Chemical Proteomics of the Tumor Suppressor Fhit Covalently Bound to the Cofactor Ap3A Elucidates Its Inhibitory Action on Translation.

The tumor suppressor protein fragile histidine triad (Fhit) is known to be associated with genomic instability and apoptosis. The tumor-suppressive function of Fhit depends on the interaction with the alarmone diadenosine triphosphate (Ap3A), a noncanonical nucleotide whose concentration increases upon cellular stress. How the Fhit-Ap3A complex exerts its signaling function is unknown. Here, guided by a chemical proteomics approach employing a synthetic stable Fhit-Ap3A complex, we found that the Fhit-Ap3A complex, but not Fhit or Ap3A alone, impedes translation. Our findings provide a mechanistic model in which Fhit translocates from the nucleolus into the cytosol upon stress to form an Fhit-Ap3A complex. The Fhit-Ap3A complex impedes translation both in vitro and in vivo, resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap3A to regulate cellular proliferation.

Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy.

As one of the most prevalent post-translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage-defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48-linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high-resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.

Thermostable protein-stabilized gold nanoclusters as a peroxidase mimic.

Protein-stabilized gold nanoclusters (AuNCs) are fascinating nanostructures with exciting properties owing to their ultra-small sizes and functional shell. However, their applications under extreme conditions are still complicated, waiting for programmable solutions. Therefore, the design of a multi-functional protein stabilizer for specific purposes gains attention to improve the stability and functionality of AuNCs. Herein, we exploited the thermostability of genetically engineered KlenTaq DNA polymerase containing five cysteine residues (KTQ5C) to synthesize heat-stable AuNCs (AuNC@KTQ5C) and characterize optical, structural, and hydrodynamic properties. Besides their excellent photophysical properties, AuNC@KTQ5C also exhibit superior peroxidase-like (POD-like) catalytic activity following typical Michaelis-Menten kinetics together with a high affinity towards the POD substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-diammonium salt (ABTS). Moreover, FTIR and relative catalytic activity analysis of AuNC@KTQ5C reveal that KTQ5C is resistant to changes in protein secondary structure while the AuNCs conserve 70-80% of their catalytic performance after heat treatments up to more than 80 °C. Our findings show that stabilizing AuNCs with thermostable KTQ5C not only preserves the advantages of protein-stabilized AuNCs but can also promote the resistance of AuNCs against aggregation due to protein denaturation under extreme reaction temperatures, protecting their fluorescent emission or catalytic activity.