Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

A homozygous hypomorphic BNIP1 variant causes an increase in autophagosomes and reduced autophagic flux and results in a spondylo-epiphyseal dysplasia.

BNIP1 (BCL2 interacting protein 1) is a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor involved in ER membrane fusion. We identified the homozygous BNIP1 intronic variant c.84+3A>T in the apparently unrelated patients 1 and 2 with disproportionate short stature. Radiographs showed abnormalities affecting both the axial and appendicular skeleton and spondylo-epiphyseal dysplasia. We detected ~80% aberrantly spliced BNIP1 pre-mRNAs, reduced BNIP1 mRNA level to ~80%, and BNIP1 protein level reduction by ~50% in patient 1 compared to control fibroblasts. The BNIP1 ortholog in Drosophila, Sec20, regulates autophagy and lysosomal degradation. We assessed lysosome positioning and identified a decrease in lysosomes in the perinuclear region and an increase in the cell periphery in patient 1 cells. Immunofluorescence microscopy and immunoblotting demonstrated an increase in LC3B-positive structures and LC3B-II levels, respectively, in patient 1 fibroblasts under steady-state condition. Treatment of serum-starved fibroblasts with or without bafilomycin A1 identified significantly decreased autophagic flux in patient 1 cells. Our data suggest a block at the terminal stage of autolysosome formation and/or clearance in patient fibroblasts. BNIP1 together with RAB33B and VPS16, disease genes for Smith-McCort dysplasia 2 and a multisystem disorder with short stature, respectively, highlight the importance of autophagy in skeletal development.

Enzyme replacement therapy in mice lacking arylsulfatase B targets bone-remodeling cells, but not chondrocytes.

Mucopolysaccharidosis type VI (MPS-VI), caused by mutational inactivation of the glycosaminoglycan-degrading enzyme arylsulfatase B (Arsb), is a lysosomal storage disorder primarily affecting the skeleton. We have previously reported that Arsb-deficient mice display high trabecular bone mass and impaired skeletal growth. In the present study, we treated them by weekly injection of recombinant human ARSB (rhARSB) to analyze the impact of enzyme replacement therapy (ERT) on skeletal growth and bone remodeling. We found that all bone-remodeling abnormalities of Arsb-deficient mice were prevented by ERT, whereas chondrocyte defects were not. Likewise, histologic analysis of the surgically removed femoral head from an ERT-treated MPS-VI patient revealed that only chondrocytes were pathologically affected. Remarkably, a side-by-side comparison with other cell types demonstrated that chondrocytes have substantially reduced capacity to endocytose rhARSB, together with low expression of the mannose receptor. We finally took advantage of Arsb-deficient mice to establish quantification of chondroitin sulfation for treatment monitoring. Our data demonstrate that bone-remodeling cell types are accessible to systemically delivered rhARSB, whereas the uptake into chondrocytes is inefficient.

Mucolipidosis type II and type III: a systematic review of 843 published cases.

PURPOSE: Mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma are rare autosomal recessive lysosomal storage disorders. Data on the natural course of the diseases are scarce. These data are important for counseling, therapies development, and improvement of outcome. The aim of this study is to gain knowledge on the natural history of ML by obtaining data on survival, symptom onset, presenting symptoms, diagnosis, and pathogenic variants associated with the MLII or MLIII phenotype. METHODS: A systematic review on all published MLII and MLIII cases between 1968 and August 2019 was performed. RESULTS: Three hundred one articles provided data on 843 patients. Median age at diagnosis: 0.7 for MLII and 9.0 years for MLIII. Median survival: 5.0 for MLII and 62.0 years for MLIIIII. Median age of death: 1.8 for MLII and 33.0 years for MLIII. Most frequent causes of death in all ML were pulmonary and/or cardiac complications. Pathogenic variants were described in 388 patients (GNPTAB: 571, GNPTG 179). CONCLUSION: This review provides unique insights into the natural history of MLII and MLIII, with a clear genotype-phenotype correlation with the most frequent pathogenic variant c.3503_3504del in MLII and in MLIII alpha/beta c.22A>G for GNPTAB. All pathogenic GNPTG variants resulted in MLIII gamma.

Pathogenic variants in GNPTAB and GNPTG encoding distinct subunits of GlcNAc-1-phosphotransferase differentially impact bone resorption in patients with mucolipidosis type II and III.

PURPOSE: Pathogenic variants in GNPTAB and GNPTG, encoding different subunits of GlcNAc-1-phosphotransferase, cause mucolipidosis (ML) II, MLIII alpha/beta, and MLIII gamma. This study aimed to investigate the cellular and molecular bases underlying skeletal abnormalities in patients with MLII and MLIII. METHODS: We analyzed bone biopsies from patients with MLIII alpha/beta or MLIII gamma by undecalcified histology and histomorphometry. The skeletal status of Gnptgko and Gnptab-deficient mice was determined and complemented by biochemical analysis of primary Gnptgko bone cells. The clinical relevance of the mouse data was underscored by systematic urinary collagen crosslinks quantification in patients with MLII, MLIII alpha/beta, and MLIII gamma. RESULTS: The analysis of iliac crest biopsies revealed that bone remodeling is impaired in patients with GNPTAB-associated MLIII alpha/beta but not with GNPTG-associated MLIII gamma. Opposed to Gnptab-deficient mice, skeletal remodeling is not affected in Gnptgko mice. Most importantly, patients with variants in GNPTAB but not in GNPTG exhibited increased bone resorption. CONCLUSION: The gene-specific impact on bone remodeling in human individuals and in mice proposes distinct molecular functions of the GlcNAc-1-phosphotransferase subunits in bone cells. We therefore appeal for the necessity to classify MLIII based on genetic in addition to clinical criteria to ensure appropriate therapy.

Distinct Modes of Balancing Glomerular Cell Proteostasis in Mucolipidosis Type II and III Prevent Proteinuria.

BACKGROUND: The mechanisms balancing proteostasis in glomerular cells are unknown. Mucolipidosis (ML) II and III are rare lysosomal storage disorders associated with mutations of the Golgi-resident GlcNAc-1-phosphotransferase, which generates mannose 6-phosphate residues on lysosomal enzymes. Without this modification, lysosomal enzymes are missorted to the extracellular space, which results in lysosomal dysfunction of many cell types. Patients with MLII present with severe skeletal abnormalities, multisystemic symptoms, and early death; the clinical course in MLIII is less progressive. Despite dysfunction of a major degradative pathway, renal and glomerular involvement is rarely reported, suggesting organ-specific compensatory mechanisms. METHODS: MLII mice were generated and compared with an established MLIII model to investigate the balance of protein synthesis and degradation, which reflects glomerular integrity. Proteinuria was assessed in patients. High-resolution confocal microscopy and functional assays identified proteins to deduce compensatory modes of balancing proteostasis. RESULTS: Patients with MLII but not MLIII exhibited microalbuminuria. MLII mice showed lysosomal enzyme missorting and several skeletal alterations, indicating that they are a useful model. In glomeruli, both MLII and MLIII mice exhibited reduced levels of lysosomal enzymes and enlarged lysosomes with abnormal storage material. Nevertheless, neither model had detectable morphologic or functional glomerular alterations. The models rebalance proteostasis in two ways: MLII mice downregulate protein translation and increase the integrated stress response, whereas MLIII mice upregulate the proteasome system in their glomeruli. Both MLII and MLIII downregulate the protein complex mTORC1 (mammalian target of rapamycin complex 1) signaling, which decreases protein synthesis. CONCLUSIONS: Severe lysosomal dysfunction leads to microalbuminuria in some patients with mucolipidosis. Mouse models indicate distinct compensatory pathways that balance proteostasis in MLII and MLIII.

Combined in vitro and in silico analyses of missense mutations in GNPTAB provide new insights into the molecular bases of mucolipidosis II and III alpha/beta.

Mucolipidosis (ML) II and III alpha/beta are inherited lysosomal storage disorders caused by mutations in GNPTAB encoding the α/ß-precursor of GlcNAc-1-phosphotransferase. This enzyme catalyzes the initial step in the modification of more than 70 lysosomal enzymes with mannose 6-phosphate residues to ensure their intracellular targeting to lysosomes. The so-called stealth domains in the α- and ß-subunit of GlcNAc-1-phosphotransferase were thought to be involved in substrate recognition and/or catalysis. Here, we performed in silico alignment analysis of stealth domain-containing phosphotransferases and showed that the amino acid residues Glu389 , Asp408 , His956 , and Arg986 are highly conserved between different phosphotransferases. Interestingly, mutations in these residues were identified in patients with MLII and MLIII alpha/beta. To further support the in silico findings, we also provide experimental data demonstrating that these four amino acid residues are strictly required for GlcNAc-1-phosphotransferase activity and thus may be directly involved in the enzymatic catalysis.

Lysosomal Proteome and Secretome Analysis Identifies Missorted Enzymes and Their Nondegraded Substrates in Mucolipidosis III Mouse Cells.

Targeting of soluble lysosomal enzymes requires mannose 6-phosphate (M6P) signals whose formation is initiated by the hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2ß2γ2). Upon proteolytic cleavage by site-1 protease, the α/ß-subunit precursor is catalytically activated but the functions of γ-subunits (Gnptg) in M6P modification of lysosomal enzymes are unknown. To investigate this, we analyzed the Gnptg expression in mouse tissues, primary cultured cells, and in Gnptg reporter mice in vivo, and found high amounts in the brain, eye, kidney, femur, vertebra and fibroblasts. Consecutively we performed comprehensive quantitative lysosomal proteome and M6P secretome analysis in fibroblasts of wild-type and Gnptgko mice mimicking the lysosomal storage disorder mucolipidosis III. Although the cleavage of the α/ß-precursor was not affected by Gnptg deficiency, the GlcNAc-1-phosphotransferase activity was significantly reduced. We purified lysosomes and identified 29 soluble lysosomal proteins by SILAC-based mass spectrometry exhibiting differential abundance in Gnptgko fibroblasts which was confirmed by Western blotting and enzymatic activity analysis for selected proteins. A subset of these lysosomal enzymes show also reduced M6P modifications, fail to reach lysosomes and are secreted, among them α-l-fucosidase and arylsulfatase B. Low levels of these enzymes correlate with the accumulation of non-degraded fucose-containing glycostructures and sulfated glycosaminoglycans in Gnptgko lysosomes. Incubation of Gnptgko fibroblasts with arylsulfatase B partially rescued glycosaminoglycan storage. Combinatorial treatments with other here identified missorted enzymes of this degradation pathway might further correct glycosaminoglycan accumulation and will provide a useful basis to reveal mechanisms of selective, Gnptg-dependent formation of M6P residues on lysosomal proteins.

The lysosomal storage disorders mucolipidosis type II, type III alpha/beta, and type III gamma: Update on GNPTAB and GNPTG mutations.

Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/ß-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.

Site-1 protease and lysosomal homeostasis.

The Golgi-resident site-1 protease (S1P) is a key regulator of cholesterol homeostasis and ER stress responses by converting latent transcription factors sterol regulatory element binding proteins (SREPBs) and activating transcription factor 6 (ATF6), as well as viral glycoproteins to their active forms. S1P is also essential for lysosome biogenesis via proteolytic activation of the hexameric GlcNAc-1-phosphotransferase complex required for modification of newly synthesized lysosomal enzymes with the lysosomal targeting signal, mannose 6-phosphate. In the absence of S1P, the catalytically inactive α/ß-subunit precursor of GlcNAc-1-phosphotransferase fails to be activated and results in missorting of newly synthesized lysosomal enzymes, and lysosomal accumulation of non-degraded material, which are biochemical features of defective GlcNAc-1-phosphotransferase subunits and the associated pediatric lysosomal diseases mucolipidosis type II and III. The early embryonic death of S1P-deficient mice and the importance of various S1P-regulated biological processes, including lysosomal homeostasis, cautioned for clinical inhibition of S1P. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Subunit interactions of the disease-related hexameric GlcNAc-1-phosphotransferase complex.

The multimeric GlcNAc-1-phosphotransferase complex catalyzes the formation of mannose 6-phosphate recognition marker on lysosomal enzymes required for receptor-mediated targeting to lysosomes. GNPTAB and GNPTG encode the α/ß-subunit precursor membrane proteins and the soluble γ-subunits, respectively. Performing extensive mutational analysis, we identified the binding regions of γ-subunits in a previously uncharacterized domain of α-subunits comprising residues 535-698, named GNPTG binding (GB) domain. Both the deletion of GB preventing γ-subunit binding and targeted deletion of GNPTG led to significant reduction in GlcNAc-1-phosphotransferase activity. We also identified cysteine 70 in α-subunits to be involved in covalent homodimerization of α-subunits which is, however, required neither for interaction with γ-subunits nor for catalytic activity of the enzyme complex. Finally, binding assays using various γ-subunit mutants revealed that residues 130-238 interact with glycosylated α-subunits suggesting a role for the mannose 6-phosphate receptor homology domain in α-subunit binding. These studies provide new insight into the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site.

Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/ß-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/ß-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/ß-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive ß-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype-phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.

Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control.

Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/ß-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/ß-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/ß-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/ß-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/ß-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different.

Mucolipidosis II-related mutations inhibit the exit from the endoplasmic reticulum and proteolytic cleavage of GlcNAc-1-phosphotransferase precursor protein (GNPTAB).

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/ß-subunit precursor protein of GlcNAc-1-phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α-subunit-specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild-type and mutant α/ß-subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/ß-subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc-1-phosphotransferase that may help to explain the clinical phenotype of MLII patients.

CNS Manifestations in Mucolipidosis Type II-A Retrospective Analysis of Longitudinal Data on Neurocognitive Development and Neuroimaging in Eleven Patients.

Mucolipidosis type II (MLII), an ultra-rare lysosomal storage disorder, manifests as a fatal multi-systemic disease. Mental inhibition and progressive neurodegeneration are commonly reported disease manifestations. Nevertheless, longitudinal data on neurocognitive testing and neuroimaging lack in current literature. This study aimed to provide details on central nervous system manifestations in MLII. All MLII patients with at least one standardized developmental assessment performed between 2005 and 2022 were included by retrospective chart review. A multiple mixed linear regression model was applied. Eleven patients with a median age of 34.0 months (range 1.6-159.6) underwent 32 neurocognitive and 28 adaptive behaviour assessments as well as 14 brain magnetic resonance imagings. The scales used were mainly BSID-III (42%) and VABS-II (47%). Neurocognitive testing (per patient: mean 2.9, standard deviation (SD) 2.0) performed over 0-52.1 months (median 12.1) revealed profound impairment with a mean developmental quotient of 36.7% (SD 20.4) at last assessment. The patients showed sustained development; on average, they gained 0.28 age-equivalent score points per month (confidence interval 0.17-0.38). Apart from common (63%) cervical spinal stenosis, neuroimaging revealed unspecific, non-progressive abnormalities (i.e., mild brain atrophy, white matter lesions). In summary, MLII is associated with profound developmental impairment, but not with neurodegeneration and neurocognitive decline.

Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype.

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

Mice heterozygous for an osteogenesis imperfecta-linked MBTPS2 variant display a compromised subchondral osteocyte lacunocanalicular network associated with abnormal articular cartilage.

Missense variants in the MBTPS2 gene, located on the X chromosome, have been associated with an X-linked recessive form of osteogenesis imperfecta (X-OI), an inherited bone dysplasia characterized by multiple and recurrent bone fractures, short stature, and various skeletal deformities in affected individuals. The role of site-2 protease, encoded by MBTPS2, and the molecular pathomechanism underlying the disease are to date elusive. This study is the first to report on the generation of two Mbtps2 mouse models, a knock-in mouse carrying one of the disease-causative MBTPS2 variants (N455S) and a Mbtps2 knock-out (ko) mouse. Because both loss-of-function variants lead to embryonic lethality in hemizygous male mutant mice, we performed a comprehensive skeletal analysis of heterozygous Mbtps2+/N455S and Mbtps2+/ko female mice. Both models displayed osteochondral abnormalities such as thinned subchondral bone, altered subchondral osteocyte interconnectivity as well as thickened articular cartilage with chondrocyte clustering, altogether resembling an early osteoarthritis (OA) phenotype. However, distant from the joints, no alterations in the bone mass and turnover could be detected in either of the mutant mice. Based on our findings we conclude that MBTPS2 haploinsufficiency results in early OA-like alterations in the articular cartilage and underlying subchondral bone, which likely precede the development of typical OI phenotype in bone. Our study provides first evidence for a potential role of site-2 protease for maintaining homeostasis of both bone and cartilage.

Non-functional ubiquitin C-terminal hydrolase L1 drives podocyte injury through impairing proteasomes in autoimmune glomerulonephritis.

Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1.

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase.

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)ß(2)γ(2)). The α- and ß-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.

Site-1 and site-2 proteases: A team of two in regulated proteolysis.

The site-1 and site-2 proteases (S1P and S2P) were identified over 20 years ago, and the functions of both have been addressed in numerous studies ever since. Whereas S1P processes a set of substrates independently of S2P, the latter acts in concert with S1P in a mechanism, called regulated intramembrane proteolysis, that controls lipid metabolism and response to unfolded proteins. This review summarizes the molecular roles that S1P and S2P jointly play in these processes. As S1P and S2P deficiencies mainly affect connective tissues, yet with varying phenotypes, we discuss the segregated functions of S1P and S2P in terms of cell homeostasis and maintenance of the connective tissues. In addition, we provide experimental data that point at S2P, but not S1P, as a critical regulator of cell adaptation to proteotoxicity or lipid imbalance. Therefore, we hypothesize that S2P can also function independently of S1P activity.