The Hippo network kinase STK38 contributes to protein homeostasis by inhibiting BAG3-mediated autophagy.

Chaperone-assisted selective autophagy (CASA) initiated by the cochaperone Bcl2-associated athanogene 3 (BAG3) represents an important mechanism for the disposal of misfolded and damaged proteins in mammalian cells. Under mechanical stress, the cochaperone cooperates with the small heat shock protein HSPB8 and the cytoskeleton-associated protein SYNPO2 to degrade force-unfolded forms of the actin-crosslinking protein filamin. This is essential for muscle maintenance in flies, fish, mice and men. Here, we identify the serine/threonine protein kinase 38 (STK38), which is part of the Hippo signaling network, as a novel interactor of BAG3. STK38 was previously shown to facilitate cytoskeleton assembly and to promote mitophagy as well as starvation and detachment induced autophagy. Significantly, our study reveals that STK38 exerts an inhibitory activity on BAG3-mediated autophagy. Inhibition relies on a disruption of the functional interplay of BAG3 with HSPB8 and SYNPO2 upon binding of STK38 to the cochaperone. Of note, STK38 attenuates CASA independently of its kinase activity, whereas previously established regulatory functions of STK38 involve target phosphorylation. The ability to exert different modes of regulation on central protein homeostasis (proteostasis) machineries apparently allows STK38 to coordinate the execution of diverse macroautophagy pathways and to balance cytoskeleton assembly and degradation.

Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas.

Recently, the conserved intracellular digestion mechanism 'autophagy' has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas.

TECPR2 Cooperates with LC3C to Regulate COPII-Dependent ER Export.

Hereditary spastic paraplegias (HSPs) are a diverse group of neurodegenerative diseases that are characterized by axonopathy of the corticospinal motor neurons. A mutation in the gene encoding for Tectonin ß-propeller containing protein 2 (TECPR2) causes HSP that is complicated by neurological symptoms. While TECPR2 is a human ATG8 binding protein and positive regulator of autophagy, the exact function of TECPR2 is unknown. Here, we show that TECPR2 associates with several trafficking components, among them the COPII coat protein SEC24D. TECPR2 is required for stabilization of SEC24D protein levels, maintenance of functional ER exit sites (ERES), and efficient ER export in a manner dependent on binding to lipidated LC3C. TECPR2-deficient HSP patient cells display alterations in SEC24D abundance and ER export efficiency. Additionally, TECPR2 and LC3C are required for autophagosome formation, possibly through maintaining functional ERES. Collectively, these results reveal that TECPR2 functions as molecular scaffold linking early secretion pathway and autophagy.

PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways.

Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy.

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection.

Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA in selective targets. However, therapeutic exosome application requires knowledge on target structures to avoid undue delivery. Previous work suggesting exosomal tetraspanin-integrin complexes to be involved in target cell binding, we aimed to control this hypothesis and to define target cell ligands. Exosomes are rich in tetraspanins that associate besides other molecules with integrins. Co-immunoprecipitation of exosome lysates from rat tumor lines that differ only with respect to Tspan8 and beta4 revealed promiscuity of tetraspanin-integrin associations, but also few preferential interactions like that of Tspan8 with alpha4 and beta4 integrin chains. These minor differences in exosomal tetraspanin-complexes strongly influence target cell selection in vitro and in vivo, efficient exosome-uptake being seen in hematopoietic cells and solid organs. Exosomes expressing the Tspan8-alpha4 complex are most readily taken up by endothelial and pancreas cells, CD54 serving as a major ligand. Selectivity of uptake was confirmed with exosomes from an alpha4 cDNA transfected Tspan8(+) lymph node stroma line. Distinct from exosomes from the parental line, the latter preferentially targeted endothelial cells and in vivo the pancreas. Importantly, pulldown experiments provided strong evidence that exosome-uptake occurs in internalization-prone membrane domains. This is the first report on the exosomal tetraspanin web contributing to target cell selection such that predictions can be made on potential targets, which will facilitate tailoring exosomes for drug delivery.