The kinase CK1ɛ controls the antiviral immune response by phosphorylating the signaling adaptor TRAF3.

The signaling adaptor TRAF3 is a highly versatile regulator of both innate immunity and adaptive immunity, but how its phosphorylation is regulated is still unknown. Here we report that deficiency in or inhibition of the conserved serine-threonine kinase CK1ɛ suppressed the production of type I interferon in response to viral infection. CK1ɛ interacted with and phosphorylated TRAF3 at Ser349, which thereby promoted the Lys63 (K63)-linked ubiquitination of TRAF3 and subsequent recruitment of the kinase TBK1 to TRAF3. Consequently, CK1ɛ-deficient mice were more susceptible to viral infection. Our findings establish CK1ɛ as a regulator of antiviral innate immune responses and indicate a novel mechanism of immunoregulation that involves CK1ɛ-mediated phosphorylation of TRAF3.

Reductive Amination Combining Dimethylation for Quantitative Analysis of Early-Stage Glycated Proteins.

Due to the critical role glycation plays in many serious pathological conditions, such as diabetes, it is of great significance to discover protein glycation at an early stage for precaution and prediction of the disease. Here, a method of reductive amination combining dimethylation (RAD) was developed for the quantification of early-stage glycated proteins. The quantitative analysis was first carried out by reducing the samples using NaBH3CN or NaBD3CN, resulting in a 1 Da mass shift and the stabilization of early-stage protein glycation. The two samples were then digested and isotopically dimethylated to achieve the mass shift of 4 m + 3 n ( m represents the number of N-termini and Lys residues, and n represents the number of glycated sites) between light- and heavy-labeled glycated peptides for quantification. Consequently, the false positive result can be removed according to the different mass shifts of glycated peptides and non-glycated peptides. In quantification of glycated myoglobin, RAD showed good linearity ( R2 > 0.99) and reproducibility (CVs ≤ 1.6%) in 2 orders of magnitude (1:10-10:1). RAD was then applied to quantify the endogenous glycated proteins in the serum of diabetic patients, revealing significant differences in the glycation level between the patients with complicated retinal detachment and those without. In conclusion, RAD is an effective method for quantifying endogenous glycated proteins.

Interlaboratory Co-validation of a UPLC-ToF MS MAM Method for Truncations of a Fc Fusion Protein.

BACKGROUND: Peptide-Fc fusion proteins are inherently heterogeneous and complex molecules. Protein post-translational modifications (PTMs) or truncation can arise during manufacturing or product storage. Some of these product attributes could potentially impact the efficacy or safety of the bio-molecule and are thus classified as critical quality attributes (CQAs). These CQAs should be controlled in order to ensure manufacturing and quality consistency. METHODS: A subunit UPLC-ToF MS based MAM method was developed for identity test and quantitatively monitored two critical quality attributes (CQAs) resulting from two truncations of that fusion protein (fragment 1 and 2). Three independent laboratories are involved in the method validation according to ICH Q2(R1), ICH Q6B, FDA and NMPA guidance. RESULTS: This developed method fully meets the pre-defined analytical target profile (ATP), including specificity, accuracy, precision, quantitation limit, linearity, range and robustness. Three independent labs co-validate a UPLC-ToF MS based MAM method for protein drug QC release and stability testing. CONCLUSION: The experimental design of method validation can be a reference for LC-HRMS-based subunit MAM methods that have been widely used in the characterization of antibodies, ADCs and other protein-based biologics. This work paves the way for implementing MAM in QC with more targeted control of product quality.

Proteomic Analysis of the Hippocampus in Mouse Models of Trigeminal Neuralgia and Inescapable Shock-Induced Depression.

To investigate the behavioral and biomolecular similarity between neuralgia and depression, a trigeminal neuralgia (TN) mouse model was established by constriction of the infraorbital nerve (CION) to mimic clinical trigeminal neuropathic pain. A mouse learned helplessness (LH) model was developed to investigate inescapable foot-shock-induced psychiatric disorders like depression in humans. Mass spectrometry was used to assess changes in the biomolecules and signaling pathways in the hippocampus from TN or LH mice. TN mice developed not only significant mechanical allodynia but also depressive-like behaviors (mainly behavioral despair) at 2 weeks after CION, similar to LH mice. MS analysis demonstrated common and distinctive protein changes in the hippocampus between groups. Many protein function families (such as cell-to-cell signaling and interaction, and cell assembly and organization,) and signaling pathways (e.g., the Huntington's disease pathway) were involved in chronic neuralgia and depression. Together, these results demonstrated that the LH and TN models both develop depressive-like behaviors, and revealed the involvement of many psychiatric disorder-related biomolecules/pathways in the pathogenesis of TN and LH.

ELISA-PLA: A novel hybrid platform for the rapid, highly sensitive and specific quantification of proteins and post-translational modifications.

Detection of low-abundance proteins and their post-translational modifications (PTMs) remains a great challenge. A conventional enzyme-linked immunosorbent assay (ELISA) is not sensitive enough to detect low-abundance PTMs and suffers from nonspecific detection. Herein, a rapid, highly sensitive and specific platform integrating ELISA with a proximity ligation assay (PLA), termed ELISA-PLA, was developed. Using ELISA-PLA, the specificity was improved by the simultaneous and proximate recognition of targets through multiple probes, and the sensitivity was significantly improved by rolling circle amplification (RCA). For GFP, the limit of detection (LOD) was decreased by two orders of magnitude compared to that of ELISA. Using site-specific phospho-antibody and pan-specific phospho-antibody, ELISA-PLA was successfully applied to quantify the phosphorylation dynamics of ERK1/2 and the overall tyrosine phosphorylation level of ERK1/2, respectively. ELISA-PLA was also used to quantify the O-GlcNAcylation of AKT, c-Fos, CREB and STAT3, which is faster and more sensitive than the conventional immunoprecipitation and western blotting (IP-WB) method. As a result, the sample consumption of ELISA-PLA was reduced 40-fold compared to IP-WB. Therefore, ELISA-PLA could be a promising platform for the rapid, sensitive and specific detection of proteins and PTMs.