Plurifaceted proteomics in studying cellular dynamics and action mechanisms of anticancer drugs

Mass spectrometry (MS)-based proteomics has developed tremendously in recent years and was the leading technology for many novel methods to study protein chemistry. Contrary to classical approaches based on Western blot, MS-based approaches are mostly unbiased. In addition to protein expression levels, today several protein chemical properties can be examined by MS-based proteomics making it unique in comparison to transcriptomics approaches. These properties include post-translational modifications (PTMs), protein localization, synthesis/degradation, and lastly protein thermal stability, adding novel dimensions or facets to characterize the proteome. However, these facets are difficult to combine as they are mainly orthogonal and are therefore often analyzed separately. This thesis presents a simplified and higher throughput version of current protein stability analyses and showcases the advantages of combined/merged analysis of proteomics facets including our new method, as well as expression and redox proteomics to study anticancer treatments and cellular dynamics. In paper I, we studied the dynamics of cancer cells in vitro with and without anticancer treatment over the course of 48 h by monitoring protein expression every 6 h. We discovered that naturally occurring proteome variations are on par with anticancer treatment killing 50% of the cells after 48 h. Then, we acquired a deep proteomics dataset of untreated HCT116 and A375 cell lines. Surprisingly, we observed downregulation of proteins involved in cell division and upregulation of proteins involved in metabolism as early as 12 h after treatment, suggesting that growth inhibition happens earlier than usually assumed and even at low cell confluence. In paper II we developed the proteome integral solubility alteration (PISA) assay that increases the throughput of pre-existing drug target deconvolution methods based on protein stability/solubility measurements and reduces the analysis time and cost as well as sample requirements. We provided theoretical calculations showing that the integral of the curve correlates well with melting temperature estimations in Thermal Proteome Profiling (TPP) and tested our assumptions with publicly available TPP datasets. Then we performed a proof of principle experiment using the well-studied methotrexate (MTX) and 5-fluorouracile (5-FU) as test drugs highlighting the targets as outliers with our method. Furthermore, we demonstrated that PISA assay can also be used for concentration series analysis as in 2D-TPP. Finally, we showcased the higher throughput of PISA compared to TPP by simultaneously analyzing nine drugs in one multiplexed analysis. In paper III we used a combination of chemical proteomics approaches to study the target and mechanism of action (MOA) of Auranofin (AF) (Ridaura®). Functional Identification of Target by Expression Proteomics (FITExP), TPP, and redox proteomics combined highlighted that thioredoxin reductase 1 (TXNRD1) is indeed a top target hit of AF, and that the main MOA of the drug is through disruption of the redox balance in the cell. Finally, we showed that protein thermal shifts can be associated with altered cysteine oxidation levels in proteins, suggesting that TPP is suitable to study disulfide bond formation/reduction and map some cysteines to the active sites of sulfiredoxin 1 (SRXN1) and peroxiredoxin 5 (PRDX5) as examples. Overall, our study demonstrates that using only one of the proteomics methods is not sufficient to accurately pinpoint drug target and MOA, but a combination of multiple complementary methods should be used instead. Following the success of our strategy in paper III, we decided to utilize the same strategy in paper IV using PISA developed in paper II instead of TPP, to study two novel inhibitors of TXNRD1, namely TRi-1 and TRi-2. For these studies, we used the mouse cancer cell lines B16-F10 and Lewis lung carcinoma (LLC) to provide a comprehensive analysis of the therapeutic effects of the inhibitors. Since we showed that Txnrd1 (the mouse version of the human TXNRD1 protein) is a main target of AF, we decided to use it as our reference point to evaluate the specificity of the two new compounds. AF had a broader effect on the proteome than TRi-1 and TRi-2 and had more target hits in PISA analyses. This suggested that AF has lower specificity than TRi-1 and TRi-2. TRi-1 was the most specific Txnrd1 inhibitor with a better target ranking in FITExP and the least target hits in PISA followed by TRi-2 and AF. Thus, we showed that TRi-1 and TRi-2 are indeed more specific inhibitors of Txnrd1. In addition to these findings, we also highlighted that only AF triggers a high nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant response, suggesting that this response is not necessarily Txnrd1-dependent. Finally, we detected selenocysteine-specific elongation factor (Eefsec), mini-chromosome maintenance complex-binding protein (Mcmbp), glycogen synthase kinase-3 (Gsk3) a and b, as target hits of AF, which would explain at least partially three different effects of AF treatment. Collectively, our data represents a resource for redox biologists interested in Txnrd1 inhibition. Our approach provides a framework for target deconvolution using proteomics approaches. Finally, in paper V we studied the dynamics of the proteome in transition between various cell types. We reprogrammed human foreskin fibroblasts (hFF) into induced pluripotent stem cells (iPSCs), which we differentiated through embryoid bodies (EBs) formation. We examined protein expression and stability after each cell type transition using PISA-Express, a new version of the PISA assay developed in paper II. We merged the readout from protein expression and thermal stability in one analysis using Sankey diagrams to detect changes in protein properties during proteome transitions resulting in the ProteoTracker web interface (http://www.proteotracker.genexplain.com/). Using this innovative analysis, we discovered that ribosomes are less stable in pluripotent stem cells (PSCs) compared to differentiated cells and that this difference stems from the deficiency of one ribosome maturation factor, Shwachman-Bodian-Diamond syndrome protein (SBDS). Knock-down (KD) of SBDS slowed down translation and increased expression of the master pluripotency markers homeobox protein NANOG (NANOG), and octamer-binding transcription factor 4 (OCT4). Collectively, we developed a new method for simultaneous analysis of protein thermal stability and expression, a new analysis and visualization tool, and provided evidence that control of translation through ribosome biogenesis is a natural mechanism used by PSCs to maintain the pluripotency state

Universality of Low-Energy Scattering in 2+1 Dimensions: The Non Symmetric Case

For a very large class of potentials, $V(\vec{x})$, $\vec{x}\in R^2$, we prove the universality of the low energy scattering amplitude, $f(\vec{k}', \vec{k})$. The result is $f=\sqrt{\frac{\pi}{2}}\{1/log k)+O(1/(log k)^2)$. The only exceptions occur if $V$ happens to have a zero energy bound state. Our new result includes as a special subclass the case of rotationally symmetric potentials, $V(|\vec{x}|)$.Comment: 65 pages, Latex, significant changes, new sections and appendice

L'élevage, le conseil, et l'écopathologie. Résultats d'une approche didactique de situations de diagnostic d'élevage en production porcine

International audienc

Dynamiques fluviales en basse vallée du Vidourle

International audienc

Relationships of Cardiorespiratory Fitness with Metabolic Risk Factors, Inflammation, and Liver Transaminases in Overweight Youths

The aim of this study was to assess the relationships of fatness and fitness with metabolic risk factors, including liver transaminases and inflammation in obese youth, taking in account gender, age, and pubertal stage. 241 children were studied (135 girls), age 11.9 ± 2.2 years (x ± SD), Body Mass Index z score 5.4 ± 2.7. For girls, VO2max was significantly associated with insulin (P = .001), Insulin resistance (HOMA-IR) (P = .005), and ALT (P = .012); a relationship was displayed between fibrinogen and age and % fat mass (FM) (P = .008); for boys, relationships were found between VO2max and diastolic blood pressure and triglycerides; independent associations were also found between age and insulin, HOMA-IR and HDL cholesterol; fibrinogen and sedimentation rate were related (P ≤ .004) with %FM. Their relationships are observed from young age and increase with the continuous increase of factors. This supports the need to treat overweight as soon as it is detected; improving CRF is one of the ways which could be used to prevent the complications of obesity

Vertex-reinforced random walk on Z eventually gets stuck on five points

Vertex-reinforced random walk (VRRW), defined by Pemantle in 1988, is a random process that takes values in the vertex set of a graph G, which is more likely to visit vertices it has visited before. Pemantle and Volkov considered the case when the underlying graph is the one-dimensional integer lattice Z. They proved that the range is almost surely finite and that with positive probability the range contains exactly five points. They conjectured that this second event holds with probability 1. The proof of this conjecture is the main purpose of this paper.Comment: Published by the Institute of Mathematical Statistics (http://www.imstat.org) in the Annals of Probability (http://www.imstat.org/aop/) at http://dx.doi.org/10.1214/00911790700000069

Influence of sample location and livestock numbers on Sporormiella concentrations and accumulation rates in surface sediments of Lake Allos, French Alps

International audienceSpores of coprophilous fungi, especially Sporormiella, are often well preserved in lake sediment cores. It has been hypothesized that such spores can be used to quantify past livestock abundance. The quantitative relationship between fungal spore abundance and livestock populations, however, is not well established, nor are the mechanisms of spore transport and deposition in lacustrine systems. Multiple cores from Lake Allos, a large high-elevation lake in the French Alps, were used to map the modern abundance of Sordaria and Sporormiella spores throughout the lake. We observed high spatial heterogeneity with respect to spore numbers. No correlation with the distance from shoreline was found. There was, however, a relation with distance from the two main lake inlets. These results were used to select two fungi-rich sediment cores to investigate grazing pressure over the last two centuries. Comparisons were made between spore influx and historic data on livestock densities in the catchment. A sharp decrease in Sporormiella influx ca. 1894-1895 was associated with a reported reduction in sheep in the Allos catchment at that time. Mean influx of Sporormiella decreased by a factor of three between the nineteenth and twentieth centuries, reflecting a reduction in the reported number of animals in the Lake Allos catchment, from 6,000 to 2,000. This study confirmed that Sporormiella spore abundance in lake sediments can be used as a proxy for catchment herbivore numbers in paleoecological reconstructions. Nevertheless, our data indicate that before spore accumulation can be used to infer past domestic herbivore density, one must understand the processes of coprophilous spore transfer from the catchment to the lake and the influence of core location on spore numbers in the sediment

Uncovering a Paleotsunami Triggered by Mass-Movement in an Alpine Lake

Mass movements and delta collapses are significant sources of tsunamis in lacustrine environments, impacting human societies enormously. Palaeotsunamis play an essential role in understanding historical events and their consequences along with their return periods. Here, we focus on a palaeo event that occurred during the Younger Dryas to Early Holocene climatic transition, ca., 12,000 years ago in the Lake Aiguebelette (NW Alps, France). Based on highresolution seismic and bathymetric surveys and sedimentological, geochemical, and magnetic analyses, a seismically induced large mass transport deposit with an initial volume of 767172 m3 was identified, dated and mapped. To investigate whether this underwater mass transport produced a palaeotsunami in the Lake Aiguebelette, this research combines sedimentary records and numerical models. Numerical simulations of tsunamis are performed using a viscoplastic landslide model for tsunami source generation and two-dimensional depth-averaged nonlinear shallow water equations for tsunami wave propagation and inundation modelling. Our simulations conclude that this sublacustrine landslide produced a tsunami wave with a maximum amplitude of approximately 2 m and run-up heights of up to 3.6 m. The modelled sediment thickness resulting from this mass transport corroborates well with the event deposits mapped in the lake. Based on our results, we suggest that this sublacustrine mass transport generated a significant tsunami wave that has not been reported previously to the best of our knowledge.Comment: Advances in Hydroinformatics, O. Delestre (Polytech Nice Sophia -- University C{\^o}te d'Azur, France), Nov 2023, Chatou, Franc