Project description:Despite the involvement of several serine hydrolases (SHs) in the metabolism of xenobiotics such as dibutyl phthalate (DBP), no study has focused on mapping this enzyme class in zebrafish, a model organism frequently used in ecotoxicology. Here, we survey and identify active SHs in zebrafish larvae and search for biological markers of SH type after exposition to DBP. Zebrafish were exposed to 0, 5, and 100 µg/L DBP from 4 to 120 h post-fertilization. A significant decrease in vitellogenin expression level of about 2-fold compared to the control was found in larvae exposed to 100 µg/L DBP for 120h. The first comprehensive profiling of active SHs in zebrafish proteome was achieved with an activity-based protein profiling (ABPP) approach. Among 49 SHs identified with high confidence, one was the carboxypeptidase ctsa overexpressed in larvae exposed to 100 µg/L DBP for 120h. To the best of our knowledge, this is the first time that a carboxypeptidase has been identified as deregulated following exposure to DBP. The overall results indicate that targeted proteomics approaches such as ABPP can therefore be an asset for understanding the mechanism of action related to xenobiotics in ecotoxicology.
Project description:Identification of hemoglobin-derived fragments generated in the anterior midgut of Rhodnius prolixus in vivo at 14 days post-feeding. The results allowed to draw the hemoglobin cleavage map by midgut-associated peptidases.
Project description:Aging studies using animal and cellular models have uncovered key proteins and pathways central to organismal aging. However, these models differ genetically and physiologically from human aging, posing challenges in translating discoveries to human contexts. In this study, we present a human normal cell aging model based on the development of cytotrophoblasts (CTBs) to syncytiotrophoblasts (STBs) in the placenta. The in vitro-derived STBs from human trophoblast stem cells (hTSCs) recapitulate the maturation and major cellular aging features of in vivo CTB-STB, including multinucleation, hormone secretion, cell cycle arrest, genome instability, epigenetic changes, activation of endogenous transposable elements, and senescence-associated secretory phenotypes (SASPs). Notably, the progressive senescence in the trophoblast system closely matches the predicted aging trajectory of other human tissue stem cells. Known anti-aging molecules, such as mTOR inhibitors and senolytics, attenuate senescence signals in STBs. The established CGA-EGFP reporter hTSC line enables scalable and quantitative screening and identified candidates with it can be further extended to other context-specific aging processes like that of skin fibroblasts. The hTSC-STB system represents a novel physiologically accelerated cellular aging model, bridges the gap between fundamental aging research and interventions, and prioritizes anti-aging candidates for clinical development.
Project description:The establishment of a replicative niche in the hostile environment of the host presents an enormous challenge for pathogens. Intracellular pathogens such as Brucella spp., the Gram-negative causative agents of Brucellosis, must subvert diverse host functions to ensure survival and replication. To schieve this, Brucella translocates effector proteins into host cells via its type 4 secretion system (T4SS), a key virulence factor. Here, we demonstrate that a subset of previously described effector proteins associates with outer membrane vesicles (OMVs) in different Brucella species. Most of these effector proteins encode predicted signal peptides for periplasmic export or transmembrane domains. Among them, BspC and VceA carry functional signal peptides that direct their export into the periplasm in a Sec-dependent manner. From the periplasm these proteins are subsequently secreted into the extracellular milieu, likely via the formation and release of OMVs. Our findings provide new insights into protein secretion by Brucella, suggesting that OMVs may represent an alternative secretion pathway to the T4SS.
Project description:N6-methyladenosine (m6A) is the most prevalent mRNA modification with diverse regulatory roles in mammalian cells. While its functions are well-documented in mouse embryonic stem cells (mESCs), its role in human pluripotent stem cells (hPSCs) remains to be fully explored. METTL3 is the main enzyme responsible for m6A deposition. Here, using a METTL3 inducible knockout (iKO) system, we uncovered that, unlike in mESCs, METTL3 was indispensable for hPSC maintenance. Importantly, loss of METTL3 caused significant upregulation of pluripotency factors including naïve pluripotency genes and failure to exit pluripotency, thus impaired stem cell differentiation towards embryonic and extraembryonic cells including trophoblasts. Mechanistically, METTL3 iKO in hPSCs substantially increased expression and enhancer activities of two primate-specific transposable elements (TEs), SVA_D and HERVK/LTR5_Hs, which are normally modified by METTL3-dependent m6A. METTL3 loss activated SVA_D by lowering H3K9me3 deposition, and increased chromatin accessibility at LTR5_Hs through the naïve and other pluripotency factors. Conversely, we discovered that the activated SVA_D and LTR5_Hs loci positively regulated naïve gene expression by directly interacted with their promoters. These findings thus reveal that METTL3-dependent m6A RNA methylation has critical roles in suppressing TE expression and in the human pluripotency regulatory network.
Project description:Treatment of Mycobacterium tuberculosis infections is a challenging task due to a growing number of resistant clinical isolates as well as an almost empty drug development pipeline. To identify new antibiotic hits, we screened a focused library of 400 synthetic compounds derived from a recently discovered molecule with promising anti-mycobacterial activity. A suite of more potent hit molecules was deciphered with sub-micromolar activity. Utilising tailored affinity-based probes for chemical proteomic investigations, we successfully pinpointed the mycolic acid transporter MmpL3 and two epoxide hydrolases, EphD and EphF, also linked to mycolic acid biosynthesis, as specific targets of the compounds. These targets were thoroughly and independently validated by activity assays, under- and overexpression, resistance generation, and proteomic studies. Structural refinement of the most potent hit molecules led to the development of a new lead compound that demonstrates enhanced biological activity in M. tuberculosis, low human cytotoxicity, and improved solubility and oral bioavailability − traits that are often challenging to achieve with anti-mycobacterial drugs. Overall, drug-likeness, as well as the dual mode of action, addressing the mycolic acid cell wall assembly at two distinct steps, holds significant potential for further in vivo applications.