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A comparative canine–human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine–canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine–murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human–murine chimeric anti-CD20 antibody Rituximab.

Despite proteotoxic stress and heat shock being implicated in diverse pathologies, currently no methodology to inflict defined, subcellular thermal damage exists. Here, we present such a single-cell method compatible with laser-scanning microscopes, adopting the plasmon resonance principle. Dose-defined heat causes protein damage in subcellular compartments, rapid heat-shock chaperone recruitment, and ensuing engagement of the ubiquitin–proteasome system, providing unprecedented insights into the spatiotemporal response to thermal damage relevant for degenerative diseases, with broad applicability in biomedicine. Using this versatile method, we discover that HSP70 chaperone and its interactors are recruited to sites of thermally damaged proteins within seconds, and we report here mechanistically important determinants of such HSP70 recruitment. Finally, we demonstrate a so-far unsuspected involvement of p97(VCP) translocase in the processing of heat-damaged proteins. Overall, we report an approach to inflict targeted thermal protein damage and its application to elucidate cellular stress-response pathways that are emerging as promising therapeutic targets., Existing methods for inflicting cellular heat shock are limited by the time delay in achieving the desired temperature and the spatial precision that can be achieved. Here the authors report a method to induce focused thermal protein damage using plasmonic silver nanoparticles.

Translocase of outer mitochondrial membrane 34 (TOMM34) orchestrates heat shock protein 70 (HSP70)/HSP90–mediated transport of mitochondrial precursor proteins. Here, using in vitro phosphorylation and refolding assays, analytical size-exclusion chromatography, and hydrogen/deuterium exchange MS, we found that TOMM34 associates with 14-3-3 proteins after its phosphorylation by protein kinase A (PKA). PKA preferentially targeted two serine residues in TOMM34: Ser(93) and Ser(160), located in the tetratricopeptide repeat 1 (TPR1) domain and the interdomain linker, respectively. Both of these residues were necessary for efficient 14-3-3 protein binding. We determined that phosphorylation-induced structural changes in TOMM34 are further augmented by binding to 14-3-3, leading to destabilization of TOMM34's secondary structure. We also observed that this interaction with 14-3-3 occludes the TOMM34 interaction interface with ATP-bound HSP70 dimers, which leaves them intact and thereby eliminates an inhibitory effect of TOMM34 on HSP70-mediated refolding in vitro. In contrast, we noted that TOMM34 in complex with 14-3-3 could bind HSP90. Both TOMM34 and 14-3-3 participated in cytosolic precursor protein transport mediated by the coordinated activities of HSP70 and HSP90. Our results provide important insights into how PKA-mediated phosphorylation and 14-3-3 binding regulate the availability of TOMM34 for its interaction with HSP70.

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)-binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the "activating" C-terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked-immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild-type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the "unfolded" conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.

Background Sustained proliferation and genetic instability of cancer cells are associated with enhanced production of mutated and conformationally unstable proteins. Excessive proteosynthesis along with increased metabolic turnover generates stress conditions that cancer cells must permanently compensate for. Tumor cells thus become dependent on the maintenance of protein homeostasis, which involves protein quality control, folding, transport and stabilization. These tasks are provided by molecular chaperones, predominantly the stress proteins HSP70 and HSP90. Their expression and activity is increased in all malignant tumors, where they associate with their cochaperones to form large multiprotein complexes. HSP70 and HSP90 maintain the malignant phenotype because they facilitate the folding of numerous oncogenic proteins, maintain proliferative potential, and inhibit apoptosis. In this regard, heat-shock proteins represent an important target for cancer therapy because their inactivation results in the simultaneous blockade of multiple signaling pathways. Although several specific HSP90 inhibitors have been developed in the past decade, their antitumor activity as single agents is limited due to the induction of HSP70, which enables cell survival. Inhibitors of HSP70 thus present new possibilities for targeting proteostatic mechanisms in cancer cells. Aim The aim of this article is to summarize information on the structure of HSP70 and its role in maintaining protein homeostasis in normal and cancer cells. The mechanisms of HSP70 inhibition by low-molecular weight compounds and their application in targeted antitumor therapy are also described. Key words: HSP70 - stress proteins - molecular chaperons - cellular stress - tumours - protein folding This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 08. 2018.

Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules in vivo, suggesting that one conserved amino acid residue in the RINGs of Mdm2 and MdmX could serve as the determinant of the differential ability of these domains to form dimers and their E3 activity. In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo. As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

Co-chaperones containing tetratricopeptide repeat (TPR) domains enable cooperation between Hsp70 and Hsp90 to maintain cellular proteostasis. Although the details of the molecular interactions between some TPR domains and heat shock proteins are known, we describe a novel mechanism by which Tomm34 interacts with and coordinates Hsp70 activities. In contrast to the previously defined Hsp70/Hsp90-organizing protein (Hop), Tomm34 interaction is dependent on the Hsp70 chaperone cycle. Tomm34 binds Hsp70 in a complex process; anchorage of the Hsp70 C terminus by the TPR1 domain is accompanied by additional contacts formed exclusively in the ATP-bound state of Hsp70 resulting in a high affinity entropically driven interaction. Tomm34 induces structural changes in determinants within the Hsp70-lid subdomain and modulates Hsp70/Hsp40-mediated refolding and Hsp40-stimulated Hsp70 ATPase activity. Because Tomm34 recruits Hsp90 through its TPR2 domain, we propose a model in which Tomm34 enables Hsp70/Hsp90 scaffolding and influences the Hsp70 chaperone cycle, providing an additional role for co-chaperones that contain multiple TPR domains in regulating protein homeostasis.

Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit “rim” of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric ReptinY340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. Significance: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a ‘hot-spot’ protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape. Agency for Science, Technology and Research (A*STAR) National Supercomputing Centre (NSCC) Singapore Accepted version The work was supported by: the Czech Science Foundation 16-20860S (PM, LH) and 16-07321S (BV, TH), the project MEYS–NPS I–LO1413, and MH CZ - DRO (MMCI, 00209805); the BBSRC RASORconsortium (BB/C511599/1; United Kingdom); Cancer Research UK(C21383/A6950); The International Centre for Cancer Vaccine Scienceproject carried out within the International Research Agendas pro-gramme of the Foundation for Polish Science co-financed by theEuropean Union under the European Regional Development Fund;A*STAR, Singapore and NSCC, Singapore.

Background The heat shock transcription factor, HSF1, is the main regulator of the proteotoxic stress response that orchestrates the adaptation of cells to stress conditions such as elevated temperature, oxidative stress, and proteotoxic stress. As such, HSF1 regulates a large number of stress response-related genes, primarily those encoding heat shock proteins (HSPs). HSPs are molecular chaperones involved in the acquisition of native protein conformations and the prevention of protein degradation, and they also contribute to the removal of denatured proteins via the proteasome. Representative members of the HSP family are HSP70 and HSP90. The stress response is a highly conserved mechanism across all eukaryotes, and HSF1 has been linked to a number of physiological processes (ribosomal biogenesis, translation, transcription, cell cycle, and metabolism) and pathological disorders (neurodegenerative disorders such as Parkinson´s and Alzheimer´s diseases). HSF1 activation is also prominent in different types of cancer (prostate, breast, colorectal carcinoma etc.) where it correlates with tumor aggressiveness and poor prognosis. HSF1 is therefore considered a diagnostic and prognostic marker and is currently being targeted to develop new cancer therapies. Several inhibitors of HSF1 have already been synthesized, but their molecular mechanism (s) of action, specificity those of HSF1, nontoxicity in healthy tissues, and their efficacy in targeting tumor cells remain to be elucidated. Purpose This review summarizes known mechanisms of HSF1 regulation and activation, the role of HSF1 during malignant transformation, and the potential of designing small molecule HSF1 inhibitors for cancer therapy. Key words: HSF1 transcription factor - molecular chaperones - cellular stress - tumor transformation - cancer This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 10. 8. 2018.

Response of tumours to Hsp90 inhibitors is highly variable and their clinical effects are unpredictable, emphasising the need for a predictive marker. We postulated that sensitivity to Hsp90 inhibitors is connected to basal proteotoxic stress that makes cells dependent on Hsp90. Therefore, we assessed HSF1 as a general sensor of proteotoxic stress and correlated its activity with sensitivity to three separate small molecule Hsp90 inhibitors in seven breast cancer cell lines representing each of the different cancer subtypes. Flow cytometry was used to analyse the viability of breast cancer cell lines after Hsp90 inhibition. HSF1 activity was characterised by Ser326 phosphorylation and the transactivation capacity of HSF1 was determined by qPCR analysis of the ratios of HSF1-dependent (HOP, Hsp70) and HSF1-independent (CHIP) chaperones and cochaperone mRNAs. We show that the sensitivity of breast cancer cell lines to Hsp90 inhibition is highly variable. The basal levels of phosphorylated HSF1 also vary between cell lines and the magnitude of change in HSF1 phosphorylation after Hsp90 inhibition showed a negative correlation with sensitivity to Hsp90 inhibitors. Similarly, the basal transactivation capacity of HSF1, determined by the ratio of Hsp70 or HOP mRNA to CHIP mRNA level, is directly proportional to sensitivity to Hsp90 inhibitors. Increasing basal HSF1 activity by prior heat shock sensitised cells to Hsp90 inhibition. These results demonstrate that endogenous HSF1 activity varies between individual cancer cell lines and inversely reflects their sensitivity to Hsp90 inhibitors, suggesting that basal proteotoxic stress is an important and generalised predictor of response. Mechanistically, the data indicate that high endogenous proteotoxic stress levels sensitise to Hsp90 inhibition due to the inability to respond adequately to further proteotoxic stress. HSF1 activity therefore represents a potential biomarker for therapy with Hsp90 inhibitors, which may be useful for the rational design of future clinical studies.

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λ(max) of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction.

Many human cancers contain missense TP53 mutations that result in p53 protein accumulation. Although generally considered as a single class of mutations that abrogate wild-type function, individual TP53 mutations may have specific properties and prognostic effects. Tumours that contain missense TP53 mutations show variable p53 stabilization patterns, which may reflect the specific mutation and/or aspects of tumour biology. We used immunohistochemistry on cell lines and human breast cancers with known TP53 missense mutations and assessed the effects of each mutation with four structure–function prediction methods. Cell lines with missense TP53 mutations show variable percentages of cells with p53 stabilization under normal growth conditions, ranging from approximately 50% to almost 100%. Stabilization is not related to structural or functional disruption, but agents that stabilize wild-type p53 increase the percentages of cells showing missense mutant p53 accumulation in cell lines with heterogeneous stabilization. The same heterogeneity of p53 stabilization occurs in primary breast cancers, independent of the effect of the mutation on structural properties or functional disruption. Heterogeneous accumulation is more common in steroid receptor-positive or HER2-positive breast cancers and cell lines than in triple-negative samples. Immunohistochemcal staining patterns associate with Mdm2 levels, proliferation, grade and overall survival, whilst the type of mutation reflects downstream target activity. Inhibiting Mdm2 activity increases the extent of p53 stabilization in some, but not all, breast cancer cell lines. The data indicate that missense mutant p53 stabilization is a complex and variable process in human breast cancers that associates with disease characteristics but is unrelated to structural or functional properties. That agents which stabilize wild-type p53 also stabilize mutant p53 has implications for patients with heterogeneous mutant p53 accumulation, where therapy may activate mutant p53 oncogenic function. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.

The TP63 gene gives rise to protein isoforms with different properties and functions due to the presence (TAp63) or absence (ΔNp63) of an N-terminal p53-like transactivation domain. Immunohistochemistry for p63 has clinical value for certain tumour types, but investigations have been hampered by a lack of well characterized antibodies and the inability to discriminate between these N-terminal isoforms with opposite functional properties. We have extensively characterized a series of monoclonal antibodies to recombinant human TAp63 and two commercial p63 monoclonals by Western blot, immunostaining and phage display epitope mapping. Twenty-eight of 29 (96.6 %) novel monoclonals that recognized all p63 isoforms showed substantial cross-reactivity with p73, as did the commercial antibody, 4A4. One novel clone, PANp63-6.1, showed slight cross-reaction with p73 by Western blotting but not immunohistochemistry and the SFI-6 monoclonal did not cross-react with p73 or p53. Phage display revealed that the PANp63-6.1 epitope has one amino acid difference between p63 and p73, the 4A4 epitope is identical in both, whereas the SFI-6 epitope is unique to p63, accounting for these findings. We also produced and characterized a TAp63-specific clone that does not recognize p53 or p73, and we prepared polyclonal sera specific for ΔNp63 isoforms. Immunohistochemistry demonstrated that TAp63 is expressed in a variety of epithelial and other cell types during development, often in a converse pattern to ΔNp63, but has a very limited expression in normal adult tissues and is independent of ΔNp63. TAp63 was expressed in 17.6 % of squamous cancers of cervix that expressed p63, unlike normal cervix where TAp63 was not expressed. TAp63 did not associate with proliferative index, but cervical carcinomas with TAp63 expression showed improved survival. These data highlight the need for rigorous antibody characterization and indicate that p63-isoform identification may improve the clinical value of p63 expression analyses.

Background Chaperone system inhibition is a recent promising strategy for cancer treatment that exploits increased metabolic needs required for rapid proliferation as well as higher level of proteotoxic stress in neoplastic cells. Chaperone HSP90 plays a key role in proper folding of many de novo synthesized proteins, so-called clients, including tumor suppressor p53 which is commonly mutated in majority of cancers. Aim of this work was therefore to understand the impact of HSP90 inhibition by NVP-AUY922 on breast cancer cell lines with wild-type and mutated p53. Methods Flow cytometry was used to analyze cell viability by fluorescein diacetate assay and changes in cell cycle. Western blotting was used to analyze expression of p53 and p21 proteins. Results Analysis of cell viability after HSP90 inhibition revealed higher sensitivity of cell line with wild-type p53. Cell cycle analysis then showed that both cell lines undergo increase in G2/M block of the cell cycle, but wild-type cell line had also substantial decrease in proliferative capacity of treated cells. We also observed increased expression of negative cell cycle regulator p21 in cell line with wild-type p53. Conclusions Since p21 is directly regulated by p53, our results suggest that mutation status of p53 can be important factor in treatment of breast cancer cells by HSP90 chaperone inhibition and that wild-type p53 can increase sensitivity to HSP90 inhibition.Key words: breast cancer - cell cycle - chaperone - HSP90 - TP53 - p21 - p53 - NVP-AUY922This work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 17. 5. 2016.

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

Heat shock proteins Hsp90 and Hsp70 facilitate protein folding but can also direct proteins for ubiquitin-mediated degradation. The mechanisms regulating these opposite activities involve Hsp binding to co-chaperones including CHIP and HOP at their C-termini. We demonstrated that the extreme C-termini of Hsp70 and Hsp90 contain phosphorylation sites targeted by kinases including CK1, CK2 and GSK3-β in vitro. The phosphorylation of Hsp90 and Hsp70 prevents binding to CHIP and thus enhances binding to HOP. Highly proliferative cells contain phosphorylated chaperones in complex with HOP and phospho-mimetic and non-phosphorylable Hsp mutant proteins show that phosphorylation is directly associated with increased proliferation rate. We also demonstrate that primary human cancers contain high levels of phosphorylated chaperones and show increased levels of HOP protein and mRNA. These data identify C-terminal phosphorylation of Hsp70 and Hsp90 as a switch for regulating co-chaperone binding and indicate that cancer cells possess an elevated protein folding environment by the concerted action of co-chaperone expression and chaperone modifications. In addition to identifying the pathway responsible for regulating chaperone-mediated protein folding/degradation balances in normal cells, the data provide novel mechanisms to account for the aberrant chaperone activities observed in human cancer cells and have implications for the application of anti-chaperone therapies in cancer treatment.

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20-40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP ( C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1.CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 ( residues 106-140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or "docking" of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase.

Objectives: SNP309 polymorphism (T-G) at the promoter region of MDM2 has been reported to cause increased binding affinity of transcriptional activator Sp1 followed by increased MDM2 both in mRNA and protein level. This model was proposed in vitro in the small panel of cell lines that indicated an on average 8-fold higher level of MDM2 mRNA in cells bearing the GG genotype. Methods: The incidence of SNP309 was determined in a cohort of 158 breast, 17 endometrium, 13 cervix and 45 ovarian cancer tissues by PCR-RFLP. The expression of p53 and MDM2 protein levels in the cohort was immunohistochemically investigated and statistically correlated with SNP309 polymorphism using Pearson χ2 and t test. Results: No significant difference was observed in the G allele incidence in breast cancer specimens compared to 149 noncancer controls. Furthermore, no statistically significant association of the G allele frequencies and p53 and MDM2 protein expression levels was observed. Conclusions: Our data clearly show neither association between SNP309 and cancer risk, nor the responsibility of G allele for increased MDM2 or decreased of p53 protein levels in human primary breast tumors.

Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 °C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and RNA polymerase II mediated transcription. Several pharmacological CDK inhibitors are currently in clinical trials as potential cancer therapeutics and some of them also exhibit antiviral effects. Olomoucine II and roscovitine, purine-based inhibitors of CDKs, were described as effective antiviral agents that inhibit replication of a broad range of wild type human viruses. Olomoucine II and roscovitine show high selectivity for CDK7 and CDK9, with important functions in the regulation of RNA polymerase II transcription. RNA polymerase II is necessary for viral transcription and following replication in cells. We analyzed the effect of inhibition of CDKs by olomoucine II on gene expression from viral promoters and compared its effect to widely-used roscovitine. We found that both roscovitine and olomoucine II blocked the phosphorylation of RNA polymerase II C-terminal domain. However the repression of genes regulated by viral promoters was strongly dependent on gene localization. Both roscovitine and olomoucine II inhibited expression only when the viral promoter was not integrated into chromosomal DNA. In contrast, treatment of cells with genome-integrated viral promoters increased their expression even though there was decreased phosphorylation of the C-terminal domain of RNA polymerase II. To define the mechanism responsible for decreased gene expression after pharmacological CDK inhibitor treatment, the level of mRNA transcription from extrachromosomal DNA was determined. Interestingly, our results showed that inhibition of RNA polymerase II C-terminal domain phosphorylation increased the number of transcribed mRNAs. However, some of these mRNAs were truncated and lacked polyadenylation, which resulted in decreased translation. These results suggest that phosphorylation of RNA polymerase II C-terminal domain is critical for linking transcription and posttrancriptional processing of mRNA expressed from extrachromosomal DNA.

Interferon-inducible protein 16 (IFI16) is a member of the HIN-200 protein family, containing two HIN domains and one PYRIN domain. IFI16 acts as a sensor of viral and bacterial DNA and is important for innate immune responses. IFI16 binds DNA and binding has been described to be DNA length-dependent, but a preference for supercoiled DNA has also been demonstrated. Here we report a specific preference of IFI16 for binding to quadruplex DNA compared to other DNA structures. IFI16 binds to quadruplex DNA with significantly higher affinity than to the same sequence in double stranded DNA. By circular dichroism (CD) spectroscopy we also demonstrated the ability of IFI16 to stabilize quadruplex structures with quadruplex-forming oligonucleotides derived from human telomere (HTEL) sequences and the MYC promotor. A novel H/D exchange mass spectrometry approach was developed to assess protein interactions with quadruplex DNA. Quadruplex DNA changed the IFI16 deuteration profile in parts of the PYRIN domain (aa 0-80) and in structurally identical parts of both HIN domains (aa 271-302 and aa 586-617) compared to single stranded or double stranded DNAs, supporting the preferential affinity of IFI16 for structured DNA. Our results reveal the importance of quadruplex DNA structure in IFI16 binding and improve our understanding of how IFI16 senses DNA. IFI16 selectivity for quadruplex structure provides a mechanistic framework for IFI16 in immunity and cellular processes including DNA damage responses and cell proliferation.

Activation of the Hsp90 chaperone system is a characteristic of cancer cells. The regulation of chaperone activities involves their interaction with cochaperones; therefore we investigated the expression of Hsp70 and Hsp90 and their specific co-chaperones HOP and CHIP in cancer cell lines and primary cancers. Inhibition of Hsp90 by 17AAG increased the levels of Hsp70, Hsp90 and HOP but not CHIP mRNA in cancer cells. These changes are linked to activation of the HSF1 transcription factor and we show that the HOP promoter contains HSF1 binding sites, and that HSF1 binding to the HOP promoter is increased following 17AAG. The lack of alteration in the co-chaperone CHIP is explained by a lack of HSF response elements in the CHIP promoter. Non-proliferating cells expressed higher levels of CHIP and lower HOP, Hsp70 and Hsp90 levels compared to proliferating cells. Decreased expression of CHIP in proliferating cancer cells is in keeping with its proposed tumor suppressor properties, while over-expression of HOP in proliferating cells may contribute to excessive Hsp90 activity and stabilization of client proteins in tumors. In a panel of colorectal cancer samples, increased expression of Hsp70 and an increased ratio of HOP to CHIP were found, and were associated with decreased median survival. These data indicate that multiple changes occur in the chaperone/co-chaperone system in cancer that impact patient survival. It is likely that the ability to identify individual alterations to this system will be beneficial for treatment strategy decisions, particularly those that employ chaperone inhibitors.

p53 missense mutant proteins commonly show increased stability compared to wild-type p53, which is thought to depend largely on the inability of mutant p53 to induce the ubiquitin ligase MDM2. However, recent work using mouse models has shown that the accumulation of mutant p53 occurs only in tumour cells, indicating that stabilization requires additional factors. To clarify the stabilization of p53 mutants in tumours, we analysed factors that affect their folding and degradation. Although all missense mutants that we studied are more stable than wild-type p53, the levels correlate with individual structural characteristics, which may be reflected in different gain-of-function properties. In the absence of Hsp90 activity, the less stable unfolded p53 mutants preferentially associate in a complex with Hsp70 and CHIP (carboxy terminus of Hsp70-interacting protein), and we show that CHIP is responsible for ubiquitination and degradation of these mutants. The demonstration of a complex interplay between Hsp90, Hsp70 and CHIP that regulate the stability of different p53 mutant proteins improves our understanding of the pro-tumorigenic effects of increased Hsp90 activity during multi-stage carcinogenesis. Understanding the roles of Hsp90, Hsp70 and CHIP in cancers may also provide an important avenue through which to target p53 to enhance treatment of human cancers.

Development of recombinant therapeutic antibodies is recently one of the fastest growing disciplines of applied biomedical research. Recombinant monoclonal antibodies are increasingly applied in biological therapy of many serious human diseases and are currently an irreplaceable part of a comprehensive cancer therapy. First mouse therapeutic antibodies had only limited applicability due to the strong immune response; however, technological advances enabled engineering of antibodies with increased specificity and efficacy, and on the other hand with reduced adverse effects due to lower antigenicity. This review provides a summary of knowledge about recombinant therapeutic antibodies, their mechanism of action and approaches how to improve their efficacy.

Background Cisplatin and its derivatives are commonly used anti-cancer drugs. However, cisplatin has clinical limitations including serious side effects and frequent emergence of intrinsic or acquired resistance. Thus, the novel platinum(IV) complex LA-12 represents a promising treatment modality, which shows increased intracellular penetration resulting in improved cytotoxicity in various cancer cell lines, including cisplatin resistant cells. Results LA-12 disrupts cellular proliferation regardless of the p53 status in the cells, however the potency of the drug is greatly enhanced by the presence of a functional p53, indicating several mechanisms of action. Similarly to cisplatin, an interaction of LA-12 with molecular chaperone Hsp90 was proposed. Binding of LA-12 to Hsp90 was demonstrated by Hsp90 immunoprecipitation followed by platinum measurement using atomic absorption spectrometry (AAS). An inhibitory effect of LA-12 on Hsp90 chaperoning function was shown by decrease of Hsp90-assisted wild-type p53 binding to p21WAF1 promoter sequence in vitro and by accelerated ubiqutination and degradation of primarily unfolded mutant p53 proteins in cells exposed to LA-12. Conclusions To generalize our findings, LA-12 induced degradation of other Hsp90 client proteins such as Cyclin D1 and estrogen receptor was shown and proved as more efficient in comparison with cisplatin. This newly characterised molecular mechanism of action opens opportunities to design new cancer treatment strategy profitable from unique LA-12 properties, which combine DNA damaging and Hsp90 inhibitory effects.