions of YdhP, YjbJ and ClsA with ProP may reflect their colocalization at the cell poles. Targeted CL synthesis may contribute to the polar localization of CL, ClsA and ProP.

Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae.

Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.

Transcriptome analysis of 20 taxonomically related benzylisoquinoline alkaloid-producing plants.

BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.

Isolation and Characterization of O-methyltransferases Involved in the Biosynthesis of Glaucine in Glaucium flavum.

Transcriptome resources for the medicinal plant Glaucium flavum were searched for orthologs showing identity with characterized O-methyltransferases (OMTs) involved in benzylisoquinoline alkaloid biosynthesis. Seven recombinant proteins were functionally tested using the signature alkaloid substrates for six OMTs: norlaudanosoline 6-OMT, 6-O-methyllaudanosoline 4'-OMT, reticuline 7-OMT, norreticuline 7-OMT, scoulerine 9-OMT, and tetrahydrocolumbamine OMT. A notable alkaloid in yellow horned poppy (G. flavum [GFL]) is the aporphine alkaloid glaucine, which displays C8-C6' coupling and four O-methyl groups at C6, C7, C3', and C4' as numbered on the 1-benzylisoquinoline scaffold. Three recombinant enzymes accepted 1-benzylisoquinolines with differential substrate and regiospecificity. GFLOMT2 displayed the highest amino acid sequence identity with norlaudanosoline 6-OMT, showed a preference for the 6-O-methylation of norlaudanosoline, and O-methylated the 3' and 4' hydroxyl groups of certain alkaloids. GFLOMT1 showed the highest sequence identity with 6-O-methyllaudanosoline 4'OMT and catalyzed the 6-O-methylation of norlaudanosoline, but more efficiently 4'-O-methylated the GFLOMT2 reaction product 6-O-methylnorlaudanosoline and its N-methylated derivative 6-O-methyllaudanosoline. GFLOMT1 also effectively 3'-O-methylated both reticuline and norreticuline. GFLOMT6 was most similar to scoulerine 9-OMT and efficiently catalyzed both 3'- and 7'-O-methylations of several 1-benzylisoquinolines, with a preference for N-methylated substrates. All active enzymes accepted scoulerine and tetrahydrocolumbamine. Exogenous norlaudanosoline was converted to tetra-O-methylated laudanosine using combinations of Escherichia coli producing (1) GFLOMT1, (2) either GFLOMT2 or GFLOMT6, and (3) coclaurine N-methyltransferase from Coptis japonica. Expression profiles of GFLOMT1, GFLOMT2, and GFLOMT6 in different plant organs were in agreement with the O-methylation patterns of alkaloids in G. flavum determined by high-resolution, Fourier-transform mass spectrometry.

Comparing system-specific chaperone interactions with their Tat dependent redox enzyme substrates.

Redox enzyme substrates of the twin-arginine translocation (Tat) system contain a RR-motif in their leader peptide and require the assistance of chaperones, redox enzyme maturation proteins (REMPs). Here various regions of the RR-containing oxidoreductase subunit (leader peptide, full preprotein with and without a leader cleavage site, mature protein) were assayed for interaction with their REMPs. All REMPs bound their preprotein substrates independent of the cleavage site. Some showed binding to either the leader or mature region, whereas in one case only the preprotein bound its REMP. The absence of Tat also influenced the amount of chaperone-substrate interaction.

DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis.

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein-protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a "node" in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

The GacS-GacA two-component regulatory system of Pseudomonas fluorescens: a bacterial two-hybrid analysis.

The two-component regulatory system comprised of the sensor kinase, GacS, and its response regulator, GacA, is involved in regulation of secondary metabolism and many other aspects of bacterial physiology. Although it is known that the sensor kinases RetS and LadS feed into the GacS/GacA system, the mechanism through which this occurs is unknown, as are the protein-protein interactions in this system. To characterize and define these interactions, we utilized a bacterial two-hybrid system to study the interactions of GacS and GacA from Pseudomonas fluorescens CHA0. Domains of GacA and GacS, identified through bioinformatics, were subcloned and their ability to interact in vivo was investigated. We found that the entire GacA molecule is required for GacA to interact with itself or GacS. Furthermore, the HisKA/HATPase/REC domains of GacS together are responsible for GacS interacting with GacA, while the HAMP domain of GacS is responsible for GacS interacting with itself. In addition, homologs of Pseudomonas aeruginosa hybrid sensor kinases, RetS and LadS, were identified in P. fluorescens, and shown to interact with GacS, but not GacA.