Small molecule screening identifies inhibitors of the Epstein-Barr virus deubiquitinating enzyme, BPLF1.

Herpesviral deubiquitinating enzymes (DUBs) were discovered in 2005, are highly conserved across the family, and are proving to be increasingly important players in herpesviral infection. EBV's DUB, BPLF1, is known to regulate both cellular and viral target activities, yet remains largely unstudied. Our work has implicated BPLF1 in a wide range of processes including infectivity, viral DNA replication, and DNA repair.

Monitoring PARKIN RBR Ubiquitin Ligase Activation States with UbFluor.

PARKIN is a RING-Between-RING (RBR) E3 ligase, which ubiquitinates mitochondrial proteins in response to mitochondrial damage. Ser of PARKIN is phosphorylated by kinase PINK1 (pPARKIN), which causes partial PARKIN activation. PINK1 also phosphorylates Ser of ubiquitin (pUb), which further activates pPARKIN.

Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8 T Cells in Chronic versus Acute Infection.

Distinct molecular pathways govern the differentiation of CD8 effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8 T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4 T cell populations.

High-Throughput Screening of HECT E3 Ubiquitin Ligases Using UbFluor.

HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases.

UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN.

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes.

UbFluor: A Fluorescent Thioester to Monitor HECT E3 Ligase Catalysis.

HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s.

Compensatory mutations restore the replication defects caused by cytotoxic T lymphocyte escape mutations in hepatitis C virus polymerase.

While human leukocyte antigen B57 (HLA-B57) is associated with the spontaneous clearance of hepatitis C virus (HCV), the mechanisms behind this control remain unclear. Immunodominant CD8(+) T cell responses against the B57-restricted epitopes comprised of residues 2629 to 2637 of nonstructural protein 5B (NS5B(2629-2637)) (KSKKTPMGF) and E2(541-549) (NTRPPLGNW) were recently shown to be crucial in the control of HCV infection. Here, we investigated whether the selection of deleterious cytotoxic T lymphocyte (CTL) escape mutations in the NS5B KSKKTPMGF epitope might impair viral replication and contribute to the B57-mediated control of HCV.

Quadratic behavior of fiber Bragg grating temperature coefficients.

We describe the characterization of the temperature and strain responses of fiber Bragg grating sensors by use of an interferometric interrogation technique to provide an absolute measurement of the grating wavelength. The fiber Bragg grating temperature response was found to be nonlinear over the temperature range -70 degrees C to 80 degrees C. The nonlinearity was observed to be a quadratic function of temperature, arising from the linear dependence on temperature of the thermo-optic coefficient of silica glass over this range, and is in good agreement with a theoretical model.