Large-scale map of RNA binding protein interactomes across the mRNA life-cycle.

Messenger RNAs (mRNAs) interact with RNA-binding proteins (RBPs) in diverse ribonucleoprotein complexes (RNPs) during distinct life-cycle stages for their processing and maturation. While substantial attention has focused on understanding RNA regulation by assigning proteins, particularly RBPs, to specific RNA substrates, there has been considerably less exploration leveraging protein-protein interaction (PPI) methodologies to identify and study the role of proteins in mRNA life-cycle stages. To address this gap, we generated an RNA-aware RBP-centric PPI map across the mRNA life-cycle by immunopurification (IP-MS) of ~100 endogenous RBPs across the life-cycle in the presence or absence of RNase, augmented by size exclusion chromatography (SEC-MS). Aside from confirming 8,700 known and discovering 20,359 novel interactions between 1125 proteins, we determined that 73% of our IP interactions are regulated by the presence of RNA. Our PPI data enables us to link proteins to life-cycle stage functions, highlighting that nearly half of the proteins participate in at least two distinct stages. We show that one of the most highly interconnected proteins, ERH, engages in multiple RNA processes, including via interactions with nuclear speckles and the mRNA export machinery. We also demonstrate that the spliceosomal protein SNRNP200 participates in distinct stress granule-associated RNPs and occupies different RNA target regions in the cytoplasm during stress. Our comprehensive RBP-focused PPI network is a novel resource for identifying multi-stage RBPs and exploring RBP complexes in RNA maturation.

SEC-TMT facilitates quantitative differential analysis of protein interaction networks.

The majority of cellular proteins interact with at least one partner or assemble into molecular-complexes to exert their function. This network of protein-protein interactions (PPIs) and the composition of macromolecular machines differ between cell types and physiological conditions. Therefore, characterizing PPI networks and their dynamic changes is vital for discovering novel biological functions and underlying mechanisms of cellular processes. However, producing an in-depth, global snapshot of PPIs from a given specimen requires measuring tens to thousands of LC-MS/MS runs. Consequently, while recent works made seminal contributions by mapping PPIs at great depth, almost all focused on just 1-2 conditions, generating comprehensive but mostly static PPI networks. In this study we report the development of SEC-TMT, a method that enables identifying and measuring PPIs in a quantitative manner from only 4-8 LC-MS/MS runs per biological sample. This was accomplished by incorporating tandem mass tag (TMT) multiplexing with a size exclusion chromatography mass spectrometry (SEC-MS) work-flow. SEC-TMT reduces measurement time by an order of magnitude while maintaining resolution and coverage of thousands of cellular interactions, equivalent to the gold standard in the field. We show that SEC-TMT provides benefits for conducting differential analyses to measure changes in the PPI network between conditions. This development makes it feasible to study dynamic systems at scale and holds the potential to drive more rapid discoveries of PPI impact on cellular processes.

Tissue engineered artificial liver model based on viscoelastic hyaluronan-collagen hydrogel and the effect of EGCG intervention on ALD.

In alcoholic liver disease (ALD) research, animal models, as one of the most popular methods to explore pathology and therapeutic drug screening, show the limitations of expensive cost and ethic, as well as long modeling time. To minimize the use of animal models in ALD research, an artificial liver model has been developed by incorporating HepG2 cells into hydrogel matrix based on difunctional hyaluronan and collagen. And on this basis an alcohol-induced ALD model in vitro by adding alcohol in the engineering process has been established. Results showed that the construct exhibited a simulated synthetic and metabolic liver function thanks to the bionic fibrillar and viscoelastic characteristics of hydrogels. And the in vitro alcohol-induced ALD model was also proved to be successfully established, even presenting equal results with ALD mice. Furthermore, epigallocatechin gallate (EGCG) as an intervention on ALD was confirmed in both in vitro and in vivo model. The findings indicate our simple artificial liver model is not only highly predictive but also easy to apply to drug screening and implantation studies, suggesting a promising alternative to animal models. Moreover, as the main active ingredient of tea, EGCG's effective intervention and reversal effect on fatty liver provides support for the theory that green tea could prevent alcoholic fatty liver.

Spatiotemporal regulation of dynamic cell microenvironment signals based on an azobenzene photoswitch.

Dynamic biochemical and biophysical signals of cellular matrix define and regulate tissue-specific cell functions and fate. To recapitulate this complex environment in vitro, biomaterials based on structural- or degradation-tunable polymers have emerged as powerful platforms for regulating the "on-demand" cell-material dynamic interplay. As one of the most prevalent photoswitch molecules, the photoisomerization of azobenzene demonstrates a unique advantage in the construction of dynamic substrates. Moreover, the development of azobenzene-containing biomaterials is particularly helpful in elucidating cells that adapt to a dynamic microenvironment or integrate spatiotemporal variations of signals. Herein, this minireview, places emphasis on the research progress of azobenzene photoswitches in the dynamic regulation of matrix signals. Some techniques and material design methods have been discussed to provide some theoretical guidance for the rational and efficient design of azopolymer-based material platforms. In addition, considering that the UV-light response of traditional azobenzene photoswitches is not conducive to biological applications, we have summarized the recent approaches to red-shifting the light wavelength for azobenzene activation.

PD-1 and BTLA regulate T cell signaling differentially and only partially through SHP1 and SHP2.

Blockade antibodies of the immunoinhibitory receptor PD-1 can stimulate the anti-tumor activity of T cells, but clinical benefit is limited to a fraction of patients. Evidence suggests that BTLA, a receptor structurally related to PD-1, may contribute to resistance to PD-1 targeted therapy, but how BTLA and PD-1 differ in their mechanisms is debated. Here, we compared the abilities of BTLA and PD-1 to recruit effector molecules and to regulate T cell signaling. While PD-1 selectively recruited SHP2 over the stronger phosphatase SHP1, BTLA preferentially recruited SHP1 to more efficiently suppress T cell signaling. Contrary to the dominant view that PD-1 and BTLA signal exclusively through SHP1/2, we found that in SHP1/2 double-deficient primary T cells, PD-1 and BTLA still potently inhibited cell proliferation and cytokine production, albeit more transiently than in wild type T cells. Thus, PD-1 and BTLA can suppress T cell signaling through a mechanism independent of both SHP1 and SHP2.

Combined non-invasive scan and biomarkers to identify independent risk factors in patients with mild coronary stenosis.

BACKGROUND: Independent risk factors for major adverse cardiovascular events (MACEs) in patients with mild coronary stenosis are uncertain. This study aims to investigate predictive biomarkers for MACEs in patients with mild coronary stenosis. METHODS: Totally 381 patients with mild coronary stenosis were included and MACE incidences were recorded through a 24-month follow-up and 91 patients with unfavorable plaques characteristic are detected by CCTA. One unfavorable characteristic was recorded for 1 point and they were divided into three groups: high-risk group (HR, score =0), intermediate-risk group (IR, score =1) and low-risk group (LR, score/2). Specific blood biomarker measurements of high-sensitivity C-reactive protein (hs-CRP), matrix metallopeptidase 9 (MMP-9), and myeloperoxidase (MPO) were taken simultaneously. RESULTS: The mean age, hs-CRP and MPO levels in the HR and IR group were significantly higher than that in the LR group. A considerably higher level of MMP-9 showed in the HR group compared to the LR group. The incidence rates of MACE were remarkably higher in HR group than LR group and IR group. Kaplan-Meier survival analysis demonstrated that the cumulative event-free survival rate of HR was significantly higher than that in LR and IR group and there was no significant difference between LR and IR group. The univariate COX regression analysis indicated that the age, hs-CRP, MPO, and unfavorable plaque scores ≥2 were independent risk factors for MACEs. CONCLUSIONS: High MPO levels were strongly correlated with MACEs in patients with mild coronary stenosis. Although confirmation is needed from larger trials, MPO could be a promising clinical tool to improve the risk stratification in patients with mild coronary stenosis and suggest strategies for the individualized prevention programs.