Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

The cochaperone BAG3 coordinates protein synthesis and autophagy under mechanical strain through spatial regulation of mTORC1.

The cochaperone BAG3 is a central protein homeostasis factor in mechanically strained mammalian cells. It mediates the degradation of unfolded and damaged forms of the actin-crosslinker filamin through chaperone-assisted selective autophagy (CASA). In addition, BAG3 stimulates filamin transcription in order to compensate autophagic disposal and to maintain the actin cytoskeleton under strain. Here we demonstrate that BAG3 coordinates protein synthesis and autophagy through spatial regulation of the mammalian target of rapamycin complex 1 (mTORC1). The cochaperone utilizes its WW domain to contact a proline-rich motif in the tuberous sclerosis protein TSC1 that functions as an mTORC1 inhibitor in association with TSC2. Interaction with BAG3 results in a recruitment of TSC complexes to actin stress fibers, where the complexes act on a subpopulation of mTOR-positive vesicles associated with the cytoskeleton. Local inhibition of mTORC1 is essential to initiate autophagy at sites of filamin unfolding and damage. At the same time, BAG3-mediated sequestration of TSC1/TSC2 relieves mTORC1 inhibition in the remaining cytoplasm, which stimulates protein translation. In human muscle, an exercise-induced association of TSC1 with the cytoskeleton coincides with mTORC1 activation in the cytoplasm. The spatial regulation of mTORC1 exerted by BAG3 apparently provides the basis for a simultaneous induction of autophagy and protein synthesis to maintain the proteome under mechanical strain.

A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes.

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.

Induction and adaptation of chaperone-assisted selective autophagy CASA in response to resistance exercise in human skeletal muscle.

Chaperone-assisted selective autophagy (CASA) is a tension-induced degradation pathway essential for muscle maintenance. Impairment of CASA causes childhood muscle dystrophy and cardiomyopathy. However, the importance of CASA for muscle function in healthy individuals has remained elusive so far. Here we describe the impact of strength training on CASA in a group of healthy and moderately trained men. We show that strenuous resistance exercise causes an acute induction of CASA in affected muscles to degrade mechanically damaged cytoskeleton proteins. Moreover, repeated resistance exercise during 4 wk of training led to an increased expression of CASA components. In human skeletal muscle, CASA apparently acts as a central adaptation mechanism that responds to acute physical exercise and to repeated mechanical stimulation.

HSP70-binding protein HSPBP1 regulates chaperone expression at a posttranslational level and is essential for spermatogenesis.

Molecular chaperones play key roles during growth, development, and stress survival. The ability to induce chaperone expression enables cells to cope with the accumulation of nonnative proteins under stress and complete developmental processes with an increased requirement for chaperone assistance. Here we generate and analyze transgenic mice that lack the cochaperone HSPBP1, a nucleotide-exchange factor of HSP70 proteins and inhibitor of chaperone-assisted protein degradation. Male HSPBP1(-/-) mice are sterile because of impaired meiosis and massive apoptosis of spermatocytes. HSPBP1 deficiency in testes strongly reduces the expression of the inducible, antiapoptotic HSP70 family members HSPA1L and HSPA2, the latter of which is essential for synaptonemal complex disassembly during meiosis. We demonstrate that HSPBP1 affects chaperone expression at a posttranslational level by inhibiting the ubiquitylation and proteasomal degradation of inducible HSP70 proteins. We further provide evidence that the cochaperone BAG2 contributes to HSP70 stabilization in tissues other than testes. Our findings reveal that chaperone expression is determined not only by regulated transcription, but also by controlled degradation, with degradation-inhibiting cochaperones exerting essential prosurvival functions.

Chaperone-assisted proteostasis is essential for mechanotransduction in mammalian cells.

Maintaining the dynamic proteome of a living cell in the face of an ever-changing environment depends on a fine-tuned balance of protein synthesis and protein degradation. Molecular chaperones exert key functions during protein homeostasis (proteostasis). They associate with nonnative client proteins following synthesis or damage and facilitate client sorting and folding. When client proteins are terminally misfolded, chaperones cooperate with protein degradation systems to dispose of such clients. This dual proteostasis activity of chaperones is essential for maintaining cell function under normal growth conditions and becomes even more important under stress conditions such as heat and oxidative stress. The recent identification of chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway highlights the critical role of molecular chaperones in mechanically strained cells and tissues. The CASA complex, assembled by the cochaperone BAG3, coordinates protein degradation and protein synthesis in response to mechanical force. Here we describe the composition and function of this chaperone complex in mammals and discuss its relevance for tissue homeostasis and the regulation of cell adhesion, migration and proliferation. We provide a unifying concept for the function of BAG3, which integrates its involvement in muscle maintenance, tumor formation and virus infection.

Tension-induced autophagy: may the chaperone be with you.

Impairment of autophagy in patients and animal models severely affects mechanically strained tissues such as skeletal muscle, heart, lung and kidney, leading for example to muscle dystrophy, cardiomyopathy and renal injury. However, the reason for this high reliance on autophagy remained largely elusive. Recent work in our lab now provides a possible explanation. We identified chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in mammalian cells.

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.