Activity-dependent neuroprotective protein constitutes a novel element in the SWI/SNF chromatin remodeling complex

Complete deficiency in activity-dependent neuroprotective protein (ADNP), a heterochromatin 1-binding protein, results in dramatic changes in gene expression, neural tube closure defects, and death at gestation day 9 in mice. To further understand the cellular roles played by ADNP, the HEK293 human embryonic kidney cell line that allows efficient transfection with recombinant DNA was used as a model for the identification of ADNP-interacting proteins. Recombinant green fluorescent protein (GFP)-ADNP was localized to cell nuclei. When nuclear extracts were subjected to immunoprecipitation with specific GFP antibodies followed by polyacrylamide gel electrophoresis, several minor protein bands were observed in addition to GFP-ADNP. In-gel protein digests followed by mass spectrometry identified BRG1, BAF250a, and BAF170, all components of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex, as proteins that co-immunoprecipitate with ADNP. These results were verified utilizing BRG1 antibodies. ADNP short hairpin RNA down-regulation resulted in microtubule reorganization and changes in cell morphology including reduction in cell process formation and cell number. These morphological changes are closely associated with the SWI/SNF complex multifunctionality. Taken together, the current study uncovers a molecular basis for the essential function of the ADNP gene and protein.     

Microtubules, schizophrenia and cognitive behavior: preclinical development of davunetide (NAP) as a peptide-drug candidate

NAP (davunetide) is an active fragment of activity-dependent neuroprotective protein (ADNP). ADNP and the homologous protein ADNP2 provide cell protection. ADNP is essential for brain formation, proper development and neuronal plasticity, all reported to be impaired in schizophrenia. ADNP haploinsufficiecy inhibits social and cognitive functions, major hallmarks in schizophrenia. Imbalance in ADNP/ADNP2 expression in the schizophrenia brain may impact disease progression. NAP treatment partly ameliorates ADNP haploinsufficiecy. The microtubule, stable tubule-only polypeptide (STOP)-deficient mice were shown to provide a reliable model for schizophrenia. Daily intranasal NAP treatment significantly decreased hyperactivity in STOP-deficient mice and protected visual memory, supporting further clinical development.     

Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP

Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.     

Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry

High-density lipoprotein (HDL) is the most abundant lipoprotein particle in the plasma and a negative risk factor of atherosclerosis. By using a proteomic approach it is possible to obtain detailed information about its protein content and protein modifications that may give new information about the physiological roles of HDL. In this study the two subfractions; HDL(2) and HDL(3), were isolated by two-step discontinuous density-gradient ultracentrifugation and the proteins were separated with two-dimensional gel electrophoresis and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionisation time of flight mass spectrometry. Identified proteins in HDL were: the dominating apo A-I as six isoforms, four of them with a glycosylation pattern and one of them with retained propeptide, apolipoprotein (apo) A-II, apo A-IV, apo C-I, apo C-II, apo C-III (two isoforms), apo E (five isoforms), the recently discovered apo M (two isoforms), serum amyloid A (two isoforms) and serum amyloid A-IV (six isoforms). Furthermore, alpha-1-antitrypsin was identified in HDL for the first time. Additionally, salivary alpha-amylase was identified as two isoforms in HDL(2), and apo L and a glycosylated apo A-II were identified in HDL(3). Besides confirming the presence of different apolipoproteins, this study indicates new patterns of glycosylated apo A-I and apo A-II. Furthermore, the study reveals new proteins in HDL; alpha-1-antitrypsin and salivary alpha-amylase. Further investigations about these proteins may give new insight into the functional role of HDL in coronary artery diseases.     

Silencing of the ADNP-family member, ADNP2, results in changes in cellular viability under oxidative stress

Activity-dependent neuroprotective protein (ADNP) 2 (KIAA0863; ZNF508) gene, a homeobox-profile containing gene, was identified in a screen for homologous proteins to ADNP. The human ADNP2 contains 1131 amino acid residues with a molecular weight of 122.8 KDa. In silico analysis indicated that ortholgs to ADNP2 exist in different phyla, suggesting that ADNP2 might be evolutionary conserved. Here, we began to explore the molecular and functional characterization of ADNP2. Results showed that the mouse ADNP2 mRNA is ubiquitously expressed in distinct normal tissues with increased expression in the brain, particularly in the cerebral cortex. During development, a relatively high level of ADNP2 gene expression was found in the embryonic mouse brain and was sustained throughout embryogenesis and adulthood. An increase in the mRNA was detected in differentiated P19 neuronal/glial-like cells as compared with the non-differentiated cells. To gain insight into ADNP2 function, ADNP2-deficient cell lines were established by the RNA silencing (small interfering RNA) technology. ADNP2 deficiency significantly changed the toxicity induced by hydrogen peroxide in P19 embryonic carcinoma cells, similar to what would be predicted for ADNP deficiency. These findings represent an initial characterization of ADNP2 and suggest that this gene product may have an important function in brain by playing a role in cellular survival pathways.     

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α₂-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α₂-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.     

A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

Summary. By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

Comparative Proteomics Analysis of Cerebrospinal Fluid of Patients with Guillain–Barré Syndrome

To better understand the pathophysiologic mechanisms underlying Guillain-Barré syndrome (GBS), Comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with GBS (the experiment group) and control subjects suffering from other neurological disorders (the control group) was carried out using two-dimensional gel electrophoresis (2-DE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and database searching to determine abnormal CSF proteins in GBS patients. Image analysis of 2-DE gels silver stained revealed that 10 protein spots showed significant differential expression between the two groups of CSF samples. The expression of cystatin C, transthyretin, apolipoprotein E and heat shock protein 70 were decreased. However, haptoglobin, alpha-1-antitrypsin, apolipoprotein A-IV and neurofilaments were elevated. The subsequent ELISA measured the concentration of cystatin C and confirmed the result of the proteomic analysis. These identified proteins may be involved in the pathophysiological process of GBS and call for further studying the role of these proteins in the pathogenesis of the disease.     

Serum differential protein identification of Xinjiang Kazakh esophageal cancer patients based on the two-dimensional liquid-phase chromatography and LTQ MS

The aim of this study was to investigate the impact of chemo-radiotherapy on serum protein expression of the esophageal cancer patients and discover potential biomarkers by detecting serum proteins mass spectrometry of the healthy Kazakh people in Xinjiang as well as the patients before and after their chemo-radiotherapy. In order to separate and compare the three serum samples (the healthy group’s, the patients’ before and after chemo-radiotherapy) with two-dimensional protein liquid chromatography system (Proteome LabTM PF-2D), then detect the differential protein spots with linear trap quadruple mass spectrometer (LTQ MS/MS). (1) The Kazakh esophageal cancer patients got 21 expressed protein spots peaks with significant difference after chemo-radiotherapy compared with before; before the treatment there were 10 different expressed protein spots compared with the healthy group, and after it there were four peaks in the expression of protein spots compared with the healthy group. (2) After LTQ mass spectrometric detection, 22 proteins were up-regulated in serum samples of the healthy group, 22 were up-regulated of the patients before medical treatment and 5 were up-regulated after chemo-radiotherapy. (3) 8 proteins including APOA1 can be served as serum markers in Kazakh esophageal cancer diagnosis, and proteins like CLU can be served as serum markers in judging the resistance and sensitivity towards chemo-radiotherapy. (4) The abnormal expressions of APOC2, APOC3, Antithrombin-III in esophageal cancer were discovered for the first time. Specific protein spots related to Xinjiang Kazakh esophageal cancer diagnosis and chemo-radiotherapy can be identified in the serum, which will probably become a maker in Kazakh esophageal cancer diagnosis and therapeutic evaluation.     

Apolipoprotein A-IV: a protein occurring in human mesenteric lymph chylomicrons and free in plasma. Isolation and quantification.

1. Human mesenteric lymph chylomicrons were isolated from chylous ascites fluid by ultra-centrifugation and agarose/gel chromatography and their apoprotein composition was analysed by dodecylsulfate/polyacrylamide gel electrophoresis, analytical isoelectric focusing and immuno-chemically. Major components of mesenteric lymph chylomicrons were apoprotein A-I, proteins of Mr less than 15 000 including the C-group apoproteins and a protein of Mr 46 000. Minor components were apoprotein E and a protein of Mr approximately equal to 200 000 (B-like protein). This apoprotein composition was qualitatively identical with that of chylomicrons from intestinal lymph of the rat, but was distinctly different from plasma chylomicrons of humans with fasting chylomicronaemia. 2. The protein of Mr approximately equal to 46 000 has been isolated by preparative dodecylsulfate/polyacrylamide gel electrophoresis from human and rat lymph chylomicrons and was compared to a protein of identical Mr present in rat high-density lipoproteins (apoplipoprotein A-IV) and in the rho less than 1.006 g/ml serum lipoprotein fraction of individual humans with alimentary hypertriglyceridaemia. In both species the 46 000-Mr proteins isolated from lymph and serum were identical according to amino acid composition and isoelectric point in 6 M urea. The human proteins from both sources were also immunologically identical. The similarities in the molecular properties of the human apolipoprotein and rat apolipoprotein A-IV indicate that these proteins are homologous. 3. Plasma levels of human apolipoprotein A-IV determined by electroimmunodiffusion were 14.15 +/- 3.66 mg/100 ml (n = 59), but greater than 90% of the protein was unassociated with the major lipoprotein fractions. It is concluded, that apolipoprotein A-IV is a main protein component of human lymph chylomicrons, that is removed from the particles in the plasma compartment.     

Time-dependent plasma protein changes in streptozotocin-induced diabetic rats before and after fungal polysaccharide treatments

Previous studies about protein modulation with chemically induced models of diabetes in animals have yielded conflicting results, in that many investigators have reported different regulation patterns for the same proteins. Therefore, it is reasonable to determine biomarkers for prognosis and diagnosis of diabetes with time profiling for the candidate proteins. In this regard, we examined the influence of hypoglycemic fungal polysaccharides (EPS) on the time-dependent plasma protein alterations in streptozotocin-induced diabetic rats. The 2-DE analysis of rat plasma demonstrated that about 50 proteins from about 900 visualized spots were found to be differentially regulated, of which 20 spots were identified as principal diabetes-associated proteins. The results of time profiling revealed that most of the identified proteins showed significant alterations in a time-dependent manner during 14 days, with notable trends. Nine out of the twenty proteins displayed very similar time profiles between normal healthy and EPS-treated diabetic rats. Interestingly, the altered profiles of several proteins by diabetes induction almost returned to control levels after EPS treatments. In particular, we found a clear distinction in differential expression of oxidative stress proteins (ceruloplasmin and transferrin) and lipid metabolism related proteins (Apo A-I, Apo A-IV, and Apo E) in the STZ-induced diabetic rats. The data presented here have identified and characterized the time-dependent changes in plasma proteins associated with EPS treatment in STZ-induced diabetic rats, thereby leading to the discovery of early-response and late-response biomarkers in diabetic and EPS-treated states.     

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.     

Serum protein profiling to identify biomarkers for small renal cell carcinoma

Diagnostic biomarkers for early detection of renal cell carcinoma (RCC) are in great need. In the present study, we compared the serum protein profiles of patients with small RCC to those of healthy individuals to identify the differentially expressed proteins with potential to serve as biomarkers. Serum samples were collected from 10 patients with small RCC and 10 healthy individuals. The serum protein expression profiles were analyzed by two-dimensional (2-D) gel electrophoresis. Twenty-seven proteins with differences in expression levels between RCC patients and healthy volunteers were identified. Of these, 19 were expressed at different levels and eight were expressed in serum from the RCC group, but not from the control group. Six differentially expressed proteins identified by using mass spectrometry included coagulation factor XIII B, complement C3 and its precursor, misato homolog 1 (isoform CRA_b), hemopexin, and alpha-1-B-glycoprotein. Some of these serum proteins are known regulators of tumor progression in human malignancies. In conclusion, we successfully applied 2-D gel electrophoresis and identified six serum proteins differentially expressed between patients with small RCC and healthy volunteers. These proteins may provide novel biomarkers for early detection and diagnosis of human RCC.     

Specific Changes of Serum Proteins in Parkinson's Disease Patients

The aim of this study is to identify and validate protein change in the serum from PD patients. We used serum samples from 21 PD patients and 20 age-matched normal people as control to conduct a comparative proteomic study. We performed 2-DE and analyzed the differentially expressed protein spots by LC-MS/MS. In PD group 13 spots were shown to be differentially expressed compared to control group. They were identified as 6 proteins. Among these, 3 proteins were confirmed by Western blot analysis. It showed that the frequency of fibrinogen γ-chain (FGG) appeared 70% in PD, which could not be detected in control group. The protein of inter-alpha-trypsin inhibitor heavy chain H4 (ITI-H4) was found to exist two forms in serum. The full size (120 kDa) of the protein was increased and the fragmented ITI-H4 (35 kDa) was decreased in PD group. The ratio of full size ITI-H4 to fragmented ITI-H4 in PD patients was 3.85±0.29-fold higher than in control group. Furthermore, fragmented Apo A-IV (∼26 kDa) was mainly detected in control group, while it was rare to be found in PD group. Above findings might be useful for diagnosis of PD. When the expressions of FGG and 120 kDa ITI-H4 are increase, as well as ∼26 kDa Apo A-IV disappear would provide strong evidence for PD.