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.

The porcine epidemic diarrhea virus (PEDV), belonging to the α-coronavirus, is the causative agent of porcine epidemic diarrhea (PED). Presently, protection from the existing PEDV vaccine is not effective. Therefore, anti-PEDV compounds should be studied. Berbamine (BBM), Fangchinoline (FAN), and (+)-Fangchinoline (+FAN), are types of bis-benzylisoquinoline alkaloids that are extracted from natural medicinal plants. These bis-benzylisoquinoline alkaloids have various biological activities, including antiviral, anticancer, and anti-inflammatory properties. In this study, we found that BBM, FAN, and +FAN suppressed PEDV activity with a 50% inhibitory concentration of 9.00 µM, 3.54 µM, and 4.68 µM, respectively. Furthermore, these alkaloids can decrease the PEDV-N protein levels and virus titers in vitro. The time-of-addition assay results showed that these alkaloids mainly inhibit PEDV entry. We also found that the inhibitory effects of BBM, FAN, and +FAN on PEDV rely on decreasing the activity of Cathepsin L (CTSL) and Cathepsin B (CTSB) by suppressing lysosome acidification. Taken together, these results indicated that BBM, FAN, and +FAN were effective anti-PEDV natural products that prevented PEDV entry and may be considered novel antiviral drugs.

Cellulose nanocrystals (CNCs) display remarkable strength and physicochemical properties with significant potential applications. To better understand the potential adjuvanticity of a nanomaterial, it is important to investigate the extent of the immunological response, the mechanisms by which they elicit this response, and how this response is associated with their physicochemical characteristics. In this study, we investigated the potential mechanisms of immunomodulation and redox activity of two chemically related cationic CNC derivatives (CNC-METAC-1B and CNC-METAC-2B), using human peripheral blood mononuclear cells and mouse macrophage cells (J774A.1). Our data demonstrated that the biological effects caused by these nanomaterials occurred mainly with short term exposure. We observed opposite immunomodulatory activity between the tested nanomaterials. CNC-METAC-2B, induced IL-1β secretion at 2 h while CNC-METAC-1B decreased it at 24 h of treatment. In addition, both nanomaterials caused more noticeable increases in mitochondrial reactive oxygen species (ROS) at early time. The differences in apparent sizes of the two cationic nanomaterials could explain, at least in part, the discrepancies in biological effects, despite their closely related surface charges. This work provides initial insights about the complexity of the in vitro mechanism of action of these nanomaterials as well as foundation knowledge for the development of cationic CNCs as potential immunomodulators.

To investigate the role of H+ /K+ ATPase in the proliferation of pepsin-induced vocal cord leukoplakia (VCL) cells.
Translation research.
Affiliated Hospital of University.
Immunohistochemistry was used to detect pepsin, H+ /K+ ATPase (ATP4A and ATP4B subunits) in VCL cells with varying degrees of dysplasia. After primary cultures of VCL cells had been established, the effects of acidified pepsin on the proliferation, autophagy, and H+ /K+ -ATPase distribution of VCL cells were investigated.
The levels of pepsin, ATP4A, and ATP4B were significantly higher in VCL tissue with moderate-to-severe dysplasia than in normal tissue (p < .05); these levels gradually increased according to dysplasia severity. The expression levels of ATP4A and ATP4B were significantly correlated with the amount of pepsin in VCL cells (p < .01). Acidified pepsin enhanced the levels of proliferation and autophagy in human VCL epithelial cells. The cloning- and autophagy-promoting effects of acidified pepsin on VCL cells were partially reversed by pantoprazole; these effects were completely blocked by the autophagy inhibitor chloroquine. Finally, acidified pepsin promoted the colocalization of H+ /K+ -ATPase and lysosomes in VCL cells; it also mediated lysosome acidification.
Pepsin and H+ /K+ -ATPase may contribute to the progression of VCL. Specifically, acidified pepsin may regulate lysosome acidification by promoting lysosomal localization of H+ /K+ -ATPase.

Lysosome acidification is a dynamic equilibrium of H+ influx and efflux across the membrane, which is crucial for cell physiology. The vacuolar H+ ATPase (V-ATPase) is responsible for the H+ influx or refilling of lysosomes. TMEM175 was identified as a novel H+ permeable channel on lysosomal membranes, and it plays a critical role in lysosome acidification. However, how TMEM175 participates in lysosomal acidification remains unknown. Here, we present evidence that TMEM175 regulates lysosomal H+ influx and efflux in enlarged lysosomes isolated from COS1 treated with vacuolin-1. By utilizing the whole-endolysosome patch-clamp recording technique, a series of integrated lysosomal H+ influx and efflux signals in a ten-of-second time scale under the physiological pH gradient (luminal pH 4.60, and cytosolic pH 7.20) was recorded from this in vitro system. Lysosomal H+ fluxes constitute both the lysosomal H+ refilling and releasing, and they are asymmetrical processes with distinct featured kinetics for each of the H+ fluxes. Lysosomal H+ fluxes are entirely abolished when TMEM175 losses of function in the F39V mutant and is blocked by the antagonist (2-GBI). Meanwhile, lysosomal H+ fluxes are modulated by the pH-buffering capacity of the lumen and the lysosomal glycosylated membrane proteins, lysosome-associated membrane protein 1 (LAMP1). We propose that the TMEM175-mediated lysosomal H+ fluxes model would provide novel thoughts for studying the pathology of Parkinson\'s disease and lysosome storage disorders.

Protective autophagy can be activated by external stimuli such as chemotherapy (CT) and photothermal therapy (PTT), leading to tumour resistance. As a key subcellular for autophagy, lysosomal dysfunction is crucial for autophagy suppression. Furthermore, lysosomal drug sequestration enhances basic drug resistance such as doxorubicin (DOX), which is trapped away from its target site, namely, the nucleus. Moreover, most of nanodrug delivery systems are internalised to lysosome for degradation, which further leads to DOX resistance. Lysosome serves as an essential organelle in drug resistance mechanisms, whose acidification arrest provides a potential strategy to inhibit autophagy and lysosomal drug sequestration simultaneously. The chloride channel-3 (ClC-3) protein is known as an important Cl--H+ transporter to maintain lysosomal pH at low values of various human cells. Herein, a black phosphorus-based theranostic nanoplatform of BP-A-S@D is constructed, and HeLa cells are used as a model to verify the effect of ClC-3 on tumour lysosomal acidification and autophagy regulation. Consequently, ClC-3 silencing inhibits not only protective autophagy to sensitise chemo-photothermal therapy, but also DOX resistance by suppressing lysosomal acidification. Therefore, ClC-3 silencing could simultaneously inhibit autophagy and lysosomal drug sequestration to improve anti-tumour efficiency.

Genetic evidence has increasingly linked lysosome dysfunction to an impaired autophagy-lysosomal pathway (ALP) flux in Alzheimer disease (AD) although the relationship of these abnormalities to other pathologies is unclear. In our recent investigation on the origin of impaired autophagic flux in AD, we established the critical early role of defective lysosomes in five mouse AD models. To assess in vivo alterations of autophagy and ALP vesicle acidification, we expressed eGFP-mRFP-LC3 specifically in neurons. We discovered that autophagy dysfunction in these models arises from exceptionally early failure of autolysosome/lysosome acidification, which then drives downstream AD pathogenesis. Extreme autophagic stress in compromised but still intact neurons causes autophagic vacuoles (AVs) containing toxic APP metabolites, Aβ/β-CTFs, to pack into huge blebs and protrude from the perikaryon membrane. Most notably, AVs also coalesce with ER tubules and yield fibrillar β-amyloid within these tubules. Collectively, amyloid immunoreactivity within these intact neurons assumes the appearance of amyloid-plaques, and indeed, their eventual death transforms them into extracellular plaque lesions. Quantitative analysis confirms that neurons undergoing this transformation are the principal source of β-amyloid-plaques in APP-AD models. These findings prompt reconsideration of the conventionally accepted sequence of events in plaque formation and may help explain the inefficacy of Aβ/amyloid vaccine therapies.

Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.

Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration.

Germline mutations in heat shock factor 4 (HSF4) cause congenital cataracts. Previously, we have shown that HSF4 is involved in regulating lysosomal pH in mouse lens epithelial cell in vitro. However, the underlying mechanism remains unclear.
HSF4-deficient mouse lens epithelial cell lines and zebrafish were used in this study. Immunoblotting and quantitative RT-PCR were used for expression analysis. The protein-protein interactions were tested with GST-pull downs. The lysosomes were fractioned by ultracentrifugation.
HSF4 deficiency or knock down of αB-crystallin elevates lysosomal pH and increases the ubiquitination and degradation of ATP6V1A by the proteasome. αB-crystallin localizes partially in the lysosome and interacts solely with the ATP6V1A protein of the V1 complex of V-ATPase. Furthermore, αB-crystallin can co-precipitate with mTORC1 and ATP6V1A in GST pull down assays. Inhibition of mTORC1 by rapamycin or siRNA can lead to dissociation of αB-crystallin from the ATP6V1A and mTORC1complex, shortening the half-life of ATP6V1A and increasing the lysosomal pH. Mutation of ATP6V1A/S441A (the predicted mTOR phosphorylation site) reduces its association with αB-crystallin. In the zebrafish model, HSF4 deficiency reduces αB-crystallin expression and elevates the lysosomal pH in lens tissues.
HSF4 regulates lysosomal acidification by controlling the association of αB-crystallin with ATP6V1A and mTOR and regulating ATP6V1A protein stabilization.
This study uncovers a novel function of αB-crystallin, demonstrating that αB-crystallin can regulate lysosomal ATP6V1A protein stabilization by complexing to ATP6V1A and mTOR. This highlights a novel mechanism by which HSF4 regulates the proteolytic process of organelles during lens development.