Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

Structural and Functional Analyses of the Shedding Protease ADAM17 in HoxB8-Immortalized Macrophages and Dendritic-like Cells.

A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.

Functional Characterization of Colon Cancer-Associated Mutations in ADAM17: Modifications in the Pro-Domain Interfere with Trafficking and Maturation.

Colorectal cancer is one of the most commonly diagnosed malignancies in the Western world and is associated with elevated expression and activity of epidermal growth factor receptors (EGF-R). The metalloproteinase ADAM17 is involved in EGF-R activation by processing EGF-R ligands from membrane-bound pro-ligands. Underlining the link between colon cancer and ADAM17, genetic intestinal cancer models in ADAM17-deficient mice show a reduced tumor burden. In this study, we characterize point mutations within the ADAM17 gene found in the tissue of colon cancer patients. In order to shed light on the role of ADAM17 in cancer development, as well as into the mechanisms that regulate maturation and cellular trafficking of ADAM17, we here perform overexpression studies of four ADAM17 variants located in the pro-, membrane-proximal- and cytoplasmic-domain of the ADAM17 protein in ADAM10/17-deficient HEK cells. Interestingly, we found a cancer-associated point mutation within the pro-domain of ADAM17 (R177C) to be most impaired in its proteolytic activity and trafficking to the cell membrane. By comparing this variant to an ADAM17 construct lacking the entire pro-domain, we discovered similar functional limitations and propose a crucial role of the pro-domain for ADAM17 maturation, cellular trafficking and thus proteolytic activity.

Late-onset Krabbe disease presenting as spastic paraplegia - implications of GCase and CTSB/D.

OBJECTIVE: Krabbe disease (KD) is a multisystem neurodegenerative disorder with severe disability and premature death, mostly with an infancy/childhood onset. In rare cases of late-onset phenotypes, symptoms are often milder and difficult to diagnose. We here present a translational approach combining diagnostic and biochemical analyses of a male patient with a progressive gait disorder starting at the age of 44 years, with a final diagnosis of late-onset KD (LOKD). METHODS: Additionally to cerebral MRI, protein structural analyses of the ß-galactocerebrosidase protein (GALC) were performed. Moreover, expression, lysosomal localization, and activities of ß-glucocerebrosidase (GCase), cathepsin B (CTSB), and cathepsin D (CTSD) were analyzed in leukocytes, fibroblasts, and lysosomes of fibroblasts. RESULTS: Exome sequencing revealed biallelic likely pathogenic variants: GALC exons 11-17: 33 kb deletion; exon 4: missense variant (c.334A>G, p.Thr112Ala). We detected a reduced GALC activity in leukocytes and fibroblasts. While histological KD phenotypes were absent in fibroblasts, they showed a significantly decreased activities of GCase, CTSB, and CTSD in lysosomal fractions, while expression levels were unaffected. INTERPRETATION: The presented LOKD case underlines the age-dependent appearance of a mildly pathogenic GALC variant and its interplay with other lysosomal proteins. As GALC malfunction results in reduced ceramide levels, we assume this to be causative for the here described decrease in CTSB and CTSD activity, potentially leading to diminished GCase activity. Hence, we emphasize the importance of a functional interplay between the lysosomal enzymes GALC, CTSB, CTSD, and GCase, as well as between their substrates, and propose their conjoined contribution in KD pathology.

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.

From Lysosomal Storage Disorders to Parkinson's Disease - Challenges and Opportunities.

Lysosomes are specialized organelles with an acidic pH that act as recycling hubs for intracellular and extracellular components. They harbour numerous different hydrolytic enzymes to degrade substrates like proteins, peptides, and glycolipids. Reduced catalytic activity of lysosomal enzymes can cause the accumulation of these substrates and loss of lysosomal integrity, resulting in lysosomal dysfunction and lysosomal storage disorders (LSDs). Post-mitotic cells, such as neurons, seem to be highly sensitive to damages induced by lysosomal dysfunction, thus LSDs often manifest with neurological symptoms. Interestingly, some LSDs and Parkinson's disease (PD) share common cellular pathomechanisms, suggesting convergence of aetiology of the two disease types. This is further underlined by genetic associations of several lysosomal genes involved in LSDs with PD. The increasing number of lysosome-associated genetic risk factors for PD makes it necessary to understand functions and interactions of lysosomal proteins/enzymes both in health and disease, thereby holding the potential to identify new therapeutic targets. In this review, we highlight genetic and mechanistic interactions between the complex lysosomal network, LSDs and PD, and elaborate on methodical challenges in lysosomal research.

Reciprocal effects of alpha-synuclein aggregation and lysosomal homeostasis in synucleinopathy models.

BACKGROUND: Lysosomal dysfunction has been implicated in a number of neurodegenerative diseases such as Parkinson's disease (PD). Various molecular, clinical and genetic studies have highlighted a central role of lysosomal pathways and proteins in the pathogenesis of PD. Within PD pathology the synaptic protein alpha-synuclein (αSyn) converts from a soluble monomer to oligomeric structures and insoluble amyloid fibrils. The aim of this study was to unravel the effect of αSyn aggregates on lysosomal turnover, particularly focusing on lysosomal homeostasis and cathepsins. Since these enzymes have been shown to be directly involved in the lysosomal degradation of αSyn, impairment of their enzymatic capacity has extensive consequences. METHODS: We used patient-derived induced pluripotent stem cells and a transgenic mouse model of PD to examine the effect of intracellular αSyn conformers on cell homeostasis and lysosomal function in dopaminergic (DA) neurons by biochemical analyses. RESULTS: We found impaired lysosomal trafficking of cathepsins in patient-derived DA neurons and mouse models with αSyn aggregation, resulting in reduced proteolytic activity of cathepsins in the lysosome. Using a farnesyltransferase inhibitor, which boosts hydrolase transport via activation of the SNARE protein ykt6, we enhanced the maturation and proteolytic activity of cathepsins and thereby decreased αSyn protein levels. CONCLUSIONS: Our findings demonstrate a strong interplay between αSyn aggregation pathways and function of lysosomal cathepsins. It appears that αSyn directly interferes with the enzymatic function of cathepsins, which might lead to a vicious cycle of impaired αSyn degradation. Lysosomal trafficking of cathepsin D (CTSD), CTSL and CTSB is disrupted when alpha-synuclein (αSyn) is aggregated. This results in a decreased proteolytic activity of cathepsins, which directly mediate αSyn clearance. Boosting the transport of the cathepsins to the lysosome increases their activity and thus contributes to efficient αSyn degradation.

Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.

Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties.

Cathepsin D (CTSD) is a lysosomal protease important for the degradation of various substrates, including disease-associated proteins like α-synuclein (a-syn), amyloid precursor protein (APP) and tau, all of which tend to aggregate if not efficiently degraded. Hence, it is not surprising that genetic variants within the CTSD gene have been linked to neurodegenerative diseases, like Parkinson's and Alzheimer's disease (PD, AD), as well as the lysosomal storage disorder neuronal ceroid lipofuscinosis type-10 (NCL10). Although recent studies have shown the molecular dependence of substrate degradation via CTSD within autophagic pathways, only little is known about the precise role of lysosomal CTSD function in disease development. We here performed biochemical, cellular and structural analyses of eleven disease-causing CTSD point mutations found in genomic sequencing data of patients to understand their role in neurodegeneration. These CTSD variants were analyzed for cellular localization, maturation and enzymatic activity in overexpression analyses. Moreover, for PD-associated mutants, intracellular degradation of a-syn was monitored. In summary, our results suggest that NCL10-associated CTSD variants are significantly impaired in lysosomal maturation and enzymatic activity, whereas the AD- and PD-associated variants seemed rather unaffected, indicating normal maturation, and lysosomal presence. Interestingly, a PD-associated CTSD variant (A239V) exhibited increased enzymatic activity accompanied by enhanced a-syn degradation. By structural analyses of this mutant utilizing molecular dynamics simulation (MDS), we identified a structural change within a loop adjacent to the catalytic center leading to a higher flexibility and potentially accelerated substrate exchange rates. Our data sheds light onto the role of CTSD in disease development and helps to understand the structural regulation of enzymatic function, which could be utilized for targeted CTSD activation. Because of the degradative function of CTSD, this enzyme is especially interesting for therapeutic strategies tackling protein aggregates in neurodegenerative disorders.

Corrigendum: Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties.

[This corrects the article DOI: 10.3389/fcell.2021.581805.].

Functional Characterization of Colon-Cancer-Associated Variants in ADAM17 Affecting the Catalytic Domain.

Although extensively investigated, cancer is still one of the most devastating and lethal diseases in the modern world. Among different types, colorectal cancer (CRC) is most prevalent and mortal, making it an important subject of research. The metalloprotease ADAM17 has been implicated in the development of CRC due to its involvement in signaling pathways related to inflammation and cell proliferation. ADAM17 is capable of releasing membrane-bound proteins from the cell surface in a process called shedding. A deficiency of ADAM17 activity has been previously shown to have protective effects against CRC in mice, while an upregulation of ADAM17 activity is suspected to facilitate tumor development. In this study, we characterize ADAM17 variants found in tissue samples of cancer patients in overexpression studies. We here focus on point mutations identified within the catalytic domain of ADAM17 and could show a functional dysregulation of the CRC-associated variants. Since the catalytic domain of ADAM17 is the only region structurally determined by crystallography, we study the effect of each point mutation not only to learn more about the role of ADAM17 in cancer, but also to investigate the structure-function relationships of the metalloprotease.