Characterization of HTRA1 and Calpain 2 interaction

In der vorliegenden Dissertation wurde die Interaktion von HTRA1 und Calpain 2 charakterisiert und deren potentielle physiologische Rolle analysiert. Die Proteasen HTRA1 und Calpain 2 zeigen beide vielfältige physiologische Funktionen. HTRA1 ist ein wichtiger Bestandteil der extrazellulären Proteinqualitätskontrolle, die Inhibierung von HTRA1 führt beispielsweise zur Akkumulation des mit der Alzheimer’schen Krankheit (AD, alzheimer’s disease) assoziierten Aβ Peptids (Grau et al., 2005). Auch intrazellulär ist HTRA1 in den Verlauf von AD involviert. HTRA1 ist dazu in der Lage das Mikrotubuli bindende Protein Tau und seine Aggregate zu proteolysieren (Tennstaedt et al., 2012). Intrazelluläre Tau Fibrillen und extrazelluläre Aβ Aggregate kennzeichnen AD (Crews & Masliah, 2010). Steigt die extrazelluläre Aβ Konzentration kommt es durch Aktivierung von NMDA (N-Methyl-D-Aspartat) Rezeptoren zu einem Calcium Einstrom in das Cytosol der Zelle. Dadurch wird Calpain 2 aktiviert, kann Tau proteolysieren und die entstandenen Produkte zeigen ein erhöhtes Potential zur Aggregation (Park & Ferreira, 2005). Außerdem aktiviert Calpain 2 verschiedene Kinasen, die durch Hyperphosphorylierung von Tau zur Aggregation von diesem und schließlich zur Apoptose führen können (Vosler et al., 2008). Eine Aktivierung von HTRA1 ist also wünschenswert, die Aktivierung von Calpain 2 ist dagegen ein negativer Faktor in von AD. Mit Hilfe eines sogenannten C-Terminus Screens konnte Calpain 2 als ein potentieller Interaktionspartner von HTRA1 identifiziert werden. Der C-Terminus Screen wurde in Kooperation mit dem Institut für medizinische Immunologie der Charité-Universitätsmedizin in Berlin durchgeführt und beinhaltete insgesamt 6223 Peptide, die den C-Termini des humanem Proteoms entsprechen. Für die Untersuchung der möglichen Interaktion beider Proteasen wurde Calpain 2 im Verlauf dieser Dissertation kloniert und die Proteinreinigung etabliert. In einem Pulldown-Assay konnte die Bindung von Calpain 2 an HTRA1 nachgewiesen werden. Nach der Vorinkubation beider Proteasen konnte in einer Größenausschlusschromatographie eine neue Proteinpopulation detektiert werden. Diese konnte bei Vorinkubation mit einer Mutante von HTRA1, der die PDZ-Domäne fehlt, nicht beobachtet werden. Die PDZ-Domäne von HTRA1 ist demnach essentiell für die Bildung des HTRA1 – Calpain 2 Komplexes. Durch Vergleiche mit bekannten HTRA1 und Calpain 2 Mengen konnte eine Stöchiometrie im Komplex von 1:1 abgeschätzt werden. Die molekulare Masse des Komplexes wurde mittels statischer Lichtstreuung nach Größenausschlusschromatographie, MALS-SEC (multi-angle light scattering with size exclusion chromatography), genauer analysiert. Der HTRA1 – Calpain 2 Komplex besitzt eine molekulare Masse von 535 ± 0,5 kDa. Der Komplex besteht wahrscheinlich aus einem HTRA1 Trimer (109 kDa), was der stabilen oligomeren Form von HTRA1 entspricht, und vier Calpain 2 Heterodimeren (je 104 kDa). Damit ergibt sich für den HTRA1 – Calpain 2 Komplex eine berechnete molekulare Masse von 525 kDa und eine Stöchiometrie von 3:4 (1:1,3). Des Weiteren wurde die Struktur des Komplexes mittels Transmissionselektronenmikroskopie untersucht. Die Partikel, die im Transmissionselektronenmikroskop detektiert werden konnten, wiesen eine Größe von etwa 18 x 15 x 11 nm auf. Die Längen eines Modells aus Röntgenkristallstrukturen eines HTRA1 Trimers zusammen mit vier Calpain 2 Heterodimeren stimmt mit diesen experimentell bestimmten Längen gut überein. Außerdem konnte festgestellt werden, dass der HTRA1 – Calpain 2 Komplex stabil ist, die Fraktionen des Komplexes der Größenausschlusschromatographie wurden vereinigt und nochmals chromatographisch aufgetrennt. Hier konnte nur der Komplex, nicht jedoch die einzelnen Proteine, detektiert werden. Der HTRA1 – Calpain 2 Komplex dissoziiert demnach nicht in seine Einzelkomponenten. Mittels Immunfluoreszenzfärbungen von HTRA1 und Calpain 2 konnte ebenfalls eine mögliche Kolokalisation und damit wahrscheinlich eine Komplexbildung beider Proteasen in HeLa und SH-SY5Y Zelllinien in vivo gezeigt werden. Eine Vorinkubation von HTRA1 und Calpain 2 führt zu einer deutlichen Aktivierung von HTRA1 nicht aber von Calpain 2. Es wurden pNA gekoppelte Peptide und das physiologisch relevante Substrat Tau sowie seine Fibrillen getestet. Massenspektrometrische Analysen der Proteolyseprodukte von Tau Fibrillen ergaben, dass der HTRA1 – Calpain 2 Komplex vor allem in der Repeat Regionen und der self assembly Region von Tau, die für die Aggregation verantwortlich ist, vermehrte Schnittstellen im Vergleich zu den einzelnen Proteasen zeigte. Der HTRA1 – Calpain 2 Komplex könnte in der Bekämpfung von AD eine Rolle spielen. Schon zu Fibrillen aggregiertes Tau könnte nach der Bildung des Komplexes schneller abgebaut und somit die Apoptose der Zelle verhindert werden.The aim of this work was the characterization of the interaction of two proteases, the serine protease HTRA1 and the cysteine protease calpain 2. Both proteases are involved in various neurodegenerative diseases such as Alzheimer’s disease (AD). More specifically, HTRA1 is a key player in protein quality control. Inhibition of HTRA1 leads to an accumulation of extracellular Aβ, a hallmark of AD (Grau et al., 2005). Intracellular HTRA1 is known to degrade tau and its aggregates (Tennstaedt et al., 2012). Elevated concentrations of the extracellular Aβ lead to an activation of NMDA (N-methyl-D-aspartate) receptors followed by an influx of calcium ions into the cytosol. Consequently, calpain 2 gets activated upon increasing calcium concentrations. Calpain 2 is involved in AD by degrading tau and activating various kinases, which promote the aggregation of tau by phosphorylation and finally lead to apoptosis (Vosler et al., 2008). A screen involving a nonredundant set of 6223 decameric peptides, which correspond to the C-termini of all human proteins revealed candidate ligands of HTRA1 (cooperation with Institut für medizinische Immunologie, Charité-Universitätsmedizin, Berlin). The hits from this screen suggest that HTRA1 and calpain 2 might interact. To test this hypothesis human calpain 2 and its small human subunit were expressed in E. coli and the calpain 2 heterodimer was purified. Interaction of HTRA1 and calpain 2 could be demonstrated by using pulldown-assays. Moreover, incubation of the calpain 2 heterodimer with HTRA1 resulted in the formation of a stable complex as detected by size exclusion chromatography. The formation of the HTRA1 – calpain 2 complex was probably mediated via the PDZ domain of HTRA1 due to the fact that PDZ-less HTRA1 failed to form a complex. The complex was stable after reinjection as it did not dissociate into the single components. 1:1 stoichiometry of the HTRA1 – calpain 2 complex was determined. Detailed analysis of the molecular weight of this complex was performed via multi-angle light scattering in combination with size exclusion chromatography (MALS-SEC). This analysis revealed a size of 535 ± 0,5 kDa. The formation of the protease complex was additionally confirmed via a negative staining technique using transmission electron microscopy whereby particles of similar size were detected. A model was introduced based on the parameters molecular weight, stoichiometry and the size of the particles. It consistent of four calpain 2 heterodimers, each 104 kDa, bound to one HTRA1 trimer (109 kDa). The calculation of the complex model showed a molecular weight of 525 kDa and a stoichiometry of 3:4 (1:1,3) and with that confirmed the obtained data. By using immunofluorescently labeled HTRA1 and calpain 2 a possible colocalization in HeLa and SH-SY5Y cells could be demonstrated. Further, digestion assays suggested that the formation of the protease complex increases the activity of HTRA1. Additionally, the use of mass spectrometry indicated more proteolysis sites in the repeat region of tau fibrils. This protease complex could play an important role in various diseases, especially in AD. Calpains are known to be an aggravating factor in AD and HTRA1 is implicated in the digestion of pathogenic aggregates like tau, therefore this protease complex where HTRA1 is activated by calpain 2 could be beneficial to cell survival

The epidermal barrier function is dependent on the serine protease CAP1/Prss8

Serine proteases are proteolytic enzymes that are involved in the regulation of various physiological processes. We generated mice lacking the membrane-anchored channel-activating serine protease (CAP) 1 (also termed protease serine S1 family member 8 [Prss8] and prostasin) in skin, and these mice died within 60 h after birth. They presented a lower body weight and exhibited severe malformation of the stratum corneum (SC). This aberrant skin development was accompanied by an impaired skin barrier function, as evidenced by dehydration and skin permeability assay and transepidermal water loss measurements leading to rapid, fatal dehydration. Analysis of differentiation markers revealed no major alterations in CAP1/Prss8-deficient skin even though the epidermal deficiency of CAP1/Prss8 expression disturbs SC lipid composition, corneocyte morphogenesis, and the processing of profilaggrin. The examination of tight junction proteins revealed an absence of occludin, which did not prevent the diffusion of subcutaneously injected tracer (∼600 D) toward the skin surface. This study shows that CAP1/Prss8 expression in the epidermis is crucial for the epidermal permeability barrier and is, thereby, indispensable for postnatal survival

Therapeutic drug monitoring (TDM) of atazanavir in pregnancy

Purpose of the study: Pregnant women experience physiological changes during pregnancy resulting in clinically significant alterations in antiretroviral pharmacokinetics (PK). Therefore, achieving and maintaining optimal plasma concentrations of antiretroviral drugs is essential for maternal health and minimising the risk of mother-to-child transmission of HIV. The aim of this study is to describe atazanavir/ritonavir (ATV/r) PK during pregnancy. Methods: Pregnant HIV-positive women received ATV/r as part of their routine pre-natal care. Demographic and clinical data were collected, and ATV plasma concentrations [ATV] were determined in the first (T1), second (T2) and third (T3) trimester using HPLC-MS/MS (LLQ=0.05 µg/mL). Postpartum (PP) sampling was performed where applicable. Antepartum (AP) and PP PK parameters were compared using a one-way ANOVA. Summary of results: From January 2007, 44 women (37 black African) were enrolled in the study. All received ATV/r at a standard dose of 1 tablet once daily (300/100 mg od). 24 women were receiving ART prior to pregnancy, and 20 women initiated ATV/r during pregnancy. Median (range) gestation at treatment initiation in these patients was 23.5 weeks (7–35). At the time nearest to delivery 31 patients had an undetectable plasma viral load (pVL), 6 patients had detectable pVL and 2 were unavailable. [ATV] were determined in 11/44 (T1); 25/44 (T2); 35/44 (T3) and 28/44 (PP) patients. Time of TDM sampling, gestation time and [ATV] (geometric mean; 95% CI) are given in the Table. 6 patients were either below or approaching the ATV MEC (0.15 µg/mL) during pregnancy; of these, 4/6 achieved undetectable pVL at the time of delivery (1=pVL of 291 copies/mL; 1 unavailable). [ATV] were significantly lower at T2/T3 relative to T1/PP. Equally, in a paired analysis of 28 patients (T2/T3 vs. PP), [ATV] were significantly reduced at T2/T3 (P=0.003). Conclusions: This study represents one of the larger cohorts of women undergoing TDM for ATV in pregnancy. Lower [ATV] were seen in T2 and T3 when compared to T1. However, such findings were not associated with viral breakthrough or HIV transmissions. Nonetheless, careful monitoring of women in pregnancy is required, and if there is concern for inadequate levels, dose adjustment of ATV upward from 300 mg to 400 mg may be an option

Central role of JC virus-specific CD4+ lymphocytes in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome

Progressive multi-focal leucoencephalopathy and progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are caused by infection of the central nervous system with the JC polyoma virus. Both are complications of monoclonal antibody therapy in multiple sclerosis and other autoimmune diseases. Progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome can obscure the diagnosis of progressive multi-focal leucoencephalopathy and lead to severe clinical disability and possibly death. Different from progressive multi-focal leucoencephalopathy, in which demyelination results from oligodendrocyte lysis by JC virus in the absence of an immune response, tissue destruction in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome is caused by a vigorous immune response within the brain. The cells and mediators that are involved in progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome are as yet poorly understood. We examined two patients with multiple sclerosis, who developed progressive multi-focal leucoencephalopathy and later progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome under natalizumab therapy. Due to initially negative JC viral deoxyribonucleic acid testing in the cerebrospinal fluid, a diagnostic brain biopsy was performed in one patient. Histopathology revealed brain inflammation characterized by a prominent T cell infiltrate (CD4+ > CD8+ T cells), but also B/plasma cells and monocytes. Despite very low JC viral load, both patients showed high intrathecal anti-JC virus antibodies. Brain-infiltrating CD4+ T cells were studied regarding antigen specificity and function. CD4+ T cells were highly specific for peptides from several JC virus proteins, particularly the major capsid protein VP1. T cell phenotyping revealed CD4+ Th1 and bifunctional Th1-2 cells. The latter secrete large amounts of interferon-γ and interleukin-4 explaining the strong brain inflammation, presence of plasma cells and secretion of intrathecal anti-VP1 antibodies. The functional phenotype of brain-infiltrating JC virus-specific CD4+ T cells was confirmed and extended by examining brain-derived JC virus-specific CD4+ T cell clones. Our data provide novel insight into the pathogenesis of progressive multi-focal leucoencephalopathy-immune reconstitution inflammatory syndrome and indicate that JC virus-specific CD4+ T cells play an important role in both eliminating JC virus from the brain, but also in causing the massive inflammation with often fatal outcom

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3PAR2/+), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases

Functional and genetic characterization of the non-lysosomal glucosylceramidase 2 as a modifier for Gaucher disease

Background: Gaucher disease (GD) is the most common inherited lysosomal storage disorder in humans, caused by mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1). GD is clinically heterogeneous and although the type of GBA1 mutation plays a role in determining the type of GD, it does not explain the clinical variability seen among patients. Cumulative evidence from recent studies suggests that GBA2 could play a role in the pathogenesis of GD and potentially interacts with GBA1. Methods: We used a framework of functional and genetic approaches in order to further characterize a potential role of GBA2 in GD. Glucosylceramide (GlcCer) levels in spleen, liver and brain of GBA2-deficient mice and mRNA and protein expression of GBA2 in GBA1-deficient murine fibroblasts were analyzed. Furthermore we crossed GBA2-deficient mice with conditional Gba1 knockout mice in order to quantify the interaction between GBA1 and GBA2. Finally, a genetic approach was used to test whether genetic variation in GBA2 is associated with GD and/or acts as a modifier in Gaucher patients. We tested 22 SNPs in the GBA2 and GBA1 genes in 98 type 1 and 60 type 2/3 Gaucher patients for single-and multi-marker association with GD. Results: We found a significant accumulation of GlcCer compared to wild-type controls in all three organs studied. In addition, a significant increase of Gba2-protein and Gba2-mRNA levels in GBA1-deficient murine fibroblasts was observed. GlcCer levels in the spleen from Gba1/Gba2 knockout mice were much higher than the sum of the single knockouts, indicating a cross-talk between the two glucosylceramidases and suggesting a partially compensation of the loss of one enzyme by the other. In the genetic approach, no significant association with severity of GD was found for SNPs at the GBA2 locus. However, in the multi-marker analyses a significant result was detected for p.L444P (GBA1) and rs4878628 (GBA2), using a model that does not take marginal effects into account. Conclusions: All together our observations make GBA2 a likely candidate to be involved in GD etiology. Furthermore, they point to GBA2 as a plausible modifier for GBA1 in patients with GD

Sphingolipids as critical players in retinal physiology and pathology

Sphingolipids have emerged as bioactive lipids involved in the regulation of many physiological and pathological processes. In the retina, they have been established toparticipate in numerousprocesses, suchas neuronal survival and death, proliferation and migration of neuronal and vascular cells, inflammation, and neovascularization. Dysregulation of sphingolipids is therefore crucial in the onset and progression of retinal diseases. This review examines the involvement of sphingolipids in retinal physiology and diseases. Ceramide (Cer) has emerged as a common mediator of inflammation and death of neuronal and retinal pigment epithelium cells in animal models of retinopathies such as glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa. Sphingosine- 1-phosphate (S1P) has opposite roles, preventing photoreceptor and ganglion cell degeneration but also promoting inflammation, fibrosis, and neovascularization in AMD, glaucoma, and pro-fibrotic disorders. Alterations in Cer, S1P, and ceramide 1- phosphate may also contribute to uveitis. Notably, use of inhibitors that either prevent Cer increase or modulate S1P signaling, such as Myriocin, desipramine, and Fingolimod (FTY720), preserves neuronal viability and retinal function. These findings underscore the relevance of alterations in the sphingolipid metabolic network in the etiology of multiple retinopathies and highlight the potential of modulating their metabolism for the design of novel therapeutic approaches.Fil: Simon, Maria Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Basu, Sandip K.. University of Tennessee; Estados UnidosFil: Qaladize, Bano. University of Tennessee; Estados UnidosFil: Grambergs, Richards. University of Tennessee; Estados UnidosFil: Rotstein, Nora Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Mandal, Nawajes .A.. University of Tennessee; Estados Unido

Acid Sphingomyelinase, a Lysosomal and Secretory Phospholipase C, Is Key for Cellular Phospholipid Catabolism

Here, we present the main features of human acid sphingomyelinase (ASM), its biosynthesis, processing and intracellular trafficking, its structure, its broad substrate specificity, and the proposed mode of action at the surface of the phospholipid substrate carrying intraendolysosomal luminal vesicles. In addition, we discuss the complex regulation of its phospholipid cleaving activity by membrane lipids and lipid-binding proteins. The majority of the literature implies that ASM hydrolyses solely sphingomyelin to generate ceramide and ignores its ability to degrade further substrates. Indeed, more than twenty different phospholipids are cleaved by ASM in vitro, including some minor but functionally important phospholipids such as the growth factor ceramide-1-phosphate and the unique lysosomal lysolipid bis(monoacylglycero)phosphate. The inherited ASM deficiency, Niemann-Pick disease type A and B, impairs mainly, but not only, cellular sphingomyelin catabolism, causing a progressive sphingomyelin accumulation, which furthermore triggers a secondary accumulation of lipids (cholesterol, glucosylceramide, GM2) by inhibiting their turnover in late endosomes and lysosomes. However, ASM appears to be involved in a variety of major cellular functions with a regulatory significance for an increasing number of metabolic disorders. The biochemical characteristics of ASM, their potential effect on cellular lipid turnover, as well as a potential impact on physiological processes will be discussed