Intermolecular Disulfide Bond Formation Research Articles

Deciphering the Cofilin Oligomers via Intermolecular Disulfide Bond Formation: A Coarse-Grained Molecular Dynamics Approach to Understanding Cofilin's Regulation on Actin Filaments.

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.

ALMS1-IT1: A Key Player in the Novel Disulfidptosis-Related LncRNA Prognostic Signature for Head and Neck Squamous Cell Carcinoma.

Disulfidptosis is a newly discovered form of programmed cell death that is induced by disulfide stress. It is closely associated with various cancers, including head and neck squamous cell carcinoma (HNSCC). However, the factors involved in the modulation of disulfidptosis-related genes (DRGs) still remain unknown. In this study, we established and validated a novel risk score model composed of 11 disulfidptosis-related lncRNAs (DRLs) based on 24 DRGs in HNSCC. The results revealed strong correlations between the 11-DRL prognostic signature and clinicopathological features, immune cell infiltration, immune-related functions, and disulfidptosis-associated pathways, including NADPH and disulfide oxidoreductase activities. Furthermore, we studied and verified the involvement of ALMS1-IT1, one of the 11 model DRLs, in the disulfidptosis of HNSCC cell lines. A series of assays demonstrated that ALMS1-IT1 modulated cell death under starvation conditions in a pentose phosphate pathway (PPP)-dependent manner. Knockdown of ALMS1-IT1 inhibited the PPP, contributing to a decline in NADPH levels, which resulted in the formation of multiple intermolecular disulfide bonds between actin cytoskeleton proteins and the collapse of F-actin in the cytoplasm. Therefore, ALMS1-IT1, which is highly expressed in SLC7A11high cells, can be considered a promising therapeutic target for disulfidptosis-focused treatment strategies for cancer and other diseases.

Self-Assembly of Platelet Lysates Proteins into Microparticles by Unnatural Disulfide Bonds for Bottom-up Tissue Engineering.

There is a demand to design microparticles holding surface topographies while presenting inherent bioactive cues for applications in the biomedical and biotechnological fields. Using the pool of proteins present in human-derived platelet lysates (PL), it is reported the production of protein-based microparticles via a simple and cost-effective method, exploring the prone redox behavior of cysteine (-SH) amino acid residues. The forced formation of new intermolecular disulfide bonds results in the precipitation of the proteins as spherical, pompon-like microparticles with adjustable sizes (15-50μm in diameter) and surface topography consisting of grooves and ridges. These PL microparticles exhibit extraordinary cytocompatibility, allowing cell-guided micro-aggregates to form, while also working as injectable systems for cell support. Early studies also suggests that the surface topography provided by these PL microparticles can support osteogenic behavior. Consequently, these PL microparticles may find use to create live tissues via bottom-up procedures or injectable tissue-defect fillers, particularly for bone regeneration, with the prospect of working under xeno-free conditions. This article is protected by copyright. All rights reserved.

Study on potential antigenicity and functional properties of whey protein treated by high hydrostatic pressure based on structural analysis

High hydrostatic pressure (HHP) is extensively utilized in the field of food processing due to its remarkable ability to preserve the freshness of food. The potential antigenicity of β-lactoglobulin (β-LG) in whey protein isolate (WPI, 3%) treated by HHP was detected by enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. Furthermore, the impact of pressure-induced structural alterations on the emulsification properties and antioxidant activity of WPI was investigated. The findings revealed that pressures exceeding 300 MPa resulted in molecular aggregation, the formation of inter-molecular disulfide bonds, and an increase in surface hydrophobicity (H0). The percentage of β-sheet decreased along with the pressure. The results showed the increment of α-helix and β-turn with pressure. ELISA demonstrated a significant reduction in the antigenicity of β-LG following HHP treatment (100–600 MPa), with a slight recovery observed at 300 MPa. These spatial structural modifications led to the unfolding of the β-LG molecule, thereby enhancing its digestibility. Moreover, HHP treatment substantially improved the antioxidant properties, with the exposure to hydrophobic amino acids contributing to increased antioxidant properties and emulsion stability.

Novel regulatory role of post-translational structural modification in modulating Src kinase activity in pulmonary arterial hypertension

Under conditions that lead to pulmonary arterial hypertension (PAH), the pro-proliferative oncoprotein Src kinase activity is elevated, contributing to pulmonary arterial smooth muscle cell remodeling, increasing vascular resistance, and leading to PAH. In addition, reactive oxygen species (ROS), including Hydrogen peroxide (H2O2), are elevated in the pulmonary vasculature under pro-PAH conditions. Therefore, we hypothesized that Src kinase is subject to redox modulation by elevated hydrogen peroxide (H2O2) under pro-PAH conditions. We intended to explore whether increased H2O2 can cause the oxidation of Src kinase thiol groups resulting in an inter-molecular disulfide bond forming Src kinase homodimer, increasing its catalytic activity. The results of this study detected evidence that Src kinase undergoes redox-induced homodimer formation as illustrated by western blot analysis under non-reducing conditions of rat MCT-induced PAH lung lysates. Furthermore, the redox-mediated Src kinase homodimer formation was associated with the increased catalytic activity of Src kinase, as seen in its intra-molecular auto-phosphorylation of its tyrosine 416 residue. This process was accompanied by activating one of Src kinase downstream substrates, STAT3, via the phosphorylation of its tyrosine 705 residue. Moreover, these mechanistic changes were associated with a PAH disease state confirmed with elevated right ventricle systolic pressure and right ventricle hypertrophy through right ventricle catheterization and the Fulton index, respectively. From the results above, we can conclude that in rat MCT-PAH lung lysates, Src kinase undergoes a redox-induced modulation that results in a structural modification associated with increased Src kinase activity, as observed in its autophosphorylation and the phosphorylation of its downstream substrate STAT3. In conclusion, under conditions that lead to PAH, Src kinase undergoes a post-translational structural modification. This modification occurs in redox-induced oxidation of Src kinase thiol groups. This process results in an inter-molecular disulfide bond formation yielding a homodimer associated with elevated Src kinase catalytic activity, further adding a mechanistic layer to Src kinase regulation in PAH. Kuwait University Research Sector This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation

BackgroundA number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods.ResultsTo avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences.ConclusionIn this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Fam96b recruits brain-type creatine kinase to fuel mitotic spindle formation

Mitosis is a complicated and ordered process with high energy demands and metabolite fluxes. Cytosolic creatine kinase (CK), an enzyme involved in ATP homeostasis, has been shown to be essential to chromosome movement during mitotic anaphase in sea urchin. However, it remains elusive for the molecular mechanism underlying the recruitment of cytosolic CK by the mitotic apparatus. In this study, Fam96b/MIP18, a component of the MMXD complex with a function in Fe/S cluster supply, was identified as a brain-type CK (CKB)-binding protein. The binding of Fam96b with CKB was independent of the presence of CKB substrates and did not interfere with CKB activity. Fam96b was prone to oligomerize via the formation of intermolecular disulfide bonds, while the binding of enzymatically active CKB could modulate Fam96b oligomerization. Oligomerized Fam96b recruited CKB and the MMXD complex to associate with the mitotic spindle. Depletion of Fam96b or CKB by siRNA in the HeLa cells led to mitotic defects, which further resulted in retarded cell proliferation, increased cell death and aberrant cell cycle progression. Rescue experiments indicated that both Fam96b oligomerization and CKB activity were essential to the proper formation of mitotic spindle. These findings suggest that Fam96b may act as a scaffold protein to coordinate the supply and homeostasis of ATP and Fe/S clusters during mitosis.

Effects of ultrasonic–microwave combination treatment on the physicochemical, structure and gel properties of myofibrillar protein in Penaeus vannamei (Litopenaeus vannamei) surimi

The objective of this study was to evaluate the effects of single ultrasound (360 W, 20 min), single microwave (10 W/g, 120 s) and ultrasonic–microwave combination treatment on shrimp surimi gel properties. The structure and physicochemical properties of myofibrillar protein (MP) were also determined. Low-field nuclear magnetic resonance showed that the fluidity of water molecules and the moisture content decreased, the stability and water holding capacity (WHC) increased after single ultrasound, single microwave and ultrasonic–microwave combination treatment. Compared with the traditional water bath treatment, ultrasound and microwave treatment reduced the total sulfhydryl content and promoted the formation of intermolecular disulfide bonds and hydrophobic interactions, which improved the compactness of the network structure of shrimp surimi gel. Moreover, Fourier transform infrared spectroscopy and sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis revealed that these treatments not only inhibited the degradation of MP, but also decreased the α-helix content and increased the β-sheet content. The three treatments also significantly reduced the particle size and decreased the solubility of MP. Overall, the effect of ultrasonic–microwave combination treatment was superior to that of either single treatment.

Nanoalbumin-prodrug conjugates prepared via a thiolation-and-conjugation method improve cancer chemotherapy and immune checkpoint blockade therapy by promoting CD8+ T-cell infiltration.

Protein-drug conjugates are emerging tools to combat cancers. Here, we adopted an indirect thiolation-and-conjugation method as a general strategy to prepare protein-drug conjugates. We found for the first time that this method led to the formation of nanometric conjugates, probably due to the formation of intermolecular disulfide bonds, which facilitated enhanced uptake by cancer cells. As a proof-of-concept application in cancer therapy, a nanometric albumin-doxorubicin prodrug conjugate (NanoAlb-proDOX) was prepared. The nanometric size promoted its uptake by cancer cells, and the prodrug characteristic defined its selective cytotoxicity toward cancer cells in vitro and reduced side effects in vivo. In multiple tumor xenograft models, nanometric NanoAlb-proDOX showed superior antitumor activity and synergy with immune checkpoint blockade, probably due to the synergistically enhanced tumor CD8+ T-cell infiltration and activation. Hence, the thiolation-and-conjugation strategy may serve as a generally applicable method for preparing drug conjugates, and the proof-of-concept nanometric albumin-doxorubicin conjugate may be a good choice for antitumor therapy with the ability to co-stimulate the efficacy of immune checkpoint blockade.

Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange

Abstractvon Willebrand factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excessive bleeding of type 2A von Willebrand disease (VWD). Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report electron cryomicroscopy (cryo-EM) structures of VWF tubules before and after intermolecular disulfide bond formation. The structures provide evidence that VWF tubulates through a charge–neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states before and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VWD-causing mutations.

Oxidation of catalytic cysteine of human deubiquitinase BAP1 triggers misfolding and aggregation in addition to functional loss

Deubiquitinating enzymes (DUBs) form a large protease family involved in a myriad of biological and pathological processes, including ROS sensors. ROS-mediated inhibition of their DUB activities is critical for fine-tuning the stress-activated signaling pathways. Here, we demonstrate that the ubiquitin C-terminal hydrolase (UCH) domain of BAP1 (BAP1-UCH) is highly sensitive to moderate oxidative stress. Oxidation of the catalytic C91 significantly destabilizes BAP1-UCH and increases the population of partially unfolded form, which is prone to aggregation. Unlike other DUBs, the oxidation-induced structural and functional loss of BAP1-UCH cannot be fully reversed by reducing agents. The oligomerization of oxidized BAP1-UCH is attributed to inter-molecular disulfide bond formation. Hydrogen-deuterium mass exchange spectrometry (HDX-MS) reveals increased fluctuations of the central β-sheet upon oxidation. Our findings suggest that oxidation-mediated functional loss and increased aggregation propensity may contribute to oncogenesis associated with BAP1.

Cysteine-based protein folding modulators for trapping intermediates and misfolded forms.

Folding is a key process to form functional conformations of proteins. Folding via on-pathway intermediates leads to the formation of native structures, while folding through off-pathways affords non-native and disease-causing forms. Trapping folding intermediates and misfolded forms is important for investigating folding mechanisms and disease-related biological properties of the misfolded proteins. We developed cysteine-containing dipeptides conjugated with amino acids possessing mono- and diamino-groups. In oxidative protein folding involving disulfide-bond formation, the addition of cysteine and oxidized glutathione readily promoted the folding to afford native forms. In contrast, despite the acceleration of disulfide-bond formation, non-native isomers formed in significantly increased yields upon the addition of the dipeptides. This study provides a molecular design of cysteine-based protein-folding modulators that afford proteins adopting non-native conformations through intermolecular disulfide-bond formation. Because of the intrinsic reversibility of the disulfide bonds upon redox reactions, the disulfide bond-based approach demonstrated here is expected to lead to the development of reversible methodologies for trapping transient and misfolded forms by intermolecular disulfide bond formation and restarting the folding processes of the trapped forms by subsequent cleavage of the intermolecular disulfide bonds.

The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics.

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

Engineering a stable complex of ERp44 with a designed peptide ligand for analyzing the mode of interaction of ERp44 with its clients

AbstractERp44, a chaperone of the protein disulfide isomerase (PDI) family cycles between the endoplasmic reticulum (ER) and cis‐Golgi compartments to act on a cohort of disparate proteins either to ensure their proper cellular localization or for the quality control of the correct assembly. This process involves intermolecular disulfide bond formation between the client protein and the conserved Cys29 in ERp44. We had identified a 9‐amino acid peptide derived from an ERp44 client, adiponectin, as the motif interacting with ERp44 via a highly conserved cysteine (Cys39 of adiponectin). However, in order to reveal detailed insight into the mode of interaction, the generation of sizeable amounts of corresponding thiol‐liganded ERp44 was unsuccessful under ambient conditions. Here we describe the production of a bromopeptide variant of this peptide ligand that led to large amounts of a pure, complex of peptide sufficient for structural studies in which Cys29 of Erp44 is covalently linked via a stable thioether bond. This method can be potentially used to rapidly probe putative, short, interacting peptide regions in other ERp44 clients to reveal their, hitherto unknown, mechanism of interaction with ERp44.

The controlled release of a novel thiolated icariin for enhanced osteoporotic bone regeneration

The regeneration of bone defects in osteoporotic patients presents a big challenge. Icariin (Ica), which is the main active component of traditional Chinese medicine - herba epimedii, has been widely loaded to scaffolds for the treatment of bone fracture and osteoporosis. To increase drug loading capacity, and meanwhile achieve a stable and safe drug delivery, in present study, a novel icariin derivative - thiolated icariin (Ica-SH) molecule with good cytocompatibility and bioactivity was incorporated into biphasic calcium phosphate (BCP) ceramic scaffold through Ica-SH intermolecular disulfide bonds (abbr., Ica-SH/BCP) to form film coating. Compared with Ica-loaded BCP scaffolds via physical adsorption (abbr., Ica/BCP), the formation of Ica-SH intermolecular disulfide bonds in Ica-SH/BCP scaffolds led to a decreased real-time drug release concentration, and a reduced drug release rate. The released drug from Ica-SH/BCP scaffolds could promote the proliferation, migration, and osteoblastic differentiation of ovariectomized rat bone mesenchymal stem cells (OVX-rBMSCs), and up-regulate the angiogenic gene expression in human umbilical vein endothelial cells (HUVECs) in vitro. The OVX-rat femoral condyle defect model also demonstrated that Ica-SH/BCP scaffolds promoted a superior bone regeneration in osteoporotic animals than BCP ones. These findings suggest that Ica-SH is a promising drug, which can be facilely incorporated into a variety of scaffolds for repairing bone defects of patients, in particular the ones with osteoporosis.

A conserved cysteine-based redox mechanism sustains TFEB/HLH-30 activity under persistent stress.

Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress, and pathogen infection. In this study, we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility in worms. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.

Structural basis of peroxidase catalytic cycle of human Prdx6

Peroxiredoxin 6 (Prdx6) is a ubiquitously expressed antioxidant non-selenium glutathione peroxidase that is known to play a major role in various physiological and pathological processes. It belongs to the family of peroxidases (referred to as Peroxiredoxins, Prdx’s) that work independently of any prosthetic groups or co-factors, and instead utilize a peroxidatic thiol residue for peroxide reduction. Mammalian Prdx’s are classified according to the number of Cys implicated in their catalytic activity by the formation of either inter-molecular (typical 2-Cys, Prdx1–4) or intra-molecular (atypical 2-Cys, Prdx5) disulfide bond, or non-covalent interactions (1-Cys, Prdx6). The typical and atypical 2-Prdx’s have been identified to show decamer/dimer and monomer/dimer transition, respectively, upon oxidation of their peroxidatic cysteine. However, the alterations in the oligomeric status of Prdx6 as a function of peroxidatic thiol’s redox state are still ambiguous. While the crystal structure of recombinant human Prdx6 is resolved as a dimer, the solution structures are reported to have both monomers and dimers. In the present study, we have employed several spectroscopic and electrophoretic probes to discern the impact of change in the redox status of peroxidatic cysteine on conformation and oligomeric status of Prdx6. Our study indicates Prdx6′s peroxidase activity to be a redox-based conformation driven process which essentially involves monomer–dimer transition.

Generation of Aggregates of α-Lactalbumin by UV-B Light Exposure.

Whey proteins are widely used as ingredients in the form of aggregates to obtain certain functionalities in food applications. The aim of this study was to understand how UV illumination generates aggregates of α-lactalbumin (α-LA) as an alternative to heat treatments traditionally used for industrial production of protein aggregates. Absorption of UV light by α-LA caused cleavage of disulfide bonds and release of thiol groups, which resulted in primarily disulfide-mediated aggregation. This process mediated efficient aggregation with up to 98% monomer conversion into aggregates through formation of intermolecular disulfide bonds, while only minor levels of nonreducible cross-links were observed. SDS-PAGE analysis revealed that illumination led to formation of dimeric, trimeric, and oligomeric forms of α-LA. LC-MS/MS analysis showed that all of the four native disulfide bonds in α-LA were cleaved by UV illumination but to different extents, and the extent of cleavage was found to be higher in the absence of calcium. Seventeen different non-native disulfides were formed after 24 h of UV illumination. Two dityrosine bonds were identified (Tyr103-Tyr103 and Tyr36-Tyr103) alongside ditryptophan (Trp118-Trp118) and tyrosine-tryptophan (Tyr50-Trp60) cross-links. In addition, Trp60, Trp118, Cys73, Cys91, Cys120, Phe80, Met90, His68, and His107 were found to be oxidized up to 12% as compared to a nonilluminated control. Our work illustrates that light exposure can be used for generation of α-LA aggregates, but optimization of the illumination conditions is required to reduce oxidative damage to Trp, Cys, Phe, Met, and His residues.

Self-assembly of a robust, reduction-sensitive camptothecin nanotube.

The self-assembly and covalent crosslinking of a camptothecin (CPT) tetrapeptide nanotube is reported. Intermolecular disulfide bond formation of a self-assembled CPT-peptide reversibly stabilized the nanotubes toward dissociation at low concentrations, resulting in inhibited release of CPT. In the presence of dithiothreitol (DTT), the release of CPT was significantly accelerated. The crosslinked nanotubes also exhibited in vitro cytotoxicity against human non-small cell lung cancer cell lines A549 and H460.

Conjugative Transfer of IncP-9 Catabolic Plasmids Requires a Previously Uncharacterized Gene, mpfK, Whose Homologs Are Conserved in Various MPFT-Type Plasmids.

Conjugative transfer of bacterial plasmids to recipient cells is often mediated by type IV secretion machinery. Experimental investigations into the minimal gene sets required for efficient conjugative transfer suggest that such gene sets are variable, depending on plasmids. We have been analyzing the conjugative transfer of Pseudomonas-derived and IncP-9 plasmids, NAH7 and pWW0, whose conjugation systems belong to the MPFT type. Our deletion analysis and synthetic biology analysis in this study showed that these plasmids require previously uncharacterized genes, mpfK (formerly orf34) and its functional homolog, kikA, respectively, for their efficient conjugative transfer. MpfK was localized in periplasm and had four cysteine residues whose intramolecular or intermolecular disulfide bond formation was suggested to be important for efficient conjugative transfer. The mpfK homologs were specifically carried by many MPFT-type plasmids, including non-IncP-9 plasmids, such as R388 and R751. Intriguingly, the mpfK homologs from the two non-IncP-9 plasmids were not required for conjugation of their plasmids, but were able to complement efficiently the transfer defect of the NAH7 mpfK mutant. Our results suggested the importance of the mpfK homologs for conjugative transfer of MPFT-type plasmids.IMPORTANCE IncP-9 plasmids are important mobile genetic elements for the degradation of various aromatic hydrocarbons. Elucidation of conjugative transfer of such plasmids is expected to greatly contribute to our understanding of its role in the bioremediation of polluted environments. The present study mainly focused on the conjugation system of NAH7, a well-studied and naphthalene-catabolic IncP-9 plasmid. Our analysis showed that the NAH7 conjugation system uniquely requires, in addition to the conserved components of the type IV secretion system (T4SS), a previously uncharacterized periplasmic protein, MpfK, for successful conjugation. Our findings collectively revealed a unique type of T4SS-associated conjugation system in the IncP-9 plasmids.