DDX50 cooperates with STAU1 to effect stabilization of pro-differentiation RNAs.

Glucose binding can alter protein oligomerization to enable differentiation. Here, we demonstrate that glucose binding is a general capacity of DExD/H-box RNA helicases, including DDX50, which was found to be essential for the differentiation of diverse cell types. Glucose binding to conserved DDX50 ATP binding sequences altered protein conformation and dissociated DDX50 dimers.

Disease-Linked Regulatory DNA Variants and Homeostatic Transcription Factors in Epidermis.

Identifying noncoding single nucleotide variants ( SNVs ) in regulatory DNA linked to polygenic disease risk, the transcription factors ( TFs ) they bind, and the target genes they dysregulate is a goal in polygenic disease research. Massively parallel reporter gene analysis ( MPRA ) of 3,451 SNVs linked to risk for polygenic skin diseases characterized by disrupted epidermal homeostasis identified 355 differentially active SNVs ( daSNVs ). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a pathomechanism shared across common polygenic skin diseases.

Glucose binds and activates NSUN2 to promote translation and epidermal differentiation.

Elevations in intracellular glucose concentrations are essential for epithelial cell differentiation by mechanisms that are not fully understood. Glucose has recently been found to directly bind several proteins to alter their functions to enhance differentiation. Among the newly identified glucose-binding proteins is NSUN2, an RNA-binding protein that we identified as indispensable for epidermal differentiation.

Mitochondrial Raf1 Regulates Glutamine Catabolism.

Raf kinases play vital roles in normal mitogenic signaling and cancer, however, the identities of functionally important Raf-proximal proteins throughout the cell are not fully known. Raf1 proximity proteomics/BioID in Raf1-dependent cancer cells unexpectedly identified Raf1-adjacent proteins known to reside in the mitochondrial matrix. Inner-mitochondrial localization of Raf1 was confirmed by mitochondrial purification and super-resolution microscopy.

Mutation of CsARC6 affects fruit color and increases fruit nutrition in cucumber.

A mutation of CsARC6 not only causes white fruit color in cucumber, but also affects plant growth and fruit quality. Fruit color of cucumber is a very important agronomic trait, but most of the genes affecting cucumber white fruit color are still unknow, and no further studies were reported on the effect of cucumber fruit quality caused by white fruit color genes. Here, we obtained a white fruit mutant em41 in cucumber by EMS mutagenesis.

Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries.

UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments.

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers.

AUTS2 Controls Neuronal Lineage Choice Through a Novel PRC1-Independent Complex and BMP Inhibition.

Despite a prominent risk factor for Neurodevelopmental disorders (NDD), it remains unclear how Autism Susceptibility Candidate 2 (AUTS2) controls the neurodevelopmental program. Our studies investigated the role of AUTS2 in neuronal differentiation and discovered that AUTS2, together with WDR68 and SKI, forms a novel protein complex (AWS) specifically in neuronal progenitors and promotes neuronal differentiation through inhibiting BMP signaling. Genomic and biochemical analyses demonstrated that the AWS complex achieves this effect by recruiting the CUL4 E3 ubiquitin ligase complex to mediate poly-ubiquitination and subsequent proteasomal degradation of phosphorylated SMAD1/5/9.

PROBER identifies proteins associated with programmable sequence-specific DNA in living cells.

DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest.

The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells.

Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases.

Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases.

Parallel-reaction monitoring revealed altered expression of a number of epitranscriptomic reader, writer, and eraser proteins accompanied with colorectal cancer metastasis.

RNA contains more than 170 types of chemical modifications, and these modified nucleosides are recognized, installed and removed by their reader, writer, and eraser (RWE) proteins, respectively. Here, we employed a parallel-reaction monitoring (PRM)-based targeted proteomic method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to examine comprehensively the differential expression of epitranscriptomic RWE proteins in a matched pair of primary/metastatic colorectal cancer (CRC) cells, namely SW480/SW620. We were able to quantify 113 nonredundant epitranscriptomic RWE proteins; among them, 48 and 5 were up- and down-regulated by >1.

Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Accompanied with Radioresistance in Breast Cancer Cells.

Epitranscriptomic reader, writer, and eraser (RWE) proteins recognize, install, and remove modified nucleosides in RNA, which are known to play crucial roles in RNA processing, splicing, and stability. Here, we established a liquid chromatography-parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of a total of 152 epitranscriptomic RWE proteins. We also applied the LC-PRM method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to quantify these proteins in two pairs of matched parental/radioresistant breast cancer cells (i.

Quantitative proteomics revealed new functions of ALKBH4.

ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N -methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation.

Proteome-Wide Characterizations of -Methyl-Adenosine Triphosphate- and -Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases.

Kinases catalyze the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to their protein and small-molecule substrates, and this phosphorylation is a crucial element of multiple cell signaling pathways. Herein, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method for proteome-wide identifications of endogenous kinases that can bind to two -modified ATP derivatives, -methyl-ATP (-Me-ATP), and -furfuryl-ATP (a.k.

Quantitative Proteomic Analysis Revealed Broad Roles of -Methyladenosine in Heat Shock Response.

As optimum temperature is essential for all living organisms, heat shock represents a challenging problem for their survival. Therefore, cellular response to heat shock is among the most extensively investigated stress response pathways; however, how the human proteome responds to heat shock has not been comprehensively investigated. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC), together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, to fulfill an in-depth analysis of the alterations in the human proteome in M14 human melanoma cells in response to heat shock stress.

Targeted Proteomic Analysis Revealed Kinome Reprogramming during Acquisition of Radioresistance in Breast Cancer Cells.

Radiotherapy constitutes a major therapeutic modality for early management of breast cancer. Despite the high efficacy in treating breast cancer (BC), radiation resistance and tumor recurrence are major hurdles in breast cancer radiotherapy. Herein, stable isotope labeling by amino acids in cell culture (SILAC) was employed, along with the parallel-reaction monitoring (PRM)-based targeted quantitative proteomic method, to examine the differences in kinase protein expression in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones.

easyCLIP analysis of RNA-protein interactions incorporating absolute quantification.

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation.

SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity.

SARS-CoV2 being highly infectious has been particularly effective in causing widespread infection globally and more variants of SARS-CoV2 are constantly being reported with increased genomic surveillance. In particular, the focus is on mutations of Spike protein, which binds human ACE2 protein enabling SARS-CoV2 entry and infection. Here we present a rapid experimental method leveraging the speed and flexibility of Mircoscale Thermopheresis (MST) to characterize the interaction between Spike Receptor Binding Domain (RBD) and human ACE2 protein.