p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress. Liquid-liquid phase separation of p62/SQSTM1 has been previously described, although the significance in vivo remains unclear. Here the authors show p62 droplets contain ubiquitin, autophagy-related proteins and Keap1 to serve as platform of not only autophagosome formation but also Nrf2 activation.Peer reviewe

p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

Immobilization of lipid nanorods onto two-dimensional crystals of protein tamavidin 2 for high-speed atomic force microscopy

Summary: High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action.For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

高速AFMによる古細菌由来MCMタンパク質複合体の観察

微生物化学研究会 微生物化学研究所 / 金沢大学六量体リングを形成し、二本鎖DNAを一本鎖へと分離するヘリカーゼとして機能する2種の古細菌由来MCM（ssoMCMとmthMCM）タンパク質を高速AFMにより観察した。マイカ上でのssoMCMタンパク質全長の観察の結果、ヘリカーゼ活性を持つと考えられている六量体リング（およびその部分構造）の他、梯子状に連なった長い複合体や六量体以上の多量体リング（およびその部分構造）の形成が確認された。また、タマビジン2の二次元結晶上に固定したビオチン化mthMCMタンパク質全長の観察の結果、横倒しの状態で固定された六量体リングおよびダブル六量体リング（十二量体）と思しき2種類の構造体の存在を確認した。Two archaeal MCM (ssoMCM and mthMCM) proteins which have helicase activities to unwind double-stranded DNA to produce single-stranded DNA were observed by high-speed AFM (HS-AFM). The observation of the full-length ssoMCM protein on mica surface revealed the formation of ladder-like long-shaped oligomers and ring (or partial ring) structures composed of more than hexamer as well as hexameric ring structure with helicase activity (or partial hexameric ring structure). In addition, observation of biotinylated full-length mthMCM protein on the 2D crystals of tamavidin 2 confirmed the formation of hexameric ring and double hexameric ring (12 mer) structures.研究課題/領域番号:17K15433, 研究期間(年度):2017-04-01 – 2019-03-3

Movie S3 from Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

HS-AFM movie of GroEL−GroES interaction during steady-state ATPase reaction cycle in the absence of substrate protein. D490C GroEL biotinylated at Cys490 was immobilized on a surface of tamavidin 2-LPI 2D crystals in a side-on orientation, while GroES was free in the bulk solution. The images were acquired at 4.35 fps for an area of 64 × 80 nm2. Z-scale, 14.5 nm

Movie S3 from Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

HS-AFM movie of GroEL−GroES interaction during steady-state ATPase reaction cycle in the absence of substrate protein. D490C GroEL biotinylated at Cys490 was immobilized on a surface of tamavidin 2-LPI 2D crystals in a side-on orientation, while GroES was free in the bulk solution. The images were acquired at 4.35 fps for an area of 64 × 80 nm2. Z-scale, 14.5 nm

Movie S1 from Substrate protein dependence of GroEL–GroES interaction cycle revealed by high-speed atomic force microscopy imaging

HS-AFM movie of GroEL−GroES interaction during steady-state ATPase reaction cycle in the presence of DM-MBP. D490C GroEL biotinylated at Cys490 was immobilized on a surface of tamavidin 2-LPI 2D crystals in a side-on orientation, while GroES and denature DM-MBP were free in the bulk solution. The images were acquired at 4.35 fps for an area of 64 × 80 nm2. Z-scale, 14.5 nm