Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

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.

Multi-Cell Line Analysis of Lysosomal Proteomes Reveals Unique Features and Novel Lysosomal Proteins.

Lysosomes, the main degradative organelles of mammalian cells, play a key role in the regulation of metabolism. It is becoming more and more apparent that they are highly active, diverse, and involved in a large variety of processes. The essential role of lysosomes is exemplified by the detrimental consequences of their malfunction, which can result in lysosomal storage disorders, neurodegenerative diseases, and cancer. Using lysosome enrichment and mass spectrometry, we investigated the lysosomal proteomes of HEK293, HeLa, HuH-7, SH-SY5Y, MEF, and NIH3T3 cells. We provide evidence on a large scale for cell type-specific differences of lysosomes, showing that levels of distinct lysosomal proteins are highly variable within one cell type, while expression of others is highly conserved across several cell lines. Using differentially stable isotope-labeled cells and bimodal distribution analysis, we furthermore identify a high confidence population of lysosomal proteins for each cell line. Multi-cell line correlation of these data reveals potential novel lysosomal proteins, and we confirm lysosomal localization for six candidates. All data are available via ProteomeXchange with identifier PXD020600.

Lack of a protective effect of the Tmem106b "protective SNP" in the Grn knockout mouse model for frontotemporal lobar degeneration.

Genetic variants in TMEM106B are a common risk factor for frontotemporal lobar degeneration and the most important modifier of disease risk in patients with progranulin (GRN) mutations (FTLD-GRN). TMEM106B is encoding a lysosomal transmembrane protein of unknown molecular function. How it mediates its disease-modifying function remains enigmatic. Several TMEM106B single nucleotide polymorphisms (SNPs) are significantly associated with disease risk in FTLD-GRN carriers, of which all except one are within intronic sequences of TMEM106B. Of note, the non-coding SNPs are in high linkage disequilibrium with the coding SNP rs3173615 located in exon six of TMEM106B, resulting in a threonine to serine change at amino acid 185 in the minor allele, which is protective in FTLD-GRN carriers. To investigate the functional consequences of this variant in vivo, we generated and characterized a knockin mouse model harboring the Tmem106bT186S variant. We analyzed the effect of this protective variant on FTLD pathology by crossing Tmem106bT186S mice with Grn-/- knockout mice, a model for GRN-mediated FTLD. We did not observe the amelioration of any of the investigated Grn-/- knockout phenotypes, including transcriptomic changes, lipid alterations, or microgliosis in Tmem106bT186S/T186S × Grn-/- mice, indicating that the Tmem106bT186S variant is not protective in the Grn-/- knockout mouse model. These data suggest that effects of the associated SNPs not directly linked to the amino acid exchange in TMEM106B are critical for the modifying effect.

Anchorless risk or released benefit? An updated view on the ADAM10-mediated shedding of the prion protein.

The prion protein (PrP) is a broadly expressed glycoprotein linked with a multitude of (suggested) biological and pathological implications. Some of these roles seem to be due to constitutively generated proteolytic fragments of the protein. Among them is a soluble PrP form, which is released from the surface of neurons and other cell types by action of the metalloprotease ADAM10 in a process termed 'shedding'. The latter aspect is the focus of this review, which aims to provide a comprehensive overview on (i) the relevance of proteolytic processing in regulating cellular PrP functions, (ii) currently described involvement of shed PrP in neurodegenerative diseases (including prion diseases and Alzheimer's disease), (iii) shed PrP's expected roles in intercellular communication in many more (patho)physiological conditions (such as stroke, cancer or immune responses), (iv) and the need for improved research tools in respective (future) studies. Deeper mechanistic insight into roles played by PrP shedding and its resulting fragment may pave the way for improved diagnostics and future therapeutic approaches in diseases of the brain and beyond.

The Solute Carrier MFSD1 Decreases the Activation Status of ß1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1-/- tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive ß1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive ß1 integrin and thereby protected ß1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient ß1 integrin recycling to suppress tumor cell dissemination.

Usher syndrome type IV: clinically and molecularly confirmed by novel ARSG variants.

Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is highly heterogeneous both clinically and genetically. Recently, variants in the arylsulfatase G (ARSG) gene have been reported to underlie USH type IV. This distinct type of USH is characterized by late-onset RP with predominantly pericentral and macular changes, and late onset SNHL without vestibular dysfunction. In this study, we describe the USH type IV phenotype in three unrelated subjects. We identified three novel pathogenic variants, two novel likely pathogenic variants, and one previously described pathogenic variant in ARSG. Functional experiments indicated a loss of sulfatase activity of the mutant proteins. Our findings confirm that ARSG variants cause the newly defined USH type IV and support the proposed extension of the phenotypic USH classification.

S-palmitoylation determines TMEM55B-dependent positioning of lysosomes.

The spatiotemporal cellular distribution of lysosomes depends on active transport mainly driven by microtubule motors such as kinesins and dynein. Different protein complexes attach these molecular motors to their vesicular cargo. TMEM55B (also known as PIP4P1), as an integral lysosomal membrane protein, is a component of such a complex that mediates the retrograde transport of lysosomes by establishing interactions with the cytosolic scaffold protein JIP4 (also known as SPAG9) and dynein-dynactin. Here, we show that TMEM55B and its paralog TMEM55A (PIP4P2) are S-palmitoylated proteins that are lipidated at multiple cysteine residues. Mutation of all cysteines in TMEM55B prevents S-palmitoylation and causes retention of the mutated protein in the Golgi. Consequently, non-palmitoylated TMEM55B is no longer able to modulate lysosomal positioning and the perinuclear clustering of lysosomes. Additional mutagenesis of the dileucine-based lysosomal sorting motif in non-palmitoylated TMEM55B leads to partial missorting to the plasma membrane instead of retention in the Golgi, implicating a direct effect of S-palmitoylation on the adaptor protein-dependent sorting of TMEM55B. Our data suggest a critical role for S-palmitoylation in the trafficking of TMEM55B and TMEM55B-dependent lysosomal positioning.

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins.

Protein AMPylation is a posttranslational modification with an emerging role in neurodevelopment. In metazoans two highly conserved protein AMP-transferases together with a diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques. However, the function of AMPylation remains largely unknown. Particularly problematic is the localization of thus far identified AMPylated proteins and putative AMP-transferases. We show that protein AMPylation is likely a posttranslational modification of luminal lysosomal proteins characteristic in differentiating neurons. Through a combination of chemical proteomics, gel-based separation of modified and unmodified proteins, and an activity assay, we determine that the modified, lysosomal soluble form of exonuclease PLD3 increases dramatically during neuronal maturation and that AMPylation correlates with its catalytic activity. Together, our findings indicate that AMPylation is a so far unknown lysosomal posttranslational modification connected to neuronal differentiation and it may provide a molecular rationale behind lysosomal storage diseases and neurodegeneration.

Ligands binding to the prion protein induce its proteolytic release with therapeutic potential in neurodegenerative proteinopathies.

The prion protein (PrPC) is a central player in neurodegenerative diseases, such as prion diseases or Alzheimer's disease. In contrast to disease-promoting cell surface PrPC, extracellular fragments act neuroprotective by blocking neurotoxic disease-associated protein conformers. Fittingly, PrPC release by the metalloprotease ADAM10 represents a protective mechanism. We used biochemical, cell biological, morphological, and structural methods to investigate mechanisms stimulating this proteolytic shedding. Shed PrP negatively correlates with prion conversion and is markedly redistributed in murine brain in the presence of prion deposits or amyloid plaques, indicating a sequestrating activity. PrP-directed ligands cause structural changes in PrPC and increased shedding in cells and organotypic brain slice cultures. As an exception, some PrP-directed antibodies targeting repetitive epitopes do not cause shedding but surface clustering, endocytosis, and degradation of PrPC. Both mechanisms may contribute to beneficial actions described for PrP-directed ligands and pave the way for new therapeutic strategies against currently incurable neurodegenerative diseases.

Decoding the consecutive lysosomal degradation of 3-O-sulfate containing heparan sulfate by Arylsulfatase G (ARSG).

The lysosomal degradation of heparan sulfate is mediated by the concerted action of nine different enzymes. Within this degradation pathway, Arylsulfatase G (ARSG) is critical for removing 3-O-sulfate from glucosamine, and mutations in ARSG are causative for Usher syndrome type IV. We developed a specific ARSG enzyme assay using sulfated monosaccharide substrates, which reflect derivatives of its natural substrates. These sulfated compounds were incubated with ARSG, and resulting products were analyzed by reversed-phase HPLC after chemical addition of the fluorescent dyes 2-aminoacridone or 2-aminobenzoic acid, respectively. We applied the assay to further characterize ARSG regarding its hydrolytic specificity against 3-O-sulfated monosaccharides containing additional sulfate-groups and N-acetylation. The application of recombinant ARSG and cells overexpressing ARSG as well as isolated lysosomes from wild-type and Arsg knockout mice validated the utility of our assay. We further exploited the assay to determine the sequential action of the different sulfatases involved in the lysosomal catabolism of 3-O-sulfated glucosamine residues of heparan sulfate. Our results confirm and extend the characterization of the substrate specificity of ARSG and help to determine the sequential order of the lysosomal catabolic breakdown of (3-O-)sulfated heparan sulfate.

CLN6 deficiency causes selective changes in the lysosomal protein composition.

Neuronal ceroid lipofuscinoses (NCLs) collectively account for the highest prevalence of inherited neurodegenerative diseases in childhood. This disease group is classified by the deposition of similar autofluorescence storage material in lysosomes that is accompanied by seizures, blindness and premature mortality in later disease stages. Defects in several genes affecting various proteins lead to NCL, one of them being CLN6, a transmembrane protein resident in the endoplasmic reticulum. Dysfunctionality of CLN6 causes variant late infantile NCL (vLINCL). The function of CLN6 and how its deficiency affects lysosomal integrity remains unknown. In this work, we performed a comparative proteomic analysis of isolated lysosomal fractions from liver tissue of nclf mice, a natural mouse model displaying a similar disease course than its human counterpart. We could identify a drastic reduction in the protein amounts of selected lysosomal proteins, amongst them several members of the NCL protein family. Most of these proteins were N-glycosylated, soluble hydrolases and their reduction in protein levels was verified by western blotting and enzymatic assays. Hereby we could directly link Cln6 dysfunction to changes in the lysosomal compartment and to other NCL forms.

LAMP3 deficiency affects surfactant homeostasis in mice.

Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.

The lysosomal membrane-export of metabolites and beyond.

Lysosomes are degradative organelles in eukaryotic cells mediating the hydrolytic catabolism of various macromolecules to small basic building blocks. These low-molecular-weight metabolites are transported across the lysosomal membrane and reused in the cytoplasm and other organelles for biosynthetic pathways. Even though in the past 20 years our understanding of the lysosomal membrane regarding various transporters, other integral and peripheral membrane proteins, the lipid composition, but also its turnover has dramatically improved, there are still many unresolved questions concerning key aspects of the function of the lysosomal membrane. These include a possible function of lysosomes as a cellular storage compartment, yet unidentified transporters mediating the export such as various amino acids, mechanisms mediating the transport of lysosomal membrane proteins from the Golgi apparatus to lysosomes, and the turnover of lysosomal membrane proteins. Here, we review the current knowledge about the lysosomal membrane and identify some of the open questions that need to be solved in the future for a comprehensive and complete understanding of how lysosomes communicate with other organelles, cellular processes, and pathways.

Aged Tmem106b knockout mice display gait deficits in coincidence with Purkinje cell loss and only limited signs of non-motor dysfunction.

Genetic variants in TMEM106B are a major risk factor for several neurodegenerative diseases including frontotemporal degeneration, limbic-predominant age-related TDP-43 encephalopathy, Parkinson's disease, late-onset-Alzheimer's disease and constitute a genetic determinant of differential aging. TMEM106B encodes an integral lysosomal membrane protein but its precise physiological function in the central nervous system remains enigmatic. Presently, we aimed to increase understanding of TMEM106B contribution to general brain function and aging. We analyzed an aged cohort of Tmem106b knockout-, heterozygote and wild-type mice in a behavioral test battery including assessments of motor function as well as, social, emotional and cognitive function. Aged Tmem106b knockout (KO) mice displayed diverse behavioral deficits including motor impairment, gait defects and reduced startle reactivity. In contrast, no prominent deficits were observed in social, emotional or cognitive behaviors. Histologically, we observed late-onset loss of Purkinje cells followed by reactive gliosis in the cerebellum, which likely contributed to progressive decline in motor function and gait defects in particular. Reactive gliosis was not restricted to the cerebellum but observed in different areas of the brain including the brain stem and parts of the cerebral cortex. Surviving Purkinje cells showed vacuolated lysosomes in the axon initial segment, implicating TMEM106B-dependent lysosomal trafficking defects as the underlying cause of axonal and more general neuronal dysfunction contributing to behavioral impairments. Our experiments help to elucidate how TMEM106B affects spatial neuronal homeostasis and exemplifies a critical role of TMEM106B in neuronal cells for survival.

New clinical and molecular evidence linking mutations in ARSG to Usher syndrome type IV.

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf-blindness syndrome, by abolishing the activity of the enzyme it encodes.

Quantification and characterization of the 5' exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay.

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer's disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5' exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher-coupled oligonucleotides for the quantitative determination of acidic 5' exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5' exonuclease in HeLa cells, where it showed a markedly higher specific activity compared with PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3 and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer's disease and investigated each variant's effect on the 5' nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely understood roles in vitro and in vivo.

Lysosomal sulfatases: a growing family.

Sulfatases constitute a family of enzymes that specifically act in the hydrolytic degradation of sulfated metabolites by removing sulfate monoesters from various substrates, particularly glycolipids and glycosaminoglycans. A common essential feature of all known eukaryotic sulfatases is the posttranslational modification of a critical cysteine residue in their active site by oxidation to formylglycine (FGly), which is mediated by the FGly-generating enzyme in the endoplasmic reticulum and is indispensable for catalytic activity. The majority of the so far described sulfatases localize intracellularly to lysosomes, where they act in different catabolic pathways. Mutations in genes coding for lysosomal sulfatases lead to an accumulation of the sulfated substrates in lysosomes, resulting in impaired cellular function and multisystemic disorders presenting as lysosomal storage diseases, which also cover the mucopolysaccharidoses and metachromatic leukodystrophy. Bioinformatics analysis of the eukaryotic genomes revealed, besides the well described and long known disease-associated sulfatases, additional genes coding for putative enzymes with sulfatases activity, including arylsulfatase G as well as the arylsulfatases H, I, J and K, respectively. In this article, we review current knowledge about lysosomal sulfatases with a special focus on the just recently characterized family members arylsulfatase G and arylsulfatase K.