Soluble fms-like tyrosine kinase 1 (sFlt-1): A novel biochemical marker for acute fatty liver of pregnancy.

INTRODUCTION: Acute fatty liver of pregnancy (AFLP) substantially contributes to maternal and neonatal morbidity and mortality. Other liver-associated pregnancy complications such as preeclampsia-associated HELLP (hemolysis, elevated liver enzyme, low platelet) syndrome may be difficult to differentiate from AFLP as these diseases overlap with regard to multiple clinical and laboratory features. The aim of this study was to investigate angiogenic profiles by measuring soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PlGF) in pregnancies compromised by AFLP and to compare them with those complicated by HELLP syndrome. MATERIAL AND METHODS: Pregnant women affected by AFLP or HELLP syndrome were enrolled. The study population of women with HELLP syndrome was part of a larger data collection obtained in our clinic that has been used for previous work. Patients' angiogenic profiles were assessed by measuring sFlt-1 and PlGF serum levels. To assess the diagnostic potential of these angiogenic markers in AFLP, as well as discriminating it from HELLP syndrome, non-parametric tests were used and receiver operating curves were calculated. RESULTS: Six women with AFLP and 48 women with HELLP syndrome were included in the study. Patients with AFLP showed significantly higher sFlt-1 levels (median: 57 570 pg/mL; range 31 609-147 170 pg/mL) than patients with HELLP syndrome (9713 pg/mL; 1348-30 781 pg/mL; p < 0.001). PlGF serum levels were higher in patients with AFLP compared with those with HELLP syndrome (197 pg/mL; 127-487 pg/mL vs. 40 pg/mL; 9-644 pg/mL, respectively; p < 0.01). sFlt-1/PlGF ratios were not significantly different between AFLP and HELLP syndrome patients (192; 157-1159 vs. 232; 3-948, respectively; NS). In our study population, an sFlt-1 cut-off value of 31 100 pg/mL allowed differentiation between these two diseases with a sensitivity and specificity of 100%. A linear correlation was found between the cumulative numbers of Swansea criteria and sFlt-1 serum levels (r = 0.97; p < 0.01). CONCLUSIONS: AFLP is associated with very high sFlt-1 serum levels in particular in women fulfilling eight or more Swansea criteria. Besides the suggested Swansea criteria to diagnose AFLP, an sFlt-1 value above 31 100 pg/mL may be an additional biochemical feature improving discrimination between AFLP and HELLP syndrome. However, because of the small number of pregnancies affected by AFLP included in this work further studies are needed to corroborate our findings.

Identification of Celecoxib-Targeted Proteins Using Label-Free Thermal Proteome Profiling on Rat Hippocampus.

Celecoxib, or Celebrex, a nonsteroidal anti-inflammatory drug, is one of the most common medicines for treating inflammatory diseases. Recently, it has been shown that celecoxib is associated with implications in complex diseases, such as Alzheimer disease and cancer as well as with cardiovascular risk assessment and toxicity, suggesting that celecoxib may affect multiple unknown targets. In this project, we detected targets of celecoxib within the nervous system using a label-free thermal proteome profiling method. First, proteins of the rat hippocampus were treated with multiple drug concentrations and temperatures. Next, we separated the soluble proteins from the denatured and sedimented total protein load by ultracentrifugation. Subsequently, the soluble proteins were analyzed by nano-liquid chromatography tandem mass spectrometry to determine the identity of the celecoxib-targeted proteins based on structural changes by thermal stability variation of targeted proteins toward higher solubility in the higher temperatures. In the analysis of the soluble protein extract at 67°C, 44 proteins were uniquely detected in drug-treated samples out of all 478 identified proteins at this temperature. Ras-associated binding protein 4a, 1 out of these 44 proteins, has previously been reported as one of the celecoxib off targets in the rat central nervous system. Furthermore, we provide more molecular details through biomedical enrichment analysis to explore the potential role of all detected proteins in the biologic systems. We show that the determined proteins play a role in the signaling pathways related to neurodegenerative disease-and cancer pathways. Finally, we fill out molecular supporting evidence for using celecoxib toward the drug-repurposing approach by exploring drug targets. SIGNIFICANCE STATEMENT: This study determined 44 off-target proteins of celecoxib, a nonsteroidal anti-inflammatory and one of the most common medicines for treating inflammatory diseases. It shows that these proteins play a role in the signaling pathways related to neurodegenerative disease and cancer pathways. Finally, the study provides molecular supporting evidence for using celecoxib toward the drug-repurposing approach by exploring drug targets.

Gestational diabetes mellitus affects placental iron homeostasis: Mechanism and clinical implications.

Clinical studies suggest that pregnant women with elevated iron levels are more vulnerable to develop gestational diabetes mellitus (GDM), but the causes and underlying mechanisms are unknown. We hypothesized that hyperglycemia induces cellular stress responses leading to dysregulated placental iron homeostasis. Hence, we compared the expression of genes/proteins involved in iron homeostasis in placentae from GDM and healthy pregnancies (n = 11 each). RT-qPCR and LC-MS/MS analyses revealed differential regulation of iron transporters/receptors (DMT1/FPN1/ZIP8/TfR1), iron sensors (IRP1), iron regulators (HEPC), and iron oxidoreductases (HEPH/Zp). To identify the underlying mechanisms, we adapted BeWo trophoblast cells to normoglycemic (N), hyperglycemic (H), and hyperglycemic-hyperlipidemic (HL) conditions and assessed Fe3+ -uptake, expression patterns, and cellular pathways involving oxidative stress (OS), ER-stress, and autophagy. H and HL induced alterations in cellular morphology, differential iron transporter expression, and reduced Fe3+ -uptake confirming the impact of hyperglycemia on iron transport observed in GDM patients. Pathway analysis and rescue experiments indicated that dysregulated OS and disturbed autophagy processes contribute to the reduced placental iron transport under hyperglycemic conditions. These adaptations could represent a protective mechanism preventing the oxidative damage for both fetus and placenta caused by highly oxidative iron. In pregnancies with risk for GDM, antioxidant treatment, and controlled iron supplementation could help to balance placental OS levels protecting mother and fetus from impaired iron homeostasis.

Reactive oxygen species-regulating proteins peroxiredoxin 2 and thioredoxin, and glyceraldehyde-3-phosphate dehydrogenase are differentially abundant in induced sputum from smokers with lung cancer or asbestos exposure.

Lung cancer is a deadly disease, typically caused by known risk factors, such as tobacco smoke and asbestos exposure. By triggering cellular oxidative stress and altering the antioxidant pathways eliminating reactive oxygen species (ROS), tobacco smoke and asbestos predispose to cancer. Despite easily recognizable high-risk individuals, lung cancer screening and its early detection are hampered by poor diagnostic tools including the absence of proper biomarkers. This study aimed to recognize potential lung cancer biomarkers using induced sputum noninvasively collected from the lungs of individuals in risk of contracting lung cancer. Study groups composed of current and former smokers, who either were significantly asbestos exposed, had lung cancer, or were unexposed and asymptomatic. Screening of potential biomarkers was performed with 52, and five differentially abundant proteins, peroxiredoxin 2 (PRDX2), thioredoxin (TXN), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), extracellular matrix protein 1 (ECM1), and protein S100 A8 (S100A8), were chosen to undergo validation, for their previously known connection with oxidative stress or cancer. Results from the validation in 123 sputa showed that PRDX2, TXN, and GAPDH were differentially abundant in sputa from individuals with lung cancer. TXN had a negative correlation with asbestos exposure, yet a positive correlation with smoking and lung cancer. Thus, tobacco smoking, asbestos exposure, and lung carcinogenesis may disturb the cellular redox state in different ways. A strong correlation was found among PRDX2, TXN, GAPDH, and S100A8, suggesting that these proteins may present a diagnostic biomarker panel to aid recognizing individuals at high risk of contracting lung cancer.

Severe elastolysis in hereditary gelsolin (AGel) amyloidosis.

AGel amyloidosis is a dominantly inherited systemic amyloidosis caused by mutations p.D214N or p.D214Y resulting in gelsolin amyloid (AGel) formation. AGel accumulates extracellularly in many tissues and alongside elastic fibres. AGel deposition associates with elastic fibre degradation leading to severe clinical manifestations, such as cutis laxa and angiopathic complications. We analysed elastic fibre pathology in dermal and vascular tissue and plasma samples from 35 patients with AGel amyloidosis and 40 control subjects by transmission electron microscopy, immunohistochemistry and ELISA methods. To clarify the pathomechanism(s) of AGel-related elastolysis, we studied the roles of MMP-2, -7, -9, -12 and -14, TIMP-1 and TGFß. We found massive accumulation of amyloid fibrils along elastic fibres as well as fragmentation and loss of elastic fibres in all dermal and vascular samples of AGel patients. Fibrils of distinct types formed fibrous matrix. The degradation pattern of elastic fibres in AGel patients was different from the age-related degradation in controls. The elastin of elastic fibres in AGel patients was strongly decreased compared to controls. MMP-9 was expressed at lower and TGFß at higher levels in AGel patients than in controls. The accumulation of amyloid fibrils with severe elastolysis characterises both dermal and vascular derangement in AGel amyloidosis.

Angiogenic profiling in HELLP syndrome cases with or without hypertension and proteinuria.

OBJECTIVES: Angiogenic profiling with the use of sFlt-1/PlGF ratio (soluble fms-like tyrosine kinase-1/placental growth factor) can be helpful to characterize women with signs of impending preeclampsia (PE). However, little is known about the angiogenic profile of pregnancies complicated by HELLP syndrome. The aim of this study was to examine the relationship of angiogenic profiles in cases of HELLP syndrome with and without classical signs of preeclampsia. STUDY DESIGN: The angiogenic profile of pregnant women with singleton gestation and isolated PE (group 1), PE associated with HELLP syndrome (group 2), and isolated HELLP syndrome (group 3) from 01/2011 to 03/2018, were compared. To overcome gestational age dependent angiogenic behavior, cases (group 3) were matched 1:2 with cases from group 1 and 2. Matching criteria was gestational age (±1 week). PE and HELLP syndrome were defined according to the international Society for the Study of Hypertension in Pregnancy (ISSHP) statement 2014. RESULTS: During the observational period, 244 women could be included in the study. Of those, 237 (97.1%) were diagnosed with PE. In 42 cases (17.2%) PE was associated with HELLP syndrome while 7 (2.9%) patients were diagnosed with isolated HELLP syndrome. Angiogenic profiles in terms of sFlt-1/PlGF ratios differed significantly between the three groups, showing highest levels in group 2 (PE/HELLP) while cases with isolated HELLP demonstrated the lowest ratios and sFlt-1 values (p = 0.01). CONCLUSION: We conclude that isolated HELLP syndrome is rare and seems to be a particular entity expressing a different angiogenic behaviour compared to classical PE or PE associated with HELLP syndrome.

Ultra-high sensitive C-reactive protein during normal pregnancy and in preeclampsia: a pilot study.

INTRODUCTION: Angiogenic and inflammatory factors have been shown to play an important role in the pathogenesis of preeclampsia. However, there is little information on their interaction. The aims of this study were to investigate the longitudinal pattern of inflammatory markers, such as interleukin-6 (IL-6) and C-reactive protein (CRP) using a novel ultra-high sensitive assay method (uhsCRP), and to explore their relationship with angiogenic factors such as placental growth factor (PLGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and vascular endothelial growth factor (VEGF) in normal pregnancies and pregnancies complicated by preeclampsia. MATERIALS AND METHODS: Serum levels of uhsCRP, IL-6, PLGF, VEGF and s-Flt-1 were longitudinally determined in 16 women with normal, singleton healthy pregnancies at 7-13, 17-22, 27-31 and 37-41 weeks of gestation by ELISA. uhsCRP was measured using a ultra-high sensitivity ELISA test. Serum of women with preeclampsia (n = 15) was available only once, usually in the third trimester of pregnancy. Women with premature rupture of membranes (PROM) or infection such as chorioamnionitis were excluded. Spearman rank correlation, logistic regression, ROC analysis, ANOVA and Mann-Whitney U-test were used for statistical purposes. RESULTS: In normal pregnancies, serum uhsCRP showed a gestational age-dependent increase (r = 0.40; P < 0.001). In women suffering from preeclampsia, uhsCRP levels were higher than in gestational age-matched controls (18010 ±â€Š4763 versus 3026 ±â€Š587 ng/ml; P < 0.001). Similarly, serum IL-6 levels increased throughout pregnancy and correlated with uhsCRP in normal pregnancies and in preeclampsia (n = 64, r = 0.37; P < 0.01 and n = 15, r = 1.00, P < 0.0001). uhsCRP levels were positively correlated with sFlt-1 levels (n = 64, r = 0.34; P < 0.01). CONCLUSION: The increases in uhsCRP (and IL-6) serum levels with advancing gestation indicate a shift towards an inflammatory state during normal pregnancy. The excessive rise in uhsCRP and sFlt-1 in preeclampsia indicate that both may be involved in its pathogenesis. uhsCRP may be useful as an early marker for preeclampsia and studies defining the pattern of its rise throughout pregnancies at risk are urgently needed.

Shrunken pore syndrome, preeclampsia, and markers of NO metabolism in pregnant women during the first trimester.

Shrunken pore syndrome (SPS) is a condition that manifests itself as the decreased renal clearance of low-molecular-weight proteins but normal clearance of creatinine. Pregnant women with evidence of SPS during the first trimester have an increased risk of developing preeclampsia (PE). The nitric oxide (NO) metabolism markers arginine and ADMA, especially their ratio (Arg/ADMA), are recognized markers of endothelial dysfunction. The aim of this nested case-control study was to establish first-trimester reference intervals (RI) for markers of NO metabolism and to study these markers in women with evidence of SPS at the end of the first trimester. Seventy-four women were stratified in the first trimester according to evidence of SPS (SPS + or SPS-) and the occurrence of PE during subsequent pregnancy (PE + or PE-), as follows: SPS-/PE-, SPS+/PE-, SPS-/PE+, and SPS+/PE+. RIs were determined according to the CLSI EP28-A3c guidelines. Serum Arg and ADMA levels were analyzed. The Arg and ADMA concentrations did not differ among the four groups. However, women in the SPS+/PE + group had a significantly lower Arg/ADMA ratio than those in the other 3 groups (p = .02). In conclusion, we defined the first-trimester RI of Arg, ADMA and the Arg/ADMA ratio as markers of NO metabolism. Our results suggest that SPS in the first trimester predicts a pathophysiological hallmark of subsequent PE, i.e. lower NO production leading to increased vessel tone. Early identification of women at risk for later PE could lead to adaptive prophylactic interventions, such as supplementation with Arg or an NO-donor drug in order to mitigate the risk of developing PE.

SETBP1 induces transcription of a network of development genes by acting as an epigenetic hub.

SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel-Giedion syndrome caused by SETBP1 mutations.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

High first-trimester maternal blood cystatin C levels despite normal serum creatinine predict pre-eclampsia in singleton pregnancies.

Early biochemical identification of women at high risk for the development of pre-eclampsia (PE) is still unsatisfactory. Renal markers measured during the first trimester were analysed to predict later occurrence of PE. A nested case-control study was conducted within the prospective predictive markers for the diagnosis of preeclampsia study. Pregnant women were included at the end of the first trimester and followed up until birth. Controls were matched to PE cases. Renal markers [i.e. creatinine, cystatin C (CysC), ß2 microglobulin (B2M), ß-trace protein (BTP), glomerular filtration rate estimations (eGFR) of the aforementioned markers, uric acid (UA), urea, and serum uromodulin (sUMOD)] were compared to placental growth factor (PlGF), a marker known to predict PE later in pregnancy. Reference intervals were determined for the different markers. In the 183 women (PE, n = 39; controls, n = 144), CysC, the CysC/PlGF ratio (p < .01) and UA were higher, whereas the eGFRCysC/eGFRCrea ratio (a marker of glomerular endothelial integrity and shrunken pore syndrome) and PlGF were lower in women who developed PE (p < .05 for all). Compromised filtration of the larger molecule CysC together with a normal creatinine, in a subset of PE cases (15.3%) was a unique, strong and independent predictor of later PE if the baseline CysC concentration was >0.85 mg/l. In conclusion, CysC and its derivatives as well as UA, indicating volume expansion, measured at the end of the first trimester are predictive of PE. Thus, women can be easily identified and followed as an early reduction in glomerular filtration quality poses a high risk for a subsequent development of PE.

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTß, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.

Gelsolin amyloid angiopathy causes severe disruption of the arterial wall.

Hereditary gelsolin amyloidosis (HGA) is a dominantly inherited systemic disease reported worldwide. HGA is characterized by ophthalmological, neurological, and dermatological manifestations. AGel amyloid accumulates at basal lamina of epithelial and muscle cells, thus amyloid angiopathy is encountered in nearly every organ. HGA patients have cardiovascular, hemorrhagic, and potentially vascularly induced neurological problems. To clarify pathomechanisms of AGel angiopathy, we performed histological, immunohistochemical, and electron microscopic analyses on facial temporal artery branches from 8 HGA patients and 13 control subjects. We demonstrate major pathological changes in arteries: disruption of the tunica media, disorganization of vascular smooth muscle cells, and accumulation of AGel fibrils in arterial walls, where they associate with the lamina elastica interna, which becomes fragmented and diminished. We also provide evidence of abnormal accumulation and localization of collagen types I and III and an increase of collagen type I degradation product in the tunica media. Vascular smooth muscle cells appear to be morphologically and semi-quantitatively normal, only their basal lamina is often thickened. In conclusion, angiopathy in HGA results in severe disruption of arterial walls, characterized by prominent AGel deposition, collagen derangement and severe elastolysis, and it may be responsible for several, particularly hemorrhagic, disease manifestations in HGA.

Placental expression of the angiogenic placental growth factor is stimulated by both aldosterone and simulated starvation.

Aldosterone is an important factor supporting placental growth and fetal development. Recently, expression of placental growth factor (PlGF) has been observed in response to aldosterone exposure in different models of atherosclerosis. Thus, we hypothesized that aldosterone up-regulates growth-adaptive angiogenesis in pregnancy, via increased placental PlGF expression. We followed normotensive pregnant women (n = 24) throughout pregnancy and confirmed these results in a second independent first trimester cohort (n = 36). Urinary tetrahydroaldosterone was measured by gas chromatography-mass spectrometry and corrected for creatinine. Circulating PlGF concentrations were determined by ELISA. Additionally, cultured cell lines, adrenocortical H295R and choriocarcinoma BeWo cells, as well as primary human third trimester trophoblasts were tested in vitro. PlGF serum concentrations positively correlated with urinary tetrahydroaldosterone corrected for creatinine in these two independent cohorts. This observation was not due to PlGF, which did not induce aldosterone production in cultured H295R cells. On the other hand, PlGF expression was specifically enhanced by aldosterone in the presence of forskolin (p < 0.01) in trophoblasts. A pronounced stimulation of PlGF expression was observed with reduced glucose concentrations simulating starvation (p < 0.001). In conclusion, aldosterone stimulates placental PlGF production, enhancing its availability during human pregnancy, a response amplified by reduced glucose supply. Given the crucial role of PlGF in maintaining a healthy pregnancy, these data support a key role of aldosterone for a healthy pregnancy outcome.

Peroxisome proliferator-activated receptor-γ coactivator-1α mediates neuroprotection against excitotoxic brain injury in transgenic mice: role of mitochondria and X-linked inhibitor of apoptosis protein.

Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator involved in the regulation of mitochondrial biogenesis and cell defense. The functions of PGC-1α in physiology of brain mitochondria are, however, not fully understood. To address this we have studied wild-type and transgenic mice with a two-fold overexpression of PGC-1α in brain neurons. Data showed that the relative number and basal respiration of brain mitochondria were increased in PGC-1α transgenic mice compared with wild-type mitochondria. These changes occurred concomitantly with altered levels of proteins involved in oxidative phosphorylation (OXPHOS) as studied by proteomic analyses and immunoblottings. Cultured hippocampal neurons from PGC-1α transgenic mice were more resistant to cell degeneration induced by the glutamate receptor agonist kainic acid. In vivo kainic acid induced excitotoxic cell death in the hippocampus at 48 h in wild-type mice but significantly less so in PGC-1α transgenic mice. However, at later time points cell degeneration was also evident in the transgenic mouse hippocampus, indicating that PGC-1α overexpression can induce a delay in cell death. Immunoblotting showed that X-linked inhibitor of apoptosis protein (XIAP) was increased in PGC-1α transgenic hippocampus with no significant changes in Bcl-2 or Bcl-X. Collectively, these results show that PGC-1α overexpression contributes to enhanced neuronal viability by stimulating mitochondria number and respiration and increasing levels of OXPHOS proteins and the anti-apoptotic protein XIAP.

Quantitative analysis of PPT1 interactome in human neuroblastoma cells.

Mutations in the CLN1 gene that encodes Palmitoyl protein thioesterase 1 (PPT1) or CLN1, cause Infantile NCL (INCL, MIM#256730). PPT1 removes long fatty acid chains such as palmitate from modified cysteine residues of proteins. The data shown here result from isolated protein complexes from PPT1-expressing SH-SY5Y stable cells that were subjected to single step affinity purification coupled to mass spectrometry (AP-MS). Prior to the MS analysis, we utilised a modified filter-aided sample preparation (FASP) protocol. Based on label free quantitative analysis of the data by SAINT, 23 PPT1 interacting partners (IP) were identified. A dense connectivity in PPT1 network was further revealed by functional coupling and extended network analyses, linking it to mitochondrial ATP synthesis coupled protein transport and thioester biosynthetic process. Moreover, the terms: inhibition of organismal death, movement disorders and concentration of lipid were predicted to be altered in the PPT1 network. Data presented here are related to Scifo et al. (J. Proteomics, 123 (2015) 42-53).

Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway. BIOLOGICAL SIGNIFICANCE: The significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Mass-tag enhanced immuno-laser desorption/ionization mass spectrometry for sensitive detection of intact protein antigens.

A new read-out method for antibody arrays using laser desorption/ionization-mass spectrometry (LDI-MS) is presented. Small, photocleavable reporter molecules with a defined mass called "mass-tags" are used for detection of immunocaptured proteins from human plasma. Using prostate specific antigen (PSA), a biomarker for prostate cancer, as a model antigen, a high sensitivity generic detection methodology based immunocapture with a primary antibody and with a biotin labeled secondary antibody coupled to mass-tagged avidin is demonstrated. As each secondary antibody can bind several avidin molecules, each having a large number of mass-tags, signal amplification can be achieved. The developed PSA sandwich mass-tag analysis method provided a limit of detection below 200 pg/mL (6 pM) for a 10 µL plasma sample, well below the clinically relevant cutoff value of 3-4 ng/mL. This brings the limit of detection (LOD) for detection of intact antigens with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) down to levels comparable to capture by anti-peptide antibodies selected reaction monitoring (SISCAPA SRM) and enzyme linked immunosorbent assay (ELISA), as 6 pM corresponds to a maximal amount of 60 amol PSA captured on-spot. We propose the potential use of LDI (laser desorption/ionization) with mass-tag read-out implemented in a sandwich assay format for low abundant and/or early disease biomarker detection.

Identification of cytoplasmic proteins interacting with unliganded estrogen receptor α and ß in human breast cancer cells.

Estrogen receptor subtypes (ERα and ERß) are transcription factors sharing a similar structure but exerting opposite roles in breast cancer cells. Besides the well-characterized genomic actions of nuclear ERs upon ligand binding, specific actions of ligand-free ERs in the cytoplasm also affect cellular functions. The identification of cytoplasmic interaction partners of unliganded ERα and ERß may help characterize the molecular basis of the extra-nuclear mechanism of action of these receptors, revealing novel mechanisms to explain their role in breast cancer response or resistance to endocrine therapy. To this aim, cytoplasmic extracts from human breast cancer MCF-7 cells stably expressing tandem affinity purification-tagged ERα and ERß and maintained in estrogen-free medium were subject to affinity-purification and MS analysis, leading to the identification of 84 and 142 proteins associated with unliganded ERα and ERß, respectively. Functional analyses of ER subtype-specific interactomes revealed significant differences in the molecular pathways targeted by each receptor in the cytoplasm. This work, reporting the first identification of the unliganded ERα and ERß cytoplasmic interactomes in breast cancer cells, provides novel experimental evidence on the nongenomic effects of ERs in the absence of hormonal stimulus. All MS data have been deposited in the ProteomeXchange with identifier PXD001202 (http://proteomecentral.proteomexchange.org/dataset/PXD001202).

CADASIL and CARASIL.

CADASIL and CARASIL are hereditary small vessel diseases leading to vascular dementia. CADASIL commonly begins with migraine followed by minor strokes in mid-adulthood. Dominantly inherited CADASIL is caused by mutations (n > 230) in NOTCH3 gene, which encodes Notch3 receptor expressed in vascular smooth muscle cells (VSMC). Notch3 extracellular domain (N3ECD) accumulates in arterial walls followed by VSMC degeneration and subsequent fibrosis and stenosis of arterioles, predominantly in cerebral white matter, where characteristic ischemic MRI changes and lacunar infarcts emerge. The likely pathogenesis of CADASIL is toxic gain of function related to mutation-induced unpaired cysteine in N3ECD. Definite diagnosis is made by molecular genetics but is also possible by electron microscopic demonstration of pathognomonic granular osmiophilic material at VSMCs or by positive immunohistochemistry for N3ECD in dermal arteries. In rare, recessively inherited CARASIL the clinical picture and white matter changes are similar as in CADASIL, but cognitive decline begins earlier. In addition, gait disturbance, low back pain and alopecia are characteristic features. CARASIL is caused by mutations (presently n = 10) in high-temperature requirement. A serine peptidase 1 (HTRA1) gene, which result in reduced function of HTRA1 as repressor of transforming growth factor-ß (TGF ß) -signaling. Cerebral arteries show loss of VSMCs and marked hyalinosis, but not stenosis.