Affinity purification coupled with mass spectrometry (AP-MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics ...studies. Whereas AP-MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP-MS and BioID data has been shown to comprehensively characterize a protein's molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP-MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform ( http://proteomics.fi ) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP-MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.
Protein-protein interactions govern almost all cellular functions. These complex networks of stable and transient associations can be mapped by affinity purification mass spectrometry (AP-MS) and ...complementary proximity-based labeling methods such as BioID. To exploit the advantages of both strategies, we here design and optimize an integrated approach combining AP-MS and BioID in a single construct, which we term MAC-tag. We systematically apply the MAC-tag approach to 18 subcellular and 3 sub-organelle localization markers, generating a molecular context database, which can be used to define a protein's molecular location. In addition, we show that combining the AP-MS and BioID results makes it possible to obtain interaction distances within a protein complex. Taken together, our integrated strategy enables the comprehensive mapping of the physical and functional interactions of proteins, defining their molecular context and improving our understanding of the cellular interactome.
Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs ...through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.
Oncogenic gene fusions are estimated to account for up-to 20% of cancer morbidity. Recently sequence-level studies have established oncofusions throughout all tissue types. However, the functional ...implications of the identified oncofusions have often not been investigated. In this study, identified oncofusions from a fusion detection approach (DEEPEST) were analyzed in detail. Of the 28,863 oncofusions, we found almost 30% are expected to produce functional proteins with features from both parent genes. Kinases and transcription factors were the main gene families of the protein producing fusions. Considering their role as initiators, actors, and termination points of cellular signaling pathways, we focused our in-depth analyses on them. Domain architecture of the fusions and their wild-type interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins. To understand overall oncofusion effects, we performed differential expression analysis using TCGA cancer project samples. Results indicated oncofusion-specific alterations in gene expression levels, and lower expression levels of components of key cellular pathways, in particular signal transduction and transcription regulation. The sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling, possibly via acquiring novel functions.
Cancer-associated gene fusions, also known as oncofusions, have emerged as influential drivers of oncogenesis across a diverse range of cancer types. These genetic events occur via chromosomal ...translocations, deletions, and inversions, leading to the fusion of previously separate genes. Due to the drastic nature of these mutations, they often result in profound alterations of cellular behavior. The identification of oncofusions has revolutionized cancer research, with advancements in sequencing technologies facilitating the discovery of novel fusion events at an accelerated pace. Oncofusions exert their effects through the manipulation of critical cellular signaling pathways that regulate processes such as proliferation, differentiation, and survival. Extensive investigations have been conducted to understand the roles of oncofusions in solid tumors, leukemias, and lymphomas. Large-scale initiatives, including the Cancer Genome Atlas, have played a pivotal role in unraveling the landscape of oncofusions by characterizing a vast number of cancer samples across different tumor types. While validating the functional relevance of oncofusions remains a challenge, even non-driver mutations can hold significance in cancer treatment. Oncofusions have demonstrated potential value in the context of immunotherapy through the production of neoantigens. Their clinical importance has been observed in both treatment and diagnostic settings, with specific fusion events serving as therapeutic targets or diagnostic markers. However, despite the progress made, there is still considerable untapped potential within the field of oncofusions. Further research and validation efforts are necessary to understand their effects on a functional basis and to exploit the new targeted treatment avenues offered by oncofusions. Through further functional and clinical studies, oncofusions will enable the advancement of precision medicine and the drive towards more effective and specific treatments for cancer patients.
Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role ...of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.
Treatment options for COVID‐19, caused by SARS‐CoV‐2, remain limited. Understanding viral pathogenesis at the molecular level is critical to develop effective therapy. Some recent studies have ...explored SARS‐CoV‐2–host interactomes and provided great resources for understanding viral replication. However, host proteins that functionally associate with SARS‐CoV‐2 are localized in the corresponding subnetwork within the comprehensive human interactome. Therefore, constructing a downstream network including all potential viral receptors, host cell proteases, and cofactors is necessary and should be used as an additional criterion for the validation of critical host machineries used for viral processing. This study applied both affinity purification mass spectrometry (AP‐MS) and the complementary proximity‐based labeling MS method (BioID‐MS) on 29 viral ORFs and 18 host proteins with potential roles in viral replication to map the interactions relevant to viral processing. The analysis yields a list of 693 hub proteins sharing interactions with both viral baits and host baits and revealed their biological significance for SARS‐CoV‐2. Those hub proteins then served as a rational resource for drug repurposing via a virtual screening approach. The overall process resulted in the suggested repurposing of 59 compounds for 15 protein targets. Furthermore, antiviral effects of some candidate drugs were observed in vitro validation using image‐based drug screen with infectious SARS‐CoV‐2. In addition, our results suggest that the antiviral activity of methotrexate could be associated with its inhibitory effect on specific protein–protein interactions.
Synopsis
A large‐scale proteomics study identifies critical host proteins for SARS‐CoV‐2 processing. Proteins from these core subnetworks are used for drug repurposing analyses, indicating drugs with antiviral effects.
A large‐scale proteomics study identifies 4,781 unique high‐confidence virus‐host protein‐protein interactions (PPIs) using 29 viral ORFs, and 4,362 unique PPIs for 18 suggested receptors/proteases/cofactors for SARS‐CoV‐2.
The characterization of 693 hub proteins that connect viral baits and host baits via a dense network reveals critical host pathways used for viral replication.
59 compounds are prioritized that could be repurposed for 15 host protein targets used by the SARS‐CoV‐2 during the infection.
Ten candidate drugs are validated using an image‐based drug screen assay, six of them demonstrating antiviral effects.
A large‐scale proteomics study identifies critical host proteins for SARS‐CoV‐2 processing. Proteins from these core subnetworks are used for drug repurposing analyses, indicating drugs with antiviral effects.
The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the ...eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as β1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling.
Chromosomal translocations creating fusion genes are common cancer drivers. The oncogenic ETV6-NTRK3 (EN) gene fusion joins the sterile alpha domain of the ETV6 transcription factor with the tyrosine ...kinase domain of the neurotrophin-3 receptor NTRK3. Four EN variants with alternating break points have since been detected in a wide range of human cancers. To provide molecular level insight into EN oncogenesis, we employed a proximity labeling mass spectrometry approach to define the molecular context of the fusions. We identify in total 237 high-confidence interactors, which link EN fusions to several key signaling pathways, including ERBB, insulin and JAK/STAT. We then assessed the effects of EN variants on these pathways, and showed that the pan NTRK inhibitor Selitrectinib (LOXO-195) inhibits the oncogenic activity of EN2, the most common variant. This systems-level analysis defines the molecular framework in which EN oncofusions operate to promote cancer and provides some mechanisms for therapeutics.
Most patients with ovarian cancer (OC) are diagnosed at a late stage when there are very few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms ...or an oncogenic addiction that can be targeted pharmacologically, unlike other types of cancer. Here, we identified protein tyrosine kinase 7 (PTK7) as a potential new therapeutic target in OC following a multiomics approach using genetic and pharmacological interventions. We performed proteomics analyses upon PTK7 knockdown in OC cells and identified novel downstream effectors such as synuclein-γ (SNCG), SALL2, and PP1γ, and these findings were corroborated in ex vivo primary samples using PTK7 monoclonal antibody cofetuzumab. Our phosphoproteomics analyses demonstrated that PTK7 modulates cell adhesion and Rho-GTPase signaling to sustain epithelial-mesenchymal transition (EMT) and cell plasticity, which was confirmed by high-content image analysis of 3D models. Furthermore, using high-throughput drug sensitivity testing (525 drugs) we show that targeting PTK7 exhibited synergistic activity with chemotherapeutic agent paclitaxel, CHK1/2 inhibitor prexasertib, and PLK1 inhibitor GSK461364, among others, in OC cells and ex vivo primary samples. Taken together, our study provides unique insight into the function of PTK7, which helps to define its role in mediating aberrant Wnt signaling in ovarian cancer.
PDF
