Research

Host-Pathogen Pathways

We are interested in understanding how pathogens perturb host cellular pathways. As my colleague Benhur Lee says, “viruses are the best cell biologists”. We often end up learning more about host cellular pathways by understanding how pathogens manipulate them.

  • Innate immune pathways can sense a pathogen infection by detecting pathogen-associated molecular patterns (e.g., LPS for bacterial infections, or cytosolic DNA for some viral infections) and activate an antiviral or antibacterial cellular state. Pathogens have evolved mechanisms to circumvent innate immune pathways and we are working to identify these mechanisms.
  • Energy homeostasis pathways are found throughout life and maintain a balance between spending energy to replicate and grow when nutrients are plentiful and conserving energy under states of starvation or stress. Pathogens require vast energy stores for maximizing replication and frequently modulate key energy signaling nodes like AMPK and mTOR. We discovered a novel mechanism by which HIV-1 regulates AMPK and mTOR activity and we are now trying to understand how this benefits HIV-1 transcription, replication, and immune evasion.
  • DNA damage pathways are frequently modulated by pathogens in order to initiate DNA repair for integration of the pathogen genome, as is the case of HIV-1, or to inhibit DNA repair, as is the case for oncogenic viruses like human papillomaviruses and herpesviruses.
    • The HIV-1 Vpr gene is a long-standing mystery in the field of virology. Although it is required for infectivity in vivo, it is dispensable in most ex vivo systems and its function remains unclear. Vpr strongly activates DNA damage response pathways, and we are working to understand how it does so and how the activation of these pathways benefits HIV-1.

Post-Translational Modification (PTM) Enzyme – Substrate Relationships

Relationships between PTM enzymes (i.e., kinases, phosphatases, ubiquitin ligases, deubiquitinases, etc.) are difficult to identify because their physical interactions are often weak and/or transient. Shotgun proteomics technologies have matured to the point that it is feasible to identify these relationships by, for example, measuring all phosphorylation changes in response to kinase inhibition to identify those phosphorylation sites that are affected. We are now developing approaches to systematically identify PTM enzyme-substrate relationships in the specific molecular pathways described above. To do so we are developing genetic and chemical screening approaches to identify PTM enzymes that function in our pathways of interest, and we will then perform shotgun proteomics to identify affected substrates. Our focus is currently on discovering novel PTM enzyme-substrate relationships that function in innate immune regulation.

Mass Spectrometry (MS)-Based Proteomics Methods Development

We are a moderately hardcore mass spectrometry lab! We love developing new proteomics technologies and bioinformatics tools that can help us discover exciting biology.

  • Top-Down Proteomics is a subfield of proteomics where intact proteins are injected and fragmented in the MS. For most proteomics applications we digest material with trypsin before MS analysis. Intact protein analysis is more challenging but it has many advantages. Intact proteins retain all of their post-translational modifications in the MS. By analyzing proteins intact that allows to see PTMs in areas of a protein that may not be amenable to trypsin, and it allows us to observe combinations of PTMs that we can’t see when they are digested into different peptides. We are especially interested in using top-down proteomics to study PTMs on viral proteins.
  • PTM Enrichment and Analysis Strategies allow us to purify specific PTM types to get deep and comprehensive coverage of that PTM in a cellular system. We are developing approaches to analyze PTM types for which there are not yet well established methods for enrichment and analysis.
  • Data-Independent Acquisition (DIA) is a rapidly growing field of mass spectrometry that allows for comprehensive coverage of measurements across large numbers of samples. We have a very fancy mass spectrometry that is capable of performing an ion mobility separation that we believe will make DIA even better, and we are developing methods to acquire DIA data with ion mobility and software to analyze that data.
    • As part of the Dengue Human Immunology Project Consortium (led by Ana Sesma-Fernandez) we are using DIA to measure protein responses in Dengue virus-affected patient cohorts in Nicaragua in order to better predict their clinical outcomes.
  • Bioinformatics approaches are fundamental to our approach to science. We are always developing new ways to interpret the data that we generate and to present that data to the scientific community.