TDP2 as a biomarker of sensitivity to TOP2 targeting agents and as a novel therapeutic target

TDP2, a DNA phosphodiesterase that removes trapped topoisomerase 2 (TOP2) from 5’-DNA termini, is required for efficient repair of TOP2-induced DNA double-strand breaks (DSBs). Cellular depletion of TDP2 was shown to result in a substantially increased sensitivity to TOP2-induced DSBs and TOP2 poisons, such as etoposide, in various types of human cancer cell lines. In addition, over-expression of TDP2 has been shown to increase resistance to etoposide. Recent data suggest that expression levels of TDP2 vary greatly in different cancer cell lines. However, there are no reported studies addressing the possible role of TDP2 as a clinical predictor of anti-cancer therapy outcome, or wider studies correlating TDP2 over-expression with resistance to TOP2 poisons. TDP2 has the potential to be a good target for pharmacological inhibition potentially increasing tumour sensitivity to TOP2 poisons, particularly those that develop resistance during the course of treatment. There is already a lot of interest in the development of TDP2 inhibitors and the in vitro results are very promising with many possible small molecule inhibitors showing selectiveness in their target. In my thesis I aim to further establish the range of TDP2 and TOP2 mRNA and protein levels in a panel of lung and breast cancer cell lines. In addition, I will explore the possibility of a correlation between TDP2 protein levels or TOP2/TDP2 protein ratios and sensitivity to the TOP2 poison etoposide. This likely complex relationship will be further defined by possible mutation effects based on available literature and studies for the cancer cell lines accessible for this project. Furthermore, as etoposide has been tested in clinical trials not only alone but also in combination with other treatments, I aim to include other accessible drugs, either currently in use for cancer treatment or at a promising clinical trial stage, such as estradiol and PARP1 inhibitors. There is a recent model, which suggests that induction of transcriptional programs by stimulating breast cancer cells with estrogens, or prostate cancer cells with androgens, can involve the formation of TOP2B mediated DSBs and the recruitment of DSB repair proteins. TOP2B is believed to be recruited with the estrogen/androgen receptor to regulatory sites on target genes. It is hypothesized that the formation of these DSBs by TOP2B could also be exploited therapeutically. In my thesis I aim to utilise a combination treatment that will first induce transcription with estradiol in breast cancer cells, which will cause transient TOP2B-mediated DSBs, and then transform those breaks into abortive breaks with etoposide. Cells will then be overwhelmed with DSBs and apoptosis will be promoted. This will also be explored in TDP2-depleted MCF7 breast cancer cells. Such a strategy could possibly find particular use in hormone dependent cancers, where other types of treatment have failed

Self-assembly of temperature-responsive protein–polymer bioconjugates

We report a simple temperature-responsive bioconjugate system comprising superfolder green fluorescent protein (sfGFP) decorated with poly[(oligo ethylene glycol) methyl ether methacrylate] (PEGMA) polymers. We used amber suppression to site-specifically incorporate the non-canonical azide-functional amino acid p-azidophenylalanine (pAzF) into sfGFP at different positions. The azide moiety on modified sfGFP was then coupled using copper-catalyzed “click” chemistry with the alkyne terminus of a PEGMA synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The protein in the resulting bioconjugate was found to remain functionally active (i.e., fluorescent) after conjugation. Turbidity measurements revealed that the point of attachment of the polymer onto the protein scaffold has an impact on the thermoresponsive behavior of the resultant bioconjugate. Furthermore, small-angle X-ray scattering analysis showed the wrapping of the polymer around the protein in a temperature-dependent fashion. Our work demonstrates that standard genetic manipulation combined with an expanded genetic code provides an easy way to construct functional hybrid biomaterials where the location of the conjugation site on the protein plays an important role in determining material properties. We anticipate that our approach could be generalized for the synthesis of complex functional materials with precisely defined domain orientation, connectivity, and composition

Alpha-synuclein ferrireductase activity is detectible in vivo, is altered in Parkinson's disease and increases the neurotoxicity of DOPAL

© 2017 Elsevier Inc. The normal cellular role of α-synuclein is of potential importance in understanding diseases in which an aggregated form of the protein has been implicated. A potential loss or change in the normal function of α-synuclein could play a role in the aetiology of diseases such as Parkinson's disease. Recently, it has been suggested that α-synuclein could cause the enzymatic reduction of iron and a cellular increase in Fe(II) levels. Experiments were carried out to determine if such activity could be measured in vivo. Experiments with rats overexpressing human α-synuclein in nigral dopaminergic neurons demonstrated a correlation between α-synuclein expression and ferrireductase activity. Furthermore, studies on tissue from Parkinson's disease patient brains showed a significant decrease in ferrireductase activity, possibly due to deposition of large amounts of inactive protein. Cellular studies suggest that increase ferrireductase activity results in increased levels of dopamine metabolites and increased sensitivity to the toxicity of DOPAL. These findings demonstrate that α-synuclein ferrireductase activity is present in vivo and its alteration may play a role in neuron loss in disease

Steady-State Kinetics of α-Synuclein Ferrireductase Activity Identifies the Catalytically Competent Species

α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson’s disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe­(III) and Fe­(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity

Nanomedicines: The magic bullets reaching their target?

Nanomedicines, since the approval of the first one in the 1950s, have been accompanied by expectations of higher efficiency and efficacy, compared to less complex drugs. The fulfilment of those expectations has been slower than anticipated, due to the high complexity of nanomedicine drugs combined with a lack of scientific understanding of nanomedicine interactions with biological systems. The unique properties of their size and their surface composition create difficulties in their physicochemical characterization, and as a consequence, difficulty in assessing the similarity of follow-on products (nanosimilars) to originator nanomedicines. During the 2018 European Federation for Pharmaceutical Sciences (EUFEPS) annual meeting "Crossing the barrier for future medicines" in Athens, there were several sessions on nanomedicines organised by the EUFEPS Nanomedicine Network. This review focuses on the session "Nanomedicines and nanosimilars: how to assess similar?", discussing the nature of nanomedicines, the regulatory aspects of the topic and the impact of practical use and handling of such medicinal products. Emphasis is put on the consequences their nanosize-related properties have on the establishment of their critical quality attributes and how this affects the demonstration of bioequivalence of nanosimilars to their originator products. The lack of an appropriate and harmonized regulatory evaluation procedure and the absence of corresponding education are also discussed, especially the uncertainty surrounding the practical use of nanosimilars, including the higher healthcare cost due to less than satisfactory number of safe and efficacious nanosimilars in the market

Autopilot: An Online Data Acquisition Control System for the Enhanced High-throughput Characterization of Intact Proteins

The ability to study organisms by direct analysis of their proteomes without digestion via mass spectrometry has benefited greatly from recent advances in separation techniques, instrumentation, and bioinformatics. However, improvements to data acquisition logic have lagged in comparison. Past workflows for Top Down Proteomics (TDPs) have focused on high throughput at the expense of maximal protein coverage and characterization. This mode of data acquisition has led to enormous overlap in the identification of highly abundant proteins in subsequent LC-MS injections. Furthermore, a wealth of data is left underutilized by analyzing each newly targeted species as unique, rather than as part of a collection of fragmentation events on a distinct proteoform. Here, we present a major advance in software for acquisition of TDP data that incorporates a fully automated workflow able to detect intact masses, guide fragmentation to achieve maximal identification and characterization of intact protein species, and perform database search online to yield real-time protein identifications. On Pseudomonas aeruginosa, the software combines fragmentation events of the same precursor with previously obtained fragments to achieve improved characterization of the target form by an average of 42 orders of magnitude in confidence. When HCD fragmentation optimization was applied to intact proteins ions, there was an 18.5 order of magnitude gain in confidence. These improved metrics set the stage for increased proteome coverage and characterization of higher order organisms in the future for sharply improved control over MS instruments in a project- and lab-wide context

Optimizing capillary electrophoresis for top-down proteomics of 30-80 kDa proteins.

The direct analysis of intact proteins via mass spectrometry offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the ‘middle mass range’, defined here as proteins from 30-80 kDa, in a robust fashion has limited the adoption of these “top-down” methods. Largely a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary and a stacking method for on-column sample concentration were developed to achieve high loading capacity and separation resolution. We achieved full width at half maximum of 8-16 seconds for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from Pseudomonas aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.This work was supported by the National Institute of General Medical Sciences and National Institute of Drug Abuse of the National Institutes of Health under award numbers R01 GM067193 and DA018310, and the National Science Foundation under award number ABI-1062432 to Indiana University. Additional support was provided by the Chicago Biomedical Consortium with support from The Searle Funds at The Chicago Community Trust, and the Robert H. Lurie Comprehensive Cancer Center