Insights into gli factors ubiquitylation methods

The Hedgehog (Hh) signaling pathway governs cell growth and tissue development. Malfunctioning of several Hh pathway components, including the key transcriptional effector Gli proteins, is responsible for the onset of several tumors. Gli proteins activity is finely controlled by multilayered regulatory mechanisms, the most prominent of which is their proteasome-dependent proteolytic cleavage or massive ubiquitin-mediated proteolysis. Here, we described multiple procedures to determine whether a Gli protein is ubiquitylated both in a cellular context and in vitro, in basal conditions or by different E3 ubiquitin ligases and whether these processes are associated to Gli proteasome degradation

Integrated proteogenomic approach for identifying degradation motifs in eukaryotic cells

Since its discovery nearly 40 years ago, many components of the ubiquitin-proteasome system (UPS) have been identified and characterized in detail. However, a key aspect of the UPS that remains largely obscure is the signals that initiate the interaction of a substrate with enzymes of the UPS machinery. Understanding these signals is of particular interest for studies that examine the mechanism of substrate recognition for proteins that have adopted a non-native structure, as part of the cellular protein quality control (PQC) defense mechanism. Such studies are quite salient as the entire proteome makes up the potential battery of PQC substrates, and yet only a limited number of ubiquitination pathways are known to handle misfolded proteins. Our current research aims at understanding how a small number of PQC ubiquitin-protein ligases specifically recognize and ubiquitinate the overwhelming assortment of misfolded proteins. Here, we present a new proteogenomic approach for identifying and characterizing recognition motifs within degradation elements (degrons) in a high-throughput manner. The method utilizes yeast growth under restrictive conditions for selecting protein fragments that confer instability. The corresponding cDNA fragments are analyzed by next-generation sequencing (NGS) that provides information about each fragment's identity, reading frame, and abundance over time. This method was used by us to identify PQC-specific and compartment-specific degrons. It can readily be modified to study protein degradation signals and pathways in other organisms and in various settings, such as different strain backgrounds and under various cell conditions, all of which can be sequenced and analyzed simultaneously

The lowdown on breakdown: Open questions in plant proteolysis

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research

FAT10 is a proteasomal degradation signal that is itself regulated by ubiquitination

The degradation of the ubiquitin-like protein FAT10 requires ubiquitination: degradation was inhibited in cells expressing a nonpolymerizable ubiquitin mutant or harboring a thermolabile ubiquitin-activating enzyme. Degradation of FAT10 is accelerated after induction of apoptosis, suggesting that it plays a role in prosurvival pathways

Structural plasticity of D3-D14 ubiquitin ligase in strigolactone signalling.

The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/β hydrolase D14-which metabolizes strigolactone-interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3-D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.Gatsby Charitable Foundatio