Bruce E. Torbett - US grants

The Protein Expression and Proteomics (PEP) Core received supplemental funding for one year starting June 1, 2006, to provide an unmet need for our community of investigators. The Protein Expression portion of the proposed pilot Core, under the direction of Dr. John Elder, will provide the appropriate vectors and cell lines needed to facilitate expression cloning of investigator-targeted proteins. The Proteomics portion of the core, under the direction of Dr. Bruce Torbett, will provide protein analysis services for CFAR investigators. These services include protein extraction from viruses, cells, and tissues; protein resolution and quantification using 1- and 2-dimensional (D) electrophoresis and imaging systems; protein identification using mass spectroscopy; and analyses of proteins using Luminex¿¿ xMAP¿¿ multiplex detection technology. The unique aspect of the PEP Core is the focus on providing services to investigators requiring expression of proteins from HIV-1 and other viral genes and protein analysis of HIV-1 and other virally infected samples. The Protein Expression portion of the Core will provide the appropriate vectors and cell lines needed to facilitate expression cloning of investigator-targeted proteins, using bacterial, insect cell, or mammalian cell vector systems, as deemed appropriate for the particular application. The Elder laboratory has compiled an extensive panel of vectors for each expression system and has extensive experience in protein expression and purification for ligand binding studies, enzymology, and crystallographic analyses. The laboratory is equipped with incubators required to grow cells at the required temperatures and also centrifuges, FPLC, and HPLC systems to aid in protein purification where necessary. The Core will aid the investigator in vector selection and amplification and cloning of targeted genes. In addition, the Core will help investigators in optimizing protein expression and selection of purification schemes most appropriate for each application. The Proteomics portion of the Core will provide all facets of protein resolution from visualization to identification. CFAR members will have access to the NCI-funded Proteomics Core housed within the Departmental of Molecular and Experimental Medicine at The Scripps Research Institute. Currently the Proteomics Core has available 1- and 2-D gel electrophoresis protein separation instrumentation, IEF Zoom fractionators for solution phase IEF, a state-of-the-art 2D Proteomic Imaging System with a Bio-Rad VersaDoc 1000 imager and imaging software. All imaging software can be utilized free of charge on resident computers housed within the Core. Technical advice is available from the Core technicians, and when needed, Core technicians provide demonstrations and seminars on analysis and imaging software. In addition, Core technicians will provide advice to investigators for optimizing protein extraction procedures and selection of purification schemes most appropriate for each application. The Protein Expression and Proteomics Core will provide informational seminars/sessions on the Core services each year and new technology seminars/sessions throughout the funding period.

DESCRIPTION (provided by applicant): Malignant glioma growth is characterized by extensive tumor infiltration associated with breakdown of the blood brain barrier (BBB). To determine the effect of reduced BBB breakdown on glioma infiltration we have developed an orthotopic xenograft mouse brain tumor model in which gliomas are implanted into immunodeficient Src knockout mice (Src KO). The Src KO mouse has been previously shown to mediate a reduction in glioma-induced BBB breakdown that was associated with reduced glioma infiltration, but independent of direct effects on glioma growth in general. In this model the focus is on effects of the host compartment (i.e. Src defects in the vascular endothelium) rather than on the more common analysis of tumor cells themselves (i.e. tumor compartment). The proposed studies are responsive to the PAS ("Understanding mechanisms of brain tumor dispersal") by outlining a novel proteomics strategy to identify and quantitate Src-mediated changes in expression and phosphorylation in response to glioma-mediated BBB breakdown in Src KO vs. wild type (WT) mouse brains. In contrast to RNA analyses, proteomics enables the detection of changes in protein expression without introducing a bias from variable translational efficiency/stability of different mRNAs. In Aim 1 endothelial cells will be isolated from tumor-bearing Src KO and WT mouse brains to identify Src-regulated proteins. For each candidate Src-mediated target molecule identified in Aim 1, we will validate the function of a candidate hit in a BBB reconstitution assay in Aim 2. The value of these studies is the identification of protein expression changes in the blood brain barrier (BBB) associated with glioma invasion using a knockout model with a well-defined phenotype exhibiting: 1) reduced VEGF-induced breakdown of the BBB;2) reduced glioma-induced breakdown of the BBB;3) reduced glioma invasion;and 4) reduced glioma-induced perivascular fibrin(-ogen) accumulation. This xenograft/proteomics approach is a novel strategy with much wider implications for tumor-host interactions, since it enables the identification of host-derived (i.e. mouse) vs. tumor-derived (i.e. human) proteins in the tumor-induced remodeling of the tumor microenvironment. PUBLIC HEALTH RELEVANCE: These studies address the PAS-06-201 (Understanding mechanisms of brain tumor dispersal) by analyzing changes in protein expression in blood vessels that mediate the breakdown of the blood brain barrier. The identification of these proteins will lead to a better understanding of the mechanisms regulating brain tumor dispersal and the future design of innovative therapies that target the host rather than tumor cells themselves.

DESCRIPTION (provided by applicant): HSC transplantations have become a standard of care for treating otherwise incurable blood cancers and genetic diseases. The curing of HIV and leukemia by transplanting HSCs from HIV-resistant patients with a CCR5-D32 mutation has demonstrated the power of stem cell-based therapies for AIDS. However, difficulties in the genetic modifications of autologous HSCs and in finding HLA-compatible CCR5-D32 donors significantly hamper the widespread use of somatic HSC-based AIDS therapies in the clinical setting. Converting adult human cells to induced pluripotent stem cells (iPSCs) provides a unique opportunity to produce immunologically matched gene-edited therapeutic cells for diseases of the blood and immune system as iPSCs can be expanded indefinitely ex vivo, genetically modified using homologous recombination and differentiated into hematopoietic cells. However, transferring this approach to the clinic requires the improvement of iPSC- derived blood cell engraftment, development of robust cGMP-compatible protocols for blood production from iPSCs, and the bi-allelic CCR5 disruption to provide an anti-HIV effect. The proposed studies capitalize on our recent advances in identification of pre-HSC hemogenic endothelium (HE) stage in human ESC/iPSC cultures and progress in locus-specific gene editing in ESC/iPSCs using ZNF-mediated homologous recombination. The three related specific aims are directed at understanding the molecular mechanisms controlling development of HSCs from human PSCs through the HE stage, with the ultimate goal to develop clinically- relevant protocols for ex vivo production of CCR5-knockout autologous HSCs for AIDS therapies. In aim 1, we will identify the biological regulators guiding the formation of engraftabl hematopoietic cells from HE with a goal to improve production of blood cells with regenerative potential from human PSCs. In aim 2, we will develop homologous recombination-based technology for the bi-allelic CCR5 knockout in iPSCs and test the engraftability and safety of genetically corrected iPSC-derived blood cells following transplantation in NOD/SCD/IL2Rg-/- (NSG) mice. In aim 3, we will test whether iPSC-derived CCR5-null cells are protected from HIV-1 challenge in NSG mice. Successful completion of the studies will validate a methodology for generation of regenerative blood cells from iPSCs and their potential use for HIV therapies. The applications of the methodology proposed here will be also useful for basic research and for future clinical applications for modification of any genomic target in iPSCs.

Project Summary Polymerases with ultra-high processivity would be exceptionally useful in biotechnology because they will make it possible to monitor long-range correlated changes in the sequence, structure and post-synthetic modifications of DNA or RNA without losing information on linkage between sites. We have discovered an ultra-processive reverse-transcriptase (MarathonRT) during the course of our structural studies on group II intron encoded proteins, and have been optimizing it for use as a new tool in the enzyme arsenal of molecular biologists and genomicists, particularly in studies of long-range correlated mutations within viral genomes such as HIV. Specifically, we are interested in understanding the patterns by which correlated mutations shift in response to antiretroviral drug treatment, thereby providing insights into mechanisms of resistance and, potentially, providing clinically valuable information on changes in therapeutic drug combinations for infected patients. While our ongoing parent grant continues to develop MarathonRT as a tool enzyme for a variety of applications in RNA science, in this supplemental study we will focus specifically on the use of MarathonRT for discovery of regulatory structures, sequences and post-transcriptional modifications within the HIV genome. We will conduct chemical probing studies, analysis of correlated mutations and studies on changes in RT dynamical behavior, such as pausing. These investigations will be carried out in conjunction with adaptations to emerging sequencing platforms, such as Oxford Nanopore MinION sequencing, enabling a unified and high- throughput approach for the discovery of specific regulatory elements in viruses. The work will have direct clinical applications to our understanding and treatment of drug resistance in patients, and it will lay the groundwork for related studies in other clinically relevant viruses, such as HCV.

Project Summary/Abstract Core H - The purpose of the Protein Expression and Proteomics Analysis (PEP) Core is to provide state-of- the-art technology, equipment and expertise for the cloning, expression and purification of recombinant proteins required by the San Diego (SD) CFAR community, as well as the tools and expertise required for molecular analysis of proteins and protein mixtures. In particular, the PEP Core will provide protein expression and analytical tools not available to most laboratories and at a reduced cost affordable for CFAR members. The PEP Core is divided into two units based on services provided: The Protein Expression Unit (Co-Director, John Elder) will aid CFAR members as needed for cloning, expression and/or purification of proteins, based on the needs of the investigator. This unit will also maintain stocks of frequently requested viral and immunological proteins such as HIV gp120 and Gag antigens and will aid in the purification of antibodies, as required. The Proteomics Unit (Co-Director, Bruce Torbett) maintains state-of-the-art equipment and capabilities for protein analysis including nanospray mass spectrometer, HPLC, Typhoon 3-color laser for analysis of protein mixtures, light cycler instrumentation for analyzing protein mixtures, and equipment for 1- and 2-dimensional protein separations. These technologies and provided expertise will aid investigators in quantitative and qualitative analysis of their proteins and protein mixtures from cell samples. In addition, the PEP Core will provide training to CFAR members in all facets of protein expression and analysis offered by the Core, as requested by the community. Information will be provided by both one-on-one interactions, as well as through seminars on various techniques, based on the interests of the members. The long-term goal of the Core is to help facilitate efficient and economical analysis of proteins required by the CFAR membership to aid in the more rapid development of an understanding of the HIV life cycle and the development of intervention strategies against the global spread of HIV.

HIV-1 enters the nucleus of non-dividing cells where the reverse transcribed viral DNA is integrated into the host genome. Whereas the nuclear pore complex (NPC) prevents passive transport of large macromolecules, HIV-1 has evolved effective strategies to penetrate this barrier. The HIV-1 nuclear import is a poorly understood process that involves complex interactions with the nuclear import machinery, including several nucleoporins, transportin-3, and CPSF6, all of which bind the viral capsid core, which comprises hundreds of copies of the capsid protein (CA). Pleiotropic effects caused by nucleoporin knockdown and the ability of HIV-1 to use alternative import pathways have impeded the mechanistic studies of nuclear import and frustrated efforts to identify the full array of host factors involved in this process. Our single particle tracking experiments revealed that HIV-1 infection progresses through CA-dependent docking at the nuclear membrane, followed by uncoating (loss of CA) and CA-dependent nuclear transport to the sites of integration. However, very little is known regarding the molecular details and dynamics of virus-NPC interactions, including the structural changes in the architecture of both HIV-1 and the NPC in the course of nuclear import. There is thus an unmet need for structural and functional studies on the molecular mechanisms of HIV-1 nuclear import in the context of productive infection. We propose to combine cutting-edge approaches developed by our highly collaborative team to delineate the molecular interactions and structural changes in the virus and NPC during the nuclear import. Specifically, we will: (1) identify the host factors involved in HIV-1 import using novel proteomics and chemical cross-linking approaches; (2) obtain cryo-electron tomography (cryo-ET) and cross-linking mass-spec structures of the HIV-1 core/NPC complexes by capturing the incoming virus through our new on-demand pore clogging assay; and (3) develop correlative fluorescence/cryo-ET imaging pipeline to structurally characterize the intermediates of HIV-1 nuclear import. Knowledge of molecular interactions during the HIV-1 nuclear import should help identify novel therapeutic targets to block infection, provide a framework for studies of the nuclear import of other viruses and further fundamental understanding of the nuclear pore function.