Systemic Convergent Multitarget Interactions of Plant Polyphenols Revealed by Affinity-Based Protein Profiling of Bone Cells Using C-Glucosidic Vescal(ag)in-Bearing Chemoproteomic Probes.

The ellagitannins vescalagin and vescalin, known as actin-dependent inhibitors of osteoclastic bone resorption, were mounted onto chemical probes to explore their interactions with bone cell proteins by means of affinity-based chemoproteomics and bioinformatics. The chemical reactivity of the pyrogallol units of these polyphenols toward oxidation into electrophilic ortho-quinones was exploited using NaIO4 to promote the covalent capture of target proteins, notably those expressed at lower abundance and those interacting with polyphenols at low-to-moderate levels of affinity. Different assays revealed the multitarget nature of both ellagitannins, with 100-370 statistically significant proteins captured by their corresponding probes. A much higher number of proteins were captured from osteoclasts than from osteoblasts. Bioinformatic analyses unveiled a preference for the capture of proteins having phosphorylated ligands and GTPase regulators and enabled the identification of 33 potential target proteins with systemic relevance to osteoclast differentiation and activity, as well as to the regulation of actin dynamics.

Synthesis of Flavonol-Bearing Probes for Chemoproteomic and Bioinformatic Analyses of Asteraceae Petals in Search of Novel Flavonoid Enzymes.

This study aimed at searching for the enzymes that are responsible for the higher hydroxylation of flavonols serving as UV-honey guides for pollinating insects on the petals of Asteraceae flowers. To achieve this aim, an affinity-based chemical proteomic approach was developed by relying on the use of quercetin-bearing biotinylated probes, which were thus designed and synthesized to selectively and covalently capture relevant flavonoid enzymes. Proteomic and bioinformatic analyses of proteins captured from petal microsomes of two Asteraceae species (Rudbeckia hirta and Tagetes erecta) revealed the presence of two flavonol 6-hydroxylases and several additional not fully characterized proteins as candidates for the identification of novel flavonol 8-hydroxylases, as well as relevant flavonol methyl- and glycosyltransferases. Generally speaking, this substrate-based proteome profiling methodology constitutes a powerful tool for the search for unknown (flavonoid) enzymes in plant protein extracts.

Targeting CAMKK2 and SOC Channels as a Novel Therapeutic Approach for Sensitizing Acute Promyelocytic Leukemia Cells to All-Trans Retinoic Acid.

Calcium ions (Ca2+) play important and diverse roles in the regulation of autophagy, cell death and differentiation. Here, we investigated the impact of Ca2+ in regulating acute promyelocytic leukemia (APL) cell fate in response to the anti-cancer agent all-trans retinoic acid (ATRA). We observed that ATRA promotes calcium entry through store-operated calcium (SOC) channels into acute promyelocytic leukemia (APL) cells. This response is associated with changes in the expression profiles of ORAI1 and STIM1, two proteins involved in SOC channels activation, as well as with a significant upregulation of several key proteins associated to calcium signaling. Moreover, ATRA treatment of APL cells led to a significant activation of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) and its downstream effector AMP-activated protein kinase (AMPK), linking Ca2+ signaling to autophagy. Pharmacological inhibition of SOC channels and CAMKK2 enhanced ATRA-induced cell differentiation and death. Altogether, our results unravel an ATRA-elicited signaling pathway that involves SOC channels/CAMKK2 activation, induction of autophagy, inhibition of cellular differentiation and suppression of cell death. We suggest that SOC channels and CAMKK2 may constitute novel drug targets for potentiating the anti-cancer effect of ATRA in APL patients.

TGFß promotes low IL10-producing ILC2 with profibrotic ability involved in skin fibrosis in systemic sclerosis.

OBJECTIVE: Innate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc). METHODS: Blood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc. RESULTS: We found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1- ILC2 (natural ILC2) were dominating over KLRG1+ ILC2 (inflammatory ILC2). The cytokine transforming growth factor-ß (TGFß), whose activity is increased in SSc, favoured the expansion of KLRG1- ILC2 simultaneously decreasing their production of interleukin 10 (IL10), which regulates negatively collagen production by dermal fibroblasts. TGFß-stimulated ILC2 also increased myofibroblast differentiation. Thus, human KLRG1- ILC2 had an enhanced profibrotic activity. In a mouse model of SSc, therapeutic intervention-combining pirfenidone with the administration of IL10 was required to reduce the numbers of skin infiltrating ILC2, enhancing their expression of KLRG1 and strongly alleviating skin fibrosis. CONCLUSION: Our results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFß and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.

Novel analytical methods to interpret large sequencing data from small sample sizes.

BACKGROUND: Targeted therapies have greatly improved cancer patient prognosis. For instance, chronic myeloid leukemia is now well treated with imatinib, a tyrosine kinase inhibitor. Around 80% of the patients reach complete remission. However, despite its great efficiency, some patients are resistant to the drug. This heterogeneity in the response might be associated with pharmacokinetic parameters, varying between individuals because of genetic variants. To assess this issue, next-generation sequencing of large panels of genes can be performed from patient samples. However, the common problem in pharmacogenetic studies is the availability of samples, often limited. In the end, large sequencing data are obtained from small sample sizes; therefore, classical statistical analyses cannot be applied to identify interesting targets. To overcome this concern, here, we described original and underused statistical methods to analyze large sequencing data from a restricted number of samples. RESULTS: To evaluate the relevance of our method, 48 genes involved in pharmacokinetics were sequenced by next-generation sequencing from 24 chronic myeloid leukemia patients, either sensitive or resistant to imatinib treatment. Using a graphical representation, from 708 identified polymorphisms, a reduced list of 115 candidates was obtained. Then, by analyzing each gene and the distribution of variant alleles, several candidates were highlighted such as UGT1A9, PTPN22, and ERCC5. These genes were already associated with the transport, the metabolism, and even the sensitivity to imatinib in previous studies. CONCLUSIONS: These relevant tests are great alternatives to inferential statistics not applicable to next-generation sequencing experiments performed on small sample sizes. These approaches permit to reduce the number of targets and find good candidates for further treatment sensitivity studies.