In the conserved autophagy pathway, autophagosomes (APs) engulf cellular components and deliver them to the lysosome for degradation. Before fusing with the lysosome, APs have to close via an unknown mechanism. We have previously shown that the endocytic Rab5-GTPase regulates AP closure. Therefore, we asked whether ESCRT, which catalyzes scission of vesicles into late endosomes, mediates the topologically similar process of AP sealing. Here, we show that depletion of representative subunits from all ESCRT complexes causes late autophagy defects and accumulation of APs. Focusing on two subunits, we show that Snf7 and the Vps4 ATPase localize to APs and their depletion results in accumulation of open APs. Moreover, Snf7 and Vps4 proteins complement their corresponding mutant defects in vivo and in vitro. Finally, a Rab5-controlled Atg17–Snf7 interaction is important for Snf7 localization to APs. Thus, we unravel a mechanism in which a Rab5-dependent Atg17–Snf7 interaction leads to recruitment of ESCRT to open APs where ESCRT catalyzes AP closure.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

cAMP responsive element binding protein 1 (CREB1) gene, has been reported to play crucial roles in tumor progression and development in various types of cancer. Little is known, however, about its role and underlying mechanism in gastric cancer (GC). Herein, we investigated the biological roles and molecular mechanism of CREB1 in GC. The expression level was determined in four GC cell lines by quantitative RT-PCR and western blotting. Recombinant expression vector carrying small interfering RNA (siRNA) targeting CREB1 was constructed and then transfected into human GC cell line (SGC-7901). Cell proliferation, colony formation, cycle distribution, migration and invasion in vitro were determined by MTT, colony forming, flow cytometry, would healing and Transwell invasion assays after CREB1 knockdown. Tumor growth in vivo was assessed by measurement of tumor volume and weight in a nude mouse model. We found that CREB1 was highly expressed in the human GC cell lines. We also showed that knockdown of CREB1 in SGC-7901 cells significantly inhibited cell proliferation, colony formation, migration and invasion and induced cell arrest at G1/G0 phase in vitro, as well as suppressed tumor growth in vivo. In addition, CREB1 knockdown was able to significantly reduce expression of its downstream target genes cyclin D1, Bcl-2 and MMP-9 in vitro and in vivo. These findings suggest that CREB1 may be a potential therapeutic target for the treatment of gastric cancer.

Cdo bridges scaffold proteins BNIP-2 and JLP to activate p38MAPK during myoblast differentiation. KIF5B is a novel interacting partner of BNIP-2 and promotes myogenic differentiation. KIF5B-dependent transport of BNIP-2 is essential for its promyogenic effects.

Objective. Chondrocyte hypertrophy and mineralization are considered to be important pathologic factors in osteoarthritis (OA). We previously reported that Rac1 was aberrantly activated to promote chondrocyte hypertrophy, mineralization, and expression of matrix metalloproteinase 13 and ADAMTS in OA. However, the underlying mechanism of aberrant Rac1 activation in OA is unclear. The present study was undertaken to identify the specific molecular regulator controlling Rac1 activity in OA, as well as to investigate its function in chondrocyte hypertrophy, mineralization, and OA development.Methods. Expression levels of 28 upstream regulators of Rac1 activity, including 8 GTPase-activating proteins (GAPs) and 20 guanine nucleotide exchange factors, in OA and normal cartilage were assessed by quantitative polymerase chain reaction. Chondrocytes were transduced with lentiviral vectors encoding OCRL1, GAP, non-GAP, CA-Rac1, and DN-Rac1, either alone or in combination. Alkaline phosphatase staining was used as a marker of chondrocyte hypertrophy. Rac1 activity was analyzed by pulldown assay. Finally, OA was established in mice by surgical transection of the anterior cruciate ligament and cutting of the medial meniscus. The mice were injected intraarticularly with OCRL1-encoding lentivirus, and whole joints were assessed histologically 6 weeks after surgery.Results. OCRL1 was abundantly expressed in normal cartilage and was the only significantly downregulated RacGAP in OA cartilage. Overexpression of OCRL1 inhibited interleukin-1b-induced Rac1 activity, chondrocyte hypertrophy, and expression of hypertrophyrelated genes. Conversely, knockdown of OCRL1 elevated Rac1 activity and promoted chondrocyte hypertrophy and mineralization. Further, OCRL1 modulated Rac1 activity via its GAP domain. Finally, intraarticular injection of OCRL1-encoding lentivirus protected against destruction and degeneration of cartilage in the mouse OA model.Conclusion. OCRL1 acts as a RacGAP in cartilage to impede chondrocyte hypertrophy and OA development through modulating Rac1 activity. This regulatory pathway might provide potential targets for the development of new therapies for OA.Osteoarthritis (OA), characterized by cartilage degradation, synovial inflammation, and subchondral bone remodeling, is the most common form of degenerative joint disease (1). There is now a general consensus that chondrocytes in OA undergo hypertrophy and mineralization (characterized by expression of matrix metalloproteinase 13 [2], type X collagen [3],

Canagliflozin is an antidiabetic medicine that inhibits sodium-glucose cotransporter 2 (SGLT2) in proximal tubules. Recently, it was reported to have several noncanonical effects other than SGLT2 inhibiting. However, the effects of canagliflozin on skeletal muscle regeneration remain largely unexplored. Thus, in vivo muscle contractile properties recovery in mice ischemic lower limbs following gliflozins treatment was evaluated. The C2C12 myoblast differentiation after gliflozins treatment was also assessed in vitro. As a result, both in vivo and in vitro data indicate that canagliflozin impairs intrinsic myogenic regeneration, thus hindering ischemic limb muscle contractile properties, fatigue resistance recovery, and tissue regeneration. Mitochondrial structure and activity are both disrupted by canagliflozin in myoblasts. Single-cell RNA sequencing of ischemic tibialis anterior reveals a decrease in leucyl-tRNA synthetase 2 (LARS2) in muscle stem cells attributable to canagliflozin. Further investigation explicates the noncanonical function of LARS2, which plays pivotal roles in regulating myoblast differentiation and muscle regeneration by affecting mitochondrial structure and activity. Enhanced expression of LARS2 restores the differentiation of canagliflozin-treated myoblasts, and accelerates ischemic skeletal muscle regeneration in canagliflozin-treated mice. Our data suggest that canagliflozin directly impairs ischemic skeletal muscle recovery in mice by downregulating LARS2 expression in muscle stem cells, and that LARS2 may be a promising therapeutic target for injured skeletal muscle regeneration.

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