The unique monoclonal antibodies and immunochemical assay for comprehensive determination of the cell-bound and soluble HER2 in different biological samples.

The expression of the HER2 (human epidermal growth factor receptor 2) protein in cancer cells is a well-established cancer marker used for diagnostic and therapeutic purposes in modern treatment protocols, especially in breast cancer. The gold-standard immunohistochemical diagnostic methods with the specific anti-HER2 antibodies are utilized in the clinic to measure expression level of the membrane-bound receptor. However, a soluble extracellular domain (ECD) of HER2 is released to the extracellular matrix, thus the blood assays for HER2 measurements present an attractive way for HER2 level determination. There is a need for accurate and validated assays that can be used to correlate the concentration of the circulating HER2 protein with disease clinical manifestations. Here we describe two monoclonal antibodies binding HER2 with a unique sequence of the complementarity-determining regions that recognize HER2 ECD. Development and validation of the sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the soluble HER2 in a variety of biological samples is also presented. The assay provides HER2 quantitation within a concentrations range from 1.56 to 100 ng/ml with sensitivity at the level of 0.5 ng/ml that meets the expectations for measurements of HER2 in the blood and tumor tissue samples. The method presents satisfactory intra- and inter-assay precision and accuracy for immunochemical quantification of biomarkers in biological samples. The utility of the generated monoclonal anti-HER2 antibodies has been confirmed for use in the precise measurement of HER2 (both cell-bound and soluble) in several types of biological material, including serum, solid tumor tissue, and cell culture medium. Additionally, the developed immunochemical tools have a potential for HER2 detection, not only in a wide range of sample types but also independently of the sample storage/pre-processing, allowing for comprehensive HER2 analysis in tissue (IHC), cultured cells (immunofluorescence) and blood (ELISA).

New bacteriophage-derived lysins, LysJ and LysF, with the potential to control Bacillus anthracis.

Bacillus anthracis is an etiological agent of anthrax, a severe zoonotic disease that can be transmitted to people and cause high mortalities. Bacteriophages and their lytic enzymes, endolysins, have potential therapeutic value in treating infections caused by this bacterium as alternatives or complements to antibiotic therapy. They can also be used to identify and detect B. anthracis. Endolysins of two B. anthracis Wbetavirus phages, J5a and F16Ba which were described by us recently, differ significantly from the best-known B. anthracis phage endolysin PlyG from Wbetavirus genus bacteriophage Gamma and a few other Wbetavirus genus phages. They are larger than PlyG (351 vs. 233 amino acid residues), contain a signal peptide at their N-termini, and, by prediction, have a different fold of cell binding domain suggesting different structural basis of cell epitope recognition. We purified in a soluble form the modified versions of these endolysins, designated by us LysJ and LysF, respectively, and depleted of signal peptides. Both modified endolysins could lyse the B. anthracis cell wall in zymogram assays. Their activity against the living cells of B. anthracis and other species of Bacillus genus was tested by spotting on the layers of bacteria in soft agar and by assessing the reduction of optical density of bacterial suspensions. Both methods proved the effectiveness of LysJ and LysF in killing the anthrax bacilli, although the results obtained by each method differed. Additionally, the lytic efficiency of both proteins was different, which apparently correlates with differences in their amino acid sequence. KEY POINTS: • LysJ and LysF are B. anthracis-targeting lysins differing from lysins studied so far • LysJ and LysF could be overproduced in E. coli in soluble and active forms • LysJ and LysF are active in killing cells of B. anthracis virulent strains.

Two interaction surfaces between XPA and RPA organize the preincision complex in nucleotide excision repair.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

A key interaction with RPA orients XPA in NER complexes.

The XPA protein functions together with the single-stranded DNA (ssDNA) binding protein RPA as the central scaffold to ensure proper positioning of repair factors in multi-protein nucleotide excision repair (NER) machinery. We previously determined the structure of a short motif in the disordered XPA N-terminus bound to the RPA32C domain. However, a second contact between the XPA DNA-binding domain (XPA DBD) and the RPA70AB tandem ssDNA-binding domains, which is likely to influence the orientation of XPA and RPA on the damaged DNA substrate, remains poorly characterized. NMR was used to map the binding interfaces of XPA DBD and RPA70AB. Combining NMR and X-ray scattering data with comprehensive docking and refinement revealed how XPA DBD and RPA70AB orient on model NER DNA substrates. The structural model enabled design of XPA mutations that inhibit the interaction with RPA70AB. These mutations decreased activity in cell-based NER assays, demonstrating the functional importance of XPA DBD-RPA70AB interaction. Our results inform ongoing controversy about where XPA is bound within the NER bubble, provide structural insights into the molecular basis for malfunction of disease-associated XPA missense mutations, and contribute to understanding of the structure and mechanical action of the NER machinery.

Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.

Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.

MAP kinase p38 is a novel target of CacyBP/SIP phosphatase.

Mitogen-activated protein (MAP) kinases are important players in cellular signaling pathways. Recently, it has been shown that CacyBP/SIP serves as a phosphatase for one of the MAP kinases, ERK1/2. Through dephosphorylation of this kinase CacyBP/SIP modulates the transcriptional activity of Elk-1 and the activity of the CREB-BDNF pathway. In this work, using NB2a cell lysate and recombinant proteins, we show that CacyBP/SIP binds and dephosphorylates another member of the MAP kinase family, p38. Analysis of recombinant full-length CacyBP/SIP and its three major domains, N-terminal, middle CS and C-terminal SGS, indicates that the middle CS domain is responsible for p38 dephosphorylation. Moreover, we show that CacyBP/SIP might be implicated in response to oxidative stress. Dephosphorylation of phospho-p38 by CacyBP/SIP in NB2a cells treated with hydrogen peroxide is much more effective than in control ones. In conclusion, involvement of CacyBP/SIP in the regulation of p38 kinase activity, in addition to that of ERK1/2, might point to the function of CacyBP/SIP in pro-survival and pro-apoptotic pathways.

CacyBP/SIP--Structure and variety of functions.

BACKGROUND: CacyBP/SIP (Calcyclin-Binding Protein and Siah-1 Interacting Protein) is a small modular protein implicated in a wide range of cellular processes. It is expressed in different tissues of mammals but homologs are also found in some lower organisms. In mammals, a high level of CacyBP/SIP is present in tumor cells and in neurons. CacyBP/SIP binds several target proteins such as members of the S100 family, components of a ubiquitin ligase complex, and cytoskeletal proteins. SCOPE OF REVIEW: CacyBP/SIP has been shown to be involved in protein de-phosphorylation, ubiquitination, cytoskeletal dynamics, regulation of gene expression, cell proliferation, differentiation, and tumorigenesis. This review focuses on very recent reports on CacyBP/SIP structure and function in these important cellular processes. MAJOR CONCLUSIONS: CacyBP/SIP is a multi-domain and multi-functional protein. Altered levels of CacyBP/SIP in several cancers implicate its involvement in the maintenance of cell homeostasis. Changes in CacyBP/SIP subcellular localization in neurons of AD brains suggest that this protein is strongly linked to neurodegenerative diseases. Elucidation of CacyBP/SIP structure and cellular function is leading to greater understanding of its role in normal physiology and disease pathologies. GENERAL SIGNIFICANCE: The available results suggest that CacyBP/SIP is a key player in multiple biological processes. Detailed characterization of the physical, biochemical and biological properties of CacyBP/SIP will provide better insight into the regulation of its diverse functions in vivo, and given the association with specific diseases, will help clarify the potential of therapeutic targeting of this protein.

Dimerization and phosphatase activity of calcyclin-binding protein/Siah-1 interacting protein: the influence of oxidative stress.

CacyBP/SIP [calcyclin-binding protein/Siah-1 [seven in absentia homolog 1 (Siah E3 ubiquitin protein ligase 1)] interacting protein] is a multifunctional protein whose activity includes acting as an ERK1/2 phosphatase. We analyzed dimerization of mouse CacyBP/SIP in vitro and in mouse neuroblastoma cell line (NB2a) cells, as well as the structure of a full-length protein. Moreover, we searched for the CacyBP/SIP domain important for dimerization and dephosphorylation of ERK2, and we analyzed the role of dimerization in ERK1/2 signaling in NB2a cells. Cell-based assays showed that CacyBP/SIP forms a homodimer in NB2a cell lysate, and biophysical methods demonstrated that CacyBP/SIP forms a stable dimer in vitro. Data obtained using small-angle X-ray scattering supported a model in which CacyBP/SIP occupies an anti-parallel orientation mediated by the N-terminal dimerization domain. Site-directed mutagenesis established that the N-terminal domain is indispensable for full phosphatase activity of CacyBP/SIP. We also demonstrated that the oligomerization state of CacyBP/SIP as well as the level of post-translational modifications and subcellular distribution of CacyBP/SIP change after activation of the ERK1/2 pathway in NB2a cells due to oxidative stress. Together, our results suggest that dimerization is important for controlling phosphatase activity of CacyBP/SIP and for regulating the ERK1/2 signaling pathway.