Stress granules plug and stabilize damaged endolysosomal membranes.

Endomembrane damage represents a form of stress that is detrimental for eukaryotic cells1,2. To cope with this threat, cells possess mechanisms that repair the damage and restore cellular homeostasis3-7. Endomembrane damage also results in organelle instability and the mechanisms by which cells stabilize damaged endomembranes to enable membrane repair remains unknown. Here, by combining in vitro and in cellulo studies with computational modelling we uncover a biological function for stress granules whereby these biomolecular condensates form rapidly at endomembrane damage sites and act as a plug that stabilizes the ruptured membrane. Functionally, we demonstrate that stress granule formation and membrane stabilization enable efficient repair of damaged endolysosomes, through both ESCRT (endosomal sorting complex required for transport)-dependent and independent mechanisms. We also show that blocking stress granule formation in human macrophages creates a permissive environment for Mycobacterium tuberculosis, a human pathogen that exploits endomembrane damage to survive within the host.

Targeting Cancer Lysosomes with Good Old Cationic Amphiphilic Drugs.

Being originally discovered as cellular recycling bins, lysosomes are today recognized as versatile signaling organelles that control a wide range of cellular functions that are essential not only for the well-being of normal cells but also for malignant transformation and cancer progression. In addition to their core functions in waste disposal and recycling of macromolecules and energy, lysosomes serve as an indispensable support system for malignant phenotype by promoting cell growth, cytoprotective autophagy, drug resistance, pH homeostasis, invasion, metastasis, and genomic integrity. On the other hand, malignant transformation reduces the stability of lysosomal membranes rendering cancer cells sensitive to lysosome-dependent cell death. Notably, many clinically approved cationic amphiphilic drugs widely used for the treatment of other diseases accumulate in lysosomes, interfere with their cancer-promoting and cancer-supporting functions and destabilize their membranes thereby opening intriguing possibilities for cancer therapy. Here, we review the emerging evidence that supports the supplementation of current cancer therapies with lysosome-targeting cationic amphiphilic drugs.

Analysis of Mitochondrial Dysfunction During Cell Death.

Mitochondria play a key role in various modes of cell death. Analysis of mitochondrial dysfunction and the release of proteins from the intermembrane space of mitochondria represent essential tools in cell death investigation. Here we describe how to evaluate release of intermembrane space proteins during apoptosis, alterations in the mitochondrial membrane potential, and oxygen consumption in apoptotic cells.

Cdc14 phosphatase downmodulates ESCRT-0 complex formation on vacuolar membranes and microautophagy after TORC1 inactivation.

Remodeling of vacuolar membranes mediated by endosomal sorting complex required for transport (ESCRT) is critical for microautophagy induction in budding yeast. Nutrient depletion and inactivation of target of rapamycin complex 1 (TORC1) protein kinase elicit recruitment of the ESCRT-0 complex (Vps27-Hse1) onto vacuolar membranes and ESCRT-mediated microautophagy induction. Mitotic protein phosphatase Cdc14 antagonizes TORC1-mediated phosphorylation in macroautophagy induction after nutrient starvation and TORC1 inactivation. Here, we report that Cdc14 downregulates microautophagy induction after TORC1 inactivation. Cdc14 dysfunction stimulated the vacuolar membrane recruitment of Hse1, but not Vps27, after TORC1 inactivation, promoting ESCRT-0 complex formation. Conversely, overexpression of CDC14 compromises Hse1 recruitment on vacuolar membranes and microautophagy induction after TORC1 inactivation. Thus, Cdc14 phosphatase regulates the fluxes of two types of autophagy in the opposite directions, namely, it elicits macroautophagy and attenuates microautophagy.

Roles of FAM134B in diseases from the perspectives of organelle membrane morphogenesis and cellular homeostasis.

Family with sequence similarity 134 member B (FAM134B)/RETREG1/JK1 is a novel gene with recently reported roles in various diseases. Understanding the function and mechanism of action of FAM134B is necessary to develop disease therapies. Notably, emerging data are clarifying the molecular mechanisms of FAM134B function in organelle membrane morphogenesis and the regulation of signaling pathways, such as the Wnt and AKT signaling pathways. In addition, transcription factors, RNA N6 -methyladenosine-mediated epigenetic regulation, microRNA, and small molecules are involved in the regulation of FAM134B expression. This review comprehensively considers recent studies on the role of FAM134B and its potential mechanisms in neurodegenerative diseases, obesity, viral diseases, cancer, and other diseases. The functions of FAM134B in maintaining cell homeostasis by regulating Golgi morphology, endoplasmic reticulum autophagy, and mitophagy are also highlighted, which may be the underlying mechanism of FAM134B gene mutation-induced diseases. Moreover, the molecular mechanisms of the FAM134B function during numerous biological processes are discussed. This review provides novel insights into the functions and mechanisms of FAM134B in various diseases, which will inform the development of effective drugs to treat diseases.

Organelle degradation in the lens by PLAAT phospholipases.

The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.

Lysosomal retargeting of Myoferlin mitigates membrane stress to enable pancreatic cancer growth.

Lysosomes must maintain the integrity of their limiting membrane to ensure efficient fusion with incoming organelles and degradation of substrates within their lumen. Pancreatic cancer cells upregulate lysosomal biogenesis to enhance nutrient recycling and stress resistance, but it is unknown whether dedicated programmes for maintaining the integrity of the lysosome membrane facilitate pancreatic cancer growth. Using proteomic-based organelle profiling, we identify the Ferlin family plasma membrane repair factor Myoferlin as selectively and highly enriched on the membrane of pancreatic cancer lysosomes. Mechanistically, lysosomal localization of Myoferlin is necessary and sufficient for the maintenance of lysosome health and provides an early acting protective system against membrane damage that is independent of the endosomal sorting complex required for transport (ESCRT)-mediated repair network. Myoferlin is upregulated in human pancreatic cancer, predicts poor survival and its ablation severely impairs lysosome function and tumour growth in vivo. Thus, retargeting of plasma membrane repair factors enhances the pro-oncogenic activities of the lysosome.

Exogenous Fatty Acids Modulate ER Lipid Composition and Metabolism in Breast Cancer Cells.

(1) Background: Lipid metabolism is a fundamental hallmark of all tumors, especially of breast cancer. Few studies describe the different lipid metabolisms and sensitivities to the microenvironment of breast cancer cell subtypes that influence the proliferation, aggressiveness, and success of therapy. This study describes the impact of lipid microenvironment on endoplasmic reticulum (ER) membrane and metabolic activity in two breast cancer cell lines with Luminal A and triple-negative breast cancer (TNBC) features. (2) Methods: We investigated the peculiar lipid phenotype of a TNBC cell line, MDA-MB-231, and a Luminal A cell line, MCF7, and their different sensitivity to exogenous fatty acids (i.e., palmitic acid (PA) and docosahexaenoic acid (DHA)). Moreover, we verified the impact of exogenous fatty acids on ER lipid composition. (3) Results: The data obtained demonstrate that MDA-MB-231 cells are more sensitive to the lipid microenvironment and that both PA and DHA are able to remodel their ER membranes with consequences on resident enzyme activity. On the contrary, MCF7 cells are less sensitive to PA, whereas they incorporate DHA, although less efficiently than MDA-MB-231 cells. (4) Conclusions: This study sustains the importance of lipid metabolism as an innovative hallmark to discriminate breast cancer subclasses and to develop personalized and innovative pharmacological strategies. The different sensitivities to the lipid environment shown by MCF7 and MDA-MB-231 cells might be related to cell malignancy and chemoresistance onset. In the future, this new approach could lead to a substantial decrease both in deleterious side effects for the patients and in the cost of entire therapeutic treatments coupled with increased therapy efficiency.

The Molecular Mechanisms Underlying Mitochondria-Associated Endoplasmic Reticulum Membrane-Induced Insulin Resistance.

Mitochondria and the endoplasmic reticulum (ER) are connected at multiple sites via what are known as mitochondria-associated ER membranes (MAMs). These associations are known to play an important role in maintaining cellular homeostasis. Impaired MAM signaling has wide-ranging effects in many diseases, such as obesity, diabetes, and neurodegenerative disorders. Accumulating evidence has suggested that MAMs influence insulin signaling through different pathways, including those associated with Ca2+ signaling, lipid metabolism, mitochondrial function, ER stress responses, and inflammation. Altered MAM signaling is a common feature of insulin resistance in different tissues, including the liver, muscle, and even the brain. In the liver, MAMs are key glucose-sensing regulators and have been proposed to be a hub for insulin signaling. Impaired MAM integrity has been reported to disrupt hepatic responses to changes in glucose availability during nutritional transition and to induce hepatic insulin resistance. Meanwhile, these effects can be rescued by the reinforcement of MAM interactions. In contrast, several studies have proposed that enhanced ER-mitochondria connections are detrimental to hepatic insulin signaling and can lead to mitochondrial dysfunction. Thus, given these contradictory results, the role played by the MAM in the regulation of hepatic insulin signaling remains elusive. Similarly, in skeletal muscle, enhanced MAM formation may be beneficial in the early stage of diabetes, whereas continuous MAM enhancement aggravates insulin resistance. Furthermore, recent studies have suggested that ER stress may be the primary pathway through which MAMs induce brain insulin resistance, especially in the hypothalamus. This review will discuss the possible mechanisms underlying MAM-associated insulin resistance as well as the therapeutic potential of targeting the MAM in the treatment of type 2 diabetes.

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

Disrupted ER membrane protein complex-mediated topogenesis drives congenital neural crest defects.

Multipass membrane proteins have a myriad of functions, including transduction of cell-cell signals, ion transport, and photoreception. Insertion of these proteins into the membrane depends on the endoplasmic reticulum (ER) membrane protein complex (EMC). Recently, birth defects have been observed in patients with variants in the gene encoding a member of this complex, EMC1. Patient phenotypes include congenital heart disease, craniofacial malformations, and neurodevelopmental disease. However, a molecular connection between EMC1 and these birth defects is lacking. Using Xenopus, we identified defects in neural crest cells (NCCs) upon emc1 depletion. We then used unbiased proteomics and discovered a critical role for emc1 in WNT signaling. Consistent with this, readouts of WNT signaling and Frizzled (Fzd) levels were reduced in emc1-depleted embryos, while NCC defects could be rescued with ß-catenin. Interestingly, other transmembrane proteins were mislocalized upon emc1 depletion, providing insight into additional patient phenotypes. To translate our findings back to humans, we found that EMC1 was necessary for human NCC development in vitro. Finally, we tested patient variants in our Xenopus model and found the majority to be loss-of-function alleles. Our findings define molecular mechanisms whereby EMC1 dysfunction causes disease phenotypes through dysfunctional multipass membrane protein topogenesis.

PLA2G4A/cPLA2-mediated lysosomal membrane damage leads to inhibition of autophagy and neurodegeneration after brain trauma.

Lysosomal membrane permeabilization (LMP) is observed under many pathological conditions, leading to cellular dysfunction and death. However, the mechanisms by which lysosomal membranes become leaky in vivo are not clear. Our data demonstrate that LMP occurs in neurons following controlled cortical impact induced (CCI) traumatic brain injury (TBI) in mice, leading to impaired macroautophagy (autophagy) and neuronal cell death. Comparison of LC-MS/MS lysosomal membrane lipid profiles from TBI and sham animals suggested a role for PLA2G4A/cPLA2 (phospholipase A2, group IVA [cytosolic, calcium-dependent]) in TBI-induced LMP. Activation of PLA2G4A caused LMP and inhibition of autophagy flux in cell lines and primary neurons. In vivo pharmacological inhibition of PLA2G4A attenuated TBI-induced LMP, as well as subsequent impairment of autophagy and neuronal loss, and was associated with improved neurological outcomes. Inhibition of PLA2G4A in vitro limited amyloid-ß-induced LMP and inhibition of autophagy. Together, our data indicate that PLA2G4A -mediated lysosomal membrane damage is involved in neuronal cell death following CCI-induced TBI and potentially in other neurodegenerative disorders.Abbreviations: AACOCF3, arachidonyl trifluoromethyl ketone; ACTB/ß-actin, actin, beta; AD, Alzheimer disease; ATG5, autophagy related 5; ATG7, autophagy related 7; ATG12, autophagy related 12; BECN1, beclin 1, autophagy related; C1P, ceramide-1-phosphate; CCI, controlled cortical impact; CTSD, cathepsin D; CTSL, cathepsin L; GFP, green fluorescent protein; IF, immunofluorescence; LAMP1, lysosomal-associated membrane protein 1; LAMP2, lysosomal-associated membrane protein 2; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LMP, Lysosomal membrane permeabilization; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; MAP1LC3/LC3, microtuble-associated protein 1 light chain 3; NAGLU, alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); PC, diacyl glycerophosphatidylcholine; PE, diacyl glycerophosphatidylethanolamine; PE-O, plasmanyl glycerophosphatidylethanolamine; PE-P, plasmenyl glycerophosphatidylethanolamine; PLA2G4A/cPLA2, phospholipase A2, group IVA (cytosolic, calcium-dependent); RBFOX3, RNA binding protein, fox-1 homolog (C. elegans) 3; RFP, red fluorescent protein; ROS, reactive oxygen species; SQSTM1, sequestosome 1; TUBA1/α-tubulin, tubulin, alpha; TBI, traumatic brain injury; TFEB, transcription factor EB; ULK1, unc-51 like kinase 1.

Lysosomal Membrane Stability in Hemocytes and Micronuclei in Gills of Perumytilus purpuratus Lamarck 1819 (Bivalvia: Mytilidae) Exposed to Copper.

The aim of this study was to determine the cytotoxic and genotoxic effects of copper on the bivalve Perumytilus purpuratus. The individuals were exposed to three copper concentrations: 1, 30 and 45 µg L-1 for 24, 48 and 96 h. Lysosomal membrane stability in hemocytes was determined through the neutral red retention time (NRRT) and micronucleus (MN) frequency tests in hemocytes and gills. The results show that the NRRT decreased significantly at 30 µg L-1 after 48 h of exposure. The frequency of MN was significantly greater in gills after 24 h in all concentrations tested. Copper is cytotoxic from 30 µg L-1 and genotoxic from 1 µg L-1. The use of these biomarkers of effects in P. purpuratus is proposed as an early warning tool for monitoring in environmental assessment of coastal ecosystems impacted by mining activities.

Effect of Cell Age on Uptake and Toxicity of Nanoparticles: The Overlooked Factor at the Nanobio Interface.

Clinical translation of nanotechnologies has limited success, at least in part, due to the existence of several overlooked factors on the nature of the nanosystem (e.g., physicochemical properties of nanoparticles), nanobio interfaces (e.g., protein corona composition), and the cellular characteristics (e.g., cell type). In the past decade, several ignored factors including personalized and disease-specific protein corona (a layer of formed biomolecules at the surface of nanoparticles upon their entrance into a biological fluid), incubating temperature, local temperature gradient, cell shape, and cell sex has been introduced. Here, it was hypothesized and validated cell age as another overlooked factor in the field of nanomedicine. To test our hypothesis, cellular toxicity and uptake profiles of our model nanoparticles (i.e., PEGylated quantum dots, QDs) were probed in young and senescent cells (i.e., IMR90 fibroblast cells from human fetal lung and CCD841CoN epithelial cells from human fetal colon) and the outcomes revealed substantial dependency of cell-nanoparticles interactions to the cell age. For example, it was observed that the PEGylated QDs were acutely toxic to senescent IMR90 and CCD841CoN cells, leading to lysosomal membrane permeabilization which caused cell necrosis; in contrast, the young cells were resilient to the exact same amount of QDs and the same incubation time. It was also found that the formation of protein corona could delay the QDs' toxicity on senescent cells. These findings suggest that the cellular aging process have a capacity to cause deteriorative effects on their organelles and normal functions. The outcomes of this study suggest the proof-of-concept that cell age may have critical role in biosystem responses to nanoparticle technologies. Therefore, the effect of cell age should be carefully considered on the nanobio interactions and the information about cellular age (e.g., passage number and age of the cell donor) should be included in the nanomedicine papers to facilitate clinical translation of nanotechnologies and to help scientists to better design and produce safe and efficient diagnostic/therapeutic age-specific nanoparticles.

Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilization.

The current clinical care of glioblastomas leaves behind invasive, radio- and chemo-resistant cells. We recently identified mammary-derived growth inhibitor (MDGI/FABP3) as a biomarker for invasive gliomas. Here, we demonstrate a novel function for MDGI in the maintenance of lysosomal membrane integrity, thus rendering invasive glioma cells unexpectedly vulnerable to lysosomal membrane destabilization. MDGI silencing impaired trafficking of polyunsaturated fatty acids into cells resulting in significant alterations in the lipid composition of lysosomal membranes, and subsequent death of the patient-derived glioma cells via lysosomal membrane permeabilization (LMP). In a preclinical model, treatment of glioma-bearing mice with an antihistaminergic LMP-inducing drug efficiently eradicated invasive glioma cells and secondary tumours within the brain. This unexpected fragility of the aggressive infiltrating cells to LMP provides new opportunities for clinical interventions, such as re-positioning of an established antihistamine drug, to eradicate the inoperable, invasive, and chemo-resistant glioma cells from sustaining disease progression and recurrence.

Organelle crosstalk in the kidney.

Organelle damage can cause various kidney diseases. In particular, organelle stress such as decreased proteostatic activity in the endoplasmic reticulum (ER) and altered energy metabolism in mitochondria contribute to glomerular and tubulointerstitial damage, resulting in the progression and development of kidney diseases. The ER regulates protein quality control via the unfolded protein response (UPR) pathway. Pathogenic ER stress leads to dysregulation of this pathway, and a maladaptive UPR is highly deleterious to renal cell function, and thereby has been implicated in the pathophysiology of various kidney diseases. The UPR pathway in the ER also regulates mitochondrial metabolic status, indicating the pathophysiological significance of organelle crosstalk between the ER and mitochondria via the UPR pathway. In recent years, it has become obvious that communication among organelles also is conducted through direct interactions at membrane contact sites (MCSs). Organelles exchange materials including lipids, ions, and proteins at the MCS. Accordingly, alterations to these networks, such as impaired ER-mitochondria MCSs, have been linked to several diseases such as neurodegeneration and diabetes. In this review, we describe the roles of organelles in kidney diseases and the mechanisms underlying organelle communication at the MCS, and especially at the mitochondria-associated ER membrane. Potential treatment options that are focused on organelle crosstalk are discussed, in addition to the relationship between this phenomenon and various diseases, especially kidney diseases.

Src in endosomal membranes promotes exosome secretion and tumor progression.

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src-transformed cells. Our results indicate that activated c-Src in the endosomal membrane promoted the secretion of exosomes, in which c-Src was encapsulated. In addition, the ESCRT-interacting molecule, Alix was identified as a c-Src-interacting protein in exosomes. We revealed that the interaction between the SH3 domain of c-Src and the proline-rich region of Alix activates ESCRT-mediated intra-luminal vesicle (ILV) formation, resulting in the upregulation of exosome secretion in c-Src-transformed cells. We observed also a correlation between malignant phenotypes and Alix-dependent aberrant exosome secretion in Src-upregulated cancer cells. Collectively, our findings provide a unique mechanism for the upregulation of exosomes in cancer cells, as well as new insights into the significance of exosome secretion in cancer progression.

Apilimod, a candidate anticancer therapeutic, arrests not only PtdIns(3,5)P2 but also PtdIns5P synthesis by PIKfyve and induces bafilomycin A1-reversible aberrant endomembrane dilation.

PIKfyve, an evolutionarily conserved kinase synthesizing PtdIns5P and PtdIns(3,5)P2, is crucial for mammalian cell proliferation and viability. Accordingly, PIKfyve inhibitors are now in clinical trials as anti-cancer drugs. Among those, apilimod is the most promising, yet its potency to inhibit PIKfyve and affect endomembrane homeostasis is only partially characterized. We demonstrate here for the first time that apilimod powerfully inhibited in vitro synthesis of PtdIns5P along with that of PtdIns(3,5)P2. HPLC-based resolution of intracellular phosphoinositides (PIs) revealed that apilimod triggered a marked reduction of both lipids in the context of intact cells. Notably, there was also a profound rise in PtdIns3P resulting from arrested PtdIns3P consumption for PtdIns(3,5)P2 synthesis. As typical for PIKfyve inhibition and the concomitant PtdIns(3,5)P2 reduction, apilimod induced the appearance of dilated endomembrane structures in the form of large translucent cytoplasmic vacuoles. Remarkably, bafilomycin A1 (BafA1) fully reversed the aberrant cell phenotype back to normal and completely precluded the appearance of cytoplasmic vacuoles when added prior to apilimod. Inspection of the PI profiles ruled out restoration of the reduced PtdIns(3,5)P2 pool as a molecular mechanism underlying BafA1 rescue. Rather, we found that BafA1 markedly attenuated the PtdIns3P elevation under PIKfyve inhibition. This was accompanied by profoundly decreased endosomal recruitment of fusogenic EEA1. Together, our data demonstrate that apilimod inhibits not only PtdIns(3,5)P2 but also PtdIns5P synthesis and that the cytoplasmic vacuolization triggered by the inhibitor is precluded or reversed by BafA1 through a mechanism associated, in part, with reduction in both PtdIns3P levels and EEA1 membrane recruitment.

SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death.

The molecular mechanisms underlying the severe lung pathology that occurs during SARS-CoV infections remain incompletely understood. The largest of the SARS-CoV accessory protein open reading frames (SARS 3a) oligomerizes, dynamically inserting into late endosomal, lysosomal, and trans-Golgi-network membranes. While previously implicated in a non-inflammatory apoptotic cell death pathway, here we extend the range of SARS 3a pathophysiologic targets by examining its effects on necrotic cell death pathways. We show that SARS 3a interacts with Receptor Interacting Protein 3 (Rip3), which augments the oligomerization of SARS 3a helping drive necrotic cell death. In addition, by inserting into lysosomal membranes SARS 3a triggers lysosomal damage and dysfunction. Consequently, Transcription Factor EB (TFEB) translocates to the nucleus increasing the transcription of autophagy- and lysosome-related genes. Finally, SARS 3a activates caspase-1 either directly or via an enhanced potassium efflux, which triggers NLRP3 inflammasome assembly. In summary, Rip3-mediated oligomerization of SARS 3a causes necrotic cell death, lysosomal damage, and caspase-1 activation-all likely contributing to the clinical manifestations of SARS-CoV infection.

γ-Secretase promotes membrane insertion of the human papillomavirus L2 capsid protein during virus infection.

Despite their importance as human pathogens, entry of human papillomaviruses (HPVs) into cells is poorly understood. The transmembrane protease γ-secretase executes a crucial function during the early stages of HPV infection, but the role of γ-secretase in infection and the identity of its critical substrate are unknown. Here we demonstrate that γ-secretase harbors a previously uncharacterized chaperone function, promoting low pH-dependent insertion of the HPV L2 capsid protein into endosomal membranes. Upon membrane insertion, L2 recruits the cytosolic retromer, which enables the L2 viral genome complex to enter the retrograde transport pathway and traffic to the Golgi en route for infection. Although a small fraction of membrane-inserted L2 is also cleaved by γ-secretase, this proteolytic event appears dispensable for HPV infection. Our findings demonstrate that γ-secretase is endowed with an activity that can promote membrane insertion of L2, thereby targeting the virus to the productive infectious pathway.