GSK3B induces autophagy by phosphorylating ULK1

Unc-51-like autophagy activating kinase 1 (ULK1), a mammalian homolog of the yeast kinase Atg1, has an essential role in autophagy induction. In nutrient and growth factor signaling, ULK1 activity is regulated by various posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. We previously identified glycogen synthase kinase 3 beta (GSK3B) as an upstream regulator of insulin withdrawal-induced autophagy in adult hippocampal neural stem cells. Here, we report that following insulin withdrawal, GSK3B directly interacted with and activated ULK1 via phosphorylation of S405 and S415 within the GABARAP-interacting region. Phosphorylation of these residues facilitated the interaction of ULK1 with MAP1LC3B and GABARAPL1, while phosphorylation-defective mutants of ULK1 failed to do so and could not induce autophagy flux. Furthermore, high phosphorylation levels of ULK1 at S405 and S415 were observed in human pancreatic cancer cell lines, all of which are known to exhibit high levels of autophagy. Our results reveal the importance of GSK3B-mediated phosphorylation for ULK1 regulation and autophagy induction and potentially for tumorigenesis. © 2021, The Author(s).1

Branching Ratio between Proton Transfer and Electron Transfer Channels of a Bidirectional Proton-Coupled Electron Transfer

Rigorous quantum dynamical study of concerted proton-coupled electron transfer (PCET) on the time scale of a few femtoseconds (fs) has been rarely reported. Herein, a time-dependent quantum wavepacket propagation method was applied to the dynamics of the charge-transfer excited electronic state of FHCl corresponding to F⁺HCl^–. The dynamics corresponds to a bidirectional PCET with two dissociation channels: the electron transfer (ET, generating FH+Cl) and proton transfer (PT, generating F+HCl) paths. The calculated branching ratio (Cl/F), 0.78, implies a surprising fact: PT prevails over ET. A detailed analysis of the proton movement and electron readjustment suggests that the proton movement starts ∼3 fs earlier than the electron movement, and the electron readjustment is triggered by the initial movement of the proton. The branching ratio drastically inverts to 1.24 because of a reduced nonadiabatic effect in the isotope-substituted system, FDCl

Dynamic Symmetry Breaking Hidden in Fano Resonance of a Molecule: S₁ State of Diazirine Using Quantum Wave Packet Propagation

Fano resonance in the predissociation of the S₁ state of diazirine was studied by applying a time-dependent wave packet propagation method, and dynamic symmetry breaking (DSB) around the stationary structure of S₁ was disclosed in a detailed analysis of this theoretical result. The DSB was found to originate in coupling between the asymmetric C–N₂ stretching and CH₂ wagging modes, suggesting that there is a slight time gap between ring opening and the concurrent dragging of two H atoms of the CH₂ moiety. Although the depth of the double well due to DSB is just 0.011 eV, its presence noticeably affects the early time dynamics and observed spectrum

Degradation of Kidney and Psoas Muscle Proteins as Indicators of Post-Mortem Interval in a Rat Model, with Use of Lateral Flow Technology

<div><p>We investigated potential protein markers of post-mortem interval (PMI) using rat kidney and psoas muscle. Tissue samples were taken at 12 h intervals for up to 96 h after death by suffocation. Expression levels of eight soluble proteins were analyzed by Western blotting. Degradation patterns of selected proteins were clearly divided into three groups: short-term, mid-term, and long-term PMI markers based on the half maximum intensity of intact protein expression. In kidney, glycogen synthase (GS) and glycogen synthase kinase-3β were degraded completely within 48 h making them short-term PMI markers. AMP-activated protein kinase α, caspase 3 and GS were short-term PMI markers in psoas muscle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was a mid-term PMI marker in both tissues. Expression levels of the typical long-term PMI markers, p53 and β-catenin, were constant for at least 96 h post-mortem in both tissues. The degradation patterns of GS and caspase-3 were verified by immunohistochemistry in both tissues. GAPDH was chosen as a test PMI protein to perform a lateral flow assay (LFA). The presence of recombinant GAPDH was clearly detected in LFA and quantified in a concentration-dependent manner. These results suggest that LFA might be used to estimate PMI at a crime scene.</p></div

Lateral flow assay platform for PMI-indicating protein.

<p>(a) Standard calibration of rGAPDH concentration by sandwich ELISA assay. (b) Typical rGAPDH strip images and the corresponding signals in the presence of 0 and 100 ng/mL rGAPDH. The peak areas of red lines on the control (C) and the test line (T) were calculated using the ImageJ software. (c) Calibration curve of different concentrations of rGAPDH. The peak areas of red bands on the test line were calculated using the ImageJ software. Error bars are the standard deviation of triplicate experiments. A.U., arbitrary unit.</p

Immunohistochemistry and degradation curves of post-mortem rat tissues.

<p>Microscopic photographs of rat (a) kidney and (b) psoas muscle at the indicated PMI time-point. Degradation curves for glycogen synthase and caspase-3 expressions were obtained from TissueFACS scattergrams. RC, renal corpus; BC, Bowman’s capsule; M, muscle fiber bundle; N, nuclear; P, perimysium; Scale bar = 100 μm.</p

SDS-PAGE analysis of recombinant GAPDH expression.

<p>Coomassie Brilliant Blue-stained proteins were shown from (a) the serial purification procedure and (b) the final dialysis of rGAPDH. Under optimized condition, <i>E</i>. <i>coli</i> strain BL21 (DE3) was transformed with rat GAPDH-pET28a, and following IPTG induction resulted in the expression of GAPDH (37 kDa). Mw, protein molecular weight; 1, total lysate of transformed <i>E</i>. <i>coli</i>; 2, soluble supernatant fraction of total lysate; 3, insoluble pellet fraction of total lysate; 4, flow-through after affinity chromatography; 5, purified rGAPDH.</p

Degradation profiles of selected rat post-mortem interval (PMI) proteins.

<p>(a) Immunoblot analysis of kidney PMI proteins up to 96 h post-mortem. (b) Linear regression fits for kidney PMI protein degradation. PMI₅₀ values (number beside the dotted arrow), indicating the 50% degradation time-point of the starting expression level, were determined by the linear regression curve. (c) Immunoblot analysis of psoas muscle PMI proteins up to 96 h post-mortem. (d) Linear regression fits for psoas muscle PMI protein degradation. Each data point represents five biological replicates.</p