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SummaryVarious hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5’s AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasome’s protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.

International audience; We report on our progress in the preparation of MgB2 and YBa2Cu3O7 (YBCO) superconducting cryomagnets consolidated by the unconventional sintering process called Spark Plasma Sintering (SPS). SPS is a promising rapid consolidation method that results in a better understanding and control of the sintering kinetics than other techniques.Three main experimental processes are reported here:(i)Preparation of intermetallic MgB2 using conventional graphite moulds at 1000°C, 50 MPa under vacuum.(ii)Preparation of intermetallic MgB2 at high pressure (600MPa) using tungsten carbide moulds (WC) at 800°C under vacuum.(iii)Preparation of oxide YBCO in air using WC moulds at quite low temperature (500-650°C) and high pressure (600MPa). The obtained YBCO and MgB2 pellets show 99% relative density. For the MgB2 compounds, a Tc onset of 38 K was obtained. At 20K, the critical current density was measured and found equal to Jc = 2.8x105 A/cm2, while the trapped field measured at the surface of a 20 mm diameter disk was equal to 3.9 T. Levitation force measurements were carried out on a MgB2 ring and a MgB2 disk with the same thickness and external diameter as a function of the temperature. The levitation force took the same values from 17 to 32 K for the disc and the ring samples, supporting the claim by E.H. Brandt [1] that the screening current in disks flow at first along the outside edge of the samples. Otherwise, the Tc determined from these measurements was equal to 38 K, in good agreement with the result of other techniques. Overall these results suggest that bulk MgB2 superconductors could be a viable variant for magnetic levitation applications.The measurement of the properties of single phase YBCO bulks consolidated using SPS is in progress and will be also discussed.[1] E.H.Brandt, Phys.Rev.58, 6506 (1998)

Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.

Various hormones, kinases, and stressors (fasting, heat shock) stimulate 26S proteasome activity. To understand how its capacity to degrade ubiquitylated protein can increase, we studied ZFAND5, which promotes protein degradation during muscle atrophy. Cryo-electron microscopy showed that ZFAND5 induces large conformational changes in the 19S regulatory particle. ZFAND5's AN1 Zn finger interacts with the Rpt5 ATPase and its C-terminus with Rpt1 ATPase and Rpn1, a ubiquitin-binding subunit. Surprisingly, these C-terminal interactions are sufficient to activate proteolysis. With ZFAND5 bound, entry into the proteasome's protein translocation channel is wider, and ZFAND5 dissociation causes opening of the 20S gate for substrate entry. Using single-molecular microscopy, we showed that ZFAND5 binds ubiquitylated substrates, prolongs their association with proteasomes, and increases the likelihood that bound substrates undergo degradation, even though ZFAND5 dissociates before substrate deubiquitylation. These changes in proteasome conformation and reaction cycle can explain the accelerated degradation and suggest how other proteasome activators may stimulate proteolysis.