TY - JOUR T1 - A novel polyubiquitin chain linkage formed by viral Ubiquitin is resistant to host deubiquitinating enzymes. JF - Biochem J Y1 - 2020 A1 - Negi, Hitendra A1 - Reddy, Pothula Purushotham A1 - Vengayil, Vineeth A1 - Patole, Chhaya A1 - Laxman, Sunil A1 - Das, Ranabir AB -

The Baculoviridae family of viruses encode a viral Ubiquitin (vUb) gene. Though the vUb is homologous to the host eukaryotic Ubiquitin (Ub), its preservation in the viral genome indicates unique functions that are not compensated by the host Ub. We report the structural, biophysical, and biochemical properties of the vUb from Autographa californica multiple nucleo-polyhedrosis virus (AcMNPV). The packing of central helix α1 to the beta-sheet β1-β5 is different between vUb and Ub. Consequently, its stability is lower compared with Ub. However, the surface properties, ubiquitination activity, and the interaction with Ubiquitin-binding domains are similar between vUb and Ub. Interestingly, vUb forms atypical polyubiquitin chain linked by lysine at the 54th position (K54), and the deubiquitinating enzymes are ineffective against the K54-linked polyubiquitin chains. We propose that the modification of host/viral proteins with the K54-linked chains is an effective way selected by the virus to protect the vUb signal from host DeUbiquitinases.

VL - 477 IS - 12 ER - TY - JOUR T1 - Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition. JF - J Proteome Res Y1 - 2020 A1 - Bowling, Heather L A1 - Kasper, Amanda A1 - Patole, Chhaya A1 - Venkatasubramani, Janani Priya A1 - Leventer, Sarah Parker A1 - Carmody, Erin A1 - Sharp, Kevin A1 - Berry-Kravis, Elizabeth A1 - Kirshenbaum, Kent A1 - Klann, Eric A1 - Bhattacharya, Aditi AB -

Aberrant protein synthesis and protein expression are a hallmark of many conditions ranging from cancer to Alzheimer's. Blood-based biomarkers indicative of changes in proteomes have long been held to be potentially useful with respect to disease prognosis and treatment. However, most biomarker efforts have focused on unlabeled plasma proteomics that include nonmyeloid origin proteins with no attempt to dynamically tag acute changes in proteomes. Herein we report a method for evaluating de novo protein synthesis in whole blood liquid biopsies. Using a modification of the "bioorthogonal noncanonical amino acid tagging" (BONCAT) protocol, rodent whole blood samples were incubated with l-azidohomoalanine (AHA) to allow incorporation of this selectively reactive non-natural amino acid within nascent polypeptides. Notably, failure to incubate the blood samples with EDTA prior to implementation of azide-alkyne "click" reactions resulted in the inability to detect probe incorporation. This live-labeling assay was sensitive to inhibition with anisomycin and nascent, tagged polypeptides were localized to a variety of blood cells using FUNCAT. Using labeled rodent blood, these tagged peptides could be consistently identified through standard LC/MS-MS detection of known blood proteins across a variety of experimental conditions. Furthermore, this assay could be expanded to measure de novo protein synthesis in human blood samples. Overall, we present a rapid and convenient de novo protein synthesis assay that can be used with whole blood biopsies that can quantify translational change as well as identify differentially expressed proteins that may be useful for clinical applications.

VL - 19 IS - 9 ER -