VGF changes during the estrous cycle: a novel endocrine role for TLQP peptides?

Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary

The Role of Esterification on Detection of Protonated and Deprotonated Peptide Ions in Matrix Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS)

Esterification was used to investigate how introduction of aliphatic chains within the peptide structure affects the MALDI response of ions analyzed in both polarity regimes. In binary mixtures containing equimolar amounts of a peptide with its correspondent alkyl ester, derivatization of the carboxylic groups has the tendency to increase MALDI detection of the modified protonated peptide ions. This positive effect on ion yield is more pronounced when longer alcohols are employed. In negative mode, the situation is antithetic and esterification produces a deleterious effect on the ion yield of the corresponding deprotonated species. From the data reported here we postulate that modifications of the acidic character of peptides prevent formation of anionic species under MALDI analysis. Furthermore, suppression of the formation pathway for anions alters the overall number of molecules which can undergo protonation. This results in an increased ion yield for the protonated esters

Deficiency in interferon type 1 receptor improves definitive erythropoiesis in Klf1 null mice

A key regulatory gene in definitive erythropoiesis is the transcription factor Krüppel-like factor 1 (Klf1). Klf1 null mice die in utero by day 15.5 (E15.5) due to impaired definitive erythropoiesis and severe anemia. Definitive erythropoiesis takes place in erythroblastic islands in mammals. Erythroblastic islands are formed by a central macrophage (Central Macrophage of Erythroblastic Island, CMEI) surrounded by maturating erythroblasts. Interferon-β (IFN-β) is activated in the fetal liver’s CMEI of Klf1 null mice. The inhibitory effect of IFN-β on erythropoiesis is known and, therefore, we speculated that IFN-β could have contributed to the impairment of definitive erythropoiesis in Klf1 knockout (KO) mice fetal liver. To validate this hypothesis, in this work we determined whether the inactivation of type I interferon receptor (Ifnar1) would ameliorate the phenotype of Klf1 KO mice by improving the lethal anemia. Our results show a prolonged survival of Klf1/Ifnar1 double KO embryos, with an improvement of the definitive erythropoiesis and erythroblast enucleation, together with a longer lifespan of CMEI in the fetal liver and also a restoration of the apoptotic program. Our data indicate that the cytotoxic effect of IFN-β activation in CMEI contribute to the impairment of definitive erythropoiesis associated with Klf1 deprivation

Profiles of VGF peptides in the rat brain and their modulations after Phencyclidine treatment

From the VGF precursor protein originate several low molecular weight peptides, whose distribution in the brain and blood circulation is not entirely known. Among the VGF peptides, those containing the N-terminus portion were altered in the cerebro-spinal fluid (CSF) and hypothalamus of schizophrenia patients. "Hence, we aimed to better investigate the involvement of the VGF peptides in schizophrenia by studying their localization in the brain regions relevant for the disease, and revealing their possible modulations in response to certain neuronal alterations occurring in schizophrenia". We produced antibodies against different VGF peptides encompassing the N-terminus, but also C-terminus-, TLQP-, GGGE- peptide sequences, and the so named NERP-3 and -4. These antibodies were used to carry out specific ELISA and immunolocalization studies while mass spectrometry (MS) analysis was also performed to recognize the intact brain VGF fragments. We used a schizophrenia rat model, in which alterations in the prepulse inhibition (PPI) of the acoustic startle response occurred after PCP treatment. In normal rats, all the VGF peptides studied were distributed in the brain areas examined including hypothalamus, prefrontal cortex, hippocampus, accumbens and amygdaloid nuclei and also in the plasma. By liquid chromatography-high resolution mass, we identified different intact VGF peptide fragments, including those encompassing the N-terminus and the NERPs. PCP treatment caused behavioral changes that closely mimic schizophrenia, estimated by us as a disruption of PPI of the acoustic startle response. The PCP treatment also induced selective changes in the VGF peptide levels within certain brain areas. Indeed, an increase in VGF C-terminus and TLQP peptides was revealed in the prefrontal cortex (p < 0.01) where they were localized within parvoalbumin and tyrosine hydroxylase (TH) containing neurons, respectively. Conversely, in the nucleus accumbens, PCP treatment produced a down-regulation in the levels of VGF C-terminus-, N-terminus- and GGGE- peptides (p < 0.01), expressed in GABAergic- (C-terminus/GGGE) and somatostatin- (N-terminus) neurons. These results confirm that VGF peptides are widely distributed in the brain and modulated in specific areas involved in schizophreni

Stress granules induced by oxidative stress in cultured fibroblast from TDP-43 mutant ALS patients

Stress granules (SGs) are transient cytoplasmic aggregates that rapidly form when cells are exposed to stress and consist of large messenger ribonucleoprotein (mRNPs) complexes. The SGs seem to function as storage depots for translation silenced complex and are implicated in stress-induced inhibition of global protein synthesis. Protein aggregation, has been observed in several neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). The protein TDP-43 (TAR DNA-Binding Protein-43), encoded by one of the ALS-causative gene (TARDBP), is a major constituent of pathological inclusions in this disease and it is seems to be implicated in the regulation of SGs. Therefore we investigated the different characteristics of SGs in human cultured fibroblasts from ALS patients carrying TARDBPA382T mutation (group 1) versus healthy subjects (group 2). The cells were exposed to stressful conditions using sodium arsenite (SA) at different concentrations (0.5 mM, 1 mM) and exposure times (30 min, 1h). Preliminary results showed, after 30 minutes, small and sporadic cytoplasmic inclusions immunostained for TIA-1(T-cell internal antigen-1), an early marker for SGs, in both groups of cells. After 1h, the TIA-1 immunostained granules were bright, copious and scattered into the cytosol. Interestingly, we observed a significantly higher number of cells exhibiting SGs in fibroblasts from healthy controls (66%) compared to ALS patients (34%). In parallel, we identified the RNA binding protein HuR-1 (Human antigen R) in a fraction of Tia-1 positive SGs, as well as TDP- 43 localized into the nucleus of all the cells. These data raise the possibility that TDP-43 may modulate the stress granule formation, contributing to the cellular response to acute stress. Moreover the TDP-43 may regulate gene expression as well as cellular recovery and survival, and consequently its mutation may contribute to the neurodegeneration

VGF peptides as novel biomarkers in Parkinson’s disease

Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra (SN). At disease onset, a diagnosis is often difficult. VGF peptides are abundant in the SN and peripheral circulation; hence, we investigate whether their plasma profile may reflect the brain dopamine reduction. Using antibodies against the VGF C-terminal portion, we analyzed the rat brain and human plasma, with immunohistochemistry and ELISA. Rats were unilaterally lesioned with 6-hyroxydopamine and sacrificed either 3 or 6 weeks later with or without levodopa treatment. Plasma samples were obtained from PD patients, either at the time of diagnosis (group 1, drug naïve, n = 23) or upon dopamine replacement (group 2, 1–6 years, n = 24; group 3, > 6 years, n = 16), compared with age-matched control subjects (group 4, n = 21). Assessment of the olfactory function was carried out in group 2 using the “Sniffin’ Sticks” test. VGF immunoreactivity was present in GABAergic neurons and, on the lesioned side, it was reduced at 3 weeks and abolished at 6 weeks after lesion. Conversely, upon levopoda, VGF labeling was restored. In PD patients, VGF levels were reduced at the time of diagnosis (1504 ± 587 vs. 643 ± 348 pmol/mL, means ± S.E.M: control vs. naïve; p < 0.05) but were comparable with the controls after long-term drug treatment (> 6 years). A linear correlation was demonstrated between VGF immunoreactivity and disease duration, levodopa equivalent dose and olfactory dysfunction. Plasma VGF levels may represent a useful biomarker, especially in the early stages of PD

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

BACKGROUND: A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate alpha-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type. CONCLUSIONS/SIGNIFICANCE: These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins

Proteomic Biomarkers for Acute Interstitial Lung Disease in Gefitinib-Treated Japanese Lung Cancer Patients

Interstitial lung disease (ILD) events have been reported in Japanese non-small-cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors. We investigated proteomic biomarkers for mechanistic insights and improved prediction of ILD. Blood plasma was collected from 43 gefitinib-treated NSCLC patients developing acute ILD (confirmed by blinded diagnostic review) and 123 randomly selected controls in a nested case-control study within a pharmacoepidemiological cohort study in Japan. We generated ∼7 million tandem mass spectrometry (MS/MS) measurements with extensive quality control and validation, producing one of the largest proteomic lung cancer datasets to date, incorporating rigorous study design, phenotype definition, and evaluation of sample processing. After alignment, scaling, and measurement batch adjustment, we identified 41 peptide peaks representing 29 proteins best predicting ILD. Multivariate peptide, protein, and pathway modeling achieved ILD prediction comparable to previously identified clinical variables; combining the two provided some improvement. The acute phase response pathway was strongly represented (17 of 29 proteins, p = 1.0×10−25), suggesting a key role with potential utility as a marker for increased risk of acute ILD events. Validation by Western blotting showed correlation for identified proteins, confirming that robust results can be generated from an MS/MS platform implementing strict quality control