Principal Investigator

Vaughn Cooper, PhD
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Location

434 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219

Research Description

The primary goal of our laboratory is to understand how bacterial populations evolve and adapt to colonize hosts and cause disease. By studying evolution-in-action, both in experimental populations and in ongoing infections, and using the latest methods in genomic sequencing, we seek to identify mechanisms of bacterial adaptation in vitro and in vivo. We are particularly focused on how bacterial populations form complex communities within biofilms and how cells perceive cues to attach or disperse. We are also developing genome-based diagnostics for bacterial infections.

Our research on the ecology and evolution of bacterial biofilms has enabled our study of two very different topics that trace to a common evolutionary conflict: 1) the origins of multicellular life and 2) evolution within various forms of cancer. We are proud to be part of a NASA Astrobiology Institute that uses experimental evolution to pursue the goal: “To discover the laws that create Darwin’s ‘tangled bank’ remains one of biology’s grand challenges, one that requires understanding how differences among forms are selected for and how interdependence among forms is enforced.”

It is also now clear in the post-genomic age that cancers evolve in crowded spaces that resemble the high variation and mutation rates often seen in bacterial biofilms. The same tension of remaining adherent to clonemates but being metabolically confined, or dispersing to pursue new environments is found both in biofilms and cancers. A long-range goal is to advance understanding of evolutionary dynamics in structured communities, relevant to biofilms, solid tumors, and transitions to multicellularity.

We maintain an active research program studying why genome regions evolve at different rates, and how the forces of mutation, selection, drift, and recombination produce these patterns. A major factor predicting this rate variation is replication timing. We are using experimental and comparative methods to improve genome legibility, understand speciation, and to guide more rational treatment of disease states. 

Lastly, and perhaps most importantly, the fact that microbial populations evolve in real time and can produce conspicuous new forms has inspired a high-school curriculum for learning evolutionary biology, ecology, and biotechnology by simple experimentation. Not only do students learn better, they become more engaged in science. We hope to share this curriculum nationwide.

Lab Members:

Christopher Marshall, Research Assistant Professor

Alfonso Santo, Postdoctoral Associate

Emily Sileo, Research Assistant

Nate Phillips, Bioinformatics Research Assistant 

 



Principal Investigator

Nara Lee, PhD
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Location

541 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219

Research Description

Noncoding RNA-mediated regulation of transcription  

With the advent of deep sequencing technology, a plethora of noncoding RNAs (ncRNAs) with as yet unknown functions has been discovered. A subset of these ncRNAs is found in the nucleus and thus has been proposed to contribute to transcription regulation. How and which ncRNAs regulate transcription is the overarching question of the Lee lab. 

Epstein-Barr virus (EBV) is an oncogenic gamma-herpesvirus with a prevalence of over 90% in the human population. It is best known as the causative agent of mononucleosis, but is also associated with several types of cancers, such as lymphomas and carcinomas. EBV expresses two highly abundant nuclear ncRNAs called EBER1 (EBV-encoded RNA 1) and EBER2. The function of EBER1 is poorly understood, while EBER2 has recently been shown to facilitate the recruitment of an interacting transcription factor, PAX5, to the viral genome. Intriguingly, the recruitment mechanism entails RNA-RNA base pairing between EBER2 and nascent transcripts that originate from the target site. Upon recruitment, the EBER2-PAX5 ribonucleoprotein complex affects the transcription of nearby genes, probably by influencing the chromatin conformation of this region of the viral genome.

Our lab is studying the RNA-RNA based recruitment mechanism utilized in EBV in greater detail with the goal to extrapolate our findings to the host cell. Since viruses often adopt existing mechanisms from their hosts, our observation suggests that cellular ncRNAs might exist that use RNA-RNA interactions to guide transcription factors to their target sites. Such in trans activity of ncRNAs could potentially enhance the binding specificity of transcription factors by providing an additional attachment site on top of the binding motifs recognized by transcription factors. Combining RNA techniques with chromatin methodology, our lab is focusing on elaborating on this novel mechanistic aspect of transcription factor recruitment. Our studies aim to further categorize the many ncRNAs that have not yet been ascribed an apparent function.

LAB MEMBERS:

Adalena Nanni, Research Assistant

Sarah Haralam, Research Assistant



Principal Investigator

Patrick Thibodeau, PhD
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Location

537 Bridgeside Point II

450 Technology Drive

Pittsburgh, PA 15219

Research Description

My lab is interested in the fundamental principles of protein structure and dynamics. The acquisition of native protein structure and the dynamics associated with the native state are critical for proper protein biosynthesis and the regulation of protein function. Specifically, we are interested in transmembrane proteins and their roles in human physiology and pathogen virulence. The majority of this work is focused on understanding the roles of the ATP-Binding Cassette (ABC) transporter family of proteins in regulating normal physiology and the virulence of bacterial pathogens.

There are two main focuses of our current research:

First, we are interested in addressing structure-function and physiological questions related to mammalian ABC transporters. There are 48 known ABC transporters in the human genome. Among these, we are interested in understanding the functional and physiological roles of two: CFTR and ABCC6. Mutations in CFTR are responsible for cystic fibrosis, while mutations in ABCC6 are causative of ectopic mineralization disorders and are associated with premature heart disease. Our studies of these proteins are focused on elucidating the folding pathways that promote the formation of native state structure, identifying the mechanisms by which disease-causing mutations impact these pathways, and developing strategies that might be useful in correcting these defects.

Second, we are interested in the physical basis of Type I secretion in gram-negative bacteria and the role of the Type I exoproteins in bacterial virulence. Multiple human pathogens, including E. coli, P. aeruginosa, and B. pertussis utilize Type I secretion systems to export virulence factors and toxins. The secreted virulence factors range in size between 10 kDa to 1 MDa and alter host-pathogen interactions by facilitating adherence and modulating host responses to the pathogen.  We are focused on understanding the structural and functional regulation of the serralysin proteases, the physical mechanisms associated with their secretion, and their impact on host tissues during bacterial infection. Specifically, we are interested in the roles of these proteases in modulating host-pathogen interactions in P. aeruginosa infection of the airway. These studies focus on the role of both native and non-native proteins structures, the regulation of protease activity, and the effects of these exoproteases on host physiology.   

All of our studies rely on a combination of biochemical and biophysical approaches to evaluate protein structure and dynamics in vitro, including spectroscopy, X-ray crystallography, NMR, and functional biochemistry. These studies are complemented by cell culture and in vivo models, which rely on microscopy, biochemistry, and electrophysiological approaches to evaluate changes in protein structure and function in cellular environments.     

MEMBERS

Aiping Zheng, Research Associate



Principal Investigator

Gary Thomas, PhD

Location

534 Bridgeside Point II

450 Technology Drive

Pittsburgh, PA 15213

Research Description

My research program focuses on signaling pathways that integrate membrane traffic with the regulation of homeostasis and the onset of disease. These studies were grounded by our identification of the proprotein convertase furin, which is the first member of a family of secretory pathway-localized endoproteases that catalyze the activation of bioactive proteins and peptide hormones. While furin catalyzes the homeostatic activation of many growth factors, receptors and cell adhesion molecules, microbial pathogens frequently exploit the furin processing pathway. Indeed, furin has an essential role in the processing of viral envelope glycoproteins expressed by pathogenic viruses as well as in the proteolytic activation of many bacterial toxins. Although furin localizes to the trans-Golgi network (TGN), it can cleave such a diverse array of protein substrates because it moves between multiple processing compartments: the TGN/biosynthetic pathway, the cell surface and early endosomes. Our analysis of the complex intracellular trafficking pathway of furin led us to the discovery of the PACS family of homeostatic regulators, which integrate secretory pathway traffic and interorganellar communication in healthy cells with key steps in death ligand-induced apoptosis in diseased cells. Moreover, the PACS proteins have key roles in the nucleus where they regulate the transcriptional activity of p53 following DNA damage. Together, these findings form the basis for our current studies in cancer biology, viral pathogenesis and metabolism as summarized below:

Microbial pathogenesis:

Furin inhibitors: Using our determination of furin’s cleavage site specificity, we generated the first potent and selective furin inhibitor, α1-PDX. We showed α1-PDX can block the furin-dependent processing of envelope glycoproteins from many pathogenic viruses as well as the activation of bacterial toxins that require furin for their activation. Our current studies are focused on generating small molecule furin inhibitors that block pathogen activation in vivo and then develop these compounds into potential therapeutics.

HIV-1 accessory proteins: HIV-1 Nef is required for the onset of AIDS and can affect cells in many ways, including alteration of T-cell activation and maturation, promotion of viral infectivity, subversion of the apoptotic machinery and downregulation of cell-surface molecules, including MHC-I. We discovered that HIV-1 Nef directs a temporally regulated program to downregulate MHC-I in virally infected cells. During the first two-days post-infection Nef binds the PACS proteins to assemble a multi-kinase complex that triggers endocytosis and sequestration of cell-surface MHC-I. By day three Nef switches to a stoichiometric mode that downregulates MHC-I by blocking the cell-surface delivery of newly synthesized MHC-I molecules. We identified small molecule inhibitors that block the ability of Nef to assemble the multi-kinase complex and thus downregulate MHC-I. Importantly, recent studies suggest the ability of Nef to assemble the multi-kinase complex is central to its ability to drive disease. Because of the key role of the PACS proteins in Nef action, we have mapped the sites on HIV-1 Nef and the PACS proteins essential for their interaction. Our future studies will determine to what extent assembly of the multi-kinase complex enables Nef to drive disease, identify small molecule inhibitors of Nef action and determine the structure of the Nef-PACS complex.

Cancer biology:

Mechanism of TRAIL action: We determined that the death ligand TRAIL switches PACS-2 from a secretory pathway trafficking protein to an apoptotic effector that promotes lysosome-mitochondria communication leading to cytochrome c release and death of cancer cells. Molecularly, this switch is manifest by binding of 14-3-3 proteins to a site on PACS-2 phosphorylated by the survival kinase Akt. We identified how cancer cells or an anti-apoptotic herpesvirus protein can block PACS-2 from inducing apoptosis, suggesting key role for PACS-2 in TRAIL-induced apoptosis. Our current studies are investigating to what extent PACS-2 mediates the ability of TRAIL to inhibit tumor metastasis in vivo and how TRAIL signals to PACS-2 to direct membrane trafficking events leading to mitochondria membrane permeabilization and executioner caspase activation.

DNA damage response: We found that PACS-2 is a key regulator of the DNA damage response in vivo. Specifically, following DNA damage triggered by ionizing radiation or chemotherapeutics, p53-induced transcription of the cell cycle inhibitor p21 is repressed both in PACS-2-/- mice as well as in PACS-2 siRNA knockdown cells. This repressed transcriptional activity correlates with the hypoacetylation of p53 bound to the p21promoter. Consistent with these findings, PACS-2 interacts with the class III histone deacetylase SIRT1, which blunts p53 action by deacetylating p53 following DNA damage. Our preliminary studies suggest PACS-2 mediates the p53-p21 axis by inhibiting SIRT1. Our future studies will identify the precise mechanism by which PACS-2 regulates SIRT1 enzyme activity, how PACS-2 traffics between the cytosol and nucleus, and how this trafficking is regulated by DNA damage.

Metabolism:

Obesity: Consistent with our findings that suggest PACS-2 is a negative regulator of SIRT1 activity, PACS-2-/- mice are resistant to diet-induced obesity but clear glucose more efficiently than WT mice. Indeed, these findings parallel reports of the effect of SIRT1 activators in vivo. Our future studies will rigorously phenotype the PACS-2-/- mice and will determine to what extent PACS-2 regulation of SIRT1 controls endocrine homeostasis.

Lab Members:

Aki Nishimura-Gasparian, Research Technician



Principal Investigator

Kathy H.Y. Shair, PhD

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Location

1.8 Hillman Cancer Center

5117 Centre Avenue

Pittsburgh, PA 15213

Research Description

Epstein-Barr virus (EBV) is an oncogenic γ-herpesvirus that is associated with human epithelial and B cell malignancies.  The Shair lab studies the molecular mechanisms of cancer induced by EBV latency with the purpose of defining how these mechanisms contribute to the oncogenic and metastatic properties of EBV-associated diseases.  

The oncogenic and cancer-associated properties of the viral latent membrane proteins (LMP) 1 and LMP2A are well established however, particularly for epithelial infections, the molecular interplay between these viral proteins and their function in EBV pathogenesis are largely undetermined.  Furthermore, metastatic nasopharyngeal carcinoma remains the most challenging condition to treat.

EBV-associated cancers have a characteristic latent gene expression pattern.  EBV immortalizes primary B cells and LMP1 is critically required for this process.  In comparison, expression of LMP1 or LMP2A proteins in epithelial cells can promote growth and migratory properties, often resulting in increased tumor-forming potential in cell lines transplanted as xenografts in mice.  Our studies in transgenic mice have shown that LMP2A complements LMP1 in tumor models, promoting carcinogen-induced carcinoma incidence and can also induce unique gene expression changes in B cells that are only apparent in the presence of both proteins.  This provided the first in vivo evidence that LMP1 and LMP2A functionally co-operate to result in unique phenotypes.  A major goal of the Shair lab is to elucidate mechanisms of LMP1 and LMP2A co-operation and to determine which interacting cellular pathways are most relevant to tumorigenesis and metastasis.

Current projects:

1. EBV mechanisms of genomic instability: This project investigates LMP1 and LMP2A mechanisms in nasopharyngeal carcinoma and pediatric post-transplant lymphoproliferative disease 

2. Determinants of EBV pathogenesis and persistence in epithelial infections: This project involves developing polarized infection models to study EBV pathogenesis in respiratory epithelia

3. Small animal models of EBV oncogenesis: This project involves testing EBV oncogenic proteins in transgenic and humanized mice

4. Discovery of cancer biomarkers: This project screens human sera for EBV biomarkers

Dr. Shair conducts research through the University of Pittsburgh Cancer Institute (UPCI) Cancer Virology Program at the Hillman Cancer Center located in Shadyside.  Learn more>

Members

Amit Kumar - Postdoctoral Associate

Elizabeth Caves – Undergraduate researcher

Akhil Reddy – Undergraduate researcher



Principal Investigator

James E. Bina, PhD
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Location

443 Bridgeside Point II
450 Technology Dr.
Pittsburgh, PA 15219

Research Description

Our research is centered on defining the molecular mechanisms used by bacteria to resist antibiotics and cause disease in humans. Our work currently focuses on two important gram negative human pathogens: Vibrio cholerae and Francisella tularensis.

Vibrio cholerae

V. cholerae is a highly motile gram-negative, facultative human pathogen that causes the potentially lethal diarrheal disease cholera. V. cholerae is a significant health threat in the developing world where this organism is responsible for an estimated 3-5 million cholera cases each year with a mortality rate of ~1.5%. Cholera is acquired by ingestion of food or water contaminated with V. cholerae. Upon ingestion, V. cholerae colonizes the small intestine where the organism produces a variety of virulence factors that lead to disease.

Virulence factor production in V. cholerae is induced in vivo in response to unknown stimuli. A key question in cholera research is to determine how these unknown stimuli effect gene expression in vivo. We recently showed that cFP, a cyclic dipeptide that is produced by V. cholerae, functioned as an inhibitor of virulence factor production. Since cFP is produced in a growth-dependent manner, and functions independently of the quorum sensing systems, we hypothesize that cFP functions as a novel cell density-dependent signaling molecule that regulates gene expression during pathogenesis. We are currently working to define the genes involved in cFP biosynthesis and to define the signal transduction pathway by which cFP inhibits virulence factor production.  We are also exploring the possible use of cFP-like chemicals as novel anti-virulence therapeutics for cholera treatment.

In addition to the production of virulence factors, to cause disease V. cholerae must also protect itself from the antibacterial effects of toxic molecules that are present in the host gastrointestinal tract. V. cholerae does this by the expression of active efflux systems belonging to the RND family. The RND family efflux systems are ubiquitous transporters found among gram negative bacteria. The RND systems function to remove toxic molecules from within the cell and thus contribute to the evolution of antibiotic resistance. We have shown that the RND systems are also required for the production of virulence factors in V. cholerae. This suggested that there is a relationship between RND efflux activity and virulence gene expression and provided the first evidence that the RND efflux systems influence pathogenesis by a mechanism other than antimicrobial resistance. We are currently studying the genetic linkage between efflux and virulence factor production.

Francisella tularensis

F. tularensis is a gram negative bacterium that causes the zoonotic disease tularemia.  F. tularensis is one of the most infectious pathogens known with an LD50 of  fewer than 10 bacteria. F. tularensis is most frequently transmitted to humans by insect vectors or the handling of contaminated material, but can also be transmitted by inhalation.  Untreated, inhalation tularemia is associated with a 30-60% mortality rate. The high infectivity, high virulence, and easy of dissemination by aerosols have led to the development of F. tularensis as a bioweapon by several nations. These properties have also let to growing concerns about the potential use of F. tularensis in bioterrorism.

Very little is known about F. tularensis pathogenesis. During infection of mammalian hosts F. tularensis is believed to grow intracellularly in macrophages. Subsequently, F. tularensis inhibits both phagosome and lysosome fusion by an unknown mechanism. Recent findings suggest that F. tularensis, like Listeria monocytogenes, rapidly escapes the phagosome and resides and replicates within the cytoplasm of host cells. Following an initial growth lag, intracellular F. tularensis enters logarithmic growth by 12 hours post-infection and eventually induces apoptosis and cell death.

My laboratory is working on the development and application of Francisella-specific genetic tools to define potential virulence factors and vaccine targets in this organism. We employ  multiple disciplinary approaches including genetics, genomics, biochemistry and immunology and are collaborating with investigators at Emory University and the University of Tennessee Health Science Center to characterize the function of F. tularensis genes in virulence  and immune evasion.

Lab Member:  Dillon Kunkle, Graduate Student



Principal Investigator

Li Lan, MD, PhD
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Location

2.6 Hillman Cancer Center
5117 Centre Avenue
Pittsburgh, PA 15213

Research Description

Dr Lan's Lab is focusing on analyzing DNA damage response chromatin remodeling mechanisms at specific genome loci.

Eukaryotic cells produce chromatin remodeling factors (CRFs) to increase the accessibility of repair enzymes to DNA lesions in the context of chromatin.
ACF1 and CHD5 are CRFs that contain ATPase remodeling and chromatin binding domains, however, their role in DNA repair is largely unknown. Defects in both the repair of  DNA damage and in CRFs are implicated in tumorigenesis, but their interplay is not understood. To understand the damage responses of repair and CRFs in cells, we have established novel systems to produce either a mixture or a specific type of DNA damage in situ: 1. a UVA Laser Micro-Irradiation System produces a mixture of SSB, DSB and base damage; 2. an XPA-UVDE cell line irradiated with local UVC specifically induces SSB; 3. the I-SCEI endonuclease specifically induces DSB in human cells. We have used these experimental systems to screen the CRFs that might be involved in damage response and repair, in order to understand how repair is affected by CRF function. We found a novel tumor suppressor gene, CHD5, which is recruited to sites of DNA damage and necessary for repair. We will identify the role chromatin remodeling and histone modifications in tumorigenesis using in situ approaches and in vitro studies. We will analyze the precise mechanisms of CRF function in repair using in vivo and in vitro methods.
 
Environmental and endogenous reactive oxygen species (ROS) induced DNA base damage and single strand breaks block DNA replication, thereby leading to genome instability and genetic alterations. Therefore, understanding the dynamics of DNA damage response (DDR) in living cells will have broad implications for our understanding of the root cause of cancer and diseases. 

We developed a visualization of DNA damage response at the single molecule level in real time using a novel method for expressing KillerRed, a marker of defined chromatin sites of DNA damage in human cells.

KillerRed photosensitizers are chromophores that generate natural ROS that induce DNA damage upon light irradiation. We have established a tetracycline responsive element (TRE) integrated U2OS cell line at defined sites of chromatin and tagged KillerRed with the tetracycline repressor (tetR). Via the interaction of tetR and TRE, KillerRed will be expressed at defined sites of the genome. We will define the DDR and mechanisms of current chromatin remodeling factors using this novel system. We also have developed a system to visualize the DNA damage response at the site of telomere. The effects of ROS damage at the sites of telomere on telomere integrity, cell death is going to be analyzed. The function of early aging Werner syndrome gene, WRN protein, will be analyzed.

Dr. Lan conducts her research at the University of Pittsburgh Cancer Institute.

Members

Xiukai Chen, Visiting Scholar

Rong Tan, Health Science Fellow

Jacqueline Welty, Graduate Student

Haibo Yang, Health Science Researcher

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