Identifying an Abnormal Phosphorylated Adaptor by Viral Kinase Using Mass Spectrometry.

Mass spectrometers are widely used to identify protein phosphorylation sites. The process usually involves selective isolation of phosphoproteins and subsequent fragmentation to identify both the peptide sequence and phosphorylation site. Immunoprecipitation could capture and purify the protein of interest, greatly reducing sample complexity before submitting it for mass spectrometry analysis. This chapter describes a method to identify an abnormal phosphorylated site of the adaptor protein by a viral kinase through immunoprecipitation followed by LC-MS/MS.

Investigation of Protein Lysine Acetylation in Antiviral Innate Immunity.

Protein lysine acetylation involved in the antiviral innate immunity contributes to the regulation of antiviral inflammation responses, including type 1 interferon production and interferon-stimulated gene expression. Thus, investigation of acetylated antiviral proteins is vital for the complete understanding of inflammatory responses to viral infections. Immunoprecipitation (IP) assay with anti-targeted-protein antibody or with acetyl-lysine affinity beads followed by immunoblot provides a classical way to determine the potential modified protein in the antiviral innate pathways, whereas mass spectrometry can be utilized to identify the accurate acetylation lysine residues or explore the acetyl-proteomics. We demonstrate here comprehensive methods of protein lysine acetylation determination in virus-infected macrophages and embryonic fibroblast cells or proteins-overexpressed HEK 293 T cells in the context of antiviral innate immunity.

Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

PAIP1 binds to pre-mRNA and regulates alternative splicing of cancer pathway genes including VEGFA.

BACKGROUND: Poly (A) binding protein interacting protein 1 (PAIP1) has been shown to causally contribute to the development and progression of cancer. However, the mechanisms of the PAIP1 regulation in tumor cells remain poorly understood. RESULTS: Here, we used a recently developed UV cross-linking and RNA immunoprecipitation method (iRIP-seq) to map the direct and indirect interaction sites between PAIP1 and RNA on a transcriptome-wide level in HeLa cells. We found that PAIP1 not only binds to 3'UTRs, but also to pre-mRNAs/mRNAs with a strong bias towards the coding region and intron. PAIP1 binding sites are enriched in splicing enhancer consensus GA-rich motifs. RNA-seq analysis revealed that PAIP1 selectively modulates the alternative splicing of genes in some cancer hallmarks including cell migration, the mTOR signaling pathway and the HIF-1 signaling pathway. PAIP1-regulated alternative splicing events were strongly associated with PAIP1 binding, demonstrating that the binding may promote selection of the nearby splice sites. Deletion of a PAIP1 binding site containing seven repeats of GA motif reduced the PAIP1-mediated suppression of the exon 6 inclusion in a VEGFA mRNA isoform. Proteomic analysis of the PAIP1-interacted proteins revealed the enrichment of the spliceosome components and splicing factors. CONCLUSIONS: These findings suggest that PAIP1 is both a polyadenylation and alternative splicing regulator, that may play a large role in RNA processing via its role in alternative splicing regulation.

PRC1 Protein Subcomplexes Architecture: Focus on the Interplay between Distinct PCGF Subunits in Protein Interaction Networks.

The six PCGF proteins (PCGF1-6) define the biochemical identity of Polycomb repressor complex 1 (PRC1) subcomplexes. While structural and functional studies of PRC1 subcomplexes have revealed their specialized roles in distinct aspects of epigenetic regulation, our understanding of the variation in the protein interaction networks of distinct PCGF subunits in different PRC1 complexes is incomplete. We carried out an affinity purification mass spectrometry (AP-MS) screening of three PCGF subunits, PCGF1 (NSPC1), PCGF2 (MEL18), and PCGF4 (BMI1), to define their interactome and potential cellular function in pluripotent human embryonal carcinoma cell "NT2". The bioinformatic analysis revealed that these interacting proteins cover a range of functional pathways, often involved in cell biology and chromatin regulation. We also found evidence of mutual regulation (at mRNA and protein level) between three distinct PCGF subunits. Furthermore, we confirmed that the disruption of these subunits results in reduced cell proliferation ability. We reveal an interplay between the compositional diversity of the distinct PCGF containing PRC1 complex and the potential role of PCGF proteins within the wider cellular network.

Lactyllysine Esterification Enables Efficient Lactylprotein Expression via Genetic Code Expansion and Supports Functional Proteomics Studies.

Lysine lactylation has recently been discovered and demonstrated to be an essential player in immunity, cancer and neurodegenerative diseases. Genetic code expansion (GCE) technique is powerful in uncovering lactylation functions, since it allows site-specific incorporation of lactyllysine (Klac) into proteins of interest (POIs) in living cells. However, the inefficient uptake of Klac into cells, due to its high hydrophilicity, results in limited expression of lactylated POIs. To address this challenge, here we designed esterified Klac derivatives, exemplified by ethylated Klac (KlacOEt), to enhance Klac's lipophilicity and improve its cellular uptake. The expression level of site-specifically lactylated POIs was doubled using KlacOEt in both Escherichia coli and HEK293T cells. Immunoprecipitation mass spectrometry analysis verified the significantly increased yield of the precisely lactylated fructose-bisphosphate aldolase A using KlacOEt. Furthermore, in conjunction with the Target Responsive Accessibility Profiling approach, we found that lactylation at ALDOA-K147 altered the protein's conformation, which may explain the lactylation-induced reduction in enzyme activity. Together, we demonstrate that, through enhancing the yield of lactylated proteins with Klac esters via GCE, we are able to site-specifically reveal the effects of lactylation on POIs' interactions, conformations and activities using a suite of functional proteomics and biochemical tools.

A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence.

Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence.

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

ATP synthase subunit ATP5B interacts with TGEV Nsp2 and acts as a negative regulator of TGEV replication.

Coronavirus nonstructural protein 2 (Nsp2) is regarded as a virulence determinant and plays a critical role in virus replication, and innate immunity. Screening and identifying host cell proteins that interact with viral proteins is an effective way to reveal the functions of viral proteins. In this study, the host proteins that interacted with transmissible gastroenteritis virus (TGEV) Nsp2 were identified using immunoprecipitation combined with LC-MS/MS. 77 host cell proteins were identified as putative Nsp2 interaction host cell proteins and a protein-protein interaction (PPI) was constructed. The identified proteins were found to be associated with various subcellular locations and functional categories through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. It is hypothesized that the host cell proteins interacting with TGEV Nsp2 are mainly involved in the formation of the cytoplasmic translation initiation complex, mRNA binding, ribosomes, and proteasomes. Among these, the ATP5B, a core subunit of the mitochondrial ATP synthase was further studied. The Coimmunoprecipitation (Co-IP) and indirect immunofluorescence (IFA) results confirmed that TGEV Nsp2 interacted with ATP5B. Furthermore, the downregulation of ATP5B expression was found to promote TGEV replication, suggesting that ATP5B might function as a negative regulator of TGEV replication. Collectively, our results offer additional insights into the functions of Nsp2 and provide a novel antiviral target against TGEV.

Downregulation of the m6A reader YTHDC2 upregulates exosome content in lung adenocarcinoma via inhibiting IFIT and OAS family members.

N6-Methyladenosine (m6A) is the most prevalent mRNA modification. Its biological function primarily relies on its "Reader" protein, such as YTHDC2. Previous studies have shown that YTHDC2 downregulation is a pro-carcinogenic phenomenon in lung adenocarcinoma (LUAD). However, further investigation is needed to understand the molecular mechanisms of downstream genes and the associated biological phenomena following YTHDC2 downregulation. Here, we found that YTHDC2 knockout upregulated exosome content in LUAD. Following YTHDC2 knockout, the mRNA levels of OAS family members (OASs) and IFIT family members (IFITs) also decreased; and inhibition of OASs and IFITs could promote exosome content. Several m6A modification sites on the NT domain of OASs and the TPR12 domain of IFITs were found to increase the stability of OASs and IFITs in a YTHDC2-dependent manner. OASs and IFITs affected exosome content through target genes including RAB5A, RAB7 and RAB11A, and three arginine (R) amino acids on IFITs were critical for combination IFITs with targeted RAB mRNAs and subsequent degradation. Simultaneously, OASs degraded targeted RABs through RNAseL. Additionally, mutual bindings between OASs and IFITs were critical for their target gene degradation. Collectively, the above findings might provide a theoretical basis for the treatment of LUAD patients with low YTHDC2 expression.