A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates.

Over 600 E3 ligases in humans execute ubiquitination of specific target proteins in a spatiotemporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions.

USP7/Maged1-mediated H2A monoubiquitination in the paraventricular thalamus: an epigenetic mechanism involved in cocaine use disorder.

The risk of developing drug addiction is strongly influenced by the epigenetic landscape and chromatin remodeling. While histone modifications such as methylation and acetylation have been studied in the ventral tegmental area and nucleus accumbens (NAc), the role of H2A monoubiquitination remains unknown. Our investigations, initially focused on the scaffold protein melanoma-associated antigen D1 (Maged1), reveal that H2A monoubiquitination in the paraventricular thalamus (PVT) significantly contributes to cocaine-adaptive behaviors and transcriptional repression induced by cocaine.

Human Paraoxonase-2 (PON2): Protein Functions and Modulation.

PON1, PON2, and PON3 belong to a family of lactone hydrolyzing enzymes endowed with various substrate specificities. Among PONs, PON2 shows the highest hydrolytic activity toward many acyl-homoserine lactones (acyl-HL) involved in bacterial quorum-sensing signaling. Accordingly, defense against pathogens, such as (), was postulated to be the principal function of PON2.

WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2.

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs.

An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination.

In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1).