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Research at the Strosberg Lab

Virology:

Our laboratory analyzes the molecular determinants involved in protein-protein interactions in host pathogen relations. Most of our work at TSRI in Florida is focused on Hepatitis C virus (HCV) and its life cycle in Huh7.5 hepatoma cells. Transfer-of-energy assays were developed to demonstrate that dimerization of core, the HCV capsid protein, is essential for viral particle assembly. Peptide and small molecule inhibitors of dimerization potently reduce HCV production and infectivity (Kota et al., 2009; 2010; Wei et al., 2009; Ni et al., 2011). Furthermore, we showed that dimerization of core is essential for the interaction with the viral NS3 helicase (Mousseau et al., 2011). A biotinylated derivative of one of our small molecule inhibitors of core dimerization was shown to bind to core and to co-localize with core and NS5A, another HCV protein involved in viral particle assembly (Kota et al., submitted). Our results show that small molecular probes of protein interactions in HCV can serve as powerful tools to investigate the role of viral components in the assembly of the virus. Similar studies have been recently initiated on the HIV capsid protein p24.

A schematic model for assembly of the HCV particle

Figure 1. A schematic (left to right) model for assembly of the HCV particle. Core-directed assembly takes place at the surface of lipid droplets (“LD”). Core, originally made as a 191 residue protein, migrates from the endoplasmic reticulum to the surface of LD’s after removal of the C-terminal 14 residues by a host signal peptide peptidase. Core oligomers recruit to the LD surface NS3, NS5A, NS5B, and possibly other HCV replicase proteins implicated in the synthesis of HCV RNA, including p7 and NS2. HCV nucleocapsids resulting from the binding of RNA to core are then associated via unknown processes with HCV glycoproteins E1 and E2 and processed through the VLDL assembly pathway (not shown here), to yield lipid encapsulation, resulting in budding of viral particles. Whether the replicase complex as such interacts with core before or after RNA is made, and whether RNA binds to core dimer / oligomer on LD’s is not yet known, nor is the stoechiometry between core, RNA and the E1, E2 glycoproteins (modified from).

Cell biology:

We have previously developed the unique PAZ6 human brown pre-adipocyte cell line which we are now evaluating as a model to study potential markers and pathways in stem cell-derived brown adipocytes. We have already confirmed that the PAZ6 cells express most of the markers recently identified in primary murine cells, including PDRM16, the master regulator of brown adipocyte differentiation (Abdelkarim et al., 2011). The PAZ6 cells express myogenin, but not hcx9, in line with the hypothesis that PAZ6 cells are derived from the adipocyte/myocyte lineage rather than from white adipocyte precursors. Our goal is to identify pathways involved in human brown adipocyte differentiation and thermogenesis. 

Figure 2, Multilocular lipid accumulation in PAZ6 adipocytes upon differentiation

Figure 2: Multilocular lipid accumulation in PAZ6 adipocytes upon differentiation. (a) PAZ6 pre-adipocytes in culture. (b) Differentiated PAZ6 adipocytes. Differentiation was carried out for 3 weeks in propagation medium supplemented with 1nM T3, 850nM Insulin, 100nM Dexamethasone and 1mM Pioglitazone (Zilberfarb et al., 1997).

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