Charakterisierung des Lysosomalen Integralen Membranproteins Typ-2 (LIMP-2) und deren Interaktion mit b-Glukozerebrosidase: Implikationen in der Parkinson und Gaucher Erkrankung

The acid β-gluco¬cerebrosidase (GC) is a lysosomal hydrolase for β-glucose-terminated sphingolipids and reaches the lysosome through binding to its transport receptor the Lysosomal Integral Membrane Protein Type-2 (LIMP-2) in a mannose-6-phosphate independent manner. Mutations in GC cause the most common lyso¬somal storage disorder, Gaucher disease and represent the highest genetic risk factor for the development of Parkinson disease caused by accumulation of α synuclein. In this work properties of LIMP-2 and GC protein structure as well as their binding characteristics were analysed. By mutation and interaction studies, the GC-binding site on the LIMP 2 protein could be narrowed down to an exposed helical bundle comprised of two prominent helices. Surprisingly, a short peptide consisting of the proposed binding region of LIMP-2 was sufficient to mediate interaction with GC. Furthermore the LIMP-2-derived peptide had an activating effect on enzyme activity in vitro as well as in cell-based studies. The respective interaction site of LIMP-2 on the GC protein could also be narrowed down to a hydrophobic patch, verified by mutagenesis, interaction studies as well as peptide pulldown assays. Furthermore, effects of LIMP-2 deficiency in mouse brain were described exhibiting reduced levels of GC protein and activity, leading to aggregation of its substrates. Dysfunction of GC has been shown to interfere with α-synuclein homeostasis and its lysosomal degradation. In brain of LIMP-2-deficient mice α-synuclein accumulation could be observed, which led to severe neurological defects. Additionally, in cell-based experiments a distinct relationship between LIMP-2 expression, GC-activity and lysosomal degradation of α-synuclein was described. Heterologous expression of LIMP-2 in human and murine cell systems resulted in an increased lysosomal trafficking and activity of GC leading to a reduction of α-synuclein levels. This underscored the importance of the common function of LIMP-2 and GC in the intracellular regulation of α synuclein...Die β-Glukozerebrosidase (GC) ist eine lysosomale Hydrolase, die β-Glukose-ständige Sphingolipide umsetzt. Durch die Bindung an den Transportrezeptor, dem lysosomalen integralen Membranprotein Typ-2 (LIMP-2) gelangt das Enzym vom Endoplasmatischen Retikulum Mannose-6 Phosphat unabhängig an seinen Wirkort –den Lysosomen. Mutationen in der GC führen zu der häufigsten lysosomalen Speichererkrankung, der Gaucher Erkrankung. Außerdem wurde beschrieben, dass Mutationen im Gen der β-Glukozere¬bro¬si-dase zu den häufigsten genetischen Risikofaktoren der Parkinson Krankheit zählen. Diese neurodegenerative Erkrankung ist durch die Akkumulation des neuronalen Proteins α-Synuklein charakterisiert. In dieser Arbeit wurden die strukturellen Eigenschaften des LIMP-2 Proteins sowie der GC untersucht. Dabei war der Fokus auf die strukturelle Untersuchung ihrer Interaktion gerichtet. Durch Mutations- und Interaktionsstudien konnte die GC-Interaktionsstelle im LIMP-2 Protein auf eine exponierte helikale Region eingeschränkt werden. Außerdem konnte gezeigt werden, dass ein Peptid, bestehend aus der potentiellen Binderegion von LIMP-2, ausreichend ist um mit GC zu interagieren und sogar einen positiven Effekt auf die enzymatische Funktion aufweist. Eine erhöhte Enzymaktivität nach Peptid-Zugabe konnte sowohl in vitro, als auch in zellkulturbasierten Experimenten bestätigt werden. Zusätzlich konnte die entsprechende LIMP-2 Bindedomäne auf dem GC Protein identifiziert und ebenfalls auf einen hydrophoben helikalen Bereich im Enzym eingeschränkt werden..

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

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

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

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

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 autophagic 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, but also restored endo-lysosome and autophagy function in human and murine neurons as well as tissue. 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.authorsversionepub_ahead_of_prin

Mannose 6-phosphate-independent Lysosomal Sorting of Limp-2

Blanz J, Zunke F, Markmann S, et al. Mannose 6-phosphate-independent Lysosomal Sorting of Limp-2. Traffic. 2015;16(10):1127-1136.The lysosomal integral membrane protein type 2 (LIMP-2/SCARB2) has been described as a mannose 6-phosphate (M6P)-independent trafficking receptor for beta-glucocerebrosidase (GC). Recently, a putative M6P residue in a crystal structure of a recombinantly expressed LIMP-2 ectodomain has been reported. Based on surface plasmon resonance and fluorescence lifetime imaging analyses, it was suggested that the interaction of soluble LIMP-2 with the cation-independent M6P receptor (MPR) results in M6P-dependent targeting of LIMP-2 to lysosomes. As the physiological relevance of this observation was not addressed, we investigated M6P-dependent delivery of LIMP-2 to lysosomes in murine liver and mouse embryonic fibroblasts. We demonstrate that LIMP-2 and GC reach lysosomes independent of the M6P pathway. In fibroblasts lacking either MPRs or the M6P-forming N-acetylglucosamine (GlcNAc)-1-phosphotransferase, LIMP-2 still localizes to lysosomes. Immunoblot analyses also revealed comparable LIMP-2 levels within lysosomes purified from liver of wild-type (wt) and GlcNAc-1-phosphotransferase-defective mice. Heterologous expression of the luminal domain of LIMP-2 in wild-type, LIMP-2-deficient and GlcNAc-1-phosphotransferase-defective cells further established that the M6P modification is dispensable for lysosomal sorting of LIMP-2. Finally, cathepsin Z, a known GlcNAc-1-phosphotransferase substrate, but not LIMP-2, could be precipitated with M6P-specific antibodies. These data prove M6P-independent lysosomal sorting of LIMP-2 and subsequently GC in vivo

LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease

The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington's and Parkinson's disease. To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions. Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.status: publishe