Whole-cell Extracts Research Articles

Nonhomologous end joining of complex DNA double-strand breaks with proximal thymine glycol and interplay with base excision repair.

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol at the first (Tg1), second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3' terminus, was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. Ligase reactions containing only purified Ku, XRCC4, ligase IV and XLF showed that ligation of Tg3 and Tg5 was efficient and only partially XLF-dependent, whereas ligation of Tg1 and Tg2 was inefficient and only detectable in the presence of XLF. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. Moreover, it appears that base excision repair of Tg can sometimes interfere with repair of DSBs that would otherwise be readily rejoined.

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.

Purification of RNA-Protein Splicing Complexes Using a Tagged Protein from In Vitro Splicing Reaction Mixture.

In eukaryotes, pre-mRNA splicing is an essential step for gene expression. Splicing reactions have been well investigated by using in vitro splicing reactions with extracts prepared from cultured cells. Here, we describe protocols for the preparation of splicing-competent extracts from cells expressing a tagged spliceosomal protein. The whole-cell extracts are able to splice exogenously added pre-mRNA and the RNA-protein complex formed in the in vitro splicing reaction can be purified by immunoprecipitation using antibodies against the peptide tag on the splicing protein. The method described here to prepare splicing-active extracts from whole cells is particularly useful when studying pre-mRNA splicing in various cell types, and the expression of a tagged spliceosomal protein allows one to purify and analyze the RNA-protein complexes by simple immunoprecipitation.

Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

SLFN11 inhibits checkpoint maintenance and homologous recombination repair.

High expression levels of SLFN11 correlate with the sensitivity of human cancer cells to DNA-damaging agents. However, little is known about the underlying mechanism. Here, we show that SLFN11 interacts directly with RPA1 and is recruited to sites of DNA damage in an RPA1-dependent manner. Furthermore, we establish that SLFN11 inhibits checkpoint maintenance and homologous recombination repair by promoting the destabilization of the RPA-ssDNA complex, thereby sensitizing cancer cell lines expressing high endogenous levels of SLFN11 to DNA-damaging agents. Finally, we demonstrate that the RPA1-binding ability of SLFN11 is required for its function in the DNA damage response. Our findings not only provide novel insight into the molecular mechanisms underlying the drug sensitivity of cancer cell lines expressing SLFN11 at high levels, but also suggest that SLFN11 expression can serve as a biomarker to predict responses to DNA-damaging therapeutic agents.

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria

Cyclodextrins are cyclic oligosaccharides widely used in the pharmaceutical industry to improve drug delivery and to increase the solubility of hydrophobic compounds. Anabaenolysins are lipopeptides produced by cyanobacteria with potent lytic activity in cholesterol-containing membranes. Here, we identified the 23- to 24-kb gene clusters responsible for the production of the lipopeptide anabaenolysin. The hybrid nonribosomal peptide synthetase and polyketide synthase biosynthetic gene cluster is encoded in the genomes of three anabaenolysin-producing strains of Anabaena. We detected previously unidentified strains producing known anabaenolysins A and B and discovered the production of new variants of anabaenolysins C and D. Bioassays demonstrated that anabaenolysins have weak antifungal activity against Candida albicans. Surprisingly, addition of the hydrophilic fraction of the whole-cell extracts increased the antifungal activity of the hydrophobic anabaenolysins. The fraction contained compounds identified by NMR as α-, β-, and γ-cyclodextrins, which undergo acetylation. Cyclodextrins have been used for decades to improve the solubility and bioavailability of many drugs including antifungal compounds. This study shows a natural example of cyclodextrins improving the solubility and efficacy of an antifungal compound in an ancient lineage of photosynthetic bacteria.

Mass spectrometry data from proteomics-based screening of immunoreactive proteins of fully virulent Brucella strains using sera from naturally infected animals.

Here, we provide the dataset associated with our research article on comprehensive screening of Brucella immunoreactive proteins using sera of naturally infected hosts published in Biochemical and Biophysical Research Communications Wareth et al., 2015 [1]. Whole-cell protein extracts were prepared from Brucella abortus and Brucella melitensis, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently western blotting was carried out using sera from bovines (cows and buffaloes) and small ruminants (goats and sheep). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [2] with the dataset identifiers PXD001270 and DOI:10.6019/PXD001270.

Reductive Dehalogenation of Oligocyclic Phenolic Bromoaromatics by Dehalococcoides mccartyi Strain CBDB1.

Dehalococcoides mccartyi strains transform many halogenated compounds and are used for bioremediation. Such anaerobic transformations were intensively studied with chlorinated and simply structured compounds such as chlorinated benzenes, ethenes, and ethanes. However, many halogenated oligocyclic aromatic compounds occur in nature as either naturally produced materials or as part of commercial products such as pharmaceuticals, pesticides, or flame retardants. Here, we demonstrate that the D. mccartyi strain CBDB1 reductively debrominated two oligocyclic aromatic phenolic compounds, tetrabromobisphenol A (TBBPA) and bromophenol blue (BPB). The strain CBDB1 completely converted TBBPA to bisphenol A and BPB to phenol red with a stepwise removal of all bromide substituents. Debromination (but no cell growth) was detected in the cultures cultivated with TBBPA. In contrast, strain CBDB1 grew when interacting with BPB, demonstrating that this substrate was used as an electron acceptor for organobromine respiration. High doses of BPB delayed debromination and inhibited growth in the early cultivation phase. A higher toxicity of TBBPA compared with that of BPB might be due to the higher lipophilicity of TBBPA. Mass spectrometric analyses of whole-cell extracts demonstrated that two proteins encoded by the reductive dehalogenase homologous genes CbdbA1092 and CbdbA1503 were specifically induced by the used oligocyclic compounds, whereas others (e.g., CbdbA84 (CbrA)) were downregulated.

Inter-domain Cooperation in INCENP Promotes Aurora B Relocation from Centromeres to Microtubules.

The chromosomal passenger complex is essential for error-free chromosome segregation and proper execution of cytokinesis. To coordinate nuclear division with cytoplasmic division, its enzymatic subunit, Aurora B, relocalizes from centromeres in metaphase to the spindle midzone in anaphase. In budding yeast, this requires dephosphorylation of the microtubule-binding (MTB) domain of the INCENP analog Sli15. The mechanistic basis for this relocalization in metazoans is incompletely understood. We demonstrate that the putative coiled-coil domain within INCENP drives midzone localization of Aurora B via a direct, electrostatic interaction with microtubules. Furthermore, we provide evidence that the CPC multimerizes via INCENP's centromere-targeting domain (CEN box), which increases the MTB affinity of INCENP. In (pro)metaphase, the MTB affinity of INCENP is outcompeted by the affinity of its CEN box for centromeres, while at anaphase onset—when the histone mark H2AT120 is dephosphorylated—INCENP and Aurora B switch from centromere to microtubule localization.

Combining a Nanosensor and ELISA for Measurement of Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Activity and Protein Amount in Cell and Tissue Extract

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) was initially discovered by its ability to remove topoisomerase I (TOPI) covalently trapped to DNA. However, it has since been found to be involved in the repair of a number of DNA lesions other than TOPI–DNA complexes e.g., damages caused by ionizing radiation and free radical-based genotoxins. TDP1 has been reported to be posttranslational regulated, and in nonsmall cell lung cancer (NSCLC) it has been reported that the enzyme activity of TDP1 can be upregulated without detectable upregulation in protein amount. Activity and protein analyses are normally performed sequentially using radioactively labeled DNA substrates analyzed using gel-electrophoresis for the activity measurement and western blot or ELISA for protein measurement. We demonstrate here that our previously developed TDP1 nanosensor due to the optical real-time readout can be combined with ELISA measurement into one assay facilitating measurement of both the enzymatic activity and the protein amount of TDP1. We show that the combined assay can be used for measurements both in cell line-based studies and for measurement in clinical tissue samples. Due to all measurements being performed in the exact same aliquot of cell or tissue extract, the combined assay allows the use of minimal amount of cells or tissue and without additional incubation steps compared to the normal ELISA procedure. Measuring protein amount and activity within the same portion of extract also minimizes the risk of variation being introduced when comparing amount to activity. Since the assay only uses small amounts of extract, it requires no advanced equipment besides a plate reader, and can work in whole-cell or tissue extract. We expect that it could be useful for analysis of posttranslational modifications influencing enzymatic activities both in basic research and in clinical studies.

Identification of the PRMT1v1 and PRMT1v2 specific interactomes by quantitative mass spectrometry in breast cancer cells.

Arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). The PRMT1 gene generates at least seven distinct alternatively spliced isoforms (PRMT v1-v7), which together contribute a significant portion of the cellular arginine methylome. The distinct biochemical and biological functions of these PRMT1 isoforms have not been well characterized. Previously we have shown that while both PRMT1v1 and PRMT1v2 are overexpressed in breast cancer cells, PRMT1v2 specifically promotes breast cancer cell survival and invasion. These isoforms also have distinct subcellular localizations, PRMT1v1 is mainly nuclear and PRMT1v2 cytosolic. To gain further knowledge into their isoform-specific roles within cells we used a SILAC-based quantitative affinity purification/MS approach to identify their individual protein interactomes in breast cancer cells. This analysis has uncovered distinct interactomes for PRMT1v1 and PRMT1v2. Consistent with their distinct subcellular localizations, PRMT1v1 enriched a mainly nuclear protein interactome, while PRMT1v2 enriched predominantly cytoplasmic interactors from whole-cell extracts. Furthermore, these interactomes revealed that PRMT1v1 has a role in regulating gene expression, while PRMT1v2 functions in cytoskeletal dynamics. These results highlight the unique functions of these isoforms and the distinct roles they may play within cells, with potential implications for breast cancer and other diseases.

Computational epigenomics: challenges and opportunities.

Computational epigenomics: challenges and opportunities.

DEAD-box RNA helicase Dbp4 is required for small-subunit processome formation and function.

DEAD-box RNA helicase Dbp4 is required for 18S rRNA synthesis: cellular depletion of Dbp4 impairs the early cleavage reactions of the pre-rRNA and causes U14 small nucleolar RNA (snoRNA) to remain associated with pre-rRNA. Immunoprecipitation experiments (IPs) carried out with whole-cell extracts (WCEs) revealed that hemagglutinin (HA)-tagged Dbp4 is associated with U3 snoRNA but not with U14 snoRNA. IPs with WCEs also showed association with the U3-specific protein Mpp10, which suggests that Dbp4 interacts with the functionally active U3 RNP; this particle, called the small-subunit (SSU) processome, can be observed at the 5' end of nascent pre-rRNA. Electron microscopy analyses indicated that depletion of Dbp4 compromised SSU processome formation and cotranscriptional cleavage of the pre-rRNA. Sucrose density gradient analyses revealed that depletion of U3 snoRNA or the Mpp10 protein inhibited the release of U14 snoRNA from pre-rRNA, just as was seen with Dbp4-depleted cells, indicating that alteration of SSU processome components has significant consequences for U14 snoRNA dynamics. We also found that the C-terminal extension flanking the catalytic core of Dbp4 plays an important role in the release of U14 snoRNA from pre-rRNA.

FT-IR 스펙트럼 데이터의 다변량 통계분석을 이용한 곶감의 원산지 및 품종 식별

This study aimed to establish a rapid system for discriminating the cultivation origins and cultivars of dry persimmons, using metabolite fingerprinting by Fourier transform infrared (FT-IR) spectroscopy combined with multivariate analysis. Whole-cell extracts from the sepals of four Korean cultivars and two different Chinese dry persimmons were subjected to FT-IR spectroscopy. Principle component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) of the FT-IR spectral data successfully discriminated six dry persimmons into two groups depending on their cultivation origins. Principal component loading values showed that the 1750-1420 and regions of the FT-IR spectra were significantly important for the discrimination of cultivation origins. The accuracy of prediction of the cultivation origins and cultivars by PLS regression was 100% (p

Proteomic analyses of membrane enriched proteins of Leishmania donovani Indian clinical isolate by mass spectrometry

Visceral leishmaniasis (VL) is a major fatal disease prevalent in North-East India, caused by a protozoan parasite Leishmania donovani. The parasite multiplies and thrives inside mammalian macrophages and is transmitted by the bite of the sandfly. Due to the unsatisfactory treatment measures, increasing drug resistance and the advent of HIV-Leishmania co-infection there has been an urgent need to develop novel drug/vaccine targets against VL. Target identification is the key step in drug discovery and proteomics seems to be a suitable strategy for it due to the availability of Leishmania major, Leishmania infantum, Leishmania braziliensis, Leishmania donovani, Leishmania mexicana and Leishmania tarentolae genome sequence. Since, majority of proteome analyses of Leishmania have, so far, been performed on whole-cell extracts; this study is dealing with the sub-proteome analysis of the membrane-enriched protein (MEP) fractions of L. donovani. The analysis of 95 protein spots of the MEPs from two dimensional (2-D) gel image through matrix asserted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) endorsed the identification of various relevant functional proteins. Out of 95 the MEP spots 72 have been identified and were classified on the basis of their biological function. Several proteins of unknown function that belong to different classes like cell signaling, transmembrane receptors, and transporters have been identified which could be the new potential therapeutic targets against VL in future. The proteome array of the MEPs contributes to further elucidation of the biological system of L. donovani as well as host–parasite relationships which may be further investigated for their crucial biological role in L. donovani for disease management.

Phylogenetic Characterization of Phosphatase-Expressing Bacterial Communities in Baltic Sea Sediments.

Phosphate release from sediments hampers the remediation of aquatic systems from a eutrophic state. Microbial phosphatases in sediments release phosphorus during organic matter degradation. Despite the important role of phosphatase-expressing bacteria, the identity of these bacteria in sediments is largely unknown. We herein presented a culture-independent method to phylogenetically characterize phosphatase-expressing bacteria in sediments. We labeled whole-cell extracts of Baltic Sea sediments with an artificial phosphatase substrate and sorted phosphatase-expressing cells with a flow cytometer. Their phylogenetic affiliation was determined by Denaturing Gradient Gel Electrophoresis. The phosphatase-expressing bacterial community coarsely reflected the whole-cell bacterial community, with a similar dominance of Alphaproteobacteria.

Proteomics-based identification of immunodominant proteins of Brucellae using sera from infected hosts points towards enhanced pathogen survival during the infection

Brucella (B.) species lack classical virulence factors, but escape effectively the immune response of the host. The species Brucella abortus and Brucella melitensis infect predominantly cattle and small ruminants such as sheep or goats, respectively, but account also for most human cases. These two species share remarkably similar genomes but different proteomes have been demonstrated. This might be one of the reasons for their host specificity. A comprehensive identification of immunodominant proteins of these two species using antibodies present in the serum of naturally infected ruminants might provide insight on the mechanism of their infection in different hosts. In the present study, whole-cell protein extracts of B. abortus and B. melitensis were separated using SDS–PAGE and western blotting was performed using field sera from cows, buffaloes, sheep and goats. Protein bands that matched with western blot signals were excised, digested with trypsin and subjected to protein identification using MALDI-TOF MS. Identified proteins included heat shock proteins, enzymes, binding proteins and hypothetical proteins. Antibodies against the same set of antigen were found for all species investigated, except for superoxide dismutase of B. melitensis for which antibodies were demonstrated only in sheep serum. Brucellae appear to express these proteins mainly for their survival in the host system during infection.

Proteomic signatures associated with p53 mutational status in lung adenocarcinoma.

p53 is commonly mutated in lung adenocarcinoma. Mutant p53 loses wild-type function and some missense mutations further acquire oncogenic functions, while p53 wild-type may also induce pro-survival signaling. Therefore identification of signatures based on p53 mutational status has relevance to our understanding of p53 signaling pathways in cancer and identification of new therapeutic targets. To this end, we compared proteomic profiles of three cellular compartments (whole-cell extract, cell surface, and media) from 28 human lung adenocarcinoma cell lines that differ based on p53 mutational status. In total, 11,598, 11,569, and 9090 protein forms were identified in whole-cell extract, cell surface, and media, respectively. Bioinformatic analysis revealed that representative pathways associated with epithelial adhesion, immune and stromal cells, and mitochondrial function were highly significant in p53 missense mutations, p53 loss and wild-type p53 cell lines, respectively. Of note, mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), a transcription coactivator that promotes mitochondrial oxidative phosphorylation and mitochondrial biogenesis, was substantially higher in p53 wild-type cell lines compared to either cell lines with p53 loss or with missense mutation. Small interfering RNA targeting PGC1-α inhibited cell proliferation in p53 wild-type cell lines, indicative of PGC1-α and its downstream molecules as potential therapeutic targets in p53 wild-type lung adenocarcinoma.

C/EBP transcription factors in human squamous cell carcinoma: selective changes in expression of isoforms correlate with the neoplastic state.

The CCAAT/Enhancer Binding Proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate physiological processes such as energy metabolism, inflammation, cell cycle, and the development and differentiation of several tissues including skin. Recently, a role for C/EBPs in tumor cell proliferation and differentiation has been proposed, but the incomplete characterization in the literature of multiple translational isoforms of these proteins has made interpretation of these roles difficult. Therefore, we have carefully reexamined C/EBP isoform expression in human non-melanoma skin cancers. C/EBPα, C/EBPβ, and C/EBPδ were analyzed histologically in squamous cell carcinomas (SCC). The individual isoforms of C/EBPα and C/EBPβ were examined by immunofluorescent digital imaging, western blotting and DNA binding activity (electrophoretic mobility shift analysis). Expression of all C/EBP family proteins was decreased in SCC tumors. Suppression was greatest for C/EBPα, less for C/EBPβ, and least for C/EBPδ. Western analyses confirmed that C/EBPα p42 and p30 isoforms were decreased. For C/EBPβ, only the abundant full-length isoform (C/EBPβ−1, LAP*, 55 kD) was reduced, whereas the smaller isoforms, C/EBPβ−2 (LAP, 48 kD) and C/EBPβ−3 (LIP, 20 kD), which are predominantly nuclear, were significantly increased in well- and moderately-differentiated SCC (up to 14-fold for C/EBPβ−3). These elevations correlated with increases in PCNA, a marker of proliferation. Although C/EBPβ displayed increased post-translational modifications in SCC, phosphorylation of C/EBPβ−1 (Thr 235) was not altered. C/EBP-specific DNA binding activity in nuclear and whole-cell extracts of cultured cells and tumors was predominantly attributable to C/EBPβ. In summary, two short C/EBPβ isoforms, C/EBPβ−2 and C/EBPβ−3, represent strong candidate markers for epithelial skin malignancy, due to their preferential expression in carcinoma versus normal skin, and their strong correlation with tumor proliferation.

Elaborate cellulosome architecture of Acetivibrio cellulolyticus revealed by selective screening of cohesin-dockerin interactions.

Cellulosic waste represents a significant and underutilized carbon source for the biofuel industry. Owing to the recalcitrance of crystalline cellulose to enzymatic degradation, it is necessary to design economical methods of liberating the fermentable sugars required for bioethanol production. One route towards unlocking the potential of cellulosic waste lies in a highly complex class of molecular machines, the cellulosomes. Secreted mainly by anaerobic bacteria, cellulosomes are structurally diverse, cell surface-bound protein assemblies that can contain dozens of catalytic components. The key feature of the cellulosome is its modularity, facilitated by the ultra-high affinity cohesin–dockerin interaction. Due to the enormous number of cohesin and dockerin modules found in a typical cellulolytic organism, a major bottleneck in understanding the biology of cellulosomics is the purification of each cohesin- and dockerin-containing component, prior to analyses of their interaction. As opposed to previous approaches, the present study utilized proteins contained in unpurified whole-cell extracts. This strategy was made possible due to an experimental design that allowed for the relevant proteins to be “purified” via targeted affinity interactions as a function of the binding assay. The approach thus represents a new strategy, appropriate for future medium- to high-throughput screening of whole genomes, to determine the interactions between cohesins and dockerins. We have selected the cellulosome of Acetivibrio cellulolyticus for this work due to its exceptionally complex cellulosome systems and intriguing diversity of its cellulosomal modular components. Containing 41 cohesins and 143 dockerins, A. cellulolyticus has one of the largest number of potential cohesin–dockerin interactions of any organism, and contains unusual and novel cellulosomal features. We have surveyed a representative library of cohesin and dockerin modules spanning the cellulosome’s total cohesin and dockerin sequence diversity, emphasizing the testing of unusual and previously-unknown protein modules. The screen revealed several novel cell-bound cellulosome architectures, thus expanding on those previously known, as well as soluble cellulose systems that are not bound to the bacterial cell surface. This study sets the stage for screening the entire complement of cellulosomal components from A. cellulolyticus and other organisms with large cellulosome systems. The knowledge gained by such efforts brings us closer to understanding the exceptional catalytic abilities of cellulosomes and will allow the use of novel cellulosomal components in artificial assemblies and in enzyme cocktails for sustainable energy-related research programs.