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Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1β was strongly impaired. The expression of pro-IL-1β, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1β production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.

Nephrocystin mutations account for the vast majority of juvenile nephronophthisis, the most common inherited cause of renal failure in children. Nephrocystin has been localized to the ciliary transition zone of epithelial cells or its analogous structure, connecting cilium of retinal photoreceptors. Thus, the retinal degeneration associated with nephronophthisis may be explained by a functional ciliary defect. However, the function of nephrocystin in cilium assembly and maintenance of common epithelial cells and photoreceptors is still obscure. Here, we used Nphp1-targeted mutant mice and transgenic mice expressing EmGFP-tagged nephrocystin to demonstrate that nephrocystin located at connecting cilium axoneme can affect the sorting mechanism and transportation efficiency of the traffic machinery between inner and outer segments of photoreceptors. This traffic machinery is now recognized as intraflagellar transport (IFT); a microtubule-based transport system consisting of motors, IFT particles and associated cargo molecules. Nephrocystin seems to control some of the IFT particle components moving along the connecting cilia so as to regulate this inter-segmental traffic. Our novel findings provide a clue to unraveling the regulatory mechanism of nephrocystin in IFT machinery.

Juvenile nephronophthisis type I is the most common genetic disorder causing end-stage renal failure in children and young adults. The defective gene responsible has been identified as NPHP1. Its gene product, nephrocystin-1, is a novel protein of uncertain function that is widely expressed in many tissues and not just confined to the kidney. To gain insight into the physiological function of nephrocystin, Nphp1-targeted mutant mice were generated by homologous recombination. Interestingly, homozygous Nphp1 mutant mice were viable without renal manifestations of nephronophthisis. They appeared normal, but males were infertile with oligoteratozoospermia. Histological analysis of the seminiferous tubules showed that spermatogenesis was blocked at the early stages of spermatid elongation, with degenerating spermatids sloughing off into the lumen. Electron microscopic analysis revealed detachment of early elongating spermatids from Sertoli cells, and a failure of sperm head and tail morphogenesis. However, a few mature spermatozoa were still deposited in the epididymis, though they were frequently dead, immotile, or malformed. These novel findings indicate that nephrocystin is critically required for the differentiation of early elongating spermatids into spermatozoa in mice. The possible roles of nephrocystin in the formation and maintenance of Sertoli-spermatid junctions are still under investigation.