Lysosomal Catabolism Research Articles

Discovery of Selective Inhibitors for the Lysosomal Parkinson's Disease Channel TMEM175.

TMEM175 is a lysosomal potassium and proton channel that is associated with the development of Parkinson's disease. Advances in understanding the physiological roles of TMEM175 have been hampered by the absence of selective inhibitors, and studies involving genetic perturbations have yielded conflicting results. Here, we report the discovery and characterization of the first reported TMEM175-selective inhibitors, 2-phenylpyridin-4-ylamine (2-PPA), and AP-6. Cryo-EM structures of human TMEM175 bound by 2-PPA and AP-6 reveal that they act as pore blockers, binding at distinct sites in the pore and occluding the ion permeation pathway. Acute inhibition of TMEM175 by 2-PPA or AP-6 increases the level of lysosomal macromolecule catabolism, thereby accelerating macropinocytosis and other digestive processes. These inhibitors may serve as valuable tools to study the roles of TMEM175 in regulating lysosomal function and provide useful templates for future therapeutic development in Parkinson's disease.

The Bis(monoacylglycero)-phosphate Hypothesis: From Lysosomal Function to Therapeutic Avenues.

Lysosomes catabolize and recycle lipids and other biological molecules to maintain cellular homeostasis in diverse nutrient environments. Lysosomal lipid catabolism relies on the stimulatory activity of bis(monoacylglycero)phosphate (BMP), an enigmatic lipid whose levels are altered across myriad lysosome-associated diseases. Here, we review the discovery of BMP over half a century ago and its structural properties that facilitate the activation of lipid hydrolases and recruitment of their coactivators. We further discuss the current, yet incomplete, understanding of BMP catabolism and anabolism. To conclude, we discuss its role in lysosome-associated diseases and the potential for modulating its levels by pharmacologically activating and inhibiting the BMP synthase to therapeutically target lysosomal storage disorders, drug-induced phospholipidosis, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, cancer, and viral infection.

JEV infection leads to dysfunction of lysosome by downregulating the expression of LAMP1 and LAMP2

Japanese Encephalitis Virus (JEV), the predominant cause of viral encephalitis in many Asian countries, affects approximately 68,000 people annually. Lysosomes are dynamic structures that regulate cellular metabolism by mediating lysosomal biogenesis and autophagy. Here, we showed that lysosome-associated membrane protein 1 (LAMP1) and LAMP2 were downregulated in cells after JEV infection, resulting in a decrease in the quantity of acidified lysosomes and impaired lysosomal catabolism. What’s more, JEV nonstructural protein 4B plays key roles in the reduction of LAMP1/2 via the autophagy-lysosome pathway. JEV NS4B also promoted abnormal aggregation of SLA-DR, an important component of the swine MHC-II molecule family involved in antigen presentation and CD4+ cell activation initiation. Mechanistically, NS4B localized to the ER during JEV infection and interacted with GRP78, leading to the activation of ER stress-mediated autophagy. The 131–204 amino acid (aa) region of NS4B is essential for autophagy induction and LAMP1/2 reduction. In summary, our findings reveal a novel pathway by which JEV induces autophagy and disrupts lysosomal function.

Design and validation of a reporter mouse to study the dynamic regulation of TFEB and TFE3 activity through in vivo imaging techniques

ABSTRACT TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor’s validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism. Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

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 thesephospholipases. 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.

Effect of AZP2006, a new treatment for Alzheimer’s Disease and Progressive Supranuclear Palsy, in the Restoration of Lysosomal Homeostasis via the Progranulin‐Prosaposin Axis

AbstractBackgroundProgranulin (PGRN) is a secreted pleiotropic growth factor primarily expressed in neurons and microglia that regulates neuroinflammation, neurite branching and outgrowth. PGRN has been identified as a Cathepsin D chaperone, suggesting a role in lysosomal proteolysis. PGRN together with Prosaposin (PSAP) have been proved to be physiologically involved in lysosomal lipid catabolism. PSAP physically interacts with PGRN and facilitates glycosphingolipid degradation upon lysosomal processing, serving as activators of various lysosomal lipases. Thus, PGRN and PSAP play an essential role in the regulation of lysosomal homeostasis. Reduced lysosomal protein levels and activity, as well as accumulation of toxic proteins, have been observed in age‐dependent neurodegenerative diseases. The impact of the disturbance of the PGRN‐PSAP axis in these diseases is not yet fully understood.AZP2006 (INN: Ezeprogind) is a small molecule currently under development for the treatment of Progressive Supranuclear Palsy (PSP, Ph2a completed) and Alzheimer’s Disease (AD). AZP2006 was proved to protect neurons, to increase synaptogenesis and to reduce neuroinflammation in different models of proteinopathies. Importantly, its neuroprotective properties were fully abolished in absence of PGRN or PSAP. Although its exact mode of action (MOA) is still unclear, AZP2006 has been proved to target lysosomes and bind the complex PGRN‐PSAP. Considering this information, the AZP2006 lysosomal MOA was further investigated.MethodAged (18mo) C57Bl6 mice receiving Aβ1‐42 oligomers bilaterally injected into the CA1 area of the hippocampus, were orally treated (gavage) with AZP2006 (1 or 2mg/kg/day) for 22 days. At the end of the treatment, the brain, CSF and plasma of the animals were collected. Lysosomal markers were investigated (LAMP‐2, LC‐3b, PGRN, PSAP, α‐Syn and glycosphingolipids contents). Histological analysis of the CA1 hippocampal area was performed (LAMP‐2, LC‐3b and α‐Syn in neurons and microglial cells). In addition, plasma and CSF samples of PSP patients generated from PSP Ph2a were analyzed.ResultAZP2006 displayed beneficial effects in the Ab1‐42 oligomers injured aged mice. AZP2006 was able to correct the lysosomal dysfunction and to restore the lysosomal homeostasis.ConclusionAZP2006 represents an original therapeutic approach to protect the PGRN‐PSAP balance that is fundamental for the neuron health.

Phospholipase D3 degrades mitochondrial DNA to regulate nucleotide signaling and APP metabolism

Phospholipase D3 (PLD3) polymorphisms are linked to late-onset Alzheimer’s disease (LOAD). Being a lysosomal 5’-3’ exonuclease, its neuronal substrates remained unknown as well as how a defective lysosomal nucleotide catabolism connects to AD-proteinopathy. We identified mitochondrial DNA (mtDNA) as a major physiological substrate and show its manifest build-up in lysosomes of PLD3-defective cells. mtDNA accretion creates a degradative (proteolytic) bottleneck that presents at the ultrastructural level as a marked abundance of multilamellar bodies, often containing mitochondrial remnants, which correlates with increased PINK1-dependent mitophagy. Lysosomal leakage of mtDNA to the cytosol activates cGAS–STING signaling that upregulates autophagy and induces amyloid precursor C-terminal fragment (APP-CTF) and cholesterol accumulation. STING inhibition largely normalizes APP-CTF levels, whereas an APP knockout in PLD3-deficient backgrounds lowers STING activation and normalizes cholesterol biosynthesis. Collectively, we demonstrate molecular cross-talks through feedforward loops between lysosomal nucleotide turnover, cGAS-STING and APP metabolism that, when dysregulated, result in neuronal endolysosomal demise as observed in LOAD.

Abstract 422: Cell intrinsic determinants of response to mTOR based therapy

Abstract Lung cancer is a leading cause of cancer death with estimated 135,000 deaths in 2021. Recent developments in the immunotherapy field have expanded treatment options available to lung cancer patients, with most patients now receiving checkpoint inhibitors as part of the first line therapy. However, while durable responses have been achieved in 20-30% of patients, the majority of patients inevitably progress on immunotherapy, chemo-immunotherapy or targeted therapy regiments. With five year survival at ~10% there is an urgent need for novel strategies for the treatment of lung cancer. Lung squamous cell carcinoma (LUSC) is an aggressive subtype of non-small cell lung cancer characterized by poor prognosis. LUSC are highly metabolically active, with uptake of high levels of glucose and glutamine supporting their growth and proliferation. In addition to fueling tumor cell growth, high levels of glycolysis and glutaminolysis help create unfavorable tumor microenvironment, contributing to the poor response to immunotherapy. The Cancer Genome Atlas (TCGA) revealed that LUSC have a high mutational burden but few mutations in oncogenes that can be targeted with selective inhibitors. This lack of clear genetic drivers in LUSC has resulted in a lack of targeted therapies for these patients. We have demonstrated that catalytic mTOR kinase inhibitor TAK228 inhibits glycolysis in LUSC cell lines, in xenografts, in patient derived xenografts and genetically engineered mouse models. However, despite inhibiting active mTOR pathway and lowering glucose uptake, LUSC tumors were able to maintain high levels of proliferation. Our work showed that tumors were able to adapt to prolonged treatment with TAK228 by relying on glutaminolysis. Inhibiting both mTOR and GLS, a key enzyme in glutaminolysis, resulted in reduced proliferation index and lower tumor volume. However, while combination therapy halted tumor growth, there was a lack of tumor regression. Therefore, tumors are poised to further adapt to combination therapy. Here we show that upregulation of lysosomal function supports adaptation to TAK228 therapy in LUSC by supporting macropinocytosis, a form of endocytosis that allows cancer cells to uptake extracellular fluids and further process solutes and macromolecules in lysosomes, leading to the release of amino acids inside the cytoplasm. This suggests that macropinocytosis might be a mechanism that is utilized by LUSC in order to escape mTORi therapy, where upon treatment with mTOR inhibitors, lysosomal catabolism of macropinocytosed proteins increases, allowing tumor cells to increase their proliferation.Resistance to targeted therapies is a major hurdle to achieving effective therapies in multiple cancer types. Identifying ways to overcome resistance to targeted therapies would allow for longer and more efficient therapeutic response for larger number of patients, ultimately leading to longer survival and better quality of life. Citation Format: Milica Momcilovic, David Shackelford. Cell intrinsic determinants of response to mTOR based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 422.

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD)

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is α-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype.

Disrupted myelin lipid metabolism differentiates frontotemporal dementia caused by GRN and C9orf72 gene mutations

Heterozygous mutations in the GRN gene and hexanucleotide repeat expansions in C9orf72 are the two most common genetic causes of Frontotemporal Dementia (FTD) with TDP-43 protein inclusions. The triggers for neurodegeneration in FTD with GRN (FTD-GRN) or C9orf72 (FTD-C9orf72) gene abnormalities are unknown, although evidence from mouse and cell culture models suggests that GRN mutations disrupt lysosomal lipid catabolism. To determine how brain lipid metabolism is affected in familial FTD with TDP-43 inclusions, and how this is related to myelin and lysosomal markers, we undertook comprehensive lipidomic analysis, enzyme activity assays, and western blotting on grey and white matter samples from the heavily-affected frontal lobe and less-affected parietal lobe of FTD-GRN cases, FTD-C9orf72 cases, and age-matched neurologically-normal controls. Substantial loss of myelin-enriched sphingolipids (sulfatide, galactosylceramide, sphingomyelin) and myelin proteins was observed in frontal white matter of FTD-GRN cases. A less-pronounced, yet statistically significant, loss of sphingolipids was also observed in FTD-C9orf72. FTD-GRN was distinguished from FTD-C9orf72 and control cases by increased acylcarnitines in frontal grey matter and marked accumulation of cholesterol esters in both frontal and parietal white matter, indicative of myelin break-down. Both FTD-GRN and FTD-C9orf72 cases showed significantly increased lysosomal and phagocytic protein markers, however galactocerebrosidase activity, required for lysosomal catabolism of galactosylceramide and sulfatide, was selectively increased in FTD-GRN. We conclude that both C9orf72 and GRN mutations are associated with disrupted lysosomal homeostasis and white matter lipid loss, but GRN mutations cause a more pronounced disruption to myelin lipid metabolism. Our findings support the hypothesis that hyperactive myelin lipid catabolism is a driver of gliosis and neurodegeneration in FTD-GRN. Since FTD-GRN is associated with white matter hyperintensities by MRI, our data provides important biochemical evidence supporting the use of MRI measures of white matter integrity in the diagnosis and management of FTD.

The effects of dietary myo-inositol on growth and physiological, biochemical, and molecular responses in the Pacific white shrimp Litopenaeus vannamei

Myo-inositol serves as an essential nutrient in many animal species. However, the optimal requirements of myo-inositol and the mechanisms by which myo-inositol is involved in nutritional regulation in crustaceans are unclear. An 8-week trial was conducted to comparatively evaluate the effects of 6 levels of dietary myo-inositol (20, 528, 1030, 2060, 3180 and 4235 mg/kg diet) on growth performance, body composition, serum metabolite composition, hepatopancreatic metabolite content, and digestive enzyme activity in juvenile Pacific white shrimp Litopenaeus vannamei (0.78 ± 0.039 g). The weight gain and conditioning factors of shrimp fed myo-inositol were significantly higher than those of the 20 mg/kg myo-inositol group. The maximal hepatosomatic index was found in the 1030 mg/kg myo-inositol group. On the other hand, serum triglyceride, total cholesterol, and low-density lipoprotein contents in the control group were significantly lower than those in the other groups, and glucose content in the hepatopancreas in the control group was significantly lower than that in the 2060 and 3180 mg/kg myo-inositol groups. Metabolome and transcriptome analyses revealed that myo-inositol significantly affected glycerophospholipid metabolism, transport and catabolism in lysosomes, pancreatic secretion, and bile secretion pathways. The highest growth was achieved with the inclusion of 528 mg/kg dietary myo-inositol. This study has suggested that myo-inositol may promote growth in L. vannamei by improving lipid utilization.

Glycosidase-targeting small molecules for biological and therapeutic applications.

Glycosidases are ubiquitous enzymes that catalyze the hydrolysis of glycosidic linkages in oligosaccharides and glycoconjugates. These enzymes play a vital role in a wide variety of biological events, such as digestion of nutritional carbohydrates, lysosomal catabolism of glycoconjugates, and posttranslational modifications of glycoproteins. Abnormal glycosidase activities are associated with a variety of diseases, particularly cancer and lysosomal storage disorders. Owing to the physiological and pathological significance of glycosidases, the development of small molecules that target these enzymes is an active area in glycoscience and medicinal chemistry. Research efforts carried out thus far have led to the discovery of numerous glycosidase-targeting small molecules that have been utilized to elucidate biological processes as well as to develop effective chemotherapeutic agents. In this review, we describe the results of research studies reported since 2018, giving particular emphasis to the use of fluorescent probes for detection and imaging of glycosidases, activity-based probes for covalent labelling of these enzymes, glycosidase inhibitors, and glycosidase-activatable prodrugs.

Neuronal Ganglioside and Glycosphingolipid (GSL) Metabolism and Disease : Cascades of Secondary Metabolic Errors Can Generate Complex Pathologies (in LSDs).

Glycosphingolipids (GSLs) are a diverse group of membrane components occurring mainly on the surfaces of mammalian cells. They and their metabolites have a role in intercellular communication, serving as versatile biochemical signals (Kaltner et al, Biochem J 476(18):2623-2655, 2019) and in many cellular pathways. Anionic GSLs, the sialic acid containing gangliosides (GGs), are essential constituents of neuronal cell surfaces, whereas anionic sulfatides are key components of myelin and myelin forming oligodendrocytes. The stepwise biosynthetic pathways of GSLs occur at and lead along the membranes of organellar surfaces of the secretory pathway. After formation of the hydrophobic ceramide membrane anchor of GSLs at the ER, membrane-spanning glycosyltransferases (GTs) of the Golgi and Trans-Golgi network generate cell type-specific GSL patterns for cellular surfaces. GSLs of the cellular plasma membrane can reach intra-lysosomal, i.e. luminal, vesicles (ILVs) by endocytic pathways for degradation. Soluble glycoproteins, the glycosidases, lipid binding and transfer proteins and acid ceramidase are needed for the lysosomal catabolism of GSLs at ILV-membrane surfaces. Inherited mutations triggering a functional loss of glycosylated lysosomal hydrolases and lipid binding proteins involved in GSL degradation cause a primary lysosomal accumulation of their non-degradable GSL substrates in lysosomal storage diseases (LSDs). Lipid binding proteins, the SAPs, and the various lipids of the ILV-membranes regulate GSL catabolism, but also primary storage compounds such as sphingomyelin (SM), cholesterol (Chol.), or chondroitin sulfate can effectively inhibit catabolic lysosomal pathways of GSLs. This causes cascades of metabolic errors, accumulating secondary lysosomal GSL- and GG- storage that can trigger a complex pathology (Breiden and Sandhoff, Int J Mol Sci 21(7):2566, 2020).

Evaluation of an ester-linked immunosuppressive payload: A case study in understanding the stability and cleavability of ester-containing ADC linkers

In spite of their value in prodrug applications, the use of esters in antibody-drug-conjugate (ADC) payloads and linkers has generally been avoided due to the ubiquitous and promiscuous nature of human esterases. ADCs generally have a long circulating half life (3–7 days) that makes them susceptible to esterase-mediated metabolism. Moreover, it is largely unclear whether lysosomal and cytosolic esterases cleave ester-containing linkers upon ADC internalization. Due to our interest in the targeted delivery of immune-modulators, our team has recently prepared a series of ester-linked dexamethasone ADCs. Herein, we report our studies of the functional activity of these ADCs, with a particular focus on their catabolism in various biological milieu. We found that esters are selectively but inefficiently cleaved upon cellular uptake, likely by cytosolic esterases. Lysosomal catabolism studies indicate that, in spite of the strong proteolytic activity, very little cleavage of ester-containing linkers occurs in the lysosome. However, ADCs bearing the ester-linked payloads are active in various immune-suppressive assays, suggesting that cytosolic cleavage is taking place. This was confirmed through LCMS quantitation of the payload following cell lysis. Finally, the stability of the ester linkage was evaluated in mouse and human plasma. We found, similar to other reports, there is a significant site-dependence on the cleavage. Esters attached at highly exposed sites, such as 443C, were rapidly cleaved in plasma while esters at more hindered sites, such at 334C, were not. Together, these results help to unravel the complexities of ester-incorporation into ADC linkers and pave a path forward for their utility in ADC applications.

Direct control of lysosomal catabolic activity by mTORC1 through regulation of V-ATPase assembly

Mammalian cells can acquire exogenous amino acids through endocytosis and lysosomal catabolism of extracellular proteins. In amino acid-replete environments, nutritional utilization of extracellular proteins is suppressed by the amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) through an unknown process. Here, we show that mTORC1 blocks lysosomal degradation of extracellular proteins by suppressing V-ATPase-mediated acidification of lysosomes. When mTORC1 is active, peripheral V-ATPase V1 domains reside in the cytosol where they are stabilized by association with the chaperonin TRiC. Consequently, most lysosomes display low catabolic activity. When mTORC1 activity declines, V-ATPase V1 domains move to membrane-integral V-ATPase Vo domains at lysosomes to assemble active proton pumps. The resulting drop in luminal pH increases protease activity and degradation of protein contents throughout the lysosomal population. These results uncover a principle by which cells rapidly respond to changes in their nutrient environment by mobilizing the latent catabolic capacity of lysosomes.

Abstract 1089: Targeting amino acid uptake in lung squamous cell carcinomas to improve response to targeted therapies

Abstract Lung squamous cell carcinomas (LUSC) are a subtype of non-small cell lung cancer characterized by poor overall survival. LUSC are highly aggressive in part due to their high metabolic activity, with uptake of high levels of glucose supporting their growth and proliferation. The main driver of high rate of glycolysis in LUSC is elevated expression of glucose transporter GLUT1, whose levels and membrane localization are regulated by activated PI3K-AKT-mTOR pathway. The Cancer Genome Atlas revealed that LUSC have high tumor mutational burden with varied genetic profile that includes amplification of receptor tyrosine kinases (RTKs), PI3KCA, and a loss of PTEN. These alterations lead to increased activity of mTOR pathway, one of the major pathways in cancer cells that drives cell proliferation and metabolism. We have recently demonstrated that catalytic mTOR kinase inhibitor TAK228 (MLN128) inhibits glycolysis in LUSC cell lines in vitro, in xenografts of human LUSC lines, in patient derived xenografts (PDXs) and genetically engineered mouse models. However, despite lowered glucose uptake measured by 18F-FDG signal LUSC tumors were able to maintain high levels of proliferation. Tumors were able to adapt to prolonged treatment with TAK228 by relying on glutaminolysis. Increased glutaminolysis was due to increased uptake of glutamine as well as increased levels of glutaminase (GLS), a key enzyme that converts glutamine to glutamate. Inhibiting both mTOR (with TAK228) and GLS (with CB-839) resulted in reduced proliferation index and lower tumor volume. However, while combination therapy halted tumor growth, there was a lack of tumor regression in xenografts and PDXs of LUSC. Therefore, tumors are poised to further adapt to combination therapy through metabolism of additional amino acids beyond glutamine. Here we show that macropinocytosis, a form of endocytosis that allows cancer cells to uptake extracellular fluids and further process solutes and macromolecules in lysosomes leading to the release of amino acids inside the cytoplasm, is upregulated in LUSC tumors that are resistant to TAK228. Macropinocytosis is induced by active RTKs, PI3KCA, Ras, loss of PTEN, and active NRF2 pathway - oncogenic alterations commonly found in LUSC tumors. This suggests that macropinocytosis might be a mechanism that supports amino acid metabolism utilized by LUSC in order to escape mTOR therapy. Basal levels of macropinocytosis in LUSC appear low, however, upon treatment with mTOR inhibitors, lysosomal catabolism of macropinocytosed proteins increases, allowing tumor cells to increase their proliferation. Resistance to targeted therapies is a major hurdle to achieving effective therapies in multiple cancer types. Identifying ways to overcome resistance to targeted therapies would allow for longer therapeutic response for larger number of patients, ultimately leading to longer survival and better quality of life. Citation Format: Milica Momcilovic, David Shackelford. Targeting amino acid uptake in lung squamous cell carcinomas to improve response to targeted therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1089.

Sialidase NEU3 and its pathological significance.

Sialidases (EC 3.2.1.18, also called neuraminidases) catalyze the removal of α-glycosidically linked sialic acid residues from glycoproteins and glycolipids; this is the initial step in the degradation of these glycoconjugates. Sialidases of mammalian origin have been implicated in not only lysosomal catabolism but also the modulation of functional molecules involved in many biological processes. To date, four types of mammalian sialidases have been cloned and designated as Neu1, Neu2, Neu3 and Neu4. These sialidases differ in their subcellular localization and enzymatic properties, as well as their chromosomal localization, and they are expressed in a tissue-specific manner. Among the sialidases, the plasma membrane-associated sialidase Neu3 appears to play particular roles in controlling transmembrane signaling through the modulation of gangliosides, and its aberrant expression is closely related to various pathogeneses, including that of cancer. Interestingly, the human orthologue NEU3 acts in two ways, catalytic hydrolysis of gangliosides and protein interactions with other signaling molecules. Aberrant NEU3 expression can induce various pathological conditions. This review briefly summarizes recent studies, focusing on the involvement of NEU3 in various pathological phenomena.

Mutation of SLC7A14 causes auditory neuropathy and retinitis pigmentosa mediated by lysosomal dysfunction.

Lysosomes contribute to cellular homeostasis via processes including macromolecule degradation, nutrient sensing, and autophagy. Defective proteins related to lysosomal macromolecule catabolism are known to cause a range of lysosomal storage diseases; however, it is unclear whether mutations in proteins involved in homeostatic nutrient sensing mechanisms cause syndromic sensory disease. Here, we show that SLC7A14, a transporter protein mediating lysosomal uptake of cationic amino acids, is evolutionarily conserved in vertebrate mechanosensory hair cells and highly expressed in lysosomes of mammalian cochlear inner hair cells (IHCs) and retinal photoreceptors. Autosomal recessive mutation of SLC7A14 caused loss of IHCs and photoreceptors, leading to presynaptic auditory neuropathy and retinitis pigmentosa in mice and humans. Loss-of-function mutation altered protein trafficking and increased basal autophagy, leading to progressive cell degeneration. This study implicates autophagy-lysosomal dysfunction in syndromic hearing and vision loss in mice and humans.

GCN2 adapts protein synthesis to scavenging-dependent growth.

Pancreatic cancer cells with limited access to free amino acids can grow by scavenging extracellular protein. In a murine model of pancreatic cancer, we performed a genome-wide CRISPR screen for genes required for scavenging-dependent growth. The screen identified key mediators of macropinocytosis, peripheral lysosome positioning, endosome-lysosome fusion, lysosomal protein catabolism, and translational control. The top hit was GCN2, a kinase that suppresses translation initiation upon amino acid depletion. Using isotope tracers, we show that GCN2 is not required for protein scavenging. Instead, GCN2 prevents ribosome stalling but without slowing protein synthesis; cells still use all of the limiting amino acids as they emerge from lysosomes. GCN2 also adapts gene expression to the nutrient-poor environment, reorienting protein synthesis away from ribosomes and toward lysosomal hydrolases, such as cathepsin L. GCN2, cathepsin L, and the other genes identified in the screen are potential therapeutic targets in pancreatic cancer.