Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein–protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein–protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein–protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 <i>N</i>-terminus (H3K4Me₃), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me₃. This study indicates that our <u>c</u>ross-<u>l</u>inking-<u>a</u>ssisted and <u>S</u>ILAC-based <u>p</u>rotein <u>i</u>dentification (CLASPI) approach can be used to profile protein–protein interactions mediated by PTMs, such as lysine methylation

Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein–protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein–protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein–protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 <i>N</i>-terminus (H3K4Me₃), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me₃. This study indicates that our <u>c</u>ross-<u>l</u>inking-<u>a</u>ssisted and <u>S</u>ILAC-based <u>p</u>rotein <u>i</u>dentification (CLASPI) approach can be used to profile protein–protein interactions mediated by PTMs, such as lysine methylation

Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein–protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein–protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein–protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 <i>N</i>-terminus (H3K4Me₃), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me₃. This study indicates that our <u>c</u>ross-<u>l</u>inking-<u>a</u>ssisted and <u>S</u>ILAC-based <u>p</u>rotein <u>i</u>dentification (CLASPI) approach can be used to profile protein–protein interactions mediated by PTMs, such as lysine methylation

Quantitative Chemical Proteomics Approach To Identify Post-translational Modification-Mediated Protein–Protein Interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein–protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein–protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein–protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 <i>N</i>-terminus (H3K4Me₃), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me₃. This study indicates that our <u>c</u>ross-<u>l</u>inking-<u>a</u>ssisted and <u>S</u>ILAC-based <u>p</u>rotein <u>i</u>dentification (CLASPI) approach can be used to profile protein–protein interactions mediated by PTMs, such as lysine methylation

Table_3_Preliminary study on early diagnosis of Alzheimer’s disease in APP/PS1 transgenic mice using multimodal magnetic resonance imaging.DOCX

Alzheimer’s disease (AD) has an insidious onset and lacks clear early diagnostic markers, and by the time overt dementia symptoms appear, the disease is already in the mid-to-late stages. The search for early diagnostic markers of AD may open a critical window for Alzheimer’s treatment and facilitate early intervention to slow the progression of AD. In this study, we aimed to explore the imaging markers for early diagnosis of AD through the combined application of structural magnetic resonance imaging (sMRI), resting-state functional magnetic resonance imaging (rs-fMRI), and 1H-magnetic resonance spectroscopy (1H-MRS) multimodal magnetic resonance imaging (MRI) techniques at the animal experimental level, with the aim to provide a certain reference for early clinical diagnosis of AD. First, sMRI scans were performed on 4-month-old amyloid beta precursor protein/presenilin 1 (APP/PS1) transgenic AD model mice and wild type mice of the same litter using a 7.0 T animal MRI scanner to analyze the differential brain regions with structural changes in the gray matter of the brain by voxel-based morphometry (VBM). Next, rs-fMRI scans were performed to analyze the differential brain regions between groups for local spontaneous brain activity and functional connectivity (FC) between brain regions. Finally, 1H-MRS scans were performed to quantify and analyze intergroup differences in the relative concentrations of different metabolites within regions of interest (cortex and hippocampus). Compared with wild type mice, the volume of the left hippocampus, and right olfactory bulb of APP/PS1 transgenic AD model mice were reduced, the functional activity of the bilateral hippocampus, right piriform cortex and right caudate putamen was reduced, the functional network connectivity of the hippocampus was impaired, and the relative content of N-acetylaspartate (NAA)in the hippocampus was decreased. In addition, this study found that imaging changes in olfactory-related brain regions were closely associated with AD diagnosis, and these findings may provide some reference for the early diagnosis of AD.</p

Table_2_Preliminary study on early diagnosis of Alzheimer’s disease in APP/PS1 transgenic mice using multimodal magnetic resonance imaging.DOCX

Alzheimer’s disease (AD) has an insidious onset and lacks clear early diagnostic markers, and by the time overt dementia symptoms appear, the disease is already in the mid-to-late stages. The search for early diagnostic markers of AD may open a critical window for Alzheimer’s treatment and facilitate early intervention to slow the progression of AD. In this study, we aimed to explore the imaging markers for early diagnosis of AD through the combined application of structural magnetic resonance imaging (sMRI), resting-state functional magnetic resonance imaging (rs-fMRI), and 1H-magnetic resonance spectroscopy (1H-MRS) multimodal magnetic resonance imaging (MRI) techniques at the animal experimental level, with the aim to provide a certain reference for early clinical diagnosis of AD. First, sMRI scans were performed on 4-month-old amyloid beta precursor protein/presenilin 1 (APP/PS1) transgenic AD model mice and wild type mice of the same litter using a 7.0 T animal MRI scanner to analyze the differential brain regions with structural changes in the gray matter of the brain by voxel-based morphometry (VBM). Next, rs-fMRI scans were performed to analyze the differential brain regions between groups for local spontaneous brain activity and functional connectivity (FC) between brain regions. Finally, 1H-MRS scans were performed to quantify and analyze intergroup differences in the relative concentrations of different metabolites within regions of interest (cortex and hippocampus). Compared with wild type mice, the volume of the left hippocampus, and right olfactory bulb of APP/PS1 transgenic AD model mice were reduced, the functional activity of the bilateral hippocampus, right piriform cortex and right caudate putamen was reduced, the functional network connectivity of the hippocampus was impaired, and the relative content of N-acetylaspartate (NAA)in the hippocampus was decreased. In addition, this study found that imaging changes in olfactory-related brain regions were closely associated with AD diagnosis, and these findings may provide some reference for the early diagnosis of AD.</p

Table_1_Preliminary study on early diagnosis of Alzheimer’s disease in APP/PS1 transgenic mice using multimodal magnetic resonance imaging.DOCX

Alzheimer’s disease (AD) has an insidious onset and lacks clear early diagnostic markers, and by the time overt dementia symptoms appear, the disease is already in the mid-to-late stages. The search for early diagnostic markers of AD may open a critical window for Alzheimer’s treatment and facilitate early intervention to slow the progression of AD. In this study, we aimed to explore the imaging markers for early diagnosis of AD through the combined application of structural magnetic resonance imaging (sMRI), resting-state functional magnetic resonance imaging (rs-fMRI), and 1H-magnetic resonance spectroscopy (1H-MRS) multimodal magnetic resonance imaging (MRI) techniques at the animal experimental level, with the aim to provide a certain reference for early clinical diagnosis of AD. First, sMRI scans were performed on 4-month-old amyloid beta precursor protein/presenilin 1 (APP/PS1) transgenic AD model mice and wild type mice of the same litter using a 7.0 T animal MRI scanner to analyze the differential brain regions with structural changes in the gray matter of the brain by voxel-based morphometry (VBM). Next, rs-fMRI scans were performed to analyze the differential brain regions between groups for local spontaneous brain activity and functional connectivity (FC) between brain regions. Finally, 1H-MRS scans were performed to quantify and analyze intergroup differences in the relative concentrations of different metabolites within regions of interest (cortex and hippocampus). Compared with wild type mice, the volume of the left hippocampus, and right olfactory bulb of APP/PS1 transgenic AD model mice were reduced, the functional activity of the bilateral hippocampus, right piriform cortex and right caudate putamen was reduced, the functional network connectivity of the hippocampus was impaired, and the relative content of N-acetylaspartate (NAA)in the hippocampus was decreased. In addition, this study found that imaging changes in olfactory-related brain regions were closely associated with AD diagnosis, and these findings may provide some reference for the early diagnosis of AD.</p

Table_4_Preliminary study on early diagnosis of Alzheimer’s disease in APP/PS1 transgenic mice using multimodal magnetic resonance imaging.DOCX

Alzheimer’s disease (AD) has an insidious onset and lacks clear early diagnostic markers, and by the time overt dementia symptoms appear, the disease is already in the mid-to-late stages. The search for early diagnostic markers of AD may open a critical window for Alzheimer’s treatment and facilitate early intervention to slow the progression of AD. In this study, we aimed to explore the imaging markers for early diagnosis of AD through the combined application of structural magnetic resonance imaging (sMRI), resting-state functional magnetic resonance imaging (rs-fMRI), and 1H-magnetic resonance spectroscopy (1H-MRS) multimodal magnetic resonance imaging (MRI) techniques at the animal experimental level, with the aim to provide a certain reference for early clinical diagnosis of AD. First, sMRI scans were performed on 4-month-old amyloid beta precursor protein/presenilin 1 (APP/PS1) transgenic AD model mice and wild type mice of the same litter using a 7.0 T animal MRI scanner to analyze the differential brain regions with structural changes in the gray matter of the brain by voxel-based morphometry (VBM). Next, rs-fMRI scans were performed to analyze the differential brain regions between groups for local spontaneous brain activity and functional connectivity (FC) between brain regions. Finally, 1H-MRS scans were performed to quantify and analyze intergroup differences in the relative concentrations of different metabolites within regions of interest (cortex and hippocampus). Compared with wild type mice, the volume of the left hippocampus, and right olfactory bulb of APP/PS1 transgenic AD model mice were reduced, the functional activity of the bilateral hippocampus, right piriform cortex and right caudate putamen was reduced, the functional network connectivity of the hippocampus was impaired, and the relative content of N-acetylaspartate (NAA)in the hippocampus was decreased. In addition, this study found that imaging changes in olfactory-related brain regions were closely associated with AD diagnosis, and these findings may provide some reference for the early diagnosis of AD.</p

Image1_TKI or TKI combined with PD-1 inhibitors as second-line treatment for HCC patients after sorafenib failure.jpeg

Background: Tyrosine kinase inhibitors (TKI) in combination with programmed cell death-1 (PD-1) inhibitors become the potential treatment modality for patients undergoing unresectable hepatocellular carcinoma (uHCC) in the first-line setting. However, the efficacy and safety of this combination regimen in patients after sorafenib failure remains unclear.Methods: Participants in this study included patients with uHCC after sorafenib failure who received TKI monotherapy (TKI group) or TKI combined with PD-1 inhibitors therapy (combination group) in our center from July 2018 to July 2021. The overall survival (OS) was used to be the primary efficacy endpoint, while progression-free survival (PFS), objective response rate (ORR), and disease control rate (DCR) were applied to be secondary endpoints. In addition, the adverse events are recorded and evaluated.Results: Among the 92 patients contained in this work, 50 patients were categorized into the TKI group, while 42 patients were in the combination group. There existed no evident differences between the two groups concerning the ORR (8.0% vs. 9.5%, p = 1.000). However, the DCR in the combined group was better in relative to that in the TKI group (71.4% vs. 50.0%, p = 0.037). In comparison with the TKI group, it was found that the combination group presented notably better median PFS (8.1 months vs. 4.7 months, p = 0.005) and median OS (21.9 months vs. 16.6 months, p = 0.042). According to multivariate analysis, PFS (HR 0.5, 95% CI: 0.3–0.8, p = 0.005) and OS (HR 0.5, 95% CI: 0.3–1.0, p = 0.051) were improved in the combination group in relative to the TKI group after the adjustment for some risk factors. Additionally, the incidence rates of grade ≥1 adverse event in the TKI group and the combination group were 96.0% and 97.6%, respectively. The most normal adverse event in the TKI group was neutropenia (n = 24,48.0%) and the combination group was hypoalbuminemia (n = 23,54.8%). All of these adverse events improved after symptomatic treatment, and no new toxic events were found to occur.Conclusion: TKI combined with PD-1 inhibitors showed better prognosis with manageable toxicity in uHCC patients after sorafenib failure compared with TKI monotherapy.</p

DHX29 functions as an RNA co-sensor for MDA5-mediated EMCV-specific antiviral immunity

<div><p>Melanoma differentiation-associated gene-5 (MDA5) recognizes distinct subsets of viruses including Encephalomyocarditis virus (EMCV) of picornavirus family, but the molecular mechanisms underlying the specificity of the viral recognition of MDA5 in immune cells remain obscure. DHX29 is an RNA helicase required for the translation of 5’ structured mRNA of host and many picornaviruses (such as EMCV). We identify that DXH29 as a key RNA co-sensor, plays a significant role for specific recognition and triggering anti-EMCV immunity. We have observed that DHX29 regulates MDA5-, but not RIG-I-, mediated type I interferon signaling by preferentially interacting with structured RNAs and specifically with MDA5 for enhancing MDA5-dsRNA binding affinity. Overall, our results identify a critical role for DHX29 in innate immune response and provide molecular insights into the mechanisms by which DHX29 recognizes 5’ structured EMCV RNA and interacts with MDA5 for potent type I interferon signaling and antiviral immunity.</p></div