Lysosomal Membrane Protein Research Articles

Impaired autophagy mechanisms associated with phospholamban-R14del disease

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq Foundation Phospholamban (PLN) is a key modulator of sarcoplasmic reticulum Ca2+ homeostasis and cardiac contractility. The PLN variant c.40_42delAGA (p.R14del) leads to arrhythmogenic cardiomyopathy characterized by life-threatening ventricular arrhythmias and sudden cardiac death. A large number of PLN-R14del carriers have been identified worldwide, although the expanding use of genetic testing and increased awareness is unveiling a higher frequency. PLN-containing aggregates are emerging as a hallmark of this disease, and are increasingly believed to be associated with disease pathogenesis. However, the mechanism leading to the formation of aggregates is unknown. We performed an in depth molecular and cellular analysis utilizing in vitro systems to determine if these aggregates result from aberrations in autophagy, and the underlying mechanisms. Utilizing cell culture systems we studied the expression level and subcellular distribution of fundamental autophagy-related proteins and evaluated the impact of autophagy inducers and inhibitors. Expression of PLN-R14del in different cell culture systems (HEK293 and H9c2 cells) was found to result in PLN aggregate formation, which were determined to contain the autophagy marker proteins sequestosome-1 (p62) and microtubule-associated protein light chain 3 (LC3), indicating inefficient degradation by the autophagy pathway. In support of this, PLN-R14del expressing cells were shown to exhibit increased p62 and LC3-II protein levels. This was associated with significantly reduced autophagic flux, as demonstrated by examination of LC3-II protein levels after autophagy inhibition. Moreover, by detailed immunofluorescence analysis, we determined reduced co-localization of the autophagosomal protein LC3 and the lysosomal associated membrane protein 1 (LAMP1) in PLN-R14del cells, suggesting impaired autophagosome-lysosome fusion. We next examined the distribution of key proteins mediating autophagosome-lysosome membrane fusion including the Ras-related protein Rab7, the ultraviolet radiation resistance-associated gene (UVRAG) and membrane tether proteins. In PLN-R14del transfected cells, significant alterations in their co-localization with LC3 were observed, indicating their impaired recruitment to the autophagosomes. Importantly, in PLN-WT cells changes in cytosolic Ca2+ levels by thapsigargin treatment were found to recapitulate the autophagic defects of PLN-R14del, suggesting that Ca2+ alterations are a contributing factor of PLN-R14del mediated aberrant autophagy. In conclusion, our findings reveal a causal relationship between PLN-R14del and autophagy dysregulation, through inhibition of autophagosome-lysosome fusion and autophagic flux reduction. This ultimately leads to aggregate formation, an early pathological sign in human patients.

Chaperone-Mediated Autophagy in Brain Injury: A Double-Edged Sword with Therapeutic Potentials.

Autophagy is an intracellular recycling process that maintains cellular homeostasis by degrading excess or defective macromolecules and organelles. Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy in which a substrate containing a KFERQ-like motif is recognized by a chaperone protein, delivered to the lysosomal membrane, and then translocated to the lysosome for degradation with the assistance of lysosomal membrane protein 2A. Normal CMA activity is involved in the regulation of cellular proteostasis, metabolism, differentiation, and survival. CMA dysfunction disturbs cellular homeostasis and directly participates in the pathogenesis of human diseases. Previous investigations on CMA in the central nervous system have primarily focus on neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Recently, mounting evidence suggested that brain injuries involve a wider range of types and severities, making the involvement of CMA in the bidirectional processes of damage and repair even more crucial. In this review, we summarize the basic processes of CMA and its associated regulatory mechanisms and highlight the critical role of CMA in brain injury such as cerebral ischemia, traumatic brain injury, and other specific brain injuries. We also discuss the potential of CMA as a therapeutic target to treat brain injury and provide valuable insights into clinical strategies.

N-Acetylneuraminic Acid Inhibits Melanogenesis via Induction of Autophagy

N-acetylneuraminic acid (Neu5Ac) is the predominant form of sialic acid present in the glossy swiftlet (Collocalia esculenta). It is also the only form of sialic acid detected in the human body. In this study, we investigated the mechanism underlying melanogenesis inhibition by Neu5Ac. We discovered that a reduction in tyrosinase protein levels led to an inhibition of melanin production by Neu5Ac. Additionally, the mRNA and protein levels of ubiquitin-specific protease (USP5) and microtubule-associated protein 1 light chain 3 (LC3)-II increased, while those of p62 decreased, indicating enhanced autophagic activity. Lysosomal cathepsin L2 protein levels also increased, and immunostaining revealed colocalization of lysosomal membrane protein (LAMP)-1 and tyrosinase. Additionally, levels of chaperonin containing T-complex polypeptide (CCT), implicated in increased autophagic flux, were elevated. Altogether, these findings suggest that tyrosinase-containing coated vesicles are transported by Neu5Ac into the autophagic degradation pathway, suppressing mature melanosome generation. This process involves increased USP5 levels preventing recognition of polyubiquitin by proteasomes. Furthermore, elevated CCT3 protein levels may enhance autophagic flux, leading to the incorporation of tyrosinase-containing coated vesicles into autophagosomes. These autophagosomes then fuse with lysosomes for cathepsin L2–mediated degradation. Thus, our findings suggest that Neu5Ac reduces tyrosinase activity and inhibits melanosome maturation by promoting selective autophagic degradation of abnormal proteins by p62.

P15 Understanding the role of cathepsin H in atopic dermatitis pathogenesis

Abstract Introduction and aims Atopic dermatitis (AD) is a chronic skin condition ranked the 15th highest global burden for all nonfatal diseases. Our lab has found that patients with AD have a reduction in the gene and protein expression of cathepsin H, a lysosomal cysteine protease with aminopeptidase activity. Ctsh-/- mice have a reduced integrity of the epidermal barrier, including reduced filaggrin processing, reduced corneocyte integrity and a change in the expression of several proinflammatory mediators including interleukin-1α. We aim to elucidate the roles of cathepsin H in keratinocytes to understand its contribution to the pathogenesis of AD. Methods We utilized rat epidermal and N/TERT keratinocytes transiently overexpressing procathepsin B, H or L in addition to incubating keratinocytes with cathepsin H inhibitor (CTSHi) for 24 h to identify potential substrates and proteins upregulated or downregulated with cathepsin H expression. We also performed colocalization studies using indirect immunofluorescence in rat epidermal keratinocytes (REKs) and N/TERTs to investigate the biology of cathepsin H. Results REKs transiently overexpressing procathepsin H-FLAG or FLAG and REKs treated with the cathepsin H inhibitor H2N-Ser(OBzl)-CHN2 were analysed by liquid chromatography-mass spectrometry. This analysis indicated that keratin 1 and keratin 9 were upregulated with cathepsin H expression and downregulated by cathepsin H inhibition. Overexpressed procathepsin H and endogenous cathepsin H do not colocalize with the lysosomal membrane protein, LAMP1, whereas endogenous cathepsin B does. Conclusions Keratin 1 and keratin 9 could be coregulated with cathepsin H. Cathepsin H may not primarily localize to lysosomes unlike cathepsin B.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Interactions between LAMP3+ dendritic cells and T-cell subpopulations promote immune evasion in papillary thyroid carcinoma

BackgroundThe incidence of papillary thyroid cancer (PTC) continues to rise all over the world, 10–15% of the patients have a poor prognosis. Although immunotherapy has been applied in clinical practice,...

Activation and Purification of ß-Glucocerebrosidase by Exploiting its Transporter LIMP-2 - Implications for Novel Treatment Strategies in Gaucher's and Parkinson's Disease.

Genetic variants of GBA1 can cause the lysosomal storage disorder Gaucher disease and are among the highest genetic risk factors for Parkinson's disease (PD). GBA1 encodes the lysosomal enzyme beta-glucocerebrosidase (GCase), which orchestrates the degradation of glucosylceramide (GluCer) in the lysosome. Recent studies have shown that GluCer accelerates α-synuclein aggregation, exposing GCase deficiency as a major risk factor in PD pathology and as a promising target for treatment. This study investigates the interaction of GCase and three disease-associated variants (p.E326K, p.N370S, p.L444P) with their transporter, the lysosomal integral membrane protein 2 (LIMP-2). Overexpression of LIMP-2 in HEK 293T cells boosts lysosomal abundance of wt, E326K, and N370S GCase and increases/rescues enzymatic activity of the wt and E326K variant. Using a novel purification approach, co-purification of untagged wt, E326K, and N370S GCase in complex with His-tagged LIMP-2 from cell supernatant of HEK 293F cells is achieved, confirming functional binding and trafficking for these variants. Furthermore, a single helix in the LIMP-2 ectodomain is exploited to design a lysosome-targeted peptide that enhances lysosomal GCase activity in PD patient-derived and control fibroblasts. These findings reveal LIMP-2 as an allosteric activator of GCase, suggesting a possible therapeutic potential of targeting this interaction.

Exosomes derived from vMIP-II-Lamp2b gene-modified M2 cells provide neuroprotection by targeting the injured spinal cord, inhibiting chemokine signals and modulating microglia/macrophage polarization in mice

Inflammation is one of the key injury factors for spinal cord injury (SCI). Exosomes (Exos) derived from M2 macrophages have been shown to inhibit inflammation and be beneficial in SCI animal models. However, lacking targetability restricts their application prospects. Considering that chemokine receptors increase dramatically after SCI, viral macrophage inflammatory protein II (vMIP-II) is a broad-spectrum chemokine receptor binding peptide, and lysosomal associated membrane protein 2b (Lamp2b) is the key membrane component of Exos, we speculated that vMIP-II-Lamp2b gene-modified M2 macrophage-derived Exos (vMIP-II-Lamp2b-M2-Exo) not only have anti-inflammatory properties, but also can target the injured area by vMIP-II. In this study, using a murine contusive SCI model, we revealed that vMIP-II-Lamp2b-M2-Exo could target the chemokine receptors which highly expressed in the injured spinal cords, inhibit some key chemokine receptor signaling pathways (such as MAPK and Akt), further inhibit proinflammatory factors (such as IL-1β, IL-6, IL-17, IL-18, TNF-α, and iNOS), and promote anti-inflammatory factors (such as IL-4 and Arg1) productions, and the transformation of microglia/macrophages from M1 into M2. Moreover, the improved histological and functional recoveries were also found. Collectively, our results suggest that vMIP-II-Lamp2b-M2-Exo may provide neuroprotection by targeting the injured spinal cord, inhibiting some chemokine signals, reducing proinflammatory factor production and modulating microglia/macrophage polarization.

Porcine reproductive and respiratory syndrome virus 2 hijacks CMA-mediated lipolysis through upregulation of small GTPase RAB18.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

Loss of TMEM106B exacerbates Tau pathology and neurodegeneration in PS19 mice.

TMEM106B, a gene encoding a lysosome membrane protein, is tightly associated with brain aging, hypomyelinating leukodystrophy, and multiple neurodegenerative diseases, including frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Recently, TMEM106B polymorphisms have been associated with tauopathy in chronic traumatic encephalopathy (CTE) and FTLD-TDP patients. However, how TMEM106B influences Tau pathology and its associated neurodegeneration, is unclear. Here we show that loss of TMEM106B enhances the accumulation of pathological Tau, especially in the neuronal soma in the hippocampus, resulting in severe neuronal loss in the PS19 Tau transgenic mice. Moreover, Tmem106b-/- PS19 mice develop significantly increased abnormalities in the neuronal cytoskeleton, autophagy-lysosome activities, as well as glial activation, compared with PS19 and Tmem106b-/- mice. Together, our findings demonstrate that loss of TMEM106B drastically exacerbates Tau pathology and its associated disease phenotypes, and provide new insights into the roles of TMEM106B in neurodegenerative diseases.

Targeting of lysosomal-bound protein mEAK-7 for cancer therapy.

mEAK-7 (mammalian EAK-7 or MTOR-associated protein, eak-7 homolog), is an evolutionarily conserved lysosomal membrane protein that is highly expressed in several cancer cells. Multiple recent studies have identified mEAK-7 as a positive activator of mTOR (mammalian/mechanistic target of rapamycin) signaling via an alternative mTOR complex, implying that mEAK-7 plays an important role in the promotion of cancer proliferation and migration. In addition, structural analyses investigating interactions between mEAK-7 and V-ATPase, a protein complex responsible for regulating pH homeostasis in cellular compartments, have suggested that mEAK-7 may contribute to V-ATPase-mediated mTORC1 activation. The C-terminal α-helix of mEAK-7 binds to the D and B subunits of the V-ATPase, creating a pincer-like grip around its B subunit. This binding undergoes partial disruption during ATP hydrolysis, potentially enabling other proteins such as mTOR to bind to the α-helix of mEAK-7. mEAK-7 also promotes chemoresistance and radiation resistance by sustaining DNA damage-mediated mTOR signaling through interactions with DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Taken together, these findings indicate that mEAK-7 may be a promising therapeutic target against tumors. However, the precise molecular mechanisms and signal transduction pathways of mEAK-7 in cancer remain largely unknown, motivating the need for further investigation. Here, we summarize the current known roles of mEAK-7 in normal physiology and cancer development by reviewing the latest studies and discuss potential future developments of mEAK-7 in targeted cancer therapy.

SRT1720 inhibits bladder cancer cell progression by impairing autophagic flux

Bladder cancer (BC) is the most common cancer of the urinary tract, with poor survival, high recurrence rates, and lacking of targeted drugs. In this study, we constructed a library to screen compounds inhibiting bladder cancer cells growth. Among them, SRT1720 was identified to inhibit bladder cancer cell proliferation in vitro and in vivo. SRT1720 treatment also suppressed bladder cancer cells migration, invasion and induced apoptosis. Mechanism studies shown that SRT1720 promoted autophagosomes accumulation by inducing early-stage autophagy but disturbed the late-stage of autophagy by blocking fusion of autophagosomes and lysosomes. SRT1720 appears to induce autophagy related proteins expression and alter autophagy-related proteins acetylation to impede the autophagy flux. LAMP2, an important lysosomal associated membrane protein, may mediate SRT1720-inhibited autophagy flux as SRT1720 treatment significantly deacetylated LAMP2 which may influence its activity. Taken together, our results demonstrated that SRT1720 mediated apoptosis and autophagy flux inhibition may be a novel therapeutic strategy for bladder cancer treatment.

Hepatomegaly and Splenomegaly: An Approach to the Diagnosis of Lysosomal Storage Diseases.

Clinical findings of hepatomegaly and splenomegaly, the abnormal enlargement of the liver and spleen, respectively, should prompt a broad differential diagnosis that includes metabolic, congestive, neoplastic, infectious, toxic, and inflammatory conditions. Among the metabolic diseases, lysosomal storage diseases (LSDs) are a group of rare and ultrarare conditions with a collective incidence of 1 in 5000 live births. LSDs are caused by genetic variants affecting the lysosomal enzymes, transporters, or integral membrane proteins. As a result, abnormal metabolites accumulate in the organelle, leading to dysfunction. Therapeutic advances, including early diagnosis and disease-targeted management, have improved the life expectancy and quality of life of people affected by certain LSDs. To access these new interventions, LSDs must be considered in patients presenting with hepatomegaly and splenomegaly throughout the lifespan. This review article navigates the diagnostic approach for individuals with hepatosplenomegaly particularly focusing on LSDs. We provide hints in the history, physical exam, laboratories, and imaging that may identify LSDs. Additionally, we discuss molecular testing, arguably the preferred confirmatory test (over biopsy), accompanied by enzymatic testing when feasible.

Uridine-Modified Ruthenium(II) Complex as Lysosomal LIMP-2 Targeting Photodynamic Therapy Photosensitizer for the Treatment of Triple-Negative Breast Cancer.

Lysosome-targeted photodynamic therapy, which enhances reactive oxygen species (ROS)-responsive tumor cell death, has emerged as a promising strategy for cancer treatment. Herein, a uridine (dU)-modified Ru(II) complex (RdU) was synthesized by click chemistry. It was found that RdU exhibits impressive photo-induced inhibition against the growth of triple-negative breast cancer (TNBC) cells in normoxic and hypoxic microenvironments through ROS production. It was further revealed that RdU induces ferroptosis of MDA-MB-231 cells under light irradiation (650 nm, 300 mW/cm2). Additional experiments showed that RdU binds to lysosomal integral membrane protein 2 (LIMP-2), which was confirmed by the fact that RdU selectively localizes in the lysosomes of MDA-MB-231 cells and significantly augments the levels of LIMP-2. Molecular docking simulations and an isothermal titration calorimetry assay also showed that RdU has a high affinity to LIMP-2. Finally, in vivo studies in tumor-bearing (MDA-MB-231 cells) nude mice showed that RdU exerts promising photodynamic therapeutic effects on TNBC tumors. In summary, the uridine-modified Ru(II) complex has been developed as a potential LIMP-2 targeting agent for TNBC treatment through enhancing ROS production and promoting ferroptosis.

14-3-3 proteins-a moonlight protein complex with therapeutic potential in neurological disorder: in-depth review with Alzheimer's disease.

Alzheimer's disease (AD) affects millions of people worldwide and is a gradually worsening neurodegenerative condition. The accumulation of abnormal proteins, such as tau and beta-amyloid, in the brain is a hallmark of AD pathology. 14-3-3 proteins have been implicated in AD pathology in several ways. One proposed mechanism is that 14-3-3 proteins interact with tau protein and modulate its phosphorylation, aggregation, and toxicity. Tau is a protein associated with microtubules, playing a role in maintaining the structural integrity of neuronal cytoskeleton. However, in the context of Alzheimer's disease (AD), an abnormal increase in its phosphorylation occurs. This leads to the aggregation of tau into neurofibrillary tangles, which is a distinctive feature of this condition. Studies have shown that 14-3-3 proteins can bind to phosphorylated tau and regulate its function and stability. In addition, 14-3-3 proteins have been shown to interact with beta-amyloid (Aβ), the primary component of amyloid plaques in AD. 14-3-3 proteins can regulate the clearance of Aβ through the lysosomal degradation pathway by interacting with the lysosomal membrane protein LAMP2A. Dysfunction of lysosomal degradation pathway is thought to contribute to the accumulation of Aβ in the brain and the progression of AD. Furthermore, 14-3-3 proteins have been found to be downregulated in the brains of AD patients, suggesting that their dysregulation may contribute to AD pathology. For example, decreased levels of 14-3-3 proteins in cerebrospinal fluid have been suggested as a biomarker for AD. Overall, these findings suggest that 14-3-3 proteins may play an important role in AD pathology and may represent a potential therapeutic target for the disease. However, further research is needed to fully understand the mechanisms underlying the involvement of 14-3-3 proteins in AD and to explore their potential as a therapeutic target.

Understanding the molecular basis of mutation-induced dysfunction of lysosomal membrane proteins

Understanding the molecular basis of mutation-induced dysfunction of lysosomal membrane proteins

Lysosomal membrane proteins LAMP1 and LIMP2 are segregated in the Golgi apparatus independently of their clathrin adaptor binding motif.

To reach the lysosome, lysosomal membrane proteins (LMPs) are translocated in the endoplasmic reticulum after synthesis and then transported to the Golgi apparatus. The existence of a direct transport from the Golgi apparatus to the endosomes but also of an indirect route through the plasma membrane has been described. Clathrin adaptor binding motifs contained in the cytosolic tail of LMPs have been described as key players in their intracellular trafficking. Here we used the RUSH assay to synchronize the biosynthetic transport of multiple LMPs. After exiting the Golgi apparatus, RUSH-synchronized LAMP1 was addressed to the cell surface both after overexpression or at endogenous level. Its YXXΦ motif was not involved in the transport from the Golgi apparatus to the plasma membrane but in its endocytosis. LAMP1 and LIMP2 were sorted from each other after reaching the Golgi apparatus. LIMP2 was incorporated in punctate structures for export from the Golgi apparatus from which LAMP1 is excluded. LIMP2-containing post-Golgi transport intermediates did not rely neither on its adaptor binding signal nor on its C-terminal cytoplasmic domain.

Abstract B052: Defining the lysosome proteome during tumor evolution

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of 12%. PDAC tumors are highly reliant on nutrient scavenging pathways such as autophagy, and the lysosome – a degradative organelle which plays an essential role in the digestion and recycling of diverse cellular material. Lysosome and autophagy processes are regulated by the MiT/TFE family of transcription factors. In PDAC, MiT/TFE are uncoupled from normal regulatory mechanisms and have been shown to be constitutively nuclear, and therefore active. The function of lysosomes and the status of MiT/TFE over the course of tumor evolution and metastasis remains unknown. To fully understand the functions of the lysosome in cancer, our lab uses biochemical approaches to isolate intact lysosomes (termed LysoIP) from cultured PDAC cells, followed by mass spectrometry-based proteomics. Using this strategy, we have uncovered unique features and functions of PDAC lysosomes that promote cellular adaptation to stress in support of tumor growth. A limitation of lysosome isolation from 2D cultured cells is that it does not recapitulate lysosomal features associated with tumors growing in vivo. To address this limitation, we generated a genetically engineered mouse model of PDAC incorporating conditional expression of our ‘LysoTag’ (TMEM192-mRFP-3xHA) that enables capture and profiling of lysosomes at different stages of tumor evolution, including early stage, late stage, and metastatic disease. I have now established purification protocols for isolation of intact lysosomes with high efficiency and purity from both normal pancreas and diseased tissue. My preliminary analysis of pancreatic tumors, liver and lung metastases shows enrichment of bonafide resident lysosomal membrane proteins (Lamp1, Cln3, Npc1), and lumenal proteins (Ctsb, Lipa, Neu1), confirming successful isolation of intact lysosomes from these complex tissues. Further analysis highlights pathways and proteins that are unique to lysosomes of each tissue. For instance, lysosomes from the primary tumor show enrichment for pathways involved in ‘antigen processing and presentation of peptide antigen’ and ‘cellular response to oxidative stress’, while the liver and lung metastasis lysosomes show enrichment for ‘cholesterol metabolism’ and ‘mitochondrial gene expression’, respectively. Our analysis of lysosomes from the primary tumor, lung and liver metastasis highlights unique differences in the lysosome proteome of tumor cells growing in different tissues. Functional validation of our proteomics datasets will be needed to uncover novel roles for lysosomes in helping tumor cells adapt and grow in distinct tumor microenvironments. My ongoing studies will provide the first comprehensive atlas of lysosomal cargo and resident proteins that co-evolve with increasing tumor stage and help to inform novel therapeutic targets for treatment of PDAC. Citation Format: Grace A. Hernandez, Thuy Nguyen, Matthew Luy, Alexander Li, Longhui Qiu, Joseph D. Mancias, Rushika M. Perera. Defining the lysosome proteome during tumor evolution [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B052.

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor

ABSTRACT Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Cell death induced by nicotine in human neuroblastoma SH-SY5Y cells is mainly attributed to cytoplasmic vacuolation originating from the trans-Golgi network

Humans are usually exposed to nicotine through the use of tobacco products. Although it is generally believed that nicotine is relatively harmless in tobacco consumption, it is, in fact, a toxic substance that warrants careful consideration of its potential toxicity. However, the current understanding of the neurotoxicity of nicotine is still very limited. In this study, we aim to reveal the toxic risk of nicotine to key target neuronal cells and its potential toxic mechanisms. The results showed that nicotine induced cell death, ROS increase, mitochondrial membrane potential decrease, and DNA damage in SH-SY5Y human neuroblastoma cells at millimolar concentrations, but did not cause toxic effects at the physiological concentration. These toxic effects were accompanied by cytoplasmic vacuolation. The inhibition of cytoplasmic vacuolation by bafilomycin A1 greatly reduced nicotine-induced cell death, indicating that cytoplasmic vacuolation is the key driving factor of cell death. These cytoplasmic vacuoles originated from the trans-Golgi network (TGN) and expressed microtubule-associated protein 1 light chain 3-II (LC3-II) and lysosomal associated membrane protein 1(LAMP1). The presence of LC3-II and LAMP1 within these vacuoles serves as evidence of compromised TGN structure and function. These findings provide valuable new insights into the potential neurotoxic risk and mechanisms of nicotine.