The interconnections between co-transcriptional regulation, chromatin environment, and transcriptional output remain poorly understood. Here, we investigate the mechanism underlying RNA 3\' processing-mediated Polycomb silencing of Arabidopsis FLOWERING LOCUS C (FLC). We show a requirement for ANTHESIS PROMOTING FACTOR 1 (APRF1), a homolog of yeast Swd2 and human WDR82, known to regulate RNA polymerase II (RNA Pol II) during transcription termination. APRF1 interacts with TYPE ONE SERINE/THREONINE PROTEIN PHOSPHATASE 4 (TOPP4) (yeast Glc7/human PP1) and LUMINIDEPENDENS (LD), the latter showing structural features found in Ref2/PNUTS, all components of the yeast and human phosphatase module of the CPF 3\' end-processing machinery. LD has been shown to co-associate in vivo with the histone H3 K4 demethylase FLOWERING LOCUS D (FLD). This work shows how the APRF1/LD-mediated polyadenylation/termination process influences subsequent rounds of transcription by changing the local chromatin environment at FLC.

Advanced-stage cutaneous T-cell lymphomas (CTCLs) are notorious for their highly aggressive behavior, resistance to conventional treatments, and poor prognosis, particularly when large-cell transformation occurs. PEG10 has been recently proposed as a potent driver for large-cell transformation in CTCL. However, the targeting of PEG10 continues to present a formidable clinical challenge that has yet to be addressed. In this study, we report an important post-translational regulatory mechanism of PEG10 in CTCL. USP9X, a deubiquitinase, interacted with and deubiquitinated PEG10, thereby stabilizing PEG10. Knockdown of USP9X or pharmacological targeting of USP9X resulted in a prominent downregulation of PEG10 and its downstream pathway in CTCL. Moreover, USP9X inhibition conferred tumor cell growth disadvantage and enhanced apoptosis in vitro, an effect that occurred in part through its regulation on PEG10. Furthermore, we demonstrated that inhibition of USP9X obviously restrained CTCL tumor growth in vivo and that high expression of USP9X is associated with poor survival in patients with CTCL. Collectively, our findings uncover USP9X as a key post-translational regulator in the stabilization of PEG10 and suggest that targeting PEG10 stabilization through USP9X inhibition may represent a promising therapeutic strategy for advanced-stage CTCL.

Background: ASB6, an E3 ubiquitin ligase, mediates the proteasomal degradation of its substrate proteins via the ubiquitin-proteasome pathway. ASB6 has been reported to play significant roles in several biological processes, including tumor stemness and endoplasmic reticulum stress. However, the underlying role and mechanism of ASB6 in colorectal cancer, particularly its association with immune infiltration levels and its prognostic significance, remain to be fully elucidated. Methods: We identified key prognostic genes in CRC patients through LASSO-penalized Cox regression, Univariate and Multivariate Cox regression analyses. Subsequently, we comprehensively analyzed the prognostic value of hub genes and constructed a prognostic nomogram. Finally, we identified ASB6 interacting proteins through immunoprecipitation-mass spectrometry (IP-MS) and constructed protein-protein interaction (PPI) networks and performed pathway enrichment analysis to explore the potential mechanisms of ASB6. Meanwhile, we evaluated the functions of ASB6 in CRC cells through in vitro cell experiments. Results: We identified ASB6 as a hub gene in CRC. ASB6 was highly expressed in CRC, and patients with high ASB6 expression had worse Disease-Free Interval (DFI), Disease-Specific Survival (DSS), Overall Survival (OS), and Progression-Free Interval (PFI). Correlation analysis showed that ASB6 expression were positively correlated with lymph node invasion and distal metastasis. Overexpression of ASB6 enhanced the migration ability of CRC cells. Multivariate Cox regression analysis revealed that ASB6 was an independent prognostic factor for OS and DSS in CRC. The nomogram model constructed based on multivariate analysis results had good predictive effects, with C-indexes of 0.811 and 0.934 for OS and DSS, respectively. Furthermore, analysis of immune infiltration levels showed that ASB6 expression were positively correlated with M2-type macrophage infiltration levels in CRC, and patients with high levels of both ASB6 and M2-type macrophages had a worse prognosis. Furthermore, pathway enrichment analysis of ASB6 interacting proteins identified by IP-MS suggested that ASB6 may play a crucial role through the response to unfolded protein pathway and protein processing in the endoplasmic reticulum pathway. Conclusions: ASB6 is significantly upregulated in CRC tissues and is a risk factor for prognosis in CRC patients. ASB6 enhances the migration ability of CRC cells. Therefore, ASB6 may be an independent prognostic biomarker and potential therapeutic target for CRC patients.

The regulation of behavioral and developmental decisions by small molecules is common to all domains of life. In plants, strigolactones and karrikins are butenolide growth regulators that influence several aspects of plant growth and development, as well as interactions with symbiotic fungi.1,2,3 DWARF14 (D14) and KARRIKIN INSENSITIVE2 (KAI2) are homologous enzyme-receptors that perceive strigolactones and karrikins, respectively, and that require hydrolase activity to effect signal transduction.4,5,6,7 RsbQ, a homolog of D14 and KAI2 from the gram-positive bacterium Bacillus subtilis, regulates growth responses to nutritional stress via the alternative transcription factor SigmaB (σB).8,9 However, the molecular function of RsbQ is unknown. Here, we show that RsbQ perceives butenolide compounds that are bioactive in plants. RsbQ is thermally destabilized by the synthetic strigolactone GR24 and its desmethyl butenolide equivalent dGR24. We show that, like D14 and KAI2, RsbQ is a functional butenolide hydrolase that undergoes covalent modification of the catalytic histidine residue. Exogenous application of both GR24 and dGR24 inhibited the endogenous signaling function of RsbQ in vivo, with dGR24 being 10-fold more potent. Application of dGR24 to B. subtilis phenocopied loss-of-function rsbQ mutations and led to a significant downregulation of σB-regulated transcripts. We also discovered that exogenous butenolides promoted the transition from planktonic to biofilm growth. Our results suggest that butenolides may serve as inter-kingdom signaling compounds between plants and bacteria to help shape rhizosphere communities.

As obligate intracellular parasites, viruses rely on the efficient manipulation of the cell they invade in order to multiply and spread. Protein-protein interactions between viral proteins (or their complexes) and cellular proteins are at the interface between virus and host and hence crucial for the outcome of the infection. Multiple techniques can be used to study protein-protein interactions in vivo in the context of the infected cell; among them, immunoprecipitation followed by mass spectrometry (IP-MS) has proven an efficient approach for the unbiased identification of protein complexes containing a viral protein of interest. In this chapter, we discuss how to employ IP-MS to define the interactome of plant virus proteins by using transient expression in the experimental host Nicotiana benthamiana, using the geminivirus tomato yellow leaf curl virus (TYLCV) as an example.

OBJECTIVE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus. We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus.

Transcription factors that act within a gene regulatory network (GRN) often interact with other proteins such as chromatin remodeling factors, histone modifiers, and other co-regulators. Characterizing these interactions is crucial for understanding the function and mechanism of action of a transcription factor. Here, a method for the identification of protein-protein interactions of nuclear-localized, transcription-associated factors is described. The method is based on the immunoprecipitation (IP) of a fluorophore-tagged target, followed by mass spectrometry (MS), peptide identification, and quantification of interacting proteins. By applying label-free quantification to IPs and their input protein extracts, statistically controlled protein enrichment ratios uncover high-confidence interaction partners of the target. A complete step-by-step procedure, including sample preparation, MS settings, data analysis, and visualization is provided.

Autism spectrum disorders (ASDs) have been linked to genes with enriched expression in the brain, but it is unclear how these genes converge into cell-type-specific networks. We built a protein-protein interaction network for 13 ASD-associated genes in human excitatory neurons derived from induced pluripotent stem cells (iPSCs). The network contains newly reported interactions and is enriched for genetic and transcriptional perturbations observed in individuals with ASDs. We leveraged the network data to show that the ASD-linked brain-specific isoform of ANK2 is important for its interactions with synaptic proteins and to characterize a PTEN-AKAP8L interaction that influences neuronal growth. The IGF2BP1-3 complex emerged as a convergent point in the network that may regulate a transcriptional circuit of ASD-associated genes. Our findings showcase cell-type-specific interactomes as a framework to complement genetic and transcriptomic data and illustrate how both individual and convergent interactions can lead to biological insights into ASDs.

Protein-protein interaction networks (PPIs) govern the majority of biological processes, but how oncogenic mutations impact these interactions and their functions at a network scale is poorly understood. Mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) is a pre-requisition for EGFR tyrosine kinase inhibitor (TKI) treatment. Identification of interaction partners that bind to mutated EGFR can help understand the mechanism of action and pathways that mediate drug resistance. In this study, we characterized the dynamic interaction network of a pair of EGFR wildtype and mutant NSCLC cell lines. We performed immunoprecipitation of endogenous EGFR at various time points following EGF treatment and analyzed the associated proteins by quantitative mass spectrometry. Our results showed that the core signaling modules and key downstream pathways are maintained in the mutant cell line, but receptor internalization and intracellular trafficking in the mutant is delayed. Furthermore, we identified mutant EGFR-associated proteins that could affect EGFR functions in lung adenocarcinoma. SIGNIFICANCE: We analyzed the dynamic EGFR interaction network in NSCLC cell lines expressing wild-type and mutant EGFR. By comparing the similarities and differences in the EGFR proteome, we gained a better understanding of EGFR signal transduction network, and identified new factors for further functional characterizations and clinical significance assessment.

Neddylation is an ubiquitin-like modification of proteins that affects the activity, stability and protein-protein interaction of its substrates. Apart from its role as a promoter for Cullin ring E3 ligase to positively regulate the ubiquitylation process, other functional studies about neddylation are still lacking. In this study, we developed a system to explore the impact of neddylation on changes in the subcellular localization of proteins at the omics level. By applying a method combining subcellular protein extraction and immunoprecipitation-mass spectrometry (IP-MS), 81 proteins with a tendency to shuttle between the cytoplasm and nucleus due to different neddylation levels were obtained. Among the 81 candidates, transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) and growth arrest and DNA damage protein 45a (Gadd45a) were confirmed as novel substrates of Nedd8, and neddylation promotes TAK1 nuclear import as well as Gadd45a nuclear export.