Principal Investigator

Zandrea Ambrose, PhD
View profile 

Location

541 Bridgeside Point 2
450 Technology Drive
Pittsburgh, PA 15219

Research Description

The Ambrose laboratory studies antiretroviral therapeutics used for HIV prevention and suppression, including the characterization of new drug targets against early steps of the virus life cycle, and the impact of drug resistance on HIV transmission, prevention, and treatment.

Understanding early HIV infection steps 

We investigate the early post-entry events in HIV infection of different cell types as part of the Pittsburgh Center for HIV Protein Interactions. Specifically, we study HIV capsid uncoating, reverse transcription, and nuclear entry using molecular and cellular biology, including innovative imaging techniques. Understanding these cellular pathways and the host-pathogen interactions associated with them may provide potential novel therapeutic targets for virus inhibition. Previously we identified a capsid mutation, N74D, that disrupts HIV interaction with the host protein CPSF6 and uses a different nuclear import pathway for viral DNA. CPSF6 binds to the karyopherin protein TNPO3 for nuclear import through the nuclear pore complex, which contains two proteins, Nup358 and Nup153, required for HIV infection. We continue to study the processes of HIV capsid uncoating and its influence on reverse transcription, trafficking and entry into the nucleus, and use of host cell factors.

 

Transmission and prevention of drug-resistant HIV 

Daily oral pre-exposure prophylaxis (PrEP) using two antiretroviral drugs is effective at preventing HIV transmission in high-risk populations. We are currently evaluating long-acting non-nucleoside reverse transcriptase inhibitors as PrEP. A concern in using antiretroviral drugs for both treatment of HIV-infected individuals and for PrEP for uninfected individuals is the potential for transmission of or development of drug-resistant HIV during PrEP. The Ambrose Lab studies the efficacy of long-acting PrEP in preventing transmission of drug-resistant HIV. In addition, we evaluate whether long-acting PrEP can lead to development of drug-resistant mutations, using single-genome sequencing methods. If resistant HIV develops or is transmitted, we investigate how this impacts subsequent antiretroviral therapy (ART).

 

Establishment and persistence of HIV reservoirs 

The Ambrose Lab studies diversity of HIV/SIV that develops in the blood and in tissues before, during, and after antiretroviral therapy to identify the nature and dynamic properties of persistent viral reservoirs at different anatomical sites. We showed that viral evolution and compart- mentalization is unique in mucosal tissues, such as the gastrointestinal and female genital tracts that are sites of mucosal transmission, compared to the blood or lymphoid tissues. For example, the composition of the viral DNA population in the blood and lymph nodes is mostly wild-type over time. However, the viral DNA population in the gastrointestinal tract becomes dominated by mutant viruses, suggesting higher turnover of infected cells in the gut compared to the blood. Recently we started investigating the influence of M. tuberculosis infection and immunity on SIV replication during co-infection, focusing on the blood and lung and using MiSeq deep sequencing.

Lab Members

Douglas Fischer, MS - Graduate Student

Chandra Nath Roy, PhD - Postdoctoral Fellow

Austin Souryavong - Undergraduate Researcher

Youya Wang - Undergraduate Researcher

Zhou Zhong - Graduate Student



Principal Investigator

Vaughn Cooper, PhD
View profile

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

Eisha Mhatre, Research Scholar

Christopher Deitrick, Bioinformatics Research Specialist

Joshua Beatty, Research Assistant



Principal Investigator

Nara Lee, PhD
View profile

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:

Belle Henry, Research Assistant

Jack Kanarek, Research Assistant



Principal Investigator

Patrick Thibodeau, PhD
View profile

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:

You Jin Choi, Postdoctoral Associate

Sabrina Villar-Pazos, Postdoctoral Associate



Principal Investigator

Kathy H.Y. Shair, PhD

View profile

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

Gabriella Zarkovic, Postdoctoral Associate

Elizabeth Caves – Undergraduate researcher

Akhil Reddy – Undergraduate researcher



Principal Investigator

James E. Bina, PhD
View profile

Location

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

Research Description

The Bina lab studies mechanisms of antimicrobial resistance and bacterial pathogenesis in the gram-negative pathogens Vibrio cholerae and Klebsiella pneumoniae. The long-term goal of our studies is to define the underlying processes that allow bacteria use to cause disease and resist antibiotics. There are two ongoing research projects:

I. Function of the RND transporters in V. cholerae pathogenesis. RND efflux systems are ubiquitous transporters in Gram-negative bacteria and have critical functions in antimicrobial resistance. Independent of antimicrobial resistance, RND transporters also effect the expression of diverse phenotypes including virulence, metabolism, and environmental adaptation. Thus, RND transporters fulfil unknown physiological functions in addition to their role in antimicrobial resistance.  

V. cholerae is an important human pathogen that causes ~5 million cases of the epidemic diarrheal disease cholera each year. V. cholerae is also a model enteric pathogen. Like most enteric pathogens, V. cholerae encodes multiple RND transporters that contribute to diverse phenotypes. Our previous studies documented that the RND systems were required for V. cholerae pathogenesis and antimicrobial resistance. Ongoing work is focused on characterizing the molecular mechanisms that link RND-mediated efflux to pathogenesis and identifying novel inhibitors of the RND efflux systems.

II. Antimicrobial resistance in K. pneumoniae. K. pneumoniae is an understudied gram-negative pathogen that causes a multitude of healthcare-associated infections including pneumonia, bloodstream infections, urinary tract infections, wound or surgical site infections, and meningitis. K. pneumoniae has rapidly evolved resistance to all clinically relevant antibiotics. This resulted in K. pneumoniae being identified as a member of the ESKAPE pathogens and included in the World Health Organization’s Priority 1 list of antibiotic-resistant pathogens for which new therapeutics are critically needed. We are working to define and characterize the intrinsic mechanisms that allow K. pneumoniae to resist antibiotics and persist in humans during infection.

Lab Member:  Dillon Kunkle, Graduate Student

Syndicate content