Activity-Based Hydrazine Probes for Protein Profiling of Electrophilic Functionality in Therapeutic Targets

Most known probes for activity-based protein profiling (ABPP) use electrophilic groups that tag a single type of nucleophilic amino acid to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electrophilic enzyme cofactors, transient intermediates, and labile regulatory modifications, but ABPP probes for such species are underdeveloped. Here, we describe a versatile class of probes for this less charted hemisphere of the proteome. The use of an electron-rich hydrazine as the common chemical modifier enables covalent targeting of multiple, pharmacologically important classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment occurs by both polar and radicaloid mechanisms, can be blocked by molecules that occupy the active sites, and depends on the proper poise of the active site for turnover. These traits will enable the probes to be used to identify specific inhibitors of individual members of these multiple enzyme classes, making them uniquely versatile among known ABPP probes

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes [preprint]

Known chemoproteomic probes generally use warheads that tag a single type of amino acid or modified form thereof to identify cases in which its hyper-reactivity underpins function. Much important biochemistry derives from electron-poor enzyme cofactors, transient intermediates and chemically-labile regulatory modifications, but probes for such species are underdeveloped. Here, we have innovated a versatile class of chemoproteomic probes for this less charted hemisphere of the proteome by using hydrazine as the common chemical warhead. Its electron-rich nature allows it to react by both polar and radicaloid mechanisms and to target multiple, pharmacologically important functional classes of enzymes bearing diverse organic and inorganic cofactors. Probe attachment can be blocked by active-site-directed inhibitors, and elaboration of the warhead supports connection of a target to a lead compound. The capacity of substituted hydrazines to profile, discover and inhibit diverse cofactor-dependent enzymes enables cell and tissue imaging and makes this platform useful for enzyme and drug discovery

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-a activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.Peer reviewe

Telocytes and putative stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy

This study describes a novel type of interstitial (stromal) cell — telocytes (TCs) — in the human and mouse respiratory tree (terminal and respiratory bronchioles, as well as alveolar ducts). TCs have recently been described in pleura, epicardium, myocardium, endocardium, intestine, uterus, pancreas, mammary gland, etc. (see www.telocytes.com). TCs are cells with specific prolongations called telopodes (Tp), frequently two to three per cell. Tp are very long prolongations (tens up to hundreds of μm) built of alternating thin segments known as podomers (≤ 200 nm, below the resolving power of light microscope) and dilated segments called podoms, which accommodate mitochondria, rough endoplasmic reticulum and caveolae. Tp ramify dichotomously, making a 3-dimensional network with complex homo- and heterocellular junctions. Confocal microscopy reveals that TCs are c-kit- and CD34-positive. Tp release shed vesicles or exosomes, sending macromolecular signals to neighboring cells and eventually modifying their transcriptional activity. At bronchoalveolar junctions, TCs have been observed in close association with putative stem cells (SCs) in the subepithelial stroma. SCs are recognized by their ultrastructure and Sca-1 positivity. Tp surround SCs, forming complex TC-SC niches (TC-SCNs). Electron tomography allows the identification of bridging nanostructures, which connect Tp with SCs. In conclusion, this study shows the presence of TCs in lungs and identifies a TC-SC tandem in subepithelial niches of the bronchiolar tree. In TC-SCNs, the synergy of TCs and SCs may be based on nanocontacts and shed vesicles

Legislative Documents

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Defining criteria for the reintroduction of locally extinct populations based on contemporary and ancient genetic diversity: The case of the Adriatic Beluga sturgeon ( Huso huso )

Aim The restoration of the extinct Adriatic population of Beluga sturgeon, an iconic species with economic and traditional relevance, is a priority in upcoming conservation strategies but it must not occur without deep prior knowledge on the current diversity distribution. We defined informed criteria for the reintroduction of Beluga in Italian rivers by analysing its contemporary and ancient diversity based on a multi\u2010markers approach. Location Ponto\u2013Caspian and Adriatic basins. Methods We examined the distribution pattern of genetic diversity of the extant beluga populations by conducting genetic analysis on the mitochondrial dLoop and 27 nuclear microsatellites in 119 individuals from 3 geographical basins (Azov, Black and Caspian seas) and genomic analysis on 893 SNPs isolated through the 2bRAD approach in a subgroup of 92 samples. Mitochondrial information was also used to evaluate the variability of the extinct Adriatic population by analysing a few available museum samples. Results The historical Adriatic sample cannot be traced back to any of the contemporary ones laying to hypothesize the presence of a past isolated population. Instead, mitochondrial data did not reveal any geographically based clustering possibly reflecting the deep paleogeographical changes experienced by those areas. The genomic approach allowed us to depict for the first time a clear and supported genetic differentiation between two areas (the Black\u2013Azov and the Caspian basins) but microsatellites also revealed a signal of differentiation of the Azov sample, possibly related to historical management activities in that area. Main conclusions The outcomes of the study revealed an unprecedented amount of information that can provide great benefits to the establishment of ex situ Beluga broodstocks and support any future translocations. The provided guidelines should be taken as a reference for the upcoming restoring of the Italian extinct Beluga population and also for any future management activity of this species in its entire distribution range

Fast genetic identification of the Beluga sturgeon and its sought-after caviar to stem illegal trade

none11siSturgeons are well known for the delicacy of their eggs, the caviar, one of the most valuable products on the food market. The high price of caviar led in the past to a severe overharvest of wild sturgeon species and to an increase in trade of counterfeit products sold with impunity in spite of the strict trade limitations. A priority in the effort to reduce illegal trading is the development of genetic tools in order to identify the species of traded products using a standardized, cheap and rapid approach. We developed the first genetic nuclear marker for the identification of the Beluga sturgeon (Huso huso), the most sought-after caviar producer. We explored the interspecific variability at the setond intron of the nuclear S6 Ribosomal Protein (RP2S6), selected among 1867 introns, predicted by aligning the transcriptome of 3 sturgeon species with 3 complete fish genomes. The Beluga-specific SNP was identified by cloning and sequencing RP2S6 in 65 individuals of 11 species, validated on 341 additional individuals and tested on 18 caviar samples. Diagnostic primers designed on the SNP successfully amplified the expected band in all Beluga specimens while no PCR product was obtained from other pure species. The marker can also contribute to the identification of interspecific hybrids in which the Beluga is one of the parent species, such as in the case of the Bester, which produces one of the most mislabeled caviars in trade. The complete identification power on this highly relevant species and the proved efficacy on caviar samples represent an essential progress towards a standardized panel of nuclear markers for the control of illegal poaching, smuggling and mislabeling of sturgeons and their products.mixedBoscari, Elisa; Vitulo, Nicola; Ludwig, Arne; Caruso, Chiara; Mugue, Nikolai S.; Suciu, Radu; Onara, Dalia F.; Papetti, Chiara; Marino, Ilaria A.M.; Zane, Lorenzo; Congiu, LeonardoBoscari, Elisa; Vitulo, Nicola; Ludwig, Arne; Caruso, Chiara; Mugue, Nikolai S.; Suciu, Radu; Onara, Dalia F.; Papetti, Chiara; Marino, ILARIA ANNA MARIA; Zane, Lorenzo; Congiu, Leonard