Active Molecular Network Discovery Links Lifestyle Variables to Breast Cancer in the Long Island Breast Cancer Study Project.

A healthy lifestyle has been associated with decreased risk of developing breast cancer. Using untargeted metabolomics profiling, which provides unbiased information regarding lifestyle choices such as diet and exercise, we aim to identify the molecular mechanisms connecting lifestyle and breast cancer through network analysis. A total of 100 postmenopausal women, 50 with breast cancer and 50 cancer-free controls, were selected from the Long Island Breast Cancer Study Project (LIBCSP). We measured untargeted plasma metabolomics using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Using the "enet" package, we retained highly correlated metabolites representing active molecular network (AMN) clusters for analysis. LASSO was used to examine associations between cancer status and AMN metabolites and covariates such as BMI, age, and reproductive factors. LASSO was then repeated to examine associations between AMN metabolites and 10 lifestyle-related variables including smoking, physical activity, alcohol consumption, meat consumption, fruit and vegetable consumption, and supplemental vitamin use. Results were displayed as a network to uncover biological pathways linking lifestyle factors to breast cancer status. After filtering, 851 "active" metabolites out of 1797 metabolomics were retained in 197 correlation AMN clusters. Using LASSO, breast cancer status was associated with 71 "active" metabolites. Several of these metabolites were associated with lifestyle variables including meat consumption, alcohol consumption, and supplemental ß-carotene, B12, and folate use. Those metabolites could potentially serve as molecular-level biological intermediaries connecting healthy lifestyle factors to breast cancer, even though direct associations between breast cancer and the investigated lifestyles at the phenotype level are not evident. In particular, DiHODE, a metabolite linked with inflammation, was associated with breast cancer status and connected to ß-carotene supplement usage through an AMN. We found several plasma metabolites associated with lifestyle factors and breast cancer status. Future studies investigating the mechanistic role of inflammation in linking supplement usage to breast cancer status are warranted.

Comparison of maternal venous blood metabolomics collected as dried blood spots, dried blood microsamplers, and plasma for integrative environmental health research.

Use of capillary blood devices for exposome research can deepen our understanding of the intricate relationship between environment and health, and open up new avenues for preventive and personalized medicine, particularly for vulnerable populations. While the potential of these whole blood devices to accurately measure chemicals and metabolites has been demonstrated, how untargeted metabolomics data from these samplers can be integrated with previous and ongoing environmental health studies that have used conventional blood collection approaches is not yet clear. Therefore, we performed a comprehensive comparison between relative-quantitative metabolite profiles measured in venous blood collected with dried whole blood microsamplers (DBM), dried whole blood spots (DBS), and plasma from 54 mothers in an ethnically diverse population. We determined that a majority of the 309 chemicals and metabolites showed similar median intensity rank, moderate correlation, and moderate agreement between participant-quantiled intraclass correlation coefficients (ICCs) for pair-wise comparisons among the three biomatrices. In particular, whole blood sample types, DBM and DBS, were in highest agreement across metabolite comparison metrics, followed by metabolites measured in DBM and plasma, and then metabolites measured in DBS and plasma. We provide descriptive characteristics and measurement summaries as a reference database. This includes unique metabolites that were particularly concordant or discordant in pairwise comparisons. Our results demonstrate that the range of metabolites from untargeted metabolomics data collected with DBM, DBS, and plasma provides biologically relevant information for use in independent exposome investigations. However, before meta-analysis with combined datasets are performed, robust statistical approaches that integrate untargeted metabolomics data collected on different blood matrices need to be developed.

Effects of storage temperature and time on metabolite profiles measured in dried blood spots, dried blood microsamplers, and plasma.

The practical advantages of capillary whole blood collection over venipuncture plasma collection for human exposome research are well known. However, before epidemiologists, clinicians, and public health researchers employ these microvolume sample collections, a rigorous evaluation of pre-analytical storage conditions is needed to develop protocols that maximize sample stability and reliability over time. Therefore, we performed a controlled experiment of dried whole blood collected on 10 µL Mitra microsamplers (DBM), 5-mm punches of whole blood from a dried blood spot (DBS), and 10 µL of plasma, and evaluated the effects of storage conditions at 4 °C, -20 °C, or -80 °C for up to 6 months on the resulting metabolite profiles measured with untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS). At -80 °C storage conditions, metabolite profiles from DBS, DBM, and plasma showed similar stability. While DBS and DBM metabolite profiles remained similarly stable at -20 °C storage, plasma profiles showed decreased stability at -20 °C compared to -80 °C storage. At refrigerated temperatures (4 °C), metabolite profiles collected on DBM were more stable than plasma or DBS, particularly for lipid classes. These results inform robust capillary blood sample storage protocols for DBM and DBS at potentially warmer temperatures than -80 °C, which may facilitate blood collections for populations outside of a clinical setting.

Per- and polyfluoroalkyl substances (PFAS) exposure and thyroid cancer risk.

BACKGROUND: Although per- and polyfluoroalkyl substances (PFAS) exposure is a potential contributor to the increasing thyroid cancer trend, limited studies have investigated the association between PFAS exposure and thyroid cancer in human populations. We therefore investigated associations between plasma PFAS levels and thyroid cancer diagnosis using a nested case-control study of patients with thyroid cancer with plasma samples collected at/before cancer diagnosis. METHODS: 88 patients with thyroid cancer using diagnosis codes and 88 healthy (non-cancer) controls pair-matched on sex, age (±5 years), race/ethnicity, body mass index, smoking status, and year of sample collection were identified in the BioMe population (a medical record-linked biobank at the Icahn School of Medicine at Mount Sinai in New York); 74 patients had papillary thyroid cancer. Eight plasma PFAS were measured using untargeted analysis with liquid chromatography-high resolution mass spectrometry and suspect screening. Associations between individual PFAS levels and thyroid cancer were evaluated using unconditional logistic regression models to estimate adjusted odds ratios (ORadj) and 95% confidence intervals (CI). FINDINGS: There was a 56% increased rate of thyroid cancer diagnosis per doubling of linear perfluorooctanesulfonic acid (n-PFOS) intensity (ORadj, 1.56, 95% CI: 1.17-2.15, P = 0.004); results were similar when including patients with papillary thyroid cancer only (ORadj, 1.56, 95% CI: 1.13-2.21, P = 0.009). This positive association remained in subset analysis investigating exposure timing including 31 thyroid cancer cases diagnosed ≥1 year after plasma sample collection (ORadj, 2.67, 95% CI: 1.59-4.88, P < 0.001). INTERPRETATION: This study reports associations between exposure to PFAS and increased rate of (papillary) thyroid cancer. Thyroid cancer risk from PFAS exposure is a global concern given the prevalence of PFAS exposure. Individual PFAS studied here are a small proportion of the total number of PFAS supporting additional large-scale prospective studies investigating thyroid cancer risk associated with exposure to PFAS chemicals. FUNDING: National Institutes of Health grants and The Andrea and Charles Bronfman Philanthropies.

Activation of mitochondrial TRAP1 stimulates mitochondria-lysosome crosstalk and correction of lysosomal dysfunction.

Numerous studies have established the involvement of lysosomal and mitochondrial dysfunction in the pathogenesis of neurodegenerative disorders such as Alzheimer's and Parkinson diseases. Building on our previous studies of the neurodegenerative lysosomal lipidosis Niemann-Pick C1 (NPC1), we have unexpectedly discovered that activation of the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) leads to the correction of the lysosomal storage phenotype in patient cells from multiple lysosomal storage disorders including NPC1. Using small compound activators specific for TRAP1, we find that activation of this chaperone leads to a generalized restoration of lysosomal and mitochondrial health. Mechanistically, we show that this process includes inhibition of oxidative phosphorylation and reduction of oxidative stress, which results in activation of AMPK and ultimately stimulates lysosome recycling. Thus, TRAP1 participates in lysosomal-mitochondrial crosstalk to maintain cellular homeostasis and could represent a potential therapeutic target for multiple disorders.

The transcriptional coactivator Eya1 exerts transcriptional repressive activity by interacting with REST corepressors and REST-binding sequences to maintain nephron progenitor identity.

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs.

Molecular Gatekeeper Discovery: Workflow for Linking Multiple Exposure Biomarkers to Metabolomics.

The exposome reflects multiple exposures across the life-course that can affect health. Metabolomics can reveal the underlying molecular basis linking exposures to health conditions. Here, we explore the concept and general data analysis framework of "molecular gatekeepers"─key metabolites that link single or multiple exposure biomarkers with correlated clusters of endogenous metabolites─to inform health-relevant biological targets. We performed untargeted metabolomics on plasma from 152 adolescent girls participating in the Growing Up Healthy Study in New York City. We then performed network analysis to link metabolites to exposure biomarkers including five trace elements (Cd, Mn, Pb, Se, and Hg) and five perfluorinated chemicals (PFCs; n-PFOS, Sm-PFOS, n-PFOA, PFHxS, and PFNA). We found 144 molecular gatekeepers and annotated 22 of them. Lysophosphatidylcholine (16:0) and taurodeoxycholate were correlated with both n-PFOA and n-PFOS, suggesting a shared dysregulation from multiple xenobiotic exposures. Sphingomyelin (d18:2/14:0) was significantly associated with age at menarche; yet, no direct association was detected between any exposure biomarkers and age at menarche. Thus, molecular gatekeepers can also discover molecular linkages between exposure biomarkers and health outcomes that may otherwise be obscured by complex interactions in direct measurements.

Reproducible untargeted metabolomics workflow for exhaustive MS2 data acquisition of MS1 features.

Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected following a secondary analysis impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for reproducible untargeted mass spectrometry MS2 fragment ion collection of unknown compounds found in MS1 full scan. Our workflow first removes redundant peaks from MS1 data and then exports a list of precursor ions for pseudo-targeted MS/MS analysis on independent peaks. This workflow provides comprehensive coverage of MS2 collection on unknown compounds found in full scan analysis using a "one peak for one compound" workflow without a priori redundant peak information. We compared pseudo-spectra formation and the number of MS2 spectra linked to MS1 data using the PMDDA workflow to that obtained using CAMERA and RAMclustR algorithms. More annotated compounds, molecular networks, and unique MS/MS spectra were found using PMDDA compared with CAMERA and RAMClustR. In addition, PMDDA can generate a preferred ion list for iterative DDA to enhance coverage of compounds when instruments support such functions. Finally, compounds with signals in both positive and negative modes can be identified by the PMDDA workflow, to further reduce redundancies. The whole workflow is fully reproducible as a docker image xcmsrocker with both the original data and the data processing template.

Lead exposure and serum metabolite profiles in pregnant women in Mexico City.

BACKGROUND: Lead (Pb) exposure is a global health hazard causing a wide range of adverse health outcomes. Yet, the mechanisms of Pb toxicology remain incompletely understood, especially during pregnancy. To uncover biological pathways impacted by Pb exposure, this study investigated serum metabolomic profiles during the third trimester of pregnancy that are associated with blood Pb and bone Pb. METHODS: We used data and specimens from 99 women enrolled in the Programming Research in Obesity, Growth, Environment, and Social Stressors birth cohort in Mexico City. Maternal Pb exposure was measured in whole blood samples from the third trimester of pregnancy and in the tibia and patella bones at 1 month postpartum. Third-trimester serum samples underwent metabolomic analysis; metabolites were identified based on matching to an in-house analytical standard library. A metabolome-wide association study was performed using multiple linear regression models. Class- and pathway-based enrichment analyses were also conducted. RESULTS: The median (interquartile range) blood Pb concentration was 2.9 (2.6) µg/dL. Median bone Pb, measured in the tibia and patella, were 2.5 (7.3) µg/g and 3.6 (9.5) µg/g, respectively. Of 215 total metabolites identified in serum, 31 were associated with blood Pb (p < 0.05). Class enrichment analysis identified significant overrepresentation of metabolites classified as fatty acids and conjugates, amino acids and peptides, and purines. Tibia and patella Pb were associated with 14 and 8 metabolites, respectively (p < 0.05). Comparing results from bone and blood Pb, glycochenodeoxycholic acid, glycocholic acid, and 1-arachidonoylglycerol were positively associated with blood Pb and tibia Pb, and 7-methylguanine was negatively associated with blood Pb and patella Pb. One metabolite, 5-aminopentanoic acid, was negatively associated with all three Pb measures. CONCLUSIONS: This study identified serum metabolites in pregnant women associated with Pb measured in blood and bone. These findings provide insights on the metabolic profile around Pb exposure in pregnancy and information to guide mechanistic studies of toxicological effects for mothers and children.

Tooth biomarkers to characterize the temporal dynamics of the fetal and early-life exposome.

BACKGROUND: Teeth have unique histology that make this biomatrix a time-capsule for retrospective exposure analysis of fetal and early life. However, most analytic methods require pulverizing the whole tooth, which eliminates exposure timing information. Further, the range of chemicals and endogenous exposures that can be measured in teeth has yet to be fully characterized. METHODS: We performed untargeted metabolomics on micro-dissected layers from naturally shed deciduous teeth. Using four liquid-chromatography high-resolution mass spectrometry analytical modes, we profiled small molecules (<1000 Da) from prenatal and postnatal tooth fractions. In addition, we employed linear regression on the tooth fraction pairs from 31 children to identify metabolites that discriminate between prenatal and postnatal exposures. RESULTS: Of over 10,000 features measured in teeth dentin, 390 unique compounds were annotated from 62 chemical classes. The class with the largest number of compounds was carboxylic acids and their derivatives (36%). Of the annotated exogenous metabolites (phthalates, parabens, perfluoroalkyl compounds, and cotinine) and endogenous metabolites (fatty acids, steroids, carnitines, amino acids, and others), 91 are linked to 256 health conditions through published literature. Differential analysis revealed 267 metabolites significantly different between the prenatal and the postnatal tooth fractions (adj. p-value < 0.05, Bonferroni correction), and 21 metabolites exclusive to the prenatal fraction. CONCLUSIONS: The prenatal and early postnatal exposome revealed from dental biomarkers represents a broad range of endogenous and exogenous metabolites for a comprehensive characterization in environmental health research. Most importantly, this technology provides a direct window into fetal exposures that is not possible by maternal biomarkers. Indeed, we identified several metabolites exclusively in the prenatal fraction, suggesting unique fetal exposures that are markedly different to postnatal exposures. Expansion of databases that include tooth matrix metabolites will strengthen biological interpretation and shed light on exposures during gestation and early life that may be causally linked with later health conditions.

Untargeted metabolomics of newborn dried blood spots reveals sex-specific associations with pediatric acute myeloid leukemia.

The etiology of pediatric acute myeloid leukemia (AML) is largely unknown, but evidence for mutations in utero and long latency periods suggests that environmental factors play a role. Therefore, we used untargeted metabolomics of archived newborn dried blood spots (DBS) to investigate neonatal exposures as potential causal risk factors for AML. Untargeted metabolomics profiling was performed on DBS punches from 48 pediatric patients with AML and 46 healthy controls as part of the California Childhood Leukemia Study (CCLS). Because sex disparities are suggested by differences in AML incidence rates, metabolite features associated with AML were identified in analyses stratified by sex. There was no overlap between the 16 predictors of AML in females and 15 predictors in males, suggesting that neonatal metabolomic profiles of pediatric AML risk are sex-specific. In females, four predictors of AML were putatively annotated as ceramides, a class of metabolites that has been linked with cancer cell proliferation. In females, two metabolite predictors of AML were strongly correlated with breastfeeding duration, indicating a possible biological link between this putative protective risk factor and childhood leukemia. In males, a heterogeneous metabolite profile of AML predictors was observed. Replication with larger participant numbers is required to validate findings.

Untargeted metabolomics profiling and hemoglobin normalization for archived newborn dried blood spots from a refrigerated biorepository.

Archived dried blood spots (DBS) following newborn screening are an attractive resource for interrogating early-life biology using untargeted metabolomics. Therefore, they have the potential to substantially aid etiological studies, particularly for rare and low-frequency childhood diseases and disorders. However, metabolite quantification in DBS is hindered by variation sources not present in serum and plasma samples such as the hematocrit effect and unknown initial blood volumes. Hemoglobin (Hb) is an appropriate correlate for hematocrit in experimentally-generated DBS punches. However, since many biorepositories worldwide archive DBS at 4-5 °C, there is a need to validate the utility of Hb for DBS archived under refrigeration. We evaluated two simple spectroscopic methods for measuring Hb in DBS stored at 4 +/- 2 °C for up to 21 years, obtained from the newborn screening program at the Karolinska University Hospital, Sweden. Spearman correlation analysis and Akaike Information Criterion model selection found that measurement of a Hb sodium lauryl sulfate complex at 540 nm better described nuisance variation than Hb measured at 404 nm, or using age of spot alone. This is the first study to profile metabolites and to propose a normalization factor for metabolite measurements from DBS archived for decades at 4 °C.

Mass defect filtering for suspect screening of halogenated environmental chemicals: A case study of chlorinated organophosphate flame retardants.

RATIONALE: Organophosphate flame retardants (OPFRs) are a class of flame retardants widely found in environmental and biological matrices that have been extensively studied due to their adverse health effects in humans. OPFRs are loosely bound chemicals that can detach from treated products and be released into indoor and outdoor environments, where they have the potential to further undergo transformation and degradation processes, in particular the chlorinated OPFRs (Cl-PFRs). Their detection remains a moving target for analysts, and traditional targeted mass spectrometry methods are suitable only for those compounds with authentic standards. METHODS: Mass defect filter (MDF) is a strategy to filter molecular features using thresholds applied to the mass defect value of a target ion or molecular feature of interest. We have developed an MDF strategy for the detection and tentative identification of twelve potential Cl-PFR transformation products in a study mixture of six known Cl-PFRs using MS/MS data acquired on a high-resolution mass spectrometer. Most compounds in the Cl-PFRs family share a ClO4 P group as a core structure, of which modification results in a significant shift in the exact masses of the resulting compounds but show only a minimal shift in their mass defects. Subsequently, the MDF strategy was employed to tentatively identify Cl-PFRs retrospectively in six human urine samples that had previously been analyzed. RESULTS: MDF in combination with product ion filtering for the characteristic [H2 O3 P]+ and [H4 O4 P]+ ions and neutral loss filtering for the characteristic Cn H2n-x Clx group resulted in revealing suspects and homologues in the Cl-PFRs family. Furthermore, the MDF of the product ions detected additional Cl-PFR-related compounds that differed significantly in the exact masses of both precursor and product ions but had minimal shift in the mass defects of product ions. The mass defect of one or more common product ions helped to detect a few Cl-PFR analogs that had not been identified by MDF of the core structure precursor ion. CONCLUSIONS: MDF helped to detect some Cl-PFRs present in lower concentrations, which went undetected without data filters. MDF also helped to detect chromatographic peaks for Cl-PFR homologues that are likely structural analogs that resulted from impurities and/or derivatives and transformation products. The methodology was applied to demonstrate and tentatively detect known and suspect Cl-PFRs in human urine samples retrospectively.

Novel Organelles with Elements of Bacterial and Eukaryotic Secretion Systems Weaponize Parasites of Drosophila.

The evolutionary success of parasitoid wasps, a highly diverse group of insects widely used in biocontrol, depends on a variety of life history strategies in conflict with those of their hosts [1]. Drosophila melanogaster is a natural host of parasitic wasps of the genus Leptopilina. Attack by L. boulardi (Lb), a specialist wasp to flies of the melanogaster group, activates NF-κB-mediated humoral and cellular immunity. Inflammatory blood cells mobilize and encapsulate Lb eggs and embryos [2-5]. L. heterotoma (Lh), a generalist wasp, kills larval blood cells and actively suppresses immune responses. Spiked virus-like particles (VLPs) in wasp venom have clearly been linked to wasps' successful parasitism of Drosophila [6], but the composition of VLPs and their biotic nature have remained mysterious. Our proteomics studies reveal that VLPs lack viral coat proteins but possess a pharmacopoeia of (1) the eukaryotic vesicular transport system, (2) immunity, and (3) previously unknown proteins. These novel proteins distinguish Lh from Lb VLPs; notably, some proteins specific to Lh VLPs possess sequence similarities with bacterial secretion system proteins. Structure-informed analyses of an abundant Lh VLP surface and spike-tip protein, p40, reveal similarities to the needle-tip invasin proteins SipD and IpaD of Gram-negative bacterial type-3 secretion systems that breach immune barriers and deliver virulence factors into mammalian cells. Our studies suggest that Lh VLPs represent a new class of extracellular organelles and share pathways for protein delivery with both eukaryotic microvesicles and bacterial surface secretion systems. Given their mixed prokaryotic and eukaryotic properties, we propose the term mixed-strategy extracellular vesicle (MSEV) to replace VLP.

Trends in the application of high-resolution mass spectrometry for human biomonitoring: An analytical primer to studying the environmental chemical space of the human exposome.

Global profiling of xenobiotics in human matrices in an untargeted mode is gaining attention for studying the environmental chemical space of the human exposome. Defined as the study of a comprehensive inclusion of environmental influences and associated biological responses, human exposome science is currently evolving out of the metabolomics science. In analogy to the latter, the development and applications of high resolution mass spectrometry (HRMS) has shown potential and promise to greatly expand our ability to capture the broad spectrum of environmental chemicals in exposome studies. HRMS can perform both untargeted and targeted analysis because of its capability of full- and/or tandem-mass spectrum acquisition at high mass accuracy with good sensitivity. The collected data from target, suspect and non-target screening can be used not only for the identification of environmental chemical contaminants in human matrices prospectively but also retrospectively. This review covers recent trends and advances in this field. We focus on advances and applications of HRMS in human biomonitoring studies, and data acquisition and mining. The acquired insights provide stepping stones to improve understanding of the human exposome by applying HRMS, and the challenges and prospects for future research.

Transmembrane Substrate Determinants for γ-Secretase Processing of APP CTFß.

The amyloid ß-peptide (Aß) of Alzheimer's disease (AD) is generated by proteolysis within the transmembrane domain (TMD) of a C-terminal fragment of the amyloid ß protein-precursor (APP CTFß) by the γ-secretase complex. This processing produces Aß ranging from 38 to 49 residues in length. Evidence suggests that this spectrum of Aß peptides is the result of successive γ-secretase cleavages, with endoproteolysis first occurring at the ε sites to generate Aß48 or Aß49, followed by C-terminal trimming mostly every three residues along two product lines to generate shorter, secreted forms of Aß: the primary Aß49-46-43-40 line and a minor Aß48-45-42-38 line. The major secreted Aß species are Aß40 and Aß42, and an increased proportion of the longer, aggregation-prone Aß42 compared to Aß40 is widely thought to be important in AD pathogenesis. We examined TMD substrate determinants of the specificity and efficiency of ε site endoproteolysis and carboxypeptidase trimming of CTFß by γ-secretase. We determined that the C-terminal negative charge of the intermediate Aß49 does not play a role in its trimming by γ-secretase. Peptidomimetic probes suggest that γ-secretase has S1', S2', and S3' pockets, through which trimming by tripeptides may be determined. However, deletion of residues around the ε sites demonstrates that a depth of three residues within the TMD is not a determinant of the location of endoproteolytic ε cleavage of CTFß. We also show that instability of the CTFß TMD helix near the ε site significantly increases endoproteolysis, and that helical instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. In addition, we found that CTFß dimers are not endoproteolyzed by γ-secretase. These results support a model in which initial interaction of the array of residues along the undimerized single helical TMD of substrates dictates the site of initial ε cleavage and that helix unwinding is essential for both endoproteolysis and carboxypeptidase trimming.

Characterization of Gonadotrope Secretoproteome Identifies Neurosecretory Protein VGF-derived Peptide Suppression of Follicle-stimulating Hormone Gene Expression.

Reproductive function is controlled by the pulsatile release of hypothalamic gonadotropin-releasing hormone (GnRH), which regulates the expression of the gonadotropins luteinizing hormone and FSH in pituitary gonadotropes. Paradoxically, Fshb gene expression is maximally induced at lower frequency GnRH pulses, which provide a very low average concentration of GnRH stimulation. We studied the role of secreted factors in modulating gonadotropin gene expression. Inhibition of secretion specifically disrupted gonadotropin subunit gene regulation but left early gene induction intact. We characterized the gonadotrope secretoproteome and global mRNA expression at baseline and after Gαs knockdown, which has been found to increase Fshb gene expression (1). We identified 1077 secreted proteins or peptides, 19 of which showed mRNA regulation by GnRH or/and Gαs knockdown. Among several novel secreted factors implicated in Fshb gene regulation, we focused on the neurosecretory protein VGF. Vgf mRNA, whose gene has been implicated in fertility (2), exhibited high induction by GnRH and depended on Gαs In contrast with Fshb induction, Vgf induction occurred preferentially at high GnRH pulse frequency. We hypothesized that a VGF-derived peptide might regulate Fshb gene induction. siRNA knockdown or extracellular immunoneutralization of VGF augmented Fshb mRNA induction by GnRH. GnRH stimulated the secretion of the VGF-derived peptide NERP1. NERP1 caused a concentration-dependent decrease in Fshb gene induction. These findings implicate a VGF-derived peptide in selective regulation of the Fshb gene. Our results support the concept that signaling specificity from the cell membrane GnRH receptor to the nuclear Fshb gene involves integration of intracellular signaling and exosignaling regulatory motifs.

A synthetic antibody fragment targeting nicastrin affects assembly and trafficking of γ-secretase.

The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid ß (Aß) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid ß peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.

Dystonia type 6 gene product Thap1: identification of a 50 kDa DNA-binding species in neuronal nuclear fractions.

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50-52 kDa in vitro and in vivo, and verified the previously identified species at 29-30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6.

Differential release of ß-amyloid from dendrite- versus axon-targeted APP.

The ß-amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer's disease. APP is processed in neurons, but little is known about the relative contributions of presynaptic or postsynaptic compartments to the release of Aß peptides. To address this issue, we transduced primary neurons from Sprague-Dawley rats or APP(-/-) mice (B6.129S7-App(tm1Dbo)/J) with lentiviral constructs expressing APP chimeras harboring targeting motifs from low-density lipoprotein receptor or neuron-glia cell-adhesion molecule to polarize expression to either dendritic or axonal membranes, respectively. Using imaging and quantitative biochemical approaches, we now report that APP selectively targeted to either axons or dendrites leads to the secretion of full-length Aß peptides with significantly elevated release from dendritic compartments. These findings reveal that the enzymatic machinery required for production of Aß peptides are operative both in presynaptic and postsynaptic compartments of primary neurons, leading to the suggestion that Aß-mediated impairments in glutamatergic neurotransmission is the result of Aß release from both local and distal neuronal compartments.