Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.     

Absence of negative feedback by oxytocin on release from magnocellular neurones in conscious rats.

Peripheral administration of vasopressin (VP) was previously shown to exert a negative feedback influence on its own release and on the release of oxytocin (OT). In this study we examined the possible influence that OT has on the function of hypothalamic magnocellular neurones. Oxytocin was administered intraperitoneally and its effects on release from VP neurones and from OT neurones were determined as indexed by plasma concentrations of vasopressin-associated neurophysin ([VP-RNP]) and oxytocin-associated neurophysin ([OT-RNP]) under basal conditions and conditions of high plasma osmolality (Posm) induced by acute salt loading. Studies were performed on conscious, chronically instrumented Long-Evans rats. Oxytocin (1 nmol or 10 nmol) dissolved in 1 mL of 0.9% saline was administered intraperitoneally to animals 1 h before they received an intravenous infusion of hypertonic saline over 60 min at a rate designed to raise Posm by approximately 0.75 mosmol.min-1. Intraperitoneal injection of vehicle or 1 nmol of OT did not significantly alter [VP-RNP], [OT-RNP], or basal Posm. Administration of 10 nmol OT also had no effect on [VP-RNP] or [OT-RNP], but this dose of peptide significantly lowered basal Posm (299 +/- 2 to 290 +/- 2 mosmol/kg H2O, p less than 0.001). Both doses of OT did not significantly alter the responsiveness of VP neurones to hyperosmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermotropic phase transitions in the plasma membrane of ram spermatozoa

A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36 degrees C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24 degrees C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24 degrees C to 30 degrees C, but the reasons for this are unclear. It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.     

Incorporation of conformationally constrained beta-amino acids into peptides.

The use of norbornene units to induce the formation of beta-sheet and beta-turn type structures in peptides is discussed. The norbornene unit is readily prepared by a desymmetrization reaction and is easily incorporated into a peptide chain. Depending upon the exact nature of the norbornene unit, it is possible to form structures which resemble parallel beta-sheets, antiparallel beta-sheets or beta-turns. Similar peptide analogues incorporating a cis-2-amino-cyclopropane carboxylic acid unit can also be prepared. As an illustration of the application of this chemistry, a short, asymmetric synthesis of conformationally constrained metalloprotease inhibitors is presented.