Optimization of Protocols for Detection of De Novo Protein Synthesis in Whole Blood Samples via Azide-Alkyne Cycloaddition.

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.

Proteomic Approaches to Dissect Neuronal Signalling Pathways.

With an increasing awareness of mental health issues and neurological disorders, "understanding the brain" is one of the biggest current challenges in biological research. This has been recognised by both governments and funding agencies, and it includes the need to understand connectivity of brain regions and coordinated network activity, as well as cellular and molecular mechanisms at play. In this chapter, we will describe how we have taken advantage of different proteomic techniques to unravel molecular mechanisms underlying two modulators of neuronal function: Neurotrophins and antipsychotics.

Proteomic approaches to dissect neuronal signaling pathways.

With an increasing awareness of mental health issues and neurological disorders, "understanding the brain" is one of the biggest current challenges in biological research. This has been recognized by both governments and funding agencies, and includes the need to understand connectivity of brain regions and coordinated network activity, as well as cellular and molecular mechanisms at play. In this chapter, we will describe how we have taken advantage of different proteomic techniques to unravel molecular mechanisms underlying two modulators of neuronal function: Neurotrophins and antipsychotics.

Opto4E-BP, an optogenetic tool for inducible, reversible, and cell type-specific inhibition of translation initiation.

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is one of the primary triggers for initiating cap-dependent translation. Amongst its functions, mTORC1 phosphorylates eIF4E-binding proteins (4E-BPs), which prevents them from binding to eIF4E and thereby enables translation initiation. mTORC1 signaling is required for multiple forms of protein synthesis-dependent synaptic plasticity and various forms of long-term memory (LTM), including associative threat memory. However, the approaches used thus far to target mTORC1 and its effectors, such as pharmacological inhibitors or genetic knockouts, lack fine spatial and temporal control. The development of a conditional and inducible eIF4E knockdown mouse line partially solved the issue of spatial control, but still lacked optimal temporal control to study memory consolidation. Here, we have designed a novel optogenetic tool (Opto4E-BP) for cell type-specific, light-dependent regulation of eIF4E in the brain. We show that light-activation of Opto4E-BP decreases protein synthesis in HEK cells and primary mouse neurons. In situ , light-activation of Opto4E-BP in excitatory neurons decreased protein synthesis in acute amygdala slices. Finally, light activation of Opto4E-BP in principal excitatory neurons in the lateral amygdala (LA) of mice after training blocked the consolidation of LTM. The development of this novel optogenetic tool to modulate eIF4E-dependent translation with spatiotemporal precision will permit future studies to unravel the complex relationship between protein synthesis and the consolidation of LTM.