Covalent Protein Adduction by Drugs of Abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Expression and purification of tau protein and its frontotemporal dementia variants using a cleavable histidine tag

Recombinant tau protein is widely used to study the biochemical, cellular and pathological aspects of tauopathies, including Alzheimer's disease and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTPD-17). Pure tau in high yield is a requirement for in vitro evaluation of the protein's physiological and toxic functions. However, the preparation of recombinant tau is complicated by the protein's propensity to aggregate and form truncation products, necessitating the use of multiple, time-consuming purification methods. In this study, we investigated parameters that influence the expression of wild type and FTPD-17 pathogenic tau, in an attempt to identify ways to maximise expression yield. Here, we report on the influence of the choice of host strain, induction temperature, duration of induction, and media supplementation with glucose on tau expression in Escherichia coli. We also describe a straightforward process to purify the expressed tau proteins using immobilised metal affinity chromatography, with favourable yields over previous reports. An advantage of the described method is that it enables high yield production of functional oligomeric and monomeric tau, both of which can be used to study the biochemical, physiological and toxic properties of the protein.

PROTEIN-TRIGGERED SUSTAINED DRUG RELEASE CONTROLLED BY APTAMERS BOUND TO OLIGOMER-CROSSLINKED ALGINATE

Sustained drug release systems provide many advantages over traditional delivery methods such as extending the time in which the drug is found to be within an effective concentration within the therapeutic window, which decreases the frequency of administration of the drug, and increases patient compliance. Research using polyacrylamide crosslinked by oligomers containing an aptamer sequence, has demonstrated a pulsatile release over 50 minutes triggered by a 2 mM target adenosine concentration. This thesis aims to build off this concept by designing a system that delivers in a sustained manner when triggered by micromolar target concentrations reflective of disease in vivo, using macromolecular targets. For example, the disease wet age related macular degeneration (wet AMD) is associated with increased concentrations of the protein vascular endothelial growth factor (VEGF-A) – a macromolecule. Patients with wet AMD would benefit from the implantation of devices or microspheres that release drugs in a sustained manner in response to local VEGF concentrations. In this thesis, we hypothesize that the protein lysozyme, used to demonstrate proof-of-concept, could trigger the increased release of drugs from oligomer-crosslinked alginate. The objectives are to (i) demonstrate sustained release from alginate, (ii) design oligomer crosslinked alginate that degrades in response to lysozyme, and then (iii) use these systems to control the release of FITC-dextran with and without lysozyme. A series of control experiments and analyses were used to optimize the crosslinking of alginate by annealed oligomers. The cumulative release of FITC-dextran (MW 20,000) from oligomer crosslinked alginate increased by 3.4 μg when lysozyme (3 μM) was introduced at 48 hours, as opposed to controls which released only 0.2 μg. FITC-loaded alginate microspheres coated by oligomer-crosslinked alginate released 15% more FITC-dextran over 120 hours when placed into 3 μM of lysozyme than without lysozyme. Controls of alginate crosslinked with PEG or control oligomers (without a lysozyme aptamer sequence) had no changes in release with lysozyme. The incorporation of a lysozyme aptamer onto oligomers used to crosslink alginate disks or alginate coatings on microspheres resulted in different diffusion and release of FITC-dextran into PBS with or without lysozyme. This approach could be adapted for the delivery of drugs to diseases with specific protein profiles such as wet AMD.

Inhibition of nonsense-mediated mRNA decay by the cytoplasmic poly(A)-binding protein

The expression of a gene from transcription of the DNA into pre-messenger RNA (pre-mRNA) over translation of messenger RNA (mRNA) into protein is constantly monitored for errors. This quality control is necessary to guarantee successful gene expression. One quality control mechanism important to this thesis is called nonsense-mediated mRNA decay (NMD). NMD is a cellular process that eliminates mRNA transcripts harboring premature translation termination codons (PTCs). Furthermore, NMD is known to regulate certain transcripts with long 3′ UTRs. However, some mRNA transcripts are known to evade NMD. The mechanism of NMD activation has been subjected to many studies whereas NMD evasion or suppression still remains rather elusive. It has previously been shown that the cytoplasmic poly(A)-binding protein (PABPC1) is able to suppress NMD of certain transcripts. In this study I show that PABPC1 is able to suppress NMD of a long 3′ UTR-carrying reporter when tethered immediately downstream of the termination codon. I further am able to show the importance of the interaction between PABPC1 and eIF4G for NMD suppression, whereas the interaction between PABPC1 and eRF3a seems dispensable. These results indicate an involvement of efficient translation termination and potentially ribosome recycling in NMD suppression. I am able to show that if PABPC1 is too far removed from the terminating ribosome NMD is activated. After showing the importance of PABPC1 recruitment directly downstream of a terminating ribosome in NMD suppression, I am further able to demonstrate several different methods by which PABPC1 can be recruited. Fold-back of the poly(A)-tail mediated by two interacting proteins on opposite ends of a 3′ UTR manages to bring PABPC1 bound to the poly(A)-tail into close proximity of the terminating ribosome and therefore suppress NMD. Furthermore, small PAM2 peptides that are known to interact with the MLLE domain of PABPC1 are able to strongly suppress NMD initiated by either a long 3′ UTR or an EJC. I am also able to show the NMD antagonizing power of recruited PABPC1 for the known endogenous NMD target β-globin PTC39, which is responsible for the disease β-thalassemia. This shows the potential medical implications and application of suppressing NMD by recruiting PABPC1 into close proximity of a terminating ribosome.

Impact of Protein Induced by Vitamin K Absence (PIVKA-II) in diagnosis and monitoring of patients with hepatocellular carcinoma

Background and Aims: Hepatocellular carcinoma (HCC) represents the second leading cause of cancer deaths worldwide. Protein induced by vitamin K absence (PIVKA-II) has been proposed as potential screening biomarker for HCC.This study has been designed to evaluate the role of PIVKA-II as diagnostic HCC marker, through the comparison between PIVKA-II and alpha-fetoprotein (AFP) serum levels on HCC patients and the two control groupsof patients with liver disease and without HCC. Methods: In an Italian prospective cohort, PIVKA-II levels were assessed on serum samplesby an automated chemiluminescent immunoassay (Abbott ARCHITECT). The study population included 65 patients with HCC (both “de novo” and recurrent), 111 with liver cirrhosis (LC) and 111 with chronic hepatitis C (CHC). Results: PIVKA-II levels were increased in patients with HCC (median 63.75, range: 12-2675 mAU/mL) compared to LC (median value: 30.95, range: 11.70–1251mAU / mL, Mann Whitney test p < 0.0001) and CHC (median value: 24.89, range: 12.98-67.68mAU / mL, p < 0.0001).The area under curve (AUC) for PIVKA-II was 0.817 (95% Confidence Interval(CI), 0.752-0.881). At the optimal threshold of 37 mAU / mL, identified by the Youden Index, the sensitivity and specificity were 79% and 76%, respectively. PIVKA-II was a better biomarker than AFP for the diagnosis of HCC, since the AUC for AFP was 0.670 (95% CI 0.585-0.754, p<0.0001) and at the best cutoff of 16.4 ng / mL AFP yielded 98% specificity but only 34% sensitivity. Conclusions:These initial data suggest the potential utility of this tool in the diagnosis of HCC.PIVKA-II alone or in combination may help to an early diagnosis of HCC and a significant optimization of patient management.

Machine learning tools for protein annotation: the cases of transmembrane β-barrel and myristoylated proteins

Biology is now a “Big Data Science” thanks to technological advancements allowing the characterization of the whole macromolecular content of a cell or a collection of cells. This opens interesting perspectives, but only a small portion of this data may be experimentally characterized. From this derives the demand of accurate and efficient computational tools for automatic annotation of biological molecules. This is even more true when dealing with membrane proteins, on which my research project is focused leading to the development of two machine learning-based methods: BetAware-Deep and SVMyr. BetAware-Deep is a tool for the detection and topology prediction of transmembrane beta-barrel proteins found in Gram-negative bacteria. These proteins are involved in many biological processes and primary candidates as drug targets. BetAware-Deep exploits the combination of a deep learning framework (bidirectional long short-term memory) and a probabilistic graphical model (grammatical-restrained hidden conditional random field). Moreover, it introduced a modified formulation of the hydrophobic moment, designed to include the evolutionary information. BetAware-Deep outperformed all the available methods in topology prediction and reported high scores in the detection task. Glycine myristoylation in Eukaryotes is the binding of a myristic acid on an N-terminal glycine. SVMyr is a fast method based on support vector machines designed to predict this modification in dataset of proteomic scale. It uses as input octapeptides and exploits computational scores derived from experimental examples and mean physicochemical features. SVMyr outperformed all the available methods for co-translational myristoylation prediction. In addition, it allows (as a unique feature) the prediction of post-translational myristoylation. Both the tools here described are designed having in mind best practices for the development of machine learning-based tools outlined by the bioinformatics community. Moreover, they are made available via user-friendly web servers. All this make them valuable tools for filling the gap between sequential and annotated data.

Development of unsupervised learning methods with applications to life sciences data

Machine Learning makes computers capable of performing tasks typically requiring human intelligence. A domain where it is having a considerable impact is the life sciences, allowing to devise new biological analysis protocols, develop patients’ treatments efficiently and faster, and reduce healthcare costs. This Thesis work presents new Machine Learning methods and pipelines for the life sciences focusing on the unsupervised field. At a methodological level, two methods are presented. The first is an “Ab Initio Local Principal Path” and it is a revised and improved version of a pre-existing algorithm in the manifold learning realm. The second contribution is an improvement over the Import Vector Domain Description (one-class learning) through the Kullback-Leibler divergence. It hybridizes kernel methods to Deep Learning obtaining a scalable solution, an improved probabilistic model, and state-of-the-art performances. Both methods are tested through several experiments, with a central focus on their relevance in life sciences. Results show that they improve the performances achieved by their previous versions. At the applicative level, two pipelines are presented. The first one is for the analysis of RNA-Seq datasets, both transcriptomic and single-cell data, and is aimed at identifying genes that may be involved in biological processes (e.g., the transition of tissues from normal to cancer). In this project, an R package is released on CRAN to make the pipeline accessible to the bioinformatic Community through high-level APIs. The second pipeline is in the drug discovery domain and is useful for identifying druggable pockets, namely regions of a protein with a high probability of accepting a small molecule (a drug). Both these pipelines achieve remarkable results. Lastly, a detour application is developed to identify the strengths/limitations of the “Principal Path” algorithm by analyzing Convolutional Neural Networks induced vector spaces. This application is conducted in the music and visual arts domains.

Taurine Supplementation Increases K(atp) Channel Protein Content, Improving Ca2+ Handling And Insulin Secretion In Islets From Malnourished Mice Fed On A High-fat Diet.

Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

Evaluation Of Different Treatment Methods Against Denture Stomatitis: A Randomized Clinical Study.

The aim of this clinical study was to determine the efficacy of Uncaria tomentosa (cat's claw) against denture stomatitis (DS). Fifty patients with DS were randomly assigned into 3 groups to receive 2% miconazole, placebo, or 2% U tomentosa gel. DS level was recorded immediately, after 1 week of treatment, and 1 week after treatment. The clinical effectiveness of each treatment was measured using Newton's criteria. Mycologic samples from palatal mucosa and prosthesis were obtained to determinate colony forming units per milliliter (CFU/mL) and fungal identification at each evaluation period. Candida species were identified with HiCrome Candida and API 20C AUX biochemical test. DS severity decreased in all groups (P < .05). A significant reduction in number of CFU/mL after 1 week (P < .05) was observed for all groups and remained after 14 days (P > .05). C albicans was the most prevalent microorganism before treatment, followed by C tropicalis, C glabrata, and C krusei, regardless of the group and time evaluated. U tomentosa gel had the same effect as 2% miconazole gel. U tomentosa gel is an effective topical adjuvant treatment for denture stomatitis.

Nat2, Xrcc1 And Hogg1 Polymorphisms, Cigarette Smoking, Alcohol Consumption And Risk Of Upper Aerodigestive Tract Cancer.

To evaluate associations between polymorphisms of the N-acetyltransferase 2 (NAT2), human 8-oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) genes and risk of upper aerodigestive tract (UADT) cancer. A case-control study involving 117 cases and 224 controls was undertaken. The NAT2 gene polymorphisms were genotyped by automated sequencing and XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms were determined by Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Slow metabolization phenotype was significantly associated as a risk factor for the development of UADT cancer (p=0.038). Furthermore, haplotype of slow metabolization was also associated with UADT cancer (p=0.014). The hOGG1 Ser326Cys polymorphism (CG or GG vs. CC genotypes) was shown as a protective factor against UADT cancer in moderate smokers (p=0.031). The XRCC1 Arg399Gln polymorphism (GA or AA vs. GG genotypes), in turn, was a protective factor against UADT cancer only among never-drinkers (p=0.048). Interactions involving NAT2, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms may modulate the risk of UADT cancer in this population.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Iis--integrated Interactome System: A Web-based Platform For The Annotation, Analysis And Visualization Of Protein-metabolite-gene-drug Interactions By Integrating A Variety Of Data Sources And Tools.

High-throughput screening of physical, genetic and chemical-genetic interactions brings important perspectives in the Systems Biology field, as the analysis of these interactions provides new insights into protein/gene function, cellular metabolic variations and the validation of therapeutic targets and drug design. However, such analysis depends on a pipeline connecting different tools that can automatically integrate data from diverse sources and result in a more comprehensive dataset that can be properly interpreted. We describe here the Integrated Interactome System (IIS), an integrative platform with a web-based interface for the annotation, analysis and visualization of the interaction profiles of proteins/genes, metabolites and drugs of interest. IIS works in four connected modules: (i) Submission module, which receives raw data derived from Sanger sequencing (e.g. two-hybrid system); (ii) Search module, which enables the user to search for the processed reads to be assembled into contigs/singlets, or for lists of proteins/genes, metabolites and drugs of interest, and add them to the project; (iii) Annotation module, which assigns annotations from several databases for the contigs/singlets or lists of proteins/genes, generating tables with automatic annotation that can be manually curated; and (iv) Interactome module, which maps the contigs/singlets or the uploaded lists to entries in our integrated database, building networks that gather novel identified interactions, protein and metabolite expression/concentration levels, subcellular localization and computed topological metrics, GO biological processes and KEGG pathways enrichment. This module generates a XGMML file that can be imported into Cytoscape or be visualized directly on the web. We have developed IIS by the integration of diverse databases following the need of appropriate tools for a systematic analysis of physical, genetic and chemical-genetic interactions. IIS was validated with yeast two-hybrid, proteomics and metabolomics datasets, but it is also extendable to other datasets. IIS is freely available online at: http://www.lge.ibi.unicamp.br/lnbio/IIS/.

The Effect Of Celastrol, A Triterpene With Antitumorigenic Activity, On Conformational And Functional Aspects Of The Human 90kda Heat Shock Protein Hsp90α, A Chaperone Implicated In The Stabilization Of The Tumor Phenotype.

Hsp90 is a molecular chaperone essential for cell viability in eukaryotes that is associated with the maturation of proteins involved in important cell functions and implicated in the stabilization of the tumor phenotype of various cancers, making this chaperone a notably interesting therapeutic target. Celastrol is a plant-derived pentacyclic triterpenoid compound with potent antioxidant, anti-inflammatory and anticancer activities; however, celastrol's action mode is still elusive. In this work, we investigated the effect of celastrol on the conformational and functional aspects of Hsp90α. Interestingly, celastrol appeared to target Hsp90α directly as the compound induced the oligomerization of the chaperone via the C-terminal domain as demonstrated by experiments using a deletion mutant. The nature of the oligomers was investigated by biophysical tools demonstrating that a two-fold excess of celastrol induced the formation of a decameric Hsp90α bound throughout the C-terminal domain. When bound, celastrol destabilized the C-terminal domain. Surprisingly, standard chaperone functional investigations demonstrated that neither the in vitro chaperone activity of protecting against aggregation nor the ability to bind a TPR co-chaperone, which binds to the C-terminus of Hsp90α, were affected by celastrol. Celastrol interferes with specific biological functions of Hsp90α. Our results suggest a model in which celastrol binds directly to the C-terminal domain of Hsp90α causing oligomerization. However, the ability to protect against protein aggregation (supported by our results) and to bind to TPR co-chaperones are not affected by celastrol. Therefore celastrol may act primarily by inducing specific oligomerization that affects some, but not all, of the functions of Hsp90α. To the best of our knowledge, this study is the first work to use multiple probes to investigate the effect that celastrol has on the stability and oligomerization of Hsp90α and on the binding of this chaperone to Tom70. This work provides a novel mechanism by which celastrol binds Hsp90α.

Treadmill Training Increases Sirt-1 And Pgc-1 α Protein Levels And Ampk Phosphorylation In Quadriceps Of Middle-aged Rats In An Intensity-dependent Manner.

The present study investigated the effects of running at 0.8 or 1.2 km/h on inflammatory proteins (i.e., protein levels of TNF- α , IL-1 β , and NF- κ B) and metabolic proteins (i.e., protein levels of SIRT-1 and PGC-1 α , and AMPK phosphorylation) in quadriceps of rats. Male Wistar rats at 3 (young) and 18 months (middle-aged rats) of age were divided into nonexercised (NE) and exercised at 0.8 or 1.2 km/h. The rats were trained on treadmill, 50 min per day, 5 days per week, during 8 weeks. Forty-eight hours after the last training session, muscles were removed, homogenized, and analyzed using biochemical and western blot techniques. Our results showed that: (a) running at 0.8 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with NE rats; (b) these responses were lower for the inflammatory proteins and higher for the metabolic proteins in young rats compared with middle-aged rats; (c) running at 1.2 km/h decreased the inflammatory proteins and increased the metabolic proteins compared with 0.8 km/h; (d) these responses were similar between young and middle-aged rats when trained at 1.2 km. In summary, the age-related increases in inflammatory proteins, and the age-related declines in metabolic proteins can be reversed and largely improved by treadmill training.

Reduced Insulin Clearance And Lower Insulin-degrading Enzyme Expression In The Liver Might Contribute To The Thrifty Phenotype Of Protein-restricted Mice.

Nutrient restriction during the early stages of life usually leads to alterations in glucose homeostasis, mainly insulin secretion and sensitivity, increasing the risk of metabolic disorders in adulthood. Despite growing evidence regarding the importance of insulin clearance during glucose homeostasis in health and disease, no information exists about this process in malnourished animals. Thus, in the present study, we aimed to determine the effect of a nutrient-restricted diet on insulin clearance using a model in which 30-d-old C57BL/6 mice were exposed to a protein-restricted diet for 14 weeks. After this period, we evaluated many metabolic variables and extracted pancreatic islet, liver, gastrocnemius muscle (GCK) and white adipose tissue samples from the control (normal-protein diet) and restricted (low-protein diet, LP) mice. Insulin concentrations were determined using RIA and protein expression and phosphorylation by Western blot analysis. The LP mice exhibited lower body weight, glycaemia, and insulinaemia, increased glucose tolerance and altered insulin dynamics after the glucose challenge. The improved glucose tolerance could partially be explained by an increase in insulin sensitivity through the phosphorylation of the insulin receptor/protein kinase B and AMP-activated protein kinase/acetyl-CoA carboxylase in the liver, whereas the changes in insulin dynamics could be attributed to reduced insulin secretion coupled with reduced insulin clearance and lower insulin-degrading enzyme (IDE) expression in the liver and GCK. In summary, protein-restricted mice not only produce and secrete less insulin, but also remove and degrade less insulin. This phenomenon has the double benefit of sparing insulin while prolonging and potentiating its effects, probably due to the lower expression of IDE in the liver, possibly with long-term consequences.