Formaldehyde Cross-linking Research Articles

Caseinate based biodegradable films with improved water resistance

AbstractProteins are considered as an interesting alternative to traditional synthetic polymers in packaging applications. Sodium caseinate based films can be used in such area, but some of their properties need to be improved. The objective of this work was to improve water resistance of caseinate based films plasticized with triethanolamine (TEA). First, plasticized films were crosslinked by formaldehyde (HCHO) or by electron beam irradiation. The crosslinking efficiency was correlated to the decrease of protein solubility in water determined from a 280 nm absorbance method. The comparison between the two crosslinking methods showed that formaldehyde crosslinking was significantly more efficient than irradiation. The HCHO crosslinking technique was selected for the following of the study. Nevertheless, even for highly crosslinked samples, the plasticizer exuded out of the film. A second part of this work was focused on the effect of surface modification on plasticizer exudation in TEA plasticized caseinate films. Considering that silicone grease coating onto the film surface was able to control TEA exudation, surface modifying additives (SMA) based on NaCAS and organo‐silicones were used to modify films surface properties. Surface wettability and energy were determined from contact angle measurements. TEA exudation ratios in water were also monitored for films containing SMA. SMA were less efficient in controlling TEA exudation rates but could significantly reduce surface energy to 42 mJ m2. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010

Direct Interaction between Quorum-sensing Regulator SmcR and RNA Polymerase Is Mediated by Integration Host Factor to Activate vvpE Encoding Elastase in Vibrio vulnificus

It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.

Analysis of HmsH and its role in plague biofilm formation

The Yersinia pestis Hms(+) phenotype is a manifestation of biofilm formation that causes adsorption of Congo red and haemin at 26 degrees C but not at 37 degrees C. This phenotype is required for blockage of the proventricular valve of the oriental rat flea and plays a role in transmission of bubonic plague from fleas to mammals. Genes responsible for this phenotype are located in three separate operons, hmsHFRS, hmsT and hmsP. HmsH and HmsF are outer membrane (OM) proteins, while the other four Hms proteins are located in the inner membrane. According to the Hidden Markov Method-based predictor, HmsH has a large N terminus in the periplasm, a beta-barrel structure with 16 beta-strands that traverse the OM, eight surface-exposed loops, and seven short turns connecting the beta-strands on the periplasmic side. Here, we demonstrate that HmsH is a heat-modifiable protein, a characteristic of other beta-barrel proteins, thereby supporting the bioinformatics analysis. Alanine scanning mutagenesis was used to identify conserved amino acids in the HmsH-like family that are critical for the function of HmsH in biofilm formation. Of 23 conserved amino acids mutated, four residues affected HmsH function and three likely caused protein instability. We used formaldehyde cross-linking to demonstrate that HmsH interacts with HmsF but not with HmsR, HmsS, HmsT or HmsP. Loss-of-function HmsH variants with single alanine substitutions retained their beta-structure and interaction with HmsF. Finally, using a polar hmsH : : mini-kan mutant, we demonstrated that biofilm development is not important for the pathogenesis of bubonic or pneumonic plague in mice.

Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrinβ1

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively.

1P209 ホルムアルデヒド架橋によるナトリウムイオン駆動型べん毛モーターのCリング・固定子タンパク質相互作用解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

1P209 ホルムアルデヒド架橋によるナトリウムイオン駆動型べん毛モーターのCリング・固定子タンパク質相互作用解析(細胞生物的課題(接着,運動,骨格,伝達,膜),第48回日本生物物理学会年会)

Ultraviolet Spectrophotometric Method for Determination of Gelatin Crosslinking in the Presence of Amino Groups

The study was carried out to develop procedure for determining concentration of formaldehyde to be used for crosslinking of gelatin in the presence of drugs having amino groups. Gentamicin sulfate was used as a drug candidate due to its high content of amino acids. Gelatin crosslinking is accelerated by aldehyde-containing compounds and inhibited by amino group-containing compounds. The major modifications from already existing procedures are that the trinitrobenzenesulphonic acid (TNBS) reaction is used to detect e-amino groups of Type A gelatin in the presence of formaldehyde and further it is supported with colorimetric analysis of free formaldehyde content using a chromotropic acid regent. Since formaldehyde crosslinks amino groups, the TNBS assay can be effectively utilized for determination of complete crosslinking of gelatin with analysis of free amino acid content in crosslinked formulation. The effect of the presence of amino groups on gelatin crosslinking was estimated in the presence of gentamicin sulfate. The ε-amino content of uncrosslinked Type A gelatin was found to be 28.6 mol/gelatin molecule of 1000 residues and in case of crosslinked gelatin it varies with varying concentration of formaldehyde. The procedure stated here should be applicable to a broad range of drugs containing amino groups which are used along with gelatin or other proteinaceous materials which are applicable after crosslinking with formaldehyde.

Polyols based on isocyanates and melamines and their applications in 1K and 2K coatings

Polyurethane polyols (PUPOs) and melamine polyols (MEPOs), invented by AkzoNobel Coatings, offer valuable alternatives to the commonly used polyester and acrylic polyols and new formulating tools for the development of novel coatings. The resin chemistry is based on the predominantly selective reactivity of α,β- and α,γ-diols with commonly used polyisocyanate and melamine formaldehyde crosslinkers. The resulting low molecular weight, hydroxyl functional resins are suitable for use in low VOC coatings. By choosing the appropriate crosslinker, diol, and modifier, the chemical structures of these resins can be altered to obtain the desired properties of the coatings. Synthesis methods for novel PUPOs and MEPOs and properties of one component and two component coatings containing them are described.

Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C)

Gene regulation in higher eukaryotes frequently involves physical interactions between genomic sequence elements tens of kilobases apart on the same chromosome but can also entail interactions between different chromosomes. Chromosome Conformation Capture (3C) is a powerful tool to identify such interactions. 3C technology is based on formaldehyde crosslinking of chromatin, followed by restriction digestion and intramolecular ligation. Quantitative detection of ligation products by PCR (qPCR; not discussed in this protocol) provides insight into the interaction frequencies between chromosomal fragments and thereby the spatial organization of a genomic region. Detailed 3C protocols have been published for yeast and mammals. However, these protocols cannot simply be transferred to plant tissues. In this paper, we provide a maize-specific 3C protocol and present a general strategy to systematically optimize the protocol for other plants. Once the technique and appropriate controls are established, the 3C procedure (including qPCR) can be completed in 5-7 d.

Cytoplasmic membrane protonmotive force energizes periplasmic interactions between ExbD and TonB

The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

CmtR, a cadmium‐sensing ArsR–SmtB repressor, cooperatively interacts with multiple operator sites to autorepress its transcription in Mycobacterium tuberculosis

CmtR is a repressor of the ArsR-SmtB family from Mycobacterium tuberculosis that has been shown to sense Cd(II) and Pb(II) in Mycobacterium smegmatis. We establish here that CmtR binds cooperatively to multiple sites in M. tuberculosis DNA and protects an unusually long 90 bp AT-rich sequence from -80 bp to +10 with respect to its own initiation codon. CmtR interacts with four hyphenated imperfect inverted repeats matching the consensus sequence TA/GTAA-N(4-5)-TT/GATA in the protected region. SDS-PAGE and formaldehyde crosslinking experiments showed that CmtR forms higher-order oligomers (up to an octamer). The oligomerization of CmtR is in agreement with the cooperative binding of CmtR to multiple sites on DNA. Two promoters transcribe cmtR, and the major promoter physically overlaps with CmtR binding sites. Autorepression of CmtR is mediated by cooperative interaction of CmtR with multiple sites on DNA that occlude the major operon promoter. The combined results of a GFP reporter assay, an electromobility shift assay and a DNase I footprinting experiment establish that Cd(II), not Pb(II), disrupts the interaction of CmtR with DNA to de-repress transcription of the cmtR-Rv1993c-cmtA operon in M. tuberculosis.

Identification and functional characterization of regions that can be crosslinked to RNA in the helicase-like domain of BaMV replicase

The helicase-like domain of the Bamboo mosaic virus replicase catalyzes the release of 5′-γ-phosphate from both ATP and 5′-triphosphated RNA by an identical set of catalytic residues with a presumably larger binding pocket for RNA. In this study, the peptidyl regions involved in RNA binding were mapped by reversible formaldehyde crosslinking and mass spectrometry. Eleven residues within these regions were examined by mutational analysis. H636A, Y704A, and K706A greatly diminished the enzymatic activities and were unable to support the viral replication in Nicotiana benthamiana protoplasts. K843A decreased activity toward the RNA substrate to 17% of WT, and ∼ 20% replication efficiency was retained in protoplasts. R597A and K610A retained ∼ 50 and ∼ 90% of the enzymatic activities, respectively. However, replication in protoplasts of these mutants was extremely limited. Proteins with the mutations K603A, R628A, R645A, H794A, and R799A were present at levels 30–69% of WT in protoplasts. However, the fates of these mutations in plants were different. Viral cell-to-cell movement was limited by the K603A and R628A mutations, while systemic movement was restricted by R645A and H794A. The implications of the helicase-like domain in the viral replication and movement are discussed.

Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states.

Detection of gene loops by 3C in yeast

“Chromosome conformation capture” (3C) is a powerful method to detect physical interaction between any two genomic loci. 3C involves formaldehyde crosslinking to stabilize transient interactions, followed by restriction digestion, ligation and locus-specific PCR. Accordingly, 3C reveals complex three-dimensional interactions between distal genetic elements within intact cells at high resolution. Here, we describe a modified 3C protocol designed for detection of transient chromatin interactions in the yeast Saccharomyces cerevisiae. Using this protocol, we are able to detect juxtaposition of promoter and terminator regions of genes with ORFs as short as 1 kb in length. We anticipate that this method will be generally applicable to detect dynamic, short-range chromatin interactions and will facilitate the characterization of gene loops and their functional consequences.

Estrogen Receptor α Enhances the Rate of Oxidative DNA Damage by Targeting an Equine Estrogen Catechol Metabolite to the Nucleus

Exposure to estrogens increases the risk of breast and endometrial cancer. It is proposed that the estrogen receptor (ER) may contribute to estrogen carcinogenesis by transduction of the hormonal signal and as a "Trojan horse" concentrating genotoxic estrogen metabolites in the nucleus to complex with DNA, enhancing DNA damage. 4-Hydroxyequilenin (4-OHEN), the major catechol metabolite of equine estrogens present in estrogen replacement formulations, autoxidizes to a redox-cycling quinone that has been shown to cause DNA damage. 4-OHEN was found to be an estrogen of nanomolar potency in cell culture using a luciferase reporter assay and, using a chromatin immunoprecipitation assay, was found to activate ERalpha binding to estrogen-responsive genes in MCF-7 cells. DNA damage was measured in cells by comparing ERalpha(+) versus ERalpha(-) cells and 4-OHEN versus menadione, a reactive oxygen species (ROS)-generating, but non-estrogenic, quinone. 4-OHEN selectively induced DNA damage in ERalpha(+) cells, whereas menadione-induced damage was not dependent on cellular ER status. The rate of 4-OHEN-induced DNA damage was significantly enhanced in ERalpha(+) cells, whereas ER status had no effect on the rate of menadione-induced damage. Imaging of ROS induced by 4-OHEN showed accumulation selective for the nucleus of ERalpha(+) cells within 5 min, whereas in ERalpha(-) or menadione-treated cells, no selectivity was observed. These data support ERalpha acting as a Trojan horse concentrating 4-OHEN in the nucleus to accelerate the rate of ROS generation and thereby amplify DNA damage. The Trojan horse mechanism may be of general importance beyond estrogen genotoxins.

The ATPase activity of an ‘essential’ Bacillus subtilis enzyme, YdiB, is required for its cellular function and is modulated by oligomerization

Characterization of 'unknown' proteins is one of the challenges of the post-genomic era. Here, we report a study of Bacillus subtilis YdiB, which belongs to an uncharted class of bacterial P-loop ATPases. Precise deletion of the ydiB gene yielded a mutant with much reduced growth rate compared to the wild-type strain. In vitro, purified YdiB was in equilibrium among different forms, monomers, dimers and oligomers, and this equilibrium was strongly affected by salts; high concentrations of NaCl favoured the monomeric over the oligomeric form of the enzyme. Interestingly, the ATPase activity of the monomer was about three times higher than that of the oligomer, and the monomer showed a K(m) of about 60 microM for ATP and a V(max) of about 10 nmol min(-1) (mg protein)(-1) (k(cat) approximately 10 h(-1)). This low ATPase activity was shown to be specific to YdiB because mutation of an invariant lysine residue in the P-loop motif (K41A) strongly attenuated this rate. This mutant was unable to restore a normal growth phenotype when introduced into a conditional knockout strain for ydiB, showing that the ATPase activity of YdiB is required for the in vivo function of the protein. Oligomerization was also observed with the purified YjeE from Escherichia coli, a YdiB orthologue, suggesting that this property is shared by all members of this family of ATPases. Importantly, dimers of YdiB were also observed in a B. subtilis extract, or when stabilized by formaldehyde cross-linking for YjeE from E. coli, suggesting that oligomerization might regulate the function of this new class of proteins in vivo.

A Temporal Threshold for Formaldehyde Crosslinking and Fixation

BackgroundFormaldehyde crosslinking is in widespread use as a biological fixative for microscopy and molecular biology. An assumption behind its use is that most biologically meaningful interactions are preserved by crosslinking, but the minimum length of time required for an interaction to become fixed has not been determined.MethodologyUsing a unique series of mutations in the DNA binding protein MeCP2, we show that in vivo interactions lasting less than 5 seconds are invisible in the microscope after formaldehyde fixation, though they are obvious in live cells. The stark contrast between live cell and fixed cell images illustrates hitherto unsuspected limitations to the fixation process. We show that chromatin immunoprecipitation, a technique in widespread use that depends on formaldehyde crosslinking, also fails to capture these transient interactions.Conclusions/SignificanceOur findings for the first time establish a minimum temporal limitation to crosslink chemistry that has implications for many fields of research.

Exploring DNA-Binding Proteins with In Vivo Chemical Cross-Linking and Mass Spectrometry

DNA-binding proteins are very important constituents of proteomes of all species and play crucial roles in transcription, DNA replication, recombination, repair, and other activities associated with DNA. Although a number of DNA-binding proteins have been identified, many proteins involved in gene regulation and DNA repair are likely still unknown because of their dynamic and/or weak interactions with DNA. In this report, we described an approach for the comprehensive identification of DNA-binding proteins with in vivo formaldehyde cross-linking and LC-MS/MS. DNA-binding proteins could be purified via the isolation of DNA-protein complexes and released from the complexes by reversing the cross-linking. By using this method, we were able to identify more than one hundred DNA-binding proteins, such as proteins involved in transcription, gene regulation, DNA replication and repair, and a large number of proteins that are potentially associated with DNA and DNA-binding proteins. This method should be generally applicable to the investigation of other nucleic acid-binding proteins, and hold great potential in the comprehensive study of gene regulation, DNA damage response and repair, as well as many other critical biological processes at proteomic level.

Optimal use of tandem biotin and V5 tags in ChIP assays

BackgroundChromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies.ResultsUsing the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking.ConclusionThe combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes.

MTERF2 is a nucleoid component in mammalian mitochondria

The mammalian MTERF family of proteins has four members, named MTERF1 to MTERF4, which were identified in homology searches using the mitochondrial transcription termination factor, mTERF (here denoted MTERF1) as query. MTERF1 and MTERF3 are known to participate in the control of mitochondrial DNA transcription, but the function of the other two proteins is not known. We here investigate the structure and function of MTERF2. Protein import experiments using isolated organelles confirm that MTERF2 is a mitochondrial protein. Edman degradation of MTERF2 isolated from stably transfected HeLa cells demonstrates that mature MTERF2 lacks a targeting peptide (amino acids 1–35) present in the precursor form of the protein. MTERF2 is a monomer in isolation and displays a non sequence-specific DNA-binding activity. In vivo quantification experiments demonstrate that MTERF2 is relatively abundant, with one monomer present per ∼265 bp of mtDNA. In comparison, the mtDNA packaging factor TFAM is present at a ratio of one molecule per ∼10–12 bp of mtDNA. Using formaldehyde cross-linking we demonstrate that MTERF2 is present in nucleoids, and therefore must be located in close proximity to mtDNA. Taken together, our work provides a basic biochemical characterization of MTERF2, paving the way for future functional studies.

Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin‐fixed paraffin‐embedded tissues using western blotting

The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross-link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, full-length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study.