The Endo-Lysosomal Damage Response.

Lysosomes are the degradative endpoints of material delivered by endocytosis and autophagy and are therefore particularly prone to damage. Membrane permeabilization or full rupture of lysosomal or late endosomal compartments is highly deleterious because it threatens cellular homeostasis and can elicit cell death and inflammatory signaling. Cells have developed a complex response to endo-lysosomal damage that largely consists of three branches. Initially, a number of repair pathways are activated to restore the integrity of the lysosomal membrane. If repair fails or if damage is too extensive, lysosomes are isolated and degraded by a form of selective autophagy termed lysophagy. Meanwhile, an mTORC1-governed signaling cascade drives biogenesis and regeneration of new lysosomal components to reestablish the full lysosomal capacity of the cell. This damage response is vital to counteract the effects of various conditions, including neurodegeneration and infection, and can constitute a critical vulnerability in cancer cells.

Leucine-Rich Repeat Kinases.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.

Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

A membrane-associated MHC-I inhibitory axis for cancer immune evasion.

Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.

Structure and mechanism of human cystine exporter cystinosin.

Lysosomal amino acid efflux by proton-driven transporters is essential for lysosomal homeostasis, amino acid recycling, mTOR signaling, and maintaining lysosomal pH. To unravel the mechanisms of these transporters, we focus on cystinosin, a prototypical lysosomal amino acid transporter that exports cystine to the cytosol, where its reduction to cysteine supplies this limiting amino acid for diverse fundamental processes and controlling nutrient adaptation. Cystinosin mutations cause cystinosis, a devastating lysosomal storage disease. Here, we present structures of human cystinosin in lumen-open, cytosol-open, and cystine-bound states, which uncover the cystine recognition mechanism and capture the key conformational states of the transport cycle. Our structures, along with functional studies and double electron-electron resonance spectroscopic investigations, reveal the molecular basis for the transporter's conformational transitions and protonation switch, show conformation-dependent Ragulator-Rag complex engagement, and demonstrate an unexpected activation mechanism. These findings provide molecular insights into lysosomal amino acid efflux and a potential therapeutic strategy.

Structural Basis of Low-pH-Dependent Lysosomal Cholesterol Egress by NPC1 and NPC2.

Lysosomal cholesterol egress requires two proteins, NPC1 and NPC2, whose defects are responsible for Niemann-Pick disease type C (NPC). Here, we present systematic structural characterizations that reveal the molecular basis for low-pH-dependent cholesterol delivery from NPC2 to the transmembrane (TM) domain of NPC1. At pH 8.0, similar structures of NPC1 were obtained in nanodiscs and in detergent at resolutions of 3.6 Å and 3.0 Å, respectively. A tunnel connecting the N-terminal domain (NTD) and the transmembrane sterol-sensing domain (SSD) was unveiled. At pH 5.5, the NTD exhibits two conformations, suggesting the motion for cholesterol delivery to the tunnel. A putative cholesterol molecule is found at the membrane boundary of the tunnel, and TM2 moves toward formation of a surface pocket on the SSD. Finally, the structure of the NPC1-NPC2 complex at 4.0 Å resolution was obtained at pH 5.5, elucidating the molecular basis for cholesterol handoff from NPC2 to NPC1(NTD).

Mitophagy in Intestinal Epithelial Cells Triggers Adaptive Immunity during Tumorigenesis.

In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.

iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.

The DNA Inflammasome in Human Myeloid Cells Is Initiated by a STING-Cell Death Program Upstream of NLRP3.

Detection of cytosolic DNA constitutes a central event in the context of numerous infectious and sterile inflammatory conditions. Recent studies have uncovered a bipartite mode of cytosolic DNA recognition, in which the cGAS-STING axis triggers antiviral immunity, whereas AIM2 triggers inflammasome activation. Here, we show that AIM2 is dispensable for DNA-mediated inflammasome activation in human myeloid cells. Instead, detection of cytosolic DNA by the cGAS-STING axis induces a cell death program initiating potassium efflux upstream of NLRP3. Forward genetics identified regulators of lysosomal trafficking to modulate this cell death program, and subsequent studies revealed that activated STING traffics to the lysosome, where it triggers membrane permeabilization and thus lysosomal cell death (LCD). Importantly, the cGAS-STING-NLRP3 pathway constitutes the default inflammasome response during viral and bacterial infections in human myeloid cells. We conclude that targeting the cGAS-STING-LCD-NLRP3 pathway will ameliorate pathology in inflammatory conditions that are associated with cytosolic DNA sensing.

Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.

Glycerophosphodiesters inhibit lysosomal phospholipid catabolism in Batten disease.

Batten disease, the most prevalent form of neurodegeneration in children, is caused by mutations in the CLN3 gene, which encodes a lysosomal transmembrane protein. CLN3 loss leads to significant accumulation of glycerophosphodiesters (GPDs), the end products of glycerophospholipid catabolism in the lysosome. Despite GPD storage being robustly observed upon CLN3 loss, the role of GPDs in neuropathology remains unclear. Here, we demonstrate that GPDs act as potent inhibitors of glycerophospholipid catabolism in the lysosome using human cell lines and mouse models. Mechanistically, GPDs bind and competitively inhibit the lysosomal phospholipases PLA2G15 and PLBD2, which we establish to possess phospholipase B activity. GPDs effectively inhibit the rate-limiting lysophospholipase activity of these phospholipases. Consistently, lysosomes of CLN3-deficient cells and tissues accumulate toxic lysophospholipids. Our work establishes that the storage material in Batten disease directly disrupts lysosomal lipid homeostasis, suggesting GPD clearance as a potential therapeutic approach to this fatal disease.

Lysosomal LAMP proteins regulate lysosomal pH by direct inhibition of the TMEM175 channel.

Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.

How Bacteria Subvert Animal Cell Structure and Function.

Bacterial pathogens encode a wide variety of effectors and toxins that hijack host cell structure and function. Of particular importance are virulence factors that target actin cytoskeleton dynamics critical for cell shape, stability, motility, phagocytosis, and division. In addition, many bacteria target organelles of the general secretory pathway (e.g., the endoplasmic reticulum and the Golgi complex) and recycling pathways (e.g., the endolysosomal system) to establish and maintain an intracellular replicative niche. Recent research on the biochemistry and structural biology of bacterial effector proteins and toxins has begun to shed light on the molecular underpinnings of these host-pathogen interactions. This exciting work is revealing how pathogens gain control of the complex and dynamic host cellular environments, which impacts our understanding of microbial infectious disease, immunology, and human cell biology.

The Lysosome as a Regulatory Hub.

The lysosome has long been viewed as the recycling center of the cell. However, recent discoveries have challenged this simple view and have established a central role of the lysosome in nutrient-dependent signal transduction. The degradative role of the lysosome and its newly discovered signaling functions are not in conflict but rather cooperate extensively to mediate fundamental cellular activities such as nutrient sensing, metabolic adaptation, and quality control of proteins and organelles. Moreover, lysosome-based signaling and degradation are subject to reciprocal regulation. Transcriptional programs of increasing complexity control the biogenesis, composition, and abundance of lysosomes and fine-tune their activity to match the evolving needs of the cell. Alterations in these essential activities are, not surprisingly, central to the pathophysiology of an ever-expanding spectrum of conditions, including storage disorders, neurodegenerative diseases, and cancer. Thus, unraveling the functions of this fascinating organelle will contribute to our understanding of the fundamental logic of metabolic organization and will point to novel therapeutic avenues in several human diseases.

Itaconate is a lysosomal inducer that promotes antibacterial innate immunity.

Lysosomes are the main organelles in macrophages for killing invading bacteria. However, the precise mechanism underlying lysosomal biogenesis upon bacterial infection remains enigmatic. We demonstrate here that LPS stimulation increases IRG1-dependent itaconate production, which promotes lysosomal biogenesis by activating the transcription factor, TFEB. Mechanistically, itaconate directly alkylates human TFEB at cysteine 212 (Cys270 in mice) to induce its nuclear localization by antagonizing mTOR-mediated phosphorylation and cytosolic retention. Functionally, abrogation of itaconate synthesis by IRG1/Irg1 knockout or expression of an alkylation-deficient TFEB mutant impairs the antibacterial ability of macrophages in vitro. Furthermore, knockin mice harboring an alkylation-deficient TFEB mutant display elevated susceptibility to Salmonella typhimurium infection, whereas in vivo treatment of OI, a cell-permeable itaconate derivative, limits inflammation. Our study identifies itaconate as an endogenous metabolite that functions as a lysosomal inducer in macrophages in response to bacterial infection, implying the potential therapeutic utility of itaconate in treating human bacterial infection.

ER-phagy: mechanisms, regulation, and diseases connected to the lysosomal clearance of the endoplasmic reticulum.

ER-phagy (reticulophagy) defines the degradation of portions of the endoplasmic reticulum (ER) within lysosomes or vacuoles. It is part of the self-digestion (i.e., autophagic) programs recycling cytoplasmic material and organelles, which rapidly mobilize metabolites in cells confronted with nutrient shortage. Moreover, selective clearance of ER subdomains participates in the control of ER size and activity during ER stress, the reestablishment of ER homeostasis after ER stress resolution, and the removal of ER parts in which aberrant and potentially cytotoxic material has been segregated. ER-phagy relies on the individual and/or concerted activation of the ER-phagy receptors, ER peripheral or integral membrane proteins that share the presence of LC3/Atg8-binding motifs in their cytosolic domains. ER-phagy involves the physical separation of portions of the ER from the bulk ER network and their delivery to the endolysosomal/vacuolar catabolic district. This last step is accomplished by a variety of mechanisms including macro-ER-phagy (in which ER fragments are sequestered by double-membrane autophagosomes that eventually fuse with lysosomes/vacuoles), micro-ER-phagy (in which ER fragments are directly engulfed by endosomes/lysosomes/vacuoles), or direct fusion of ER-derived vesicles with lysosomes/vacuoles. ER-phagy is dysfunctional in specific human diseases, and its regulators are subverted by pathogens, highlighting its crucial role for cell and organism life.

Connexin43 promotes exocytosis of damaged lysosomes through actin remodelling.

A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes.

Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation.

The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.

The G3BP1-Family-USP10 Deubiquitinase Complex Rescues Ubiquitinated 40S Subunits of Ribosomes Stalled in Translation from Lysosomal Degradation.

Ribosome-associated quality control (RQC) purges aberrant mRNAs and nascent polypeptides in a multi-step molecular process initiated by the E3 ligase ZNF598 through sensing of ribosomes collided at aberrant mRNAs and monoubiquitination of distinct small ribosomal subunit proteins. We show that G3BP1-family-USP10 complexes are required for deubiquitination of RPS2, RPS3, and RPS10 to rescue modified 40S subunits from programmed degradation. Knockout of USP10 or G3BP1 family proteins increased lysosomal ribosomal degradation and perturbed ribosomal subunit stoichiometry, both of which were rescued by a single K214R substitution of RPS3. While the majority of RPS2 and RPS3 monoubiquitination resulted from ZNF598-dependent sensing of ribosome collisions initiating RQC, another minor pathway contributed to their monoubiquitination. G3BP1 family proteins have long been considered RNA-binding proteins, however, our results identified 40S subunits and associated mRNAs as their predominant targets, a feature shared by stress granules to which G3BP1 family proteins localize under stress.

Termination of STING responses is mediated via ESCRT-dependent degradation.

cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.