Oscar Alzate

Proteomic and immunologic analyses of brain tumor exosomes

Brain tumors are horrific diseases with almost universally fatal outcomes; new therapeutics are desperately needed and will come from improved understandings of glioma biology. Exosomes are endo‐ somally derived 30‐100 nm membranous vesicles released from many cell types into the extracellular milieu; surprisingly’ exosomes are virtually unstudied in neuro‐oncology. These microvesicles were used as vaccines in other tumor settings’ but their immunological significance is unevaluated in brain tumors. Our purpose here is to report the initial biochemical’ proteomic’ and immunological studies on murine brain tumor exosomes’ following known procedures to isolate exosomes. Our findings show that these vesicles have biophysical characteristics and proteomic profiles similar to exosomes from other cell types but that brain tumor exosomes have unique features (e.g.’ very basic isoelectric points’ expressing the mutated tumor antigen EGFRVIII and the putatively immunosuppressive cytokine TGF‐β). Administration of such exosomes into syngeneic animals produced both humoral and cellular immune responses in immunized hosts capable of rejecting subsequent tumor challenges but failed to prolong survival in established orthotopic models. Control animals received saline or cell lysate vaccines and showed no antitumor responses. Exosomes and microvesicles isolated from sera of patients with brain tumors also possess EGFR’ EGFRVIII’ and TGF‐β. We conclude that exosomes released from brain tumor cells are biochemically/biophysically like other exosomes and have immune‐modulating properties. They can escape the blood‐brain barrier’ with potential systemic and distal signaling and immune consequences.— Graner M. W. Alzate’ O. Dechkovskaia A. M. Keene J. D. Sampson J. H. Mitchell D. A. Bigner D. D. Proteomic and immunologic analyses of brain tumor exosomes. FASEBJ. 23 1541–1557 (2009)

Aroclor 1254, a developmental neurotoxicant, alters energy metabolism- and intracellular signaling-associated protein networks in rat cerebellum and hippocampus

The vast literature on the mode of action of polychlorinated biphenyls (PCBs) indicates that PCBs are a unique model for understanding the mechanisms of toxicity of environmental mixtures of persistent chemicals. PCBs have been shown to adversely affect psychomotor function and learning and memory in humans. Although the molecular mechanisms for PCB effects are unclear, several studies indicate that the disruption of Ca(2+)-mediated signal transduction plays significant roles in PCB-induced developmental neurotoxicity. Culminating events in signal transduction pathways include the regulation of gene and protein expression, which affects the growth and function of the nervous system. Our previous studies showed changes in gene expression related to signal transduction and neuronal growth. In this study, protein expression following developmental exposure to PCB is examined. Pregnant rats (Long Evans) were dosed with 0.0 or 6.0mg/kg/day of Aroclor-1254 from gestation day 6 through postnatal day (PND) 21, and the cerebellum and hippocampus from PND14 animals were analyzed to determine Aroclor 1254-induced differential protein expression. Two proteins were found to be differentially expressed in the cerebellum following PCB exposure while 18 proteins were differentially expressed in the hippocampus. These proteins are related to energy metabolism in mitochondria (ATP synthase, sub unit β (ATP5B), creatine kinase, and malate dehydrogenase), calcium signaling (voltage-dependent anion-selective channel protein 1 (VDAC1) and ryanodine receptor type II (RyR2)), and growth of the nervous system (dihydropyrimidinase-related protein 4 (DPYSL4), valosin-containing protein (VCP)). Results suggest that Aroclor 1254-like persistent chemicals may alter energy metabolism and intracellular signaling, which might result in developmental neurotoxicity.

AMPK directly inhibits NDPK through a phosphoserine switch to maintain cellular homeostasis.

Nucleoside diphosphate kinase (NDPK) is a direct target of AMP-activated protein kinase (AMPK) and is inhibited by AMPK-mediated phosphorylation at a conserved serine residue. This serine residue in NDPK is mutated in neuroblastoma, making the enzyme constitutively active.

CLN5 and CLN8 protein association with ceramide synthase: Biochemical and proteomic approaches

Four patients with juvenile neuronal ceroid lipofuscinoses, a childhood neurodegenerative disorder that was previously described as CLN9 variant, are reclassified as CLN5 disease. CLN5‐deficient (CLN5−/−) fibroblasts demonstrate adhesion defects, increased growth, apoptosis, and decreased levels of ceramide, sphingomyelin, and glycosphingolipids. The CLN8 protein (CLN8p) corrects growth and apoptosis in CLN5−/− cells. Related proteins containing a Lag1 motif (CerS1/2/4/5/6) partially corrected these deficits, with CerS1, which is primarily expressed in brain, providing the best complementation, suggesting CLN5p activates CerS1 and may co‐immunoprecipitate with it. CLN8p complements CLN5‐deficient cells, consolidating the interrelationship of CLN5p/CLN8p, whose potential roles are explored as activators of (dihydro)ceramide synthases. Homozygosity mapping using microarray technology led to identification of CLN5 as the culprit gene in previously classified CLN9‐defective cases. Similar to CLN5−/−cells, ceramide synthase activity, C16/C18:0/C24:0/C24:1 ceramide species, measured by MS is decreased in CLN8−/− cells. Comparison of normal versus CLN5−/− cell CerS1‐bound proteins by immunoprecipitation, differential gel electrophoresis, and MS revealed absence of γ‐actin in CLN5−/− cells. The γ‐actin gene sequence is normal in CLN5−/− derived DNA. The γ‐actin‐bound proteins, vimentin and histones H2Afz/H3F3A/Hist1H4, were absent from the γ‐actin protein complex in CLN5−/− cells. The function of CLN5p may require vimentin and the histone proteins to bind γ‐actin. Defective binding could explain the CLN5−/− cellular phenotype. We explore the role of the CLN5/CLN8 proteins in ceramide species specific sphingolipid de novo synthesis, and suggest that CLN5/CLN8 proteins are more closely related than previously believed.

Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability

Impaired adipogenesis renders an adipose tissue unable to expand, leading to lipotoxicity and conditions such as diabetes and cardiovascular disease. While factors important for adipogenesis have been studied extensively, those that set the limits of adipose tissue expansion remain undetermined. Feeding a Western‐type diet to apolipoprotein E2 knock‐in mice, a model of metabolic syndrome, produced 3 groups of equally obese mice: mice with normal glucose tolerance, hyperinsulinemic yet glucose‐tolerant mice, and prediabetic mice with impaired glucose tolerance and reduced circulating insulin. Using proteomics, we compared subcutaneous adipose tissues from mice in these groups and found that the expression of PTRF (polymerase I and transcript release factor) associated selectively with their glucose tolerance status. Lentiviral and pharmacologically overexpressed PTRF, whose function is critical for caveola formation, compromised adipocyte differentiation of cultured 3T3‐L1cells. In human adipose tissue, PTRF mRNA levels positively correlated with markers of lipolysis and cellular senescence. Furthermore, a negative relationship between telomere length and PTRF mRNA levels was observed in human subcutaneous fat. PTRF is associated with limited adipose tissue expansion underpinning the key role of caveolae in adipocyte regulation. Furthermore, PTRF may be a suitable adipocyte marker for predicting pathological obesity and inform clinical management.—Perez‐Diaz, S., Johnson, L. A., DeKroon, R. M., Moreno‐Navarrete, J. M., Alzate, O., Fernandez‐Real, J. M., Maeda, N., Arbones‐Mainar, J. M. Polymerase I and transcript release factor (PTRF) regulates adipocyte differentiation and determines adipose tissue expandability. FASEB J. 28, 3769–3779 (2014). www.fasebj.org

Acute phase response, inflammation and metabolic syndrome biomarkers of Libby asbestos exposure

Identification of biomarkers assists in the diagnosis of disease and the assessment of health risks from environmental exposures. We hypothesized that rats exposed to Libby amphibole (LA) would present with a unique serum proteomic profile which could help elucidate epidemiologically-relevant biomarkers. In four experiments spanning varied protocols and temporality, healthy (Wistar Kyoto, WKY; and F344) and cardiovascular compromised (CVD) rat models (spontaneously hypertensive, SH; and SH heart failure, SHHF) were intratracheally instilled with saline (control) or LA. Serum biomarkers of cancer, inflammation, metabolic syndrome (MetS), and the acute phase response (APR) were analyzed. All rat strains exhibited acute increases in α-2-macroglobulin, and α1-acid glycoprotein. Among markers of inflammation, lipocalin-2 was induced in WKY, SH and SHHF and osteopontin only in WKY after LA exposure. While rat strain- and age-related changes were apparent in MetS biomarkers, no LA effects were evident. The cancer marker mesothelin was increased only slightly at 1 month in WKY in one of the studies. Quantitative Intact Proteomic profiling of WKY serum at 1 day or 4 weeks after 4 weekly LA instillations indicated no oxidative protein modifications, however APR proteins were significantly increased. Those included serine protease inhibitor, apolipoprotein E, α-2-HS-glycoprotein, t-kininogen 1 and 2, ceruloplasmin, vitamin D binding protein, serum amyloid P, and more 1 day after last LA exposure. All changes were reversible after a short recovery regardless of the acute or long-term exposures. Thus, LA exposure induces an APR and systemic inflammatory biomarkers that could have implications in systemic and pulmonary disease in individuals exposed to LA.

Analysis of proteins using DIGE and MALDI mass spectrometry.

Difference gel electrophoresis (DIGE) is a common technique for characterizing differential protein expression in quantitative proteomics. Usually a combination of enzymatic digestion and peptide analysis by mass spectrometry is used to identify differentially expressed proteins following separation and statistical analysis by DIGE. In this chapter, methods for gel spot picking, enzymatic digestion, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) for protein identification of DIGE-analyzed proteins are discussed. Two examples are given: first, a specific protein is used to test the sensitivity of the 2D DIGE/MALDI MS combination for protein quantification and identification, and second, several proteins with and without the labels typically used in DIGE are identified to demonstrate that these labels do not alter MS-based protein identification. Technical variations of protein gel spot preparation, in-gel digestion, and mass spectral protein identification are discussed.

Diggin′ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

The ubiquitin–proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing tandem ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.

Analysis of Protein Posttranslational Modifications Using DIGE-Based Proteomics

Difference gel electrophoresis (DIGE) is most often used to assess relative changes in the expression levels of individual proteins in multiple complex samples, and this information is valuable in making inferences about relative protein activity. However, a proteins activity is not solely dependent upon its expression level. A change in activity may also be influenced by myriad posttranslational modifications (PTMs), including palmitoylation, ubiquitination, oxidation, and phosphorylation. In this chapter, we describe the use of DIGE to determine specific PTMs by introducing specific labels or changes in pI and/or molecular weight.

Diabetes mellitus in patients withAlzheimer´s disease: clinical descriptionand correlation with the APOEgenotype in a sample population from the province of Antioquia, Colombia

INTRODUCTION The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. OBJECTIVE Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. MATERIALS AND METHODS The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. RESULTS On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). CONCLUSION This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.INTRODUCTION Alzheimers disease is a multifactorial disease affecting approximately twenty million people worldwide. Numerous variables are associated with increased risk of developing this severe neurological disorder. Among the risk factors, diabetes mellitus, and the ε4 isoform of the APOE gene have been amply demonstrated as increasing the risk of developing this disease. OBJECTIVE To determine if a correlation exists between APOE genotype, diabetes mellitus and Alzheimers disease. MATERIALS AND METHODS Clinical studies were carried out by surveying the clinical histories in a group of patients in the province of Antioquia, Colombia. Forty-three Alzheimers patients were compared with 43 control subjects, paired by age and gender. Commercially available methods were used to determine whether the patients had diabetes, and restriction enzyme-based genotyping was used to determine the APOE genotypes. RESULTS The most common non-neurological comorbidities were: arterial hypertension, acute myocardial infarction, chronic obstructive pulmonary disease and hypothyroidism. From the many variables investigated, two were conclusive: (1) the presence of Alzheimers disease was higher in patients with diabetes mellitus, and (2) no correlation between late-onset sporadic Alzheimers disease and APOE was found in the target population. CONCLUSIONS To detect any association with the APOE genotype, a study involving much a larger population samples must be undertaken.