LAMTOR1 ubiquitination restricts its interaction with the vacuolar-type H+-ATPase, promotes autophagy and is controlled by USP32.

Among the various signals governing autophagy, ubiquitination plays a critical role both by controlling the stability of upstream regulators or components of macroautophagy/autophagy pathways and by facilitating the recruitment of cargo to autophagy receptors. As such, modulators of ubiquitin signaling can influence autophagic substrate degradation. Recently, we identified a non-proteolytic ubiquitin signal at the Ragulator complex subunit LAMTOR1 that is reversed by the deubiquitinase USP32. Loss of USP32 promotes ubiquitination within the unstructured N-terminal region of LAMTOR1 and prevents its efficient interaction with the vacuolar-type H+-ATPase, a prerequisite for full activation of MTORC1 at lysosomes. Consequently, MTORC1 activity is decreased and autophagy is upregulated in USP32 knockout cells. This phenotype is conserved in Caenorhabditis elegans. Depletion of USP32 homolog CYK-3 in worms results in LET-363/MTOR inhibition and autophagy induction. Based on our data, we propose an additional control layer of the MTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction.

The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.

The deubiquitinase USP11 is a versatile and conserved regulator of autophagy.

Autophagy is a major cellular quality control system responsible for the degradation of proteins and organelles in response to stress and damage to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase ubiquitin-specific protease 11 (USP11) restricts autophagy and that KO of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human amyloid-ß peptide 42 aggregation-induced paralysis. USP11 coprecipitated with autophagy-specific class III phosphatidylinositol 3-kinase complex I and limited its interaction with nuclear receptor-binding factor 2, thus decreasing lipid kinase activity of class III phosphatidylinositol 3-kinase complex I and subsequent recruitment of effectors such as WD-repeat domain phosphoinositide-interacting proteins to the autophagosomal membrane. Accordingly, more WD-repeat domain phosphoinositide-interacting protein 2 puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mechanistic target of rapamycin, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.

All-arthroscopic intramedullary nailing of 2- and 3-part proximal humeral fractures: a new arthroscopic technique and preliminary results.

PURPOSE: The most criticism of antegrade humeral nailing is the potentially deleterious effect on the shoulder function, which is caused by the trauma to the M. supraspinatus (SSP) at the nail insertion site. We describe a new technique of all-arthroscopical intramedullary nailing, which preserves the rotator cuff, and compare it with the conventional open procedure. METHODS: From 11/2009 to 12/2010 82 patients with unstable, displaced proximal humeral fractures were treated surgically. Twenty-one of these patients received an intramedullary nailing. Sixteen of 21 met the inclusion criteria. Based on the surgeon's arthroscopic experience, patients were assigned to the arthroscopic (group I, n = 8) or open group (group II, n = 8). Both groups were compared due to the replacement results, complications, time of surgery and fluoroscopy. Concomitant intraarticular pathologies were assessed (group I). First clinical results after a median follow-up of 13 months (group I) and 14 months (group II) were reported. RESULTS: Between group I and II, no significant differences were seen in patients age [77 years (range 45-90 years) vs. 76 years (range 65-92 years)], gender (6 female/2 male vs. 5 female/3 male) and fracture pattern (six 2-/two 3-part fractures vs. five 2-/three 3-part fractures). The reduction was evaluated by the caput-diaphysis-angle, which was median 137° (range 120-147°) in group I and 132° (range 120-158°) in group II (p = 0.959). Postoperatively, group I showed one varus-, group II two varus- and valgus deformities. Median time of surgery was 75 min (range 45-182 min) versus 70 min (range 40-146 min) (p = 0.442), fluoroscopy time 1.5 min (range, 0.6-3.7 min) versus 1.2 min (range 0.3-2.2 min) in group I and II (p = 0.336). Concomitant pathologies like one traumatic bicipital tendon-lesion and three partial lesions of the SSP were observed and treated in group I. Constant Scores and Visual Analogue Scale did not differ significantly between both groups at the time of follow-up. CONCLUSIONS: All-arthroscopical humeral nailing is possible, preserves the rotator cuff and provides equal replacement and functional results like the open technique. An arthroscopically visualized optimal nail insertion point provides less frequent head deformities. Level of evidence Level III.