Weili Miao
Basic Life Research Scientist, Dermatology
All Publications
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Glucose binds and activates NSUN2 to promote translation and epidermal differentiation.
Nucleic acids research
2024
Abstract
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. Glucose was found to bind conserved sequences within NSUN2, enhancing its binding to S-adenosyl-L-methionine and boosting its enzymatic activity. Additionally, glucose enhanced NSUN2's proximity to proteins involved in mRNA translation, with NSUN2 modulating global messenger RNA (mRNA) translation, particularly that of key pro-differentiation mRNAs containing m5C modifications, such as GRHL3. Glucose thus engages diverse molecular mechanisms beyond its energetic roles to facilitate cellular differentiation processes.
View details for DOI 10.1093/nar/gkae1097
View details for PubMedID 39565212
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Mitochondrial Raf1 Regulates Glutamine Catabolism.
bioRxiv : the preprint server for biology
2024
Abstract
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. Inside mitochondria, Raf1 associated with glutaminase (GLS) in diverse human cancers and enabled glutaminolysis, an important source of biosynthetic precursors in cancer. These impacts required Raf1 kinase activity and were independent of canonical MAP kinase pathway signaling. Kinase-dead mitochondrial matrix-localized Raf1 impaired glutaminolysis and tumorigenesis in vivo. These data indicate that Raf1 localizes inside mitochondria where it interacts with GLS to engage glutamine catabolism and support tumorigenesis.
View details for DOI 10.1101/2024.03.08.581297
View details for PubMedID 38496616
View details for PubMedCentralID PMC10942467
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Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.
Cell
2023; 186 (1): 80
Abstract
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. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
View details for DOI 10.1016/j.cell.2022.12.004
View details for PubMedID 36608661
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Analyzing RNA-Protein Interactions by Cross-Link Rates and CLIP-seq Libraries.
Current protocols
2023; 3 (1): e659
Abstract
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. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.
View details for DOI 10.1002/cpz1.659
View details for PubMedID 36705610
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PROBER identifies proteins associated with programmable sequence-specific DNA in living cells.
Nature methods
2022; 19 (8): 959-968
Abstract
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. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.
View details for DOI 10.1038/s41592-022-01552-w
View details for PubMedID 35927480
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The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.
Nature communications
2022; 13 (1): 4121
Abstract
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. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.
View details for DOI 10.1038/s41467-022-31810-6
View details for PubMedID 35840578
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Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases.
Journal of proteome research
2022
Abstract
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. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.
View details for DOI 10.1021/acs.jproteome.2c00225
View details for PubMedID 35820187
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The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.
PLoS pathogens
2021; 17 (10): e1009412
Abstract
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. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.
View details for DOI 10.1371/journal.ppat.1009412
View details for PubMedID 34597346
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SARS-CoV-2 B.1.1.7 and B.1.351 spike variants bind human ACE2 with increased affinity.
The Lancet. Infectious diseases
2021
View details for DOI 10.1016/S1473-3099(21)00262-0
View details for PubMedID 34022142
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easyCLIP analysis of RNA-protein interactions incorporating absolute quantification.
Nature communications
2021; 12 (1): 1569
Abstract
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. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
View details for DOI 10.1038/s41467-021-21623-4
View details for PubMedID 33692367
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SARS-CoV-2 B.1.1.7 and B.1.351 Spike variants bind human ACE2 with increased affinity.
bioRxiv : the preprint server for biology
2021
Abstract
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. The B.1.351 variant harboring three mutations, (E484K, N501Y, and K417N) binds the ACE2 at nearly five-fold greater affinity than the original SARS-COV-2 RBD. We also find that the B.1.1.7 variant, binds two-fold more tightly to ACE2 than the SARS-COV-2 RBD.
View details for DOI 10.1101/2021.02.22.432359
View details for PubMedID 33655251
View details for PubMedCentralID PMC7924271
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The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.
bioRxiv : the preprint server for biology
2021
Abstract
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. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.
View details for DOI 10.1101/2021.02.23.432450
View details for PubMedID 33655243
View details for PubMedCentralID PMC7924263