Disulfide Bridge Formation Research Articles

Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system

Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

Native to designed: microbial -amylases for industrial applications.

Background-amylases catalyze the endo-hydrolysis of -1,4-D-glycosidic bonds in starch into smaller moieties. While industrial processes are usually performed at harsh conditions, -amylases from mainly the bacteria, fungi and yeasts are preferred for their stabilities (thermal, pH and oxidative) and specificities (substrate and product). Microbial -amylases can be purified and characterized for industrial applications. While exploring novel enzymes with these properties in the nature is time-costly, the advancements in protein engineering techniques including rational design, directed evolution and others have privileged their modifications to exhibit industrially ideal traits. However, the commentary on the strategies and preferably mutated residues are lacking, hindering the design of new mutants especially for enhanced substrate specificity and oxidative stability. Thus, our review ensures wider accessibility of the previously reported experimental findings to facilitate the future engineering work.Survey methodology and objectivesA traditional review approach was taken to focus on the engineering of microbial -amylases to enhance industrially favoured characteristics. The action mechanisms of - and -amylases were compared to avoid any bias in the research background. This review aimed to discuss the advances in modifying microbial -amylases via protein engineering to achieve longer half-life in high temperature, improved resistance (acidic, alkaline and oxidative) and enhanced specificities (substrate and product). Captivating results were discussed in depth, including the extended half-life at 100C, pH 3.5 and 10, 1.8 M hydrogen peroxide as well as enhanced substrate (65.3%) and product (42.4%) specificities. These shed light to the future microbial -amylase engineering in achieving paramount biochemical traits ameliorations to apt in the industries.ConclusionsMicrobial -amylases can be tailored for specific industrial applications through protein engineering (rational design and directed evolution). While the critical mutation points are dependent on respective enzymes, formation of disulfide bridge between cysteine residues after mutations is crucial for elevated thermostability. Amino acids conversion to basic residues was reported for enhanced acidic resistance while hydrophobic interaction resulted from mutated hydrophobic residues in carbohydrate-binding module or surface-binding sites is pivotal for improved substrate specificity. Substitution of oxidation-prone methionine residues with non-polar residues increases the enzyme oxidative stability. Hence, this review provides conceptual advances for the future microbial -amylases designs to exhibit industrially significant characteristics. However, more attention is needed to enhance substrate specificity and oxidative stability since they are least reported.

A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function1-4. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts5,6. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes7,8. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch9,10.

A starch-based zwitterionic hydrogel coating for blood-contacting devices with durability and bio-functionality

• Dihydrolipoic acid-modified sulfobetaine derived starch hydrogel coatings are fabricated. • Hydrogel coatings can resist cell and tissue adhesion, prevent thrombosis, and alleviate inflammation response in vivo . • Hydrogel coatings exhibit excellent long-term durability and stability. • Hydrogel coatings can selectively promote the adhesion, proliferation, and migration of HUVECs. Zwitterionic polymers coating shows significant advantages in preventing thrombosis for the treatment of cardiovascular diseases. However, their limited bio-functionality and low durability struggle to meet requirements from clinical applications. In this study, a dihydrolipoic acid-modified sulfobetaine-derived starch (SB-ST-D) hydrogel coating is developed for polyethylene terephthalate (PET) based blood-contacting devices. First, polydopamine (PDA) is deposited on the PET surface (PDA/PET). Then, SB-ST-D is covalently immobilized on the surface of the PDA/PET (SSD/PET) via Michael addition reaction and the hydrogel is formed by the disulfide bridge formation. The obtained SB-ST-D hydrogel coatings not only exhibit excellent biocompatibility, but also effectively resist non-specific protein adsorption, inhibit cell and platelet adhesion, prevent thrombosis, and alleviate inflammation response in vivo . Moreover, the SB-ST-D hydrogel coating can maintain good anti-fouling properties after 45 d of immersion in PBS, 7 d of PBS buffer shearing, and even under mechanical damage. To improve the specific adhesion capacity of human umbilical vein endothelial cells (HUVECs), the REDV is introduced on the SB-ST-D coating surface via thiol–ene click reaction. Results suggest that the obtained coating can promote the adhesion, proliferation, and migration of HUVECs, and decrease the adhesion of human aortic smooth muscle cells simultaneously. Therefore, the prepared SB-ST-D hydrogel coatings with combined durable, antithrombotic, and bio-functional properties will be a milestone in the development of long-term blood-contacting devices.

Influence of pH, Temperature and Protease Inhibitors on Kinetics and Mechanism of Thermally Induced Aggregation of Potato Proteins.

Understanding aggregation in food protein systems is essential to control processes ranging from the stabilization of colloidal dispersions to the formation of macroscopic gels. Patatin rich potato protein isolates (PPI) have promising techno-functionality as alternatives to established proteins from egg white or milk. In this work, the influence of pH and temperature on the kinetics of PPI denaturation and aggregation was investigated as an option for targeted functionalization. At a slightly acidic pH, rates of denaturation and aggregation of the globular patatin in PPI were fast. These aggregates were shown to possess a low amount of disulfide bonds and a high amount of exposed hydrophobic amino acids (S0). Gradually increasing the pH slowed down the rate of denaturation and aggregation and alkaline pH levels led to an increased formation of disulfide bonds within these aggregates, whereas S0 was reduced. Aggregation below denaturation temperature (Td) favored aggregation driven by disulfide bridge formation. Aggregation above Td led to fast unfolding, and initial aggregation was less determined by disulfide bridge formation. Inter-molecular disulfide formation occurred during extended heating times. Blocking different protein interactions revealed that the formation of disulfide bond linked aggregation is preceded by the formation of non-covalent bonds. Overall, the results help to control the kinetics, morphology, and interactions of potato protein aggregation for potential applications in food systems.

An Investigation of the Disulfide Bridge Formation of a Thylakoid Protease using Nanosecond-Level MD Simulations

Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoidal processing peptidase. Intramolecular disulfide bonds are required for for in vitro activity, unlike for in vivo activity. Previous experimental studies have revealed key mechanism of disulfide bond formation in both the structure and function of Plsp1. A Cys pair (C166 and C286) form a non-catalytic disulfide bond, inducing a large loop in Plsp1. The disulfide bond aids in maintenance of Plsp1 structure and activity, regulating changes in lumen acidity which is important for reconstitutive folding.

Plasma membrane phylloquinone biosynthesis in nonphotosynthetic parasitic plants.

Nonphotosynthetic holoparasites exploit flexible targeting of phylloquinone biosynthesis to facilitate plasma membrane redox signaling. Phylloquinone is a lipophilic naphthoquinone found predominantly in chloroplasts and best known for its function in photosystem I electron transport and disulfide bridge formation of photosystem II subunits. Phylloquinone has also been detected in plasma membrane (PM) preparations of heterotrophic tissues with potential transmembrane redox function, but the molecular basis for this noncanonical pathway is unknown. Here, we provide evidence of PM phylloquinone biosynthesis in a nonphotosynthetic holoparasite Phelipanche aegyptiaca. A nonphotosynthetic and nonplastidial role for phylloquinone is supported by transcription of phylloquinone biosynthetic genes during seed germination and haustorium development, by PM-localization of alternative terminal enzymes, and by detection of phylloquinone in germinated seeds. Comparative gene network analysis with photosynthetically competent parasites revealed a bias of P. aegyptiaca phylloquinone genes toward coexpression with oxidoreductases involved in PM electron transport. Genes encoding the PM phylloquinone pathway are also present in several photoautotrophic taxa of Asterids, suggesting an ancient origin of multifunctionality. Our findings suggest that nonphotosynthetic holoparasites exploit alternative targeting of phylloquinone for transmembrane redox signaling associated with parasitism.

Regulacja układu zależnego od tioredoksyny jako element farmakoterapii w chorobach z zaburzeniami równowagi redoks

Streszczenie Działanie wielu czynników egzogennych, a także zaburzone procesy metaboliczne komórek przyczyniają się do nasilonego wytwarzania oksydantów, a to zaburza równowagę redoks, wywołując zmiany metaboliczne, w tym śmierci lub transformacji nowotworowej komórek. Jednak każda komórka zawiera antyoksydanty, które mają zapobiegać tego typu sytuacjom. Jednym z układów antyoksydacyjnych, funkcjonujących w komórkach, jest układ zależny od tioredoksyny, w skład którego wchodzą: tioredoksyna (Trx), reduktaza tioredoksyny (TrxR) oraz peroksydaza tioredoksyny (TPx), które mogą redukować utlenione składniki komórek kosztem fosforanu dinukleotydu nikotynoamidoadeninowego (NADPH). Działanie takie wynika z budowy przestrzennej Trx oraz TrxR, która umożliwia wytworzenie wewnątrzcząsteczkowego mostka disulfidowego w obrębie cząsteczki tioredoksyny oraz dwóch międzycząsteczkowych mostków selenosulfidowych w obrębie dimeru reduktazy tioredoksyny. Inną, równie istotną funkcją układu zależnego od tioredoksyny jest regulowanie ekspresji wielu białek za pośrednictwem takich czynników jak czynnik transkrypcyjnego NF-κB oraz kinaza regulująca apoptozę (ASK-1), które uruchamiają kaskady przemian metabolicznych prowadzących ostatecznie do proliferacji lub apoptozy komórek. Wzrost ekspresji/aktywności składników systemu zależnego od Trx obserwuje się w rozwoju wielu nowotworów. Dlatego też poszukiwanie selektywnych inhibitorów tioredoksyny lub reduktazy tioredoksyny jest obecnie jednym z głównych kierunków badań w farmakoterapii nowotworów. Wykazano, że wiele naturalnie występujących związków polifenolowych pochodzenia naturalnego o działaniu antyoksydacyjnym (np. kwercetyna czy kurkumina) powoduje inaktywację układu Trx-TrxR. Jednocześnie wiele syntetycznych związków, w tym związki kompleksowe, które stosowane są w terapii przeciwnowotworowej (np. cisplatyna, auranofina, moteksafina gadolinu), również hamują działanie układu zależnego od Trx.

Generowanie MostkÓW Disiarczkowych W BiaŁKach – RÓŻNorodnoŚĆ Strukturalna I Funkcjonalna BiaŁEk Dsba

Abstract Bacterial proteins of the Dsb (disulfide bond) system catalyze the formation of disulfide bridges, a post-translational modification of extra-cytoplasmic proteins, which leads to stabilization of their tertiary and quaternary structures and often influences their activity. DsbA – Escherichia coli monomeric oxidoreductase is the best studied protein involved in this process. Recent rapid advances in global analysis of bacteria have thrown light on the enormous diversity among bacterial Dsb systems. The set of Dsb proteins involved in the oxidative pathway, varies, depending on the microorganism. In this article we have focused on characterization of structural and phylogenetic groups of monomeric DsbAs. This review discuss their physicochemical features and interactions with redox partners as well as with substrate proteins. The last part of the review concentrates on dimeric oxidoreductases responsible for disulfide generation. Many virulence factors are the substrates of the Dsb proteins. Thus unraveling the machinery that introduces disulfide bonds and expanding knowledge about Dsb protein structures and their activities may facilitate the discovery of an effective anti-bacterial drugs. 1. Introduction. 2. Escherichia coli Dsb system. 2.1. Characteristic of the E. coli thiol oxidoreductase – DsbA. 2.2. Izomerization / reduction pathway proteins. 3. Classification of the monomeric DsbAs. 3.1. Physicochemical features of different classes of DsbAs. 4. DsbA interactions with redox partner and substrates. 4.1. DsbA interactions with redox partner. 4.2. DsbA interactions with substrates. 5. Dimeric Dsb proteins with oxidative activity. 6. Conclusions. 7. References

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Antibody CD133 Biofunctionalization of Ammonium Acryloyldimethyltaurate and Vinylpyrrolidone Co-Polymer-Based Coating of the Vascular Implants.

Current vascular stents, such as drug eluting stents (DES), have some serious drawbacks, like in stent restenosis and thrombosis. Therefore, other solutions are sought to overcome these post-implantations complications. These include the strategy of biofunctionalization of the stent surface with antibodies that facilitate adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs). Rapid re-endothelialization of the surface minimizes the risk of possible complications. In this study, we proposed ammonium acryloyldimethyltaurate/vinylpyrrolidone co-polymer-based surface (AVC), which was mercaptosilanized in order to expose free thiol groups. The presence of free thiol groups allowed for the covalent attachment of CD133 antibodies by disulfide bridges formation between mercaptosilanized surface and cysteine of the protein molecule thiol groups. Various examinations were performed in order to validate the procedure, including attenuated total reflection–Fourier transform infrared spectroscopy (ATR-FTIR) and Fourier transform Raman spectroscopy (FT-Raman), atomic force microscopy (AFM) and scanning electron microscopy (SEM). By means of ATR-FTIR spectroscopy presence of the CD133 antibody within coating was confirmed. In vitro studies proved good biocompatibility for blood cells without induction of hemolytic response. Thus, proposed biofunctionalized CD133 antibody AVC surface has shown sufficient stability for adapting as cardiovascular implant coating and biocompatibility. According to conducted in vitro studies, the modified surface can be further tested for applications in various biological systems.

FGF23 contains two distinct high-affinity binding sites enabling bivalent interactions with α-Klotho

The three members of the endocrine-fibroblast growth factor (FGF) family, FGF19, 21, and 23 are circulating hormones that regulate critical metabolic processes. FGF23 stimulates the assembly of a signaling complex composed of α-Klotho (KLA) and FGF receptor (FGFR) resulting in kinase activation, regulation of phosphate homeostasis, and vitamin D levels. Here we report that the C-terminal tail of FGF23, a region responsible for KLA binding, contains two tandem repeats, repeat 1 (R1) and repeat 2 (R2) that function as two distinct ligands for KLA. FGF23 variants with a single KLA binding site, FGF23-R1, FGF23-R2, or FGF23-wild type (WT) with both R1 and R2, bind to KLA with similar binding affinity and stimulate FGFR1 activation and MAPK response. R2 is flanked by two cysteines that form a disulfide bridge in FGF23-WT; disulfide bridge formation in FGF23-WT is dispensable for KLA binding and for cell signaling via FGFRs. We show that FGF23-WT stimulates dimerization and activation of a chimeric receptor molecule composed of the extracellular domain of KLA fused to the cytoplasmic domain of FGFR and employ total internal reflection fluorescence microscopy to visualize individual KLA molecules on the cell surface. These experiments demonstrate that FGF23-WT can act as a bivalent ligand of KLA in the cell membrane. Finally, an engineered Fc-R2 protein acts as an FGF23 antagonist offering new pharmacological intervention for treating diseases caused by excessive FGF23 abundance or activity.

Stress-dependent conformational changes of artemin: Effects of heat and oxidant.

Artemin is an abundant thermostable protein in Artemia embryos and it is considered as a highly efficient molecular chaperone against extreme environmental stress conditions. The conformational dynamics of artemin have been suggested to play a critical role in its biological functions. In this study, we have investigated the conformational and functional changes of artemin under heat and oxidative stresses to identify the relationship between its structure and function. The tertiary and quaternary structures of artemin were evaluated by fluorescence measurements, protein cross-linking analysis, and dynamic light scattering. Based on the structural analysis, artemin showed irreversible substantial conformational lability in responses to heat and oxidant, which was mainly mediated through the hydrophobic interactions and dimerization of the chaperone. In addition, the chaperone-like activity of heated and oxidized artemin was examined using lysozyme refolding assay and the results showed that although both factors, i.e. heat and oxidant, at specific levels improved artemin potency, simultaneous incubation with both stressors significantly triggered the chaperone activation. Moreover, the heat-induced dimerization of artemin was found to be the most critical factor for its activation. It was suggested that oxidation presumably acts through stabilizing the dimer structures of artemin through formation of disulfide bridges between the subunits and strengthens its chaperoning efficacy. Accordingly, it is proposed that artemin probably exists in a monomer-oligomer equilibrium in Artemia cysts and environmental stresses and intracellular portion of protein substrates may shift the equilibrium towards the active dimer forms of the chaperone.

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II–III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling.

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2–C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.

Generation and testing of engineered multimeric Fabs of trastuzumab

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules.Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells.Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody.Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.

Switching the Switch: Ligand Induced Disulfide Formation in HDAC8.

Human histone deacetylase 8 is a well‐recognized target for T‐cell lymphoma and particularly childhood neuroblastoma. PD‐404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys102 and Cys153. This study on the distinct effects of PD‐404,182 on HDAC8 reveals that this compound induces the dose‐dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD‐404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges.

Activation of cell-penetrating peptide fragments by disulfide formation

Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.

Structural, functional and phylogenetic analysis of a beta defensin gene from the Whipfin silverbiddy, Gerres filamentosus (Cuvier, 1829)

Beta defensins have been considered as a major class of Antimicrobial peptides (AMPs) that play an important role in the innate immunity of fishes. In this study, we have identified and characterized a β-defensin gene sequence from the Whipfin silverbiddy, Gerres filamentosus which was termed as Gf-Def. The Gf-Def open reading frame (ORF) consists of 192 bp that encodes 63 amino acid residues. It includes a signal peptide region of 20 amino acids followed by a mature peptide sequence of 43 amino acids. Gerres filamentosus beta defensin (Gf-Def) has a molecular weight of 5.2 KDa and an isoelectric point of 8.90 with a GRAVY score of −0.872. Also, the characteristic six cysteine residues involved in pattern specific formation of intramolecular disulfide bridges could be deduced from the peptide sequence using Cys_Rec software. Three-dimensional modeling of Gf-Def carried out using DNASTAR protean software revealed β strands stabilized by cysteine sulfide bonds along with a short N-terminal α helix spanning the signal peptide sequence. Four Conserved motifs were identified in Gf-Def, all of which were unexceptionally and extensively shared by other fish defensins suggesting a possible functional similarity and evolutionary selection of the sequence. Phylogenetic analysis revealed that Gf-Def was close to Oplegnathus fasciatus, Siniperca chautsi, Trachinotus ovatus and Lates calcarifer defensins which probably suggests that Gf-Def peptide sequence is quite similar to β-defensin sequences of other Perciformes. This being the first report of a beta defensin from whipfin silverbiddy, G. filamentosus and the comparative analysis shall provide a background data for the functional and evolutionary studies on fish beta defensins.

3D Electron Microscopy Gives a Clue: Maize Zein Bodies Bud From Central Areas of ER Sheets.

Zeins are the main storage proteins in maize seed endosperm, and the onset of zein synthesis in young seeds challenges the endomembrane system and results in the formation of storage organelles. Even though zeins lack a conventional endoplasmic reticulum (ER) retention signal, they accumulate within the ER and assemble in conspicuous ER-derived protein bodies (PBs) stabilized by disulfide bridge formation and hydrophobic interaction between zein chains. Zein body formation during seed development has been extensively studied, as well as the mechanisms that lead to the initiation of PBs. However, the exact course of the PB formation process and the spatial relationship with the ER remain unclear. The development of serial block face scanning electron microscopy (SBF-SEM) techniques that allow three-dimensional imaging combined with the high resolution of electron microscopy provides new perspectives on the study of the plant endomembrane system. Here, we demonstrate that (i) the ER of maize seeds is mainly formed by massive sheets and (ii) PBs are not budding from tubules or the edge of sheets, but protrude from the entire surface of the ER sheet.