Cell Non-autonomous Proteostasis Regulation in Aging and Disease.

Aging is a risk factor for a number of diseases, being the more notorious ones perhaps neurodegenerative diseases such as Alzheimer's and Parkinson's. These and other age-related pathologies are often associated with accumulation of proteotoxic material inside cells, as well as with the accumulation of protein deposits extracellularly. It is widely accepted that this accumulation of toxic proteins trails a progressive decline in the mechanisms that regulate protein homeostasis, or proteostasis, during aging. However, despite significant efforts, the progress in terms of novel or improved therapies targeting accumulation of proteotoxic material has been rather limited. For example, clinical trials for new drugs aimed at treating Alzheimer's disease, by preventing accumulation of toxic proteins, have notoriously failed. On the other hand, it is becoming increasingly apparent that regulation of proteostasis is not a cell autonomous process. In fact, cells rely on complex transcellular networks to maintain tissue and organ homeostasis involving endocrine and paracrine signaling pathways. In this review we will discuss the impact of cell non-autonomous proteostasis mechanisms and their impact in aging and disease. We will focus on how transcellular proteostasis networks can shed new light into stablished paradigms about the aging of organisms.

LAMP2A mediates the loading of proteins into endosomes and selects exosomal cargo.

Exosomes are a subtype of extracellular vesicles (EVs), released by all cell types, that originate from the invagination of the endosomal limiting membrane. These EVs can transport biological information in the form of proteins and RNA and have been the focus of intensive research over the last decade. It is becoming apparent that EVs can have important roles in health and disease. EVs are also promising noninvasive biomarkers of disease (liquid biopsies) and valuable vectors for innovative therapies. However, little is known about the mechanisms that regulate the loading of cytosolic proteins into exosomes. We recently showed that soluble proteins containing amino acid sequences biochemically related to the KFERQ motif are loaded into nascent exosomes at the endosomal limiting membrane, in a process mediated by LAMP2A. Because of the subcellular localization and machinery involved, this mechanism has many similarities with chaperone-mediated autophagy (CMA) and endosomal microautophagy (e-Mi), but also some important differences. In this punctum we will focus on the mechanistic details of exosomal LAMP2A loading of cargo (e-LLoC) as well as on its implications for intercellular and interorgan communication.

LAMP2A regulates the loading of proteins into exosomes.

Exosomes are extracellular vesicles of endosomal origin that are released by practically all cell types across metazoans. Exosomes are active vehicles of intercellular communication and can transfer lipids, RNAs, and proteins between different cells, tissues, or organs. Here, we describe a mechanism whereby proteins containing a KFERQ motif pentapeptide are loaded into a subpopulation of exosomes in a process that is dependent on the membrane protein LAMP2A. Moreover, we demonstrate that this mechanism is independent of the ESCRT machinery but dependent on HSC70, CD63, Alix, Syntenin-1, Rab31, and ceramides. We show that the master regulator of hypoxia HIF1A is loaded into exosomes by this mechanism to transport hypoxia signaling to normoxic cells. In addition, by tagging fluorescent proteins with KFERQ-like sequences, we were able to follow the interorgan transfer of exosomes. Our findings open new avenues for exosome engineering by allowing the loading of bioactive proteins by tagging them with KFERQ-like motifs.

Exosomes and STUB1/CHIP cooperate to maintain intracellular proteostasis.

Deregulation of proteostasis is a main feature of many age-related diseases, often leading to the accumulation of toxic oligomers and insoluble protein aggregates that accumulate intracellularly or in the extracellular space. To understand the mechanisms whereby toxic or otherwise unwanted proteins are secreted to the extracellular space, we inactivated the quality-control and proteostasis regulator ubiquitin ligase STUB1/CHIP. Data indicated that STUB1 deficiency leads both to the intracellular accumulation of protein aggregates and to an increase in the secretion of small extracellular vesicles (sEVs), including exosomes. Secreted sEVs are enriched in ubiquitinated and/or undegraded proteins and protein oligomers. Data also indicates that oxidative stress induces an increase in the release of sEVs in cells depleted from STUB1. Overall, the results presented here suggest that cells use exosomes to dispose of damaged and/or undegraded proteins as a means to reduce intracellular accumulation of proteotoxic material.

Gap junctional protein Cx43 is involved in the communication between extracellular vesicles and mammalian cells.

Intercellular communication is vital to ensure tissue and organism homeostasis and can occur directly, between neighbour cells via gap junctions (GJ), or indirectly, at longer distances, through extracellular vesicles, including exosomes. Exosomes, as intercellular carriers of messenger molecules, mediate the transfer of biological information between donor and acceptor cells. Although the biological effects of exosomes in target cells have been intensively studied, the mechanisms that govern exosomal uptake are not fully understood. Here, we show that Connexin 43 (Cx43), the most widely expressed GJ protein, is present in exosomes in the form of hexameric channels and, more importantly, that exosomal Cx43 is able to modulate the interaction and transfer of information between exosomes and acceptor cells. This study envisions a new paradigm where Cx43-containing channels mediate the release of exosomal content into cells, which constitutes a novel and unanticipated mechanism to modulate intercellular communication.

K63 linked ubiquitin chain formation is a signal for HIF1A degradation by Chaperone-Mediated Autophagy.

Chaperone-Mediated Autophagy is a selective form of autophagy. Recently, the degradation of a newly identified CMA substrate, the HIF1A transcription factor, was found to be regulated by the ubiquitin ligase STUB1. In this study we show, for the first time, that K63 ubiquitination is necessary for CMA degradation of HIF1A in vitro and in vivo. Additionally, STUB1 mediates K63 linked ubiquitination of HIF1A. Our findings add a new regulatory step and increase the specificity of the molecular mechanism involved in CMA degradation of HIF1A, expanding the role of ubiquitination to yet another biological process, since the same mechanism might be applicable to other CMA substrates.

STUB1/CHIP is required for HIF1A degradation by chaperone-mediated autophagy.

The transcription factor HIF1 is mostly regulated by the oxygen-dependent proteasomal degradation of the labile subunit HIF1A. Recent data showed degradation of HIF1A in the lysosome through chaperone-mediated autophagy (CMA). However the molecular mechanism involved has not been elucidated. This study shows that the KFERQ-like motif, that has been identified in all CMA substrates, is required to mediate the interaction between HIF1A and the chaperone HSPA8. Moreover, mutations in the KFERQ-like motif of HIF1A preclude the interaction with the CMA receptor LAMP2A, thus inhibiting its lysosomal degradation. Importantly, we show for the first time that the ubiquitin ligase STUB1 is required for degradation of HIF1A in the lysosome by CMA. Indeed, mutations in STUB1 that inhibit either the ubiquitin ligase activity or its ability to bind to HSPA8, both prevent degradation of HIF1A by CMA. Moreover, we show that HIF1A binds to and is translocated into intact lysosomes isolated from rat livers. This new pathway for degradation of HIF1A does not depend on the presence of oxygen and is activated in response to nutrient deprivation such that the levels of HIF1A bound to CMA positive lysosomes significantly increase in starved animal livers and the binding of HIF1A to LAMP2A increases in response to serum deprivation. Moreover, excessive degradation of HIF1A by CMA compromises cells' ability to respond to and survive under hypoxia, suggesting that this pathway might be of pathophysiological importance in conditions that combine hypoxia with starvation.