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Folkerd E.J.,Institute for Cancer Research | Dowsett M.,Royal Marsden Hospital
Journal of Clinical Oncology | Year: 2010

To review the influence of sex hormones on the progression of breast, prostate, gynecologic, and colorectal cancer. The literature was reviewed in an informal manner utilizing the authors' prior knowledge to collate the current evidence for the involvement of sex hormones, particularly estrogens and androgens in the progression of a range of hormonally responsive cancers. In particular, the effect of treatment involving hormone withdrawal treatment was considered strong evidence for involvement. The impact of basal levels of endogenous steroids was considered. Data from clinical trials indicate the efficacy of therapeutic interventions that result in ablation or antagonism of host steroids for a range of cancers. Demonstration of the correlation of the completeness of withdrawal with clinical outcome together with direct evidence of progression from studies looking at the influence of tissue and circulating levels of sex hormones more recently in conjunction with gene expression profiles all provide compelling evidence for the involvement of steroids in the progression of disease. The involvement of steroids in the progression of cancer in hormone-sensitive tissues is well established and an important target for therapy. © 2010 by American Society of Clinical Oncology.

Seeger C.,Institute for Cancer Research | Sohn J.A.,Institute for Cancer Research
Molecular Therapy - Nucleic Acids | Year: 2014

Hepatitis B virus persistence in infected hepatocytes is due to the presence of covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs. Antiviral therapies with nucleoside analogues inhibit replication of HBV DNA in capsids present in the cytoplasm of infected cells, but do not reduce or destroy nuclear cccDNA. To investigate whether cccDNA derived from infectious HBV could be directly targeted for destruction, we used the CRISPR/Cas9 system in HepG2 cells expressing the HBV receptor sodium taurocholate cotransporting polypeptide (NTCP). We tested different HBV-specific guide RNAs and demonstrated that they could inhibit HBV infections up to eightfold. Inhibition was due to mutations and deletions in cccDNA similar to those observed with chromosomal DNA cleaved by Cas9 and repaired by nonhomologous end joining (NHEJ). Interferon alpha (IFN-α) did not have a measurable effect on the antiviral activity of the CRISPR/Cas9 system, suggesting that Cas9 and NHEJ activities are not affected by induction of an innate immune response with the cytokine. Taken together, our results demonstrated that Cas9 can be recruited to cccDNA, opening the possibility for the development of future antiviral strategies aimed at targeting cccDNA for endonucleolytic cleavage with small molecules. © 2014 The American Society of Gene & Cell Therapy All rights reserved.

Kristensen V.N.,Institute for Cancer Research
EMBO Molecular Medicine | Year: 2011

Since the luminal B tumours are associated with poor recurrence-free and disease-specific survivals in all adjuvant systemic treatment categories including hormone therapy, the identification of specific signalling pathways driving luminal B biology is paramount to improve treatment. Sircoulomb et al. and Holland et al. have independently identified the ZNF703 gene, located in chromosomal region 8p12, as preferentially amplified in luminal B tumours. © 2011 EMBO Molecular Medicine.

Xu Q.,Institute for Cancer Research | Dunbrack R.L.,Institute for Cancer Research
Bioinformatics | Year: 2012

Motivation: Automating the assignment of existing domain and protein family classifications to new sets of sequences is an important task. Current methods often miss assignments because remote relationships fail to achieve statistical significance. Some assignments are not as long as the actual domain definitions because local alignment methods often cut alignments short. Long insertions in query sequences often erroneously result in two copies of the domain assigned to the query. Divergent repeat sequences in proteins are often missed.Results: We have developed a multilevel procedure to produce nearly complete assignments of protein families of an existing classification system to a large set of sequences. We apply this to the task of assigning Pfam domains to sequences and structures in the Protein Data Bank (PDB). We found that HHsearch alignments frequently scored more remotely related Pfams in Pfam clans higher than closely related Pfams, thus, leading to erroneous assignment at the Pfam family level. A greedy algorithm allowing for partial overlaps was, thus, applied first to sequence/HMM alignments, then HMM-HMM alignments and then structure alignments, taking care to join partial alignments split by large insertions into single-domain assignments. Additional assignment of repeat Pfams with weaker E-values was allowed after stronger assignments of the repeat HMM. Our database of assignments, presented in a database called PDBfam, contains Pfams for 99.4 of chains >50 residues. © 2012 The Author.

Xu Q.,Institute for Cancer Research | Dunbrack Jr. R.L.,Institute for Cancer Research
Nucleic Acids Research | Year: 2011

The protein common interface database (ProtCID) is a database that contains clusters of similar homodimeric and heterodimeric interfaces observed in multiple crystal forms (CFs). Such interfaces, especially of homologous but non-identical proteins, have been associated with biologically relevant interactions. In ProtCID, protein chains in theprotein data bank (PDB) are grouped based on their PFAM domain architectures. For a single PFAM architecture, all the dimers present in each CF are constructed and compared with those in other CFs that contain the same domain architecture. Interfaces occurring in two or more CFs comprise an interface cluster in the database. Thesame process is used to compare heterodimers of chains with different domain architectures. By examining interfaces that are shared by many homologous proteins in different CFs, we find that the PDB and the Protein Interfaces, Surfaces, and Assemblies (PISA) are not always consistent in their annotations of biological assemblies in a homologous family. Our data therefore provide an independent check on publicly available annotations of the structures of biological interactions for PDB entries. Common interfaces may also be useful in studies of protein evolution. Coordinates for allinterfaces in a cluster are downloadable for further analysis. ProtCiD is available at http://dunbrack2. © The Author(s) 2010.

Shapovalov M.V.,Institute for Cancer Research | Dunbrack Jr. R.L.,Institute for Cancer Research
Structure | Year: 2011

Rotamer libraries are used in protein structure determination, prediction, and design. The backbone-dependent rotamer library consists of rotamer frequencies, mean dihedral angles, and variances as a function of the backbone dihedral angles. Structure prediction and design methods that employ backbone flexibility would strongly benefit from smoothly varying probabilities and angles. A new version of the backbone-dependent rotamer library has been developed using adaptive kernel density estimates for the rotamer frequencies and adaptive kernel regression for the mean dihedral angles and variances. This formulation allows for evaluation of the rotamer probabilities, mean angles, and variances as a smooth and continuous function of phi and psi. Continuous probability density estimates for the nonrotameric degrees of freedom of amides, carboxylates, and aromatic side chains have been modeled as a function of the backbone dihedrals and rotamers of the remaining degrees of freedom. New backbone-dependent rotamer libraries at varying levels of smoothing are available from © 2011 Elsevier Ltd.

Agency: NSF | Branch: Continuing grant | Program: | Phase: | Award Amount: 196.26K | Year: 2013

Intellectual merit: The first step in gene expression (RNA synthesis, or transcription) of the vast majority of eukaryotic genes is regulated by enhancers, which are DNA sequences that bind specific proteins and activate transcription over large distances. Enhancer-target communication (ETC) leads to direct interaction of enhancers with the target promoters, the sites of transcription initiation, via formation of a DNA loop. ETC can be prevented by insulators, which are DNA elements that form alternative DNA loops and can isolate enhancers from the targets. Thus, gene regulation in human cells involves formation of DNA loops of variable sizes. The mechanisms of efficient communication between enhancers and promoters and the mechanisms by which such communication is prevented by insulators remain unknown. In particular, neither structural nor dynamic aspects of DNA looping have been rationalized. Recently, it has been shown that DNA loops bound to histone proteins are a highly efficient communication device. These observations indicate that ETC could constitute a critical step in gene regulation and raise the following important questions: What elements of the DNA-histone complexes mediate efficient ETC, and how can ETC be inhibited or facilitated by various factors? In this project, a highly purified and functionally active experimental system has been established that allows quantitative analysis of the rate of ETC in the DNA-histone complexes. This system will be used as a tool to analyze structural and dynamic properties of the DNA-histone complexes that dictate the rate of ETC. The objectives of this research are to evaluate the mechanisms of efficient distant communication and inhibition of communication by insulators and to identify factors affecting distant communication and evaluate their effect on the dynamics of the DNA-histone complexes.
Broader impacts: This project will provide an enhanced learning experience for several undergraduate and two graduate students involved in the project. They will participate weekly in four different meetings; namely (1) laboratory meetings in which they will have the opportunity to present their own research every 2-3 months, (2) laboratory discussion of recent publications on chromatin structure/dynamics/function, (3) problem solving sessions with a small group of students and senior researchers that focuses on technical challenges, and (4) Departmental Progress Reports where all Departmental students present their research annually. This project is well suited for student participation, as it provides a solid conceptual foundation and utilizes methodologies applicable to many areas of research, and at the same time is paradigm-driven, providing an intuitive transition from the classroom to the bench. The laboratory regularly trains underrepresented minority undergraduates (within the UMDNJ Summer Research Program). New technologies/strategies for analysis of structure/dynamics of the DNA-histone complexes will be developed. Some of them will be of general use in the field of biology. The project is a multi- disciplinary study to examine a complex biological question, and incorporates recent advances in computational science and structural biology at the interface between chemistry, physics and biology.

Agency: NSF | Branch: Standard Grant | Program: | Phase: Molecular Biophysics | Award Amount: 1.02M | Year: 2014

Non-technical explanation
An in-depth understanding of the mechanisms of protein folding has implications beyond the immediate field. For example, non-native protein states are critical for understanding protein aggregation, which has major practical implications in biotechnology and medicine. The rapid kinetics techniques to be developed will not only benefit protein folding research, but also studies of ligand binding and enzymatic reaction mechanisms. Critical testing and refinement of computational models will benefit other areas of computational biology, such as structure prediction, modeling of protein interactions and functional conformational changes.

Although major progress has been made in recent years in understanding how small proteins fold, deciphering the mechanisms of folding of larger proteins remains a daunting challenge. This project is aimed at moving beyond small proteins to larger ones with more complex folding behavior by joining cutting edge experimental approaches with powerful computational strategies for exploring early stages of folding of apomyoglobin, a medium size protein. A rich set of experimental data on the structural and dynamic properties of intermediate states populated on the microsecond time scale will provide benchmarks for validating and refining simulation techniques. The findings will provide a critical test of our understanding of protein folding and the power of molecular simulation to accurately model the folding reaction, yielding structural and mechanistic insight with unprecedented spatial and temporal resolution.

This project offers training opportunities for future scientists in a wide range of experimental, computational and theoretical approaches. Students will benefit from the close collaboration between experimental and theoretical groups. Dr. Roder is a member of the NSF-sponsored Protein Folding Consortium whose main mission is to foster scientific exchange and collaboration. Dr. Voelz is a participating researcher in the Folding@home distributed computing project, a unique platform for scientific outreach that promotes public awareness for the importance of basic research in protein folding.

Technical description
The objectives of this project entitled Collaborative Research: Early Stages of Protein Folding Explored by Experimental and Computational Approaches are: (i) to elucidate the folding mechanism of a prototypic alpha-helical protein by detailed experimental and computational analysis of the kinetic network of states encountered during folding of apomyoglobin (apoMb); (ii) to understand key features of the amino acid sequence important for initiating folding, defining chain topology and directing the search for the native structure. The group of Heinrich Roder at the Fox Chase Cancer Center will combine ultrafast mixing methods with fluorescence and NMR-detected H/D exchange labeling to elucidate the kinetic folding dynamics of apoMb with single-residue resolution. The results will provide a basis for validating computational models to be developed by the group of Vincent Voelz at Temple University. Modeling of apoMb folding dynamics by molecular dynamics (MD) simulation, combined with Markov State Model approaches, will yield atomic-resolution structural insight and testable predictions of experimental observables. Recent kinetic studies have shown that folding of apoMb under acidic conditions (pH 4.2) is a multi-stage process completed within 250 microseconds of initiation. This time scale is computationally accessible and makes large-scale MD simulations a realistic proposition, using the Folding@home distributed computer network. Effects of mutations on experimental observables and the simulated network of states will inform on the sequence determinants for folding initiation and pathway selection.

By combining advanced experimental techniques, including kinetic analysis with microsecond resolution, mutagenesis and NMR-based hydrogen-deuterium exchange methods, with state ofthe-art computational methods, it will be possible to describe early stages of folding of the 153 residues apoMb with a level of detail that has previously been achieved only for much smaller proteins. Experimental observables, including rate constants, mutational perturbations, fluorescence properties and NH protection patterns, will serve as benchmarks for testing and refining computational models, which in turn will provide structural and mechanistic insight with atomic resolution and make predictions to be tested in a next round of experiments. The results will extend our understanding of the principles of protein folding beyond small two-state folders to a larger helical protein with complex multi-state folding behavior and address long-standing questions concerning the sequence determinants for folding initiation and propagation, and the structural features and kinetic roles of protein folding intermediates.

Agency: NSF | Branch: Continuing grant | Program: | Phase: Genetic Mechanisms | Award Amount: 200.00K | Year: 2016

This project will investigate how the DNA genome is packaged in the cellular nucleus to create alternate gene configurations that either permit or prevent retrieval and use of the genetic information. Understanding the dynamic nature of such DNA packaging may help explain how organisms successfully coordinate normal growth and development in response to ever-changing environmental signals. The project will have broad educational impacts by providing research and training opportunities for high school and undergraduate students, many of whom will be from groups traditionally underrepresented in STEM disciplines.

Histone proteins in nucleosome-associated DNA can be reversibly modified by addition of poly(ADP-ribose), so-called PAR, molecules, leading to alterations in chromatin conformation and changes in gene expression. Whereas much as been learned about the action and regulation of the enzyme that adds this modification, PAR polymerase (PARP), not much is yet known about the enzyme, PAR glycohydrolase (PARG), that degrades it. Changes in poly(ADP-ribosyl)ation levels have been attributed to up- and downregulation of PARP activity, with PARG acting constitutively to cleaving PAR at a constant rate. This project challenges that idea and poses the hypothesis that regulation of PARG activity is crucial for tissue- specific and cell cycle-specific differences in poly(ADP-ribosyl)ation rates. Preliminary results suggest that transient phosphorylation of PARG domains may lead to conformational changes in the protein, in turn affecting its activity. Using Drosophila as a model, a combination of in vivo and in vitro studies will be used to detect phosphorylation of the PARG protein and to analyze its nuclear localization and activity. The results are expected to provide new understanding of the role of PARG in regulating steady-state levels of poly(ADP-ribosyl)ation and how this poorly studied histone modification controlls cell-cycle-regulated and tissue-specific gene expression.

Kappes D.J.,Institute for Cancer Research
Immunological Reviews | Year: 2010

The role of the zinc finger transcription factor ThPOK (T-helper-inducing POZ-Kruppel-like factor) in promoting commitment of αβ T cells to the CD4 lineage is now well established. New results indicate that ThPOK is also important for the development and/or acquisition of effector functions by other T cell subsets, including several not marked by CD4 expression, i.e. double-negative invariant natural killer T (iNKT) cells, γδ cells, and even memory CD8+ T cells. There is compelling evidence that ThPOK expression in most or all of these cases is dependent on T-cell receptor signaling and that differences in relative TCR signal strength/length may induce different levels of ThPOK expression. The developmental consequences of ThPOK expression vary according to cell type, which may partly reflect differences in ThPOK levels and/or in transcriptional networks between cell types. © 2010 John Wiley & Sons A/S.

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