Nuclear magnetic resonance studies of HIV TAR RNA mutants

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1996.Includes bibliographical references (leaves 191-215).by Heidi A. Erlacher.Ph.D

Biosensors for Diagnosis and Monitoring

Biosensor technologies have received a great amount of interest in recent decades, and this has especially been the case in recent years due to the health alert caused by the COVID-19 pandemic. The sensor platform market has grown in recent decades, and the COVID-19 outbreak has led to an increase in the demand for home diagnostics and point-of-care systems. With the evolution of biosensor technology towards portable platforms with a lower cost on-site analysis and a rapid selective and sensitive response, a larger market has opened up for this technology. The evolution of biosensor systems has the opportunity to change classic analysis towards real-time and in situ detection systems, with platforms such as point-of-care and wearables as well as implantable sensors to decentralize chemical and biological analysis, thus reducing industrial and medical costs. This book is dedicated to all the research related to biosensor technologies. Reviews, perspective articles, and research articles in different biosensing areas such as wearable sensors, point-of-care platforms, and pathogen detection for biomedical applications as well as environmental monitoring will introduce the reader to these relevant topics. This book is aimed at scientists and professionals working in the field of biosensors and also provides essential knowledge for students who want to enter the field

IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

Rotational symmetry in ribonucleotide strand requirements for binding of HIV-1 Tat protein to TAR RNA.

Transactivation of human immunodeficiency virus (HIV) gene expression requires binding of the viral Tat protein to a RNA hairpin-loop structure (TAR) which contains a two or three-nucleotide bulge. Tat binds in the vicinity of the bulge and the two adjacent duplex stems, recognising both specific sequence and structural features of TAR. Binding is mediated by an arginine-rich domain, placing Tat in the family of arginine-rich RNA binding proteins that includes other transactivators, virus capsid proteins and ribosome binding proteins. In order to determine what features of TAR allow Tat to bind efficiently to RNA but not DNA forms, we examined Tat binding to a series of RNA-DNA hybrids. We found that only one specific strand in each duplex stem region needs to be RNA, implying that interaction between Tat and a given stem may be solely or predominantly with one of the two strands. However, the essential strand is not the same one for each stem, suggesting a switch in the bound strand on opposing sides of the bulge

Regulation and function of AGR2 and p53 pathways

Inactivation of p53 by mutation occurs in half of human tumours. The majority of these mutations affect the DNA-binding core domain and hence impair DNA binding and hinder transcription of p53 target genes. A wealth of data indicates that even cancers carrying wild type p53 protein, evolve mechanisms to render the p53 pathway inactive. Thus, inactivation of the p53 response, either by mutation or the alternative mechanisms, allows unpurturbed tumour growth. Recent work identified Anterior Gradient-2 (AGR2) as a protein overexpressed in wild type p53 expressing tumours and it was subsequently shown that AGR2 inhibits p53 pathway. In this study I confirmed that AGR2 protein inhibits p53 and AGR2 depletion potentiates p53-dependent DNA damage response. As there were no physiological signals known that regulate the AGR2-p53 axis, here I set out to identify pathways that activate or inhibit AGR2. I found that transforming growth factor β(TGF- β) triggers AGR2 protein reduction and this is concomitant with the stabilisation and increased activity of p53 protein. TGF-β halts AGR2 transcription in a SMAD4- dependent manner and triggers AGR2 protein degradation involving an ATM kinase. I found that SMAD nuclear interacting protein (SNIP1) mediated the ATMdependent AGR2 degradation. Interestingly, SNIP1 overexpression by itself promoted AGR2 protein degradation. I found that AGR2 protein degradation was proteasome independent and involed autophagy-lysosomal degradation pathway. As the mechanism of p53 inhibition by AGR2 is not known, I reasoned that identifying interactors of AGR2 may potentially further our understanding of the mechanism accounting for AGR2-mediated p53 inhibtion. I isolated the ATP binding protein Reptin in the yeast two-hybrid system and subsequently validated it as an AGR2 binding partner. Mutations of the two ATP binding motifs in Reptin resulted in altered oligomerization and thermostability of Reptin and affected the AGR2-Reptin complex stability. I also identified the Reptin docking site and it was mapped to a divergent octapeptide loop. I found that AGR2-Reptin complex coimmunoprecipitated with the p53 protein. Subsequently, I showed that Reptin protein can influence p53 activity, and depending on local concentration, either inhibit the transcription of p53-genes or chaperone its DNA binding activity. Interestingly, I found that Reptin formed a stable complex, independent of AGR2, with p53 R175H, p53 F270A, p53 S269D and p53 S269A, which has implication for the Reptin function in cancers bearing mutant form of p53 protein

Cancer Nanomedicine

This special issue brings together cutting edge research and insightful commentary on the currentl state of the Cancer Nanomedicine field

Computational Approaches: Drug Discovery and Design in Medicinal Chemistry and Bioinformatics

This book is a collection of original research articles in the field of computer-aided drug design. It reports the use of current and validated computational approaches applied to drug discovery as well as the development of new computational tools to identify new and more potent drugs

Allosteric regulation of MDM2 protein

The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket