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Background/aim Carbonic anhydrase 12 (CA12) is a membrane-associated enzyme that is highly expressed on many human cancers. It is a poor prognostic marker and hence an attractive target for cancer therapy. This study aimed to develop a humanized CA12-antibody with anti-cancer activity. Materials and methods Antibody libraries were constructed and screened by the Retrocyte display®. Antibody binding and blocking properties were determined by ELISA, flow cytometry and enzymatic activity assays. Spheroid viability was determined by Cell-Titer-Fluor assay. Results We developed a novel humanized CA12-specific antibody, 4AG4, which recognized CA12 as an antigen and blocked CA12 enzymatic activity. Our humanized CA12-antibody significantly inhibited spheroid growth of lung adenocarcinoma A549-cells in vitro by blocking CA12 enzymatic activity. Similar anti-tumor effects were recapitulated with CA12-gene knockout of A549-cells. Conclusion Our newly identified humanized CA12-antibody with anti-cancer activity, represents a new tool for the treatment of CA12-positive tumors.

Adoptive T cell therapy using patient T cells redirected to recognize tumor-specific antigens by expressing genetically engineered high-affinity T-cell receptors (TCRs) has therapeutic potential for melanoma and other solid tumors. Clinical trials implementing genetically modified TCRs in melanoma patients have raised concerns regarding off-target toxicities resulting in lethal destruction of healthy tissue, highlighting the urgency of assessing which off-target peptides can be recognized by a TCR. As a model system we used the clinically efficacious NY-ESO-1-specific TCR C259, which recognizes the peptide epitope SLLMWITQC presented by HLA-A*02:01. We investigated which amino acids at each position enable a TCR interaction by sequentially replacing every amino acid position outside of anchor positions 2 and 9 with all 19 possible alternative amino acids, resulting in 134 peptides (133 altered peptides plus epitope peptide). Each peptide was individually evaluated using three different in vitro assays: binding of the NY-ESOc259 TCR to the peptide, peptide-dependent activation of TCR-expressing cells, and killing of peptide-presenting target cells. To represent the TCR recognition kernel, we defined Position Weight Matrices (PWMs) for each assay by assigning normalized measurements to each of the 20 amino acids in each position. To predict potential off-target peptides, we applied a novel algorithm projecting the PWM-defined kernel into the human proteome, scoring NY-ESOc259 TCR recognition of 336,921 predicted human HLA-A*02:01 binding 9-mer peptides. Of the 12 peptides with high predicted score, we confirmed 7 (including NY-ESO-1 antigen SLLMWITQC) strongly activate human primary NY-ESOc259-expressing T cells. These off-target peptides include peptides with up to 7 amino acid changes (of 9 possible), which could not be predicted using the recognition motif as determined by alanine scans. Thus, this replacement scan assay determines the “TCR fingerprint” and, when coupled with the algorithm applied to the database of human 9-mer peptides binding to HLA-A*02:01, enables the identification of potential off-target antigens and the tissues where they are expressed. This platform enables both screening of multiple TCRs to identify the best candidate for clinical development and identification of TCR-specific cross-reactive peptide recognition and constitutes an improved methodology for the identification of potential off-target peptides presented on MHC class I molecules.

Over the last nearly three decades in vitro display technologies have played an important role in the discovery and optimization of antibodies and other proteins for therapeutic applications. Here we describe the use of retroviral expression technology for the display of full-length IgG on B lineage cells in vitro with a hallmark of a tight and stable genotype to phenotype coupling. We describe the creation of a high-diversity (>1.0E09 different heavy- and light-chain combinations) cell displayed fully human antibody library from healthy donor-derived heavy- and light-chain gene libraries, and demonstrate the recovery of high affinity target-specific antibodies from this library by staining of cells with a labeled target antigen and their magnetic- and flow cytometry-based cell sorting. The present technology represents a further evolution in the discovery of full-length, fully human antibodies using mammalian display, and is termed Retrocyte Display® (Retroviral B lymphocyte Display).

Gastric cancer mortality is second only to lung cancer, and its prognosis is dismal. Using surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry, we previously identified a single best mass, which could separate gastric cancer from patients without cancer, with a sensitivity of 89.9% and a specificity of 90%. Using protein liquid chromatography systems with various chromatography media and MS/MS analysis, we were able to identify thrombin light chain A, a proteolytic fragment of prothrombin, as the single best mass for early detection of gastric cancer patients. These findings indicate that disturbances in the coagulation-system are early events in gastric cancer biology and that a decrease or loss of thrombin light chain A, which we termed negative serum protein profiling, may contribute to the diagnosis of cancer patients. Keywords: gastric • carcinoma • stomach • SELDI • biomarker • thrombin • thrombosis • diagnosis

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc- and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).

Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. We developed a successful method that addresses these different problems. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (> 95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed.

Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase's. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases.

Glucocorticoid-induced TNFR family related protein (GITR, CD357 or TNFRSF18) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). Like other T cell co-stimulatory TNFR family members, GITR utilizes multiple oligomerization states to regulate the initiation of downstream signaling during T cell activation by antigen presenting cells (APCs). The formation of receptor superclusters, comprised of two or more trimeric molecules, has been defined for multiple TNFRs as a means of regulating downstream signal amplification. For co-stimulatory TNFRs, like GITR, CD137 and OX40, signaling outcomes in T cells are primarily mediated via the NFκB pathway that promotes cell survival and effector cell activities in response to suboptimal T cell receptor (TCR) stimulation. It has been hypothesized that the manipulation of the oligomeric states of co-stimulatory TNFRs using antibodies may have therapeutic utility in enhancing the activity of tumor-reactive T cells, either as single agents or in combination with other immunomodulatory or immune education strategies. Here we describe a structure-based analysis of two functionally distinct classes of anti-human GITR antibodies that stabilize unique conformational states of the receptor. INCAGN1876, a human IgG1 monoclonal anti-GITR antibody, was found to engage a conformational epitope located within a β-turn of the extracellular domain of GITR. This antibody binding site modified the equilibrium of GITR monomer, dimer and trimers to promote receptor oligomerization, resulting in downstream NFκB signaling. Notably, this mode of INCAGN1876 receptor engagement enabled it to effectively activate the GITR pathway in recently primed T cells. By contrast, a second reference anti-GITR antibody required concomitant TCR co-engagement in order to modulate the GITR pathway. High content confocal analysis was used to evaluate the kinetics of GITR clustering by both classes of anti-GITR antibody, confirming our T cell functional analysis. The ability of INCAGN1876 to engage and effectively activate GITR on recently primed T cells may enable them to overcome suppressive features of the tumor microenvironment. Notably, INCAGN1876 was shown to promote T cell co-stimulation both as a single agent and in combination with other antibodies targeting PD-1, CTLA-4 and OX40. Finally, we compared the pharmacologic activity of INCAGN1876 to Fc variants of this antibody with diminished binding to the inhibitory Fcγ receptor (FcγR), CD32B. The superiority of an IgG1 antibody in these assays was consistent with the potential to achieve optimal GITR clustering by FcγRs, while maintaining the potential for FcγR-mediated effector cell activity directed toward intratumoral GITRhigh regulatory T cells. INCAGN1876 is currently under evaluation in Phase 1/2 studies in subjects with advanced metastatic solid tumors (NCT02697591). Citation Format: Ana M. Gonzalez, Mariana L. Manrique, Lukasz Swiech, Thomas Horn, Jeremy Waight, Yuqi Liu, Shiwen Lin, Dennis Underwood, Ekaterina Breous, Olivier Leger, Volker Seibert, Taha Merghoub, Roberta Zappasodi, Gerd Ritter, David Schaer, Kevin N. Heller, Kimberli Brill, Peggy Scherle, Gregory Hollis, Reid Huber, Marc van Dijk, Jennifer Buell, Robert Stein, Nicholas S. Wilson. INCAGN1876, a unique GITR agonist antibody that facilitates GITR oligomerization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3643. doi:10.1158/1538-7445.AM2017-3643

The tip and tilt M5 mirror of the European Extremly Large Telescope (E-ELT) requires a demanding approach in light weighting. The approximately 3 m x 2.5 m elliptical plano mirror is specified to a weight of less than 500 kg with high Eigenfrequencies and low deformation under different inclination angles. In 2011 SCHOTT has presented a study to develop a design for the M5 mirror blank of the ESO E-ELT. The design presented was based on a radial square design to achieve the best compromise between performance and manufacturability. With the fabrication of a prototype section SCHOTT demonstrated its capability to manufacture the demanding features including pockets with 350 mm depth, thin walls and sloped pocket bottoms. Now 3 years later SCHOTT presents an iso-grid based design that is in accordance with the manufacturability progress that has been demonstrated in various ELZM (Extremely Lightweighted ZERODUR Mirrors) publications in the last two years. The achievements on the specified mechanical parameters are compared to the first approach from 2011. In this paper the results are presented and the performance parameters are discussed.

Activation of costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily in T cells is considered a promising alternative approach to potentiate anti-tumor immunity that may complement strategies focused on the blockade of co-inhibitory pathways such PD-1/PDL1. Glucocorticoid-induced TNFR-related protein (GITR, CD357 or TNFRSF18) is an important T cell costimulatory receptor that can potentiate T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In humans GITR expression is generally restricted to subsets of T cells responding to TCR stimulation, and is co-expressed with OX40. Like other TNFR family members, GITR co-stimulation can enhance T cell responsiveness to suboptimal TCR signaling by activating the NFκB pathway, leading to enhanced cytokine responses and survival. GITR signaling in T cells may also promote resistance to the immune suppressive effects of regulatory T cells, thereby enhancing T cell responsiveness to weakly immunogenic tumor-associated antigens. INCAGN01876 is a humanized IgG1 monoclonal antibody being developed for the treatment of advanced malignancies. INCAGN01876 potently binds to human and non-human primate GITR but does not cross-react with related TNFR family members. INCAGN01876 has been optimized to mediate receptor forward signaling under suboptimal TCR stimulatory conditions, leading to increased production of TNFα and IFNγ by both CD4+ and CD8+ T cells. INCAGN01876 achieves this functionality by virtue of its ability to facilitate GITR clustering in TCR-stimulated T lymphocytes. In mouse preclinical tumor models, GITR was found to be selectively overexpressed by intratumoral regulatory T cells, a finding that was also observed in primary human tumor samples from diverse tumor types. In mouse models, this feature enabled a surrogate anti-GITR antibody to co-engage activating Fcγ receptors expressed by tumor-associated effector cells, and mediate the selective depletion of intratumoral regulatory T cells. Consistent with this, INCAGN01876 was designed to co-engage activating Fcγ receptors and was shown to efficiently mediate immune effector cell mechanisms, including ADCC and ADCP. Taken together, the biophysical and functional attributes of INCAGN01876 make it ideally suited for clinical development, both as a single agent and in combination with other immunomodulatory agents. Citation Format: Ana Maria Gonzalez, Ekaterina Breous, Mariana L. Manrique, David Savitsky, Jeremy Waight, Randi Gombos, Yuqi Liu, Shiwen Lin, Olivier Leger, Volker Seibert, Takemasa Tsuji, Taha Merghoub, Sadna Budha, Roberta Zappasodi, Gerd Ritter, Jedd Wolchok, Peggy Scherle, Gregory Hollis, Reid Huber, Marc Van Dijk, Robert Stein, Nicholas Wilson. A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3220.

Previously we established that the combination of new machining parameters for openbacked monolithic lightweighted ZERODUR® mirror substrates, coupled with new methods for optical finishing of aggressively lightweighted mirrors, have relieved classical mirror design constraints imposed upon 1.2m diameter lightweight mirrors. We demonstrate that openback mirror substrates now offer comparable mass, Eigenfrequency and substructure print-through performance to the sandwich-mirror architecture, but with considerably less manufacturing effort than for sandwich mirrors. Here we extend the analyses of the first paper from 1.2m diameter down to 0.6m in diameter and up to 2.4m diameter and 4m diameter mirrors.

Recent game-changing technology greatly extends the design possibilities and range of applications for aggressively lightweighted open-back Zerodur® mirrors. We have compared several lightweighting design approaches under this new technology. Analytic comparisons are for 1.2m mirrors, all constrained to have a free-free first Eigenfrequency of 200 Hz. Figures of merit include resulting mass, thickness and relative cost. Much more aggressive masses are now available in open-back mirrors, competitive with the more expensive closed-back sandwich mirrors. These breakthroughs are relevant to spaceborne implementation of lightweight mirrors ranging from a few tenths of a meter in diameter to up to 4 meters in diameter.

In the recent past, SCHOTT has demonstrated its ability to manufacture large light weighted ZERODUR® mirror blanks for telescope projects like the GREGOR solar-telescope, for example. In 2010, SCHOTT was commissioned with a study aimed at developing a design for the M5 mirror blank of the ESO E-ELT. The tip and tilt M5 mirror of the European Extremely Large Telescope (E-ELT) requires a demanding approach in light weighting. The approximately 3.1 m x 2.5 m elliptical plano mirror is specified to a weight of less than 500 kg with high Eigenfrequencies and low deformation under different inclination angles. The study was divided into two parts. The first part focused on coming up with an optimized light weighted design with respect to performance and processability with finite element modeling. In the second part of the study, a concept for the processing sequence including melting, cold-processing, acid etching and handling of the M5 blank was developed. By producing a prototype section, SCHOTT demonstrated its ability to manufacture the demanding features, including pockets 350 mm in depth, thin walls and sloped pocket bottoms. This paper outlines the results of the design work, processing concept and demonstrator fabrication.

For most cancers, survival rates depend on the early detection of the disease. So far, no biomarkers exist to cope with this difficult task. New proteomic technologies have brought the hope of discovering novel early cancer-specific biomarkers in complex biological samples and/ or of the setting up of new clinically relevant test systems. Novel mass spectrometry-(MS) based technologies in particular, such as surface-enhanced laser desorption/ionisation time of flight (SELDI-ToF-MS), have shown promising results in the recent literature. Here, proteomic profiles of control and disease states are compared to find biomarkers for diagnosis. This paper aims to address the authors' own work and that of other groups in clinical cancer proteomics based on SELDI-ToF-MS. Shortcomings and hopes for the future are discussed.

In this review article, we describe some of the studies that have been performed using the surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry and ProteinChip® technology over the past few years, and highlight both their findings as well as limitations. Proteomic applications, such as target or marker identification and target validation or toxicology, will be addressed. We will also provide an examination of SELDI technology and go into the question of where possible future research may lead us.

A gene cluster containing a gene for maleylacetate reductase (EC 1.3.1.32) was cloned from Ralstonia eutropha 335T (DSM 531T), which is able to utilize 4-fluorobenzoate as sole carbon source. Sequencing of this gene cluster showed that the R. eutropha 335T maleylacetate reductase gene, macA, is part of a novel gene cluster, which is not related to the known maleylacetate-reductase-encoding gene clusters. It otherwise comprises a gene for a hypothetical membrane transport protein, macB, possibly co-transcribed with macA, and a presumed regulatory gene, macR, which is divergently transcribed from macBA. MacA was found to be most closely related to TftE, the maleylacetate reductase from Burkholderia cepacia AC1100 (62 % identical positions) and to a presumed maleylacetate reductase from a dinitrotoluene catabolic gene cluster from B. cepacia R34 (61 % identical positions). By expressing macA in Escherichia coli, it was confirmed that macA encodes a functional maleylacetate reductase. Purification of maleylacetate reductase from 4-fluorobenzoate-grown R. eutropha 335T cells allowed determination of the N-terminal sequence of the purified protein, which was shown to be identical to that predicted from the cloned macA gene, thus proving that the gene is, in fact, recruited for growth of R. eutropha 335T with this substrate.

Background SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes. Results We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity. Conclusion Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.

Airbag igniters are small assemblies consisting of a sealing component with specified electrical properties and an explosive. Robust igniters must guarantee the reliability of the electrical properties and of the explosive for many years. Thus, the components must not exhibit any form of corrosion and the assembly must be air-tight. Otherwise, the explosive could degrade by moisture, for example.

The chloromuconate cycloisomerase of Pseudomonas sp. B13 was purified from 3-chlorobenzoate-grown wild-type cells while the chloromuconate cycloisomerases of Ralstonia eutropha JMP134 (pJP4) and Pseudomonas sp. P51 (pP51) were purified from Escherichia coli strains expressing the corresponding gene. Kinetic studies were performed with various chloro-, fluoro-, and methylsubstituted cis,cis-muconates. 2,4-Dichloro-cis,cis-muconate proved to be the best substrate for all three chloromuconate cycloisomerases. Of the three enzymes, TfdD of Ralstonia eutropha JMP134 (pJP4) was most specific, since its specificity constant for 2,4-dichloro-cis,cis-muconate was the highest, while the constants for cis,cis-muconate, 2-chloro- and 2,5-dichloro-cis,cis-muconate were especially poor. The sequence of ClcB of the 3-chloro-benzoate-utilizing strain Pseudomonas sp. B13 was determined and turned out to be identical to that of the corresponding enzyme of pAC27 (though slightly different from the published sequences). Corresponding to 2-chloro-cis,cis-muconate being a major metabolite of 3-chlorobenzoate degradation, the kcat/K(m) with 2-chloro-cis,cis-muconate was relatively high, while that with the still preferred substrate 2,4-dichloro-cis,cis-muconate was relatively low. This enzyme was thus the least specific and the least active among the three compared enzymes. TcbD of Pseudomonas sp. P51 (pP51) took an intermediate position with respect to both the degree of specificity and the activity with the preferred substrate.

Maleylacetate reductases (EC 1.3.1.32 ) have been shown to contribute not only to the bacterial catabolism of some usual aromatic compounds like quinol or resorcinol but also to the degradation of aromatic compounds carrying unusual substituents, such as halogen atoms or nitro groups. Genes coding for maleylacetate reductases so far have been analyzed mainly in chloroaromatic compound-utilizing proteobacteria, in which they were found to belong to specialized gene clusters for the turnover of chlorocatechols or 5-chlorohydroxyquinol. We have now cloned the gene macA , which codes for one of apparently (at least) two maleylacetate reductases in the gram-positive, chlorophenol-degrading strain Rhodococcus opacus 1CP. Sequencing of macA showed the gene product to be relatively distantly related to its proteobacterial counterparts (ca. 42 to 44% identical positions). Nevertheless, like the known enzymes from proteobacteria, the cloned Rhodococcus maleylacetate reductase was able to convert 2-chloromaleylacetate, an intermediate in the degradation of dichloroaromatic compounds, relatively fast and with reductive dehalogenation to maleylacetate. Among the genes ca. 3 kb up- and downstream of macA , none was found to code for an intradiol dioxygenase, a cycloisomerase, or a dienelactone hydrolase. Instead, the only gene which is likely to be cotranscribed with macA encodes a protein of the short-chain dehydrogenase/reductase family. Thus, the R. opacus maleylacetate reductase gene macA clearly is not part of a specialized chlorocatechol gene cluster.

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively). NADH was preferred to NADPH as the cosubstrate. Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.

Background SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes.

Background: The function of the fission yeast cullins Pcu1p and Pcu4p requires modification by the ubiquitin-related peptide Ned8p. A recent report by Lyapina et al. shows that the COP9/ signalosome (CSN), a multifunctional eight subunit complex, regulates Ned8p modification of Pcu1p. Disruption of caa1/csn1, which encodes subunit 1 of the putative S. pombe CSN, results in accumulation of Pcu1p exclusively in the modified form. However, it remained unclear whether this reflects global control of all cullins by the entire CSN complex. Results: We demonstrate that multiple CSN subunits control Ned8p modification of Pcu3p, another fission yeast cullin, which, in complex with the RING domain protein Pip1p, forms a ubiquitin ligase that functions in cellular stress response. Pcu3p is modified by Ned8p on Lys 729 and accumulates exclusively in the neddylated form in cells lacking the CSN subunits 1, 3, 4, and 5. These CSN subunits co-elute with Pcu3p in gel filtration fractions corresponding to ∼ 550 kDa and specifically bind both native and Ned8p-modified Pcu3p in vivo. While CSN does not influence the subcellular localization of Pcu3p, Pcu3p-associated in vitro ubiquitin ligase activity is stimulated in the absence of CSN. Conclusions: Taken together, our data suggest that CSN is a global regulator of Ned8p modification of multiple cullins and potentially other proteins involved in cellular regulation.