The AAA+-type ATPase p97 governs an ever-expanding number of cellular processes reaching from degradation of damaged proteins and organelles to key signaling events and chromatin regulation with thousands of client proteins. With its relevance for cellular homeostasis and genome stability, it is linked to muscular and neuronal degeneration and, conversely, constitutes an attractive anti-cancer drug target. Its molecular function is ATP-driven protein unfolding, which is directed by ubiquitin and assisted by a host of cofactor proteins. This activity underlies p97's diverse ability to pull proteins out of membranes, unfold proteins for proteasomal degradation, or segregate proteins from partners for downstream activity. Recent advances in structural analysis and biochemical reconstitution have underscored this notion, resolved detailed molecular motions within the p97 hexamer, and suggested substrate threading through the central channel of the p97 hexamer as the driving mechanism. We will discuss the mechanisms and open questions in the context of the diverse cellular activities.

All Hedgehog (Hh) proteins are released from producing cells despite being synthesized as N- and C-terminally lipidated, membrane-tethered molecules. Thus, a cellular mechanism is needed for Hh solubilization. We previously suggested that a disintegrin and metalloprotease (ADAM)-mediated shedding of Sonic hedgehog (ShhNp) from its lipidated N and C termini results in protein solubilization. This finding, however, seemed at odds with the established role of N-terminal palmitoylation for ShhNp signaling activity. We now resolve this paradox by showing that N-palmitoylation of ShhNp N-terminal peptides is required for their proteolytic removal during solubilization. These peptides otherwise block ShhNp zinc coordination sites required for ShhNp binding to its receptor Patched (Ptc), explaining the essential yet indirect role of N-palmitoylation for ShhNp function. We suggest a functional model in which membrane-tethered multimeric ShhNp is at least partially autoinhibited in trans but is processed into fully active, soluble multimers upon palmitoylation-dependent cleavage of inhibitory N-terminal peptides.

Summary During DNA double strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase, p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin adapter complex and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97, or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end-joining of DSBs. Moreover, it attenuates early steps in homologous recombination consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data solve a central question regarding regulation of Ku in DSB repair, and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

Graphical Abstract Highlights d SDS22 and I3 form a transient inactive complex with PP1 during PP1 biogenesis d SDS22-PP1-I3 disassembly by the p97 AAA-ATPase allows holoenzyme formation d Direct binding of I3 by the SEP domain of the p37 adapter mediates p97 recruitment d Disassembly involves ATP-driven pulling of I3 into the channel of the p97 hexamer Correspondence hemmo.meyer@uni-due.de In BriefWeith et al. demonstrate that newly synthesized PP1 is first held in an inactive complex by SDS22 and inhibitor-3 that needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Disassembly involves direct interaction of the p37 adapter with inhibitor-3, followed by its ATP-driven unfolding. SUMMARYThe functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes.Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange. and helped with supervision of the project. J.H. generated adapter cell lines. I.P. and A.M. generated and provided baculoviruses coding for PP1 components. F.K. and M. Kaiser performed mass spectrometry measurements. J.d.P.G. and M.B. advised and analyzed data. H.M. conceived and supervised the project and wrote the manuscript. DECLARATION OF INTERESTSThe authors declare no competing interests.

We present the calibration strategy for the 20 kton liquid scintillator central detector of the Jiangmen Underground Neutrino Observatory (JUNO). By utilizing a comprehensive multiple-source and multiple-positional calibration program, in combination with a novel dual calorimetry technique exploiting two independent photosensors and readout systems, we demonstrate that the JUNO central detector can achieve a better than 1% energy linearity and a 3% effective energy resolution, required by the neutrino mass ordering determination.

Background: Humans have two enzyme isoforms to produce the universal sulfate donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS).Results: The main difference between the two PAPS synthases is their stability, which is modulated by nucleotides.Conclusion: Protein stability is a major contributing factor for PAPS availability.Significance: Naturally occurring changes in APS concentrations may be sensed by the labile PAPS synthase 2 that might act as a novel biosensor.

The Jiangmen Underground Neutrino Observatory (JUNO) features a 20 kt multi-purpose underground liquid scintillator sphere as its main detector. Some of JUNO's features make it an excellent location for B solar neutrino measurements, such as its low-energy threshold, high energy resolution compared with water Cherenkov detectors, and much larger target mass compared with previous liquid scintillator detectors. In this paper, we present a comprehensive assessment of JUNO's potential for detecting B solar neutrinos via the neutrino-electron elastic scattering process. A reduced 2 MeV threshold for the recoil electron energy is found to be achievable, assuming that the intrinsic radioactive background U and Th in the liquid scintillator can be controlled to 10 g/g. With ten years of data acquisition, approximately 60,000 signal and 30,000 background events are expected. This large sample will enable an examination of the distortion of the recoil electron spectrum that is dominated by the neutrino flavor transformation in the dense solar matter, which will shed new light on the inconsistency between the measured electron spectra and the predictions of the standard three-flavor neutrino oscillation framework. If eV , JUNO can provide evidence of neutrino oscillation in the Earth at approximately the 3 (2 ) level by measuring the non-zero signal rate variation with respect to the solar zenith angle. Moreover, JUNO can simultaneously measure using B solar neutrinos to a precision of 20% or better, depending on the central value, and to sub-percent precision using reactor antineutrinos. A comparison of these two measurements from the same detector will help understand the current mild inconsistency between the value of reported by solar neutrino experiments and the KamLAND experiment.

No abstract