Affinity Purification Mass Spectrometry Research Articles

FBXO34 promotes latent HIV-1 activation by post-transcriptional modulation

ABSTRACT Acquired immunodeficiency syndrome (AIDS) cannot be completely cured, mainly due to the existence of a latent HIV-1 reservoir. However, our current understanding of the molecular mechanisms underlying the establishment and maintenance of HIV-1 latent reservoir is not comprehensive. Here, using a genome-wide CRISPR-Cas9 activation library screening, we identified E3 ubiquitin ligase F-box protein 34 (FBXO34) and the substrate of FBXO34, heterogeneous nuclear ribonucleoprotein U (hnRNP U) was identified by affinity purification mass spectrometry, as new host factors related to HIV-1 latent maintenance. Overexpression of FBXO34 or knockout of hnRNP U can activate latent HIV-1 in multiple latent cell lines. FBXO34 mainly promotes hnRNP U ubiquitination, which leads to hnRNP U degradation and abolishment of the interaction between hnRNP U and HIV-1 mRNA. In a latently infected cell line, hnRNP U interacts with the ReV region of HIV-1 mRNA through amino acids 1-339 to hinder HIV-1 translation, thereby, promoting HIV-1 latency. Importantly, we confirmed the role of the FBXO34/hnRNP U axis in the primary CD4+ T lymphocyte model, and detected differences in hnRNP U expression levels in samples from patients treated with antiretroviral therapy (ART) and healthy people, which further suggests that the FBXO34/hnRNP U axis is a new pathway involved in HIV-1 latency. These results provide mechanistic insights into the critical role of ubiquitination and hnRNP U in HIV-1 latency. This novel FBXO34/hnRNP U axis in HIV transcription may be directly targeted to control HIV reservoirs in patients in the future.

Development of functional anti-Gn nanobodies specific for SFTSV based on next-generation sequencing and proteomics.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by novel bunyavirus (SFTSV), with a mortality rate of 6.3% ~ 30%. To date, there is no specific treatment for SFTS. Previously, we demonstrated that SFTSV surface glycoprotein (Glycoprotein N, Gn) was a potential target for the development of SFTS vaccine or therapeutic antibodies, and anti-Gn neutralizing antibodies played a protective role in SFTS infection. Compared with traditional antibodies, nanobodies from camelids have various advantages, including small molecular weight, high affinity, low immunogenicity, convenient production by gene engineering, etc. In this study, we combined next-generation sequencing (NGS) with proteomics technology based on affinity purification-mass spectrometry (AP-MS) and bioinformatics analysis to high-throughput screen monoclonal anti-Gn nanobodies from camel immunized with Gn protein. We identified 19 anti-Gn monoclonal nanobody sequences, of which six sequences were selected for recombinant protein expression and purification. Among these six anti-Gn nanobodies, nanobody 57,493 was validated to be highly specific for Gn. The innovative high-throughput technical route developed in this study could also be expanded to the production of nanobodies specific for other viruses like SARS-CoV-2.

Open Modification Searching of SARS-CoV-2–Human Protein Interaction Data Reveals Novel Viral Modification Sites

The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus 2019 disease, has led to an ongoing global pandemic since 2019. Mass spectrometry can be used to understand the molecular mechanisms of viral infection by SARS-CoV-2, for example, by determining virus–host protein–protein interactions through which SARS-CoV-2 hijacks its human hosts during infection, and to study the role of post-translational modifications. We have reanalyzed public affinity purification–mass spectrometry data using open modification searching to investigate the presence of post-translational modifications in the context of the SARS-CoV-2 virus–host protein–protein interaction network. Based on an over twofold increase in identified spectra, our detected protein interactions show a high overlap with independent mass spectrometry-based SARS-CoV-2 studies and virus–host interactions for alternative viruses, as well as previously unknown protein interactions. In addition, we identified several novel modification sites on SARS-CoV-2 proteins that we investigated in relation to their interactions with host proteins. A detailed analysis of relevant modifications, including phosphorylation, ubiquitination, and S-nitrosylation, provides important hypotheses about the functional role of these modifications during viral infection by SARS-CoV-2.

Oncogenic role of a developmentally regulated NTRK2 splice variant.

Temporally regulated alternative splicing choices are vital for proper development, yet the wrong splice choice may be detrimental. Here, we highlight a previously unidentified role for the neurotrophin receptor splice variant TrkB.T1 in neurodevelopment, embryogenesis, transformation, and oncogenesis across multiple tumor types in humans and mice. TrkB.T1 is the predominant NTRK2 isoform across embryonic organogenesis, and forced overexpression of this embryonic pattern causes multiple solid and nonsolid tumors in mice in the context of tumor suppressor loss. TrkB.T1 also emerges as the predominant NTRK isoform expressed in a wide range of adult and pediatric tumors, including those harboring tropomyosin receptor kinase fusions. Affinity purification–mass spectrometry proteomic analysis reveals distinct interactors with known developmental and oncogenic signaling pathways such as Wnt, transforming growth factor–β, Sonic Hedgehog, and Ras. From alterations in splicing factors to changes in gene expression, the discovery of isoform specific oncogenes with embryonic ancestry has the potential to shape the way we think about developmental systems and oncology.

Identification of sitagliptin binding proteins by affinity purification mass spectrometry.

Type 2 diabetes mellitus (T2DM) is recognized as a serious public health concern with increasing incidence. The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin has been used for the treatment of T2DM worldwide. Although sitagliptin has excellent therapeutic outcome, adverse effects are observed. In addition, previous studies have suggested that sitagliptin may have pleiotropic effects other than treating T2DM. These pieces of evidence point to the importance of further investigation of the molecular mechanisms of sitagliptin, starting from the identification of sitagliptin-binding proteins. In this study, by combining affinity purification mass spectrometry (AP-MS) and stable isotope labeling by amino acids in cell culture (SILAC), we discover seven high-confidence targets that can interact with sitagliptin. Surface plasmon resonance (SPR) assay confirms the binding of sitagliptin to three proteins, i. e., LYPLAL1, TCP1, and CCAR2, with binding affinities (K D) ranging from 50.1 μM to 1490 μM. Molecular docking followed by molecular dynamic (MD) simulation reveals hydrogen binding between sitagliptin and the catalytic triad of LYPLAL1, and also between sitagliptin and the P-loop of ATP-binding pocket of TCP1. Molecular mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis indicates that sitagliptin can stably bind to LYPLAL1 and TCP1 in active sites, which may have an impact on the functions of these proteins. SPR analysis validates the binding affinity of sitagliptin to TCP1 mutant D88A is ~10 times lower than that to the wild-type TCP1. Our findings provide insights into the sitagliptin-targets interplay and demonstrate the potential of sitagliptin in regulating gluconeogenesis and in anti-tumor drug development.

Spike S1 domain interactome in non-pulmonary systems: A role beyond the receptor recognition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which, since 2019 in China, has rapidly become a worldwide pandemic. The aggressiveness and global spread were enhanced by the many SARS-CoV-2 variants that have been isolated up to now. These mutations affect mostly the viral glycoprotein Spike (S), the capsid protein mainly involved in the early stages of viral entry processes, through the recognition of specific receptors on the host cell surface. In particular, the subunit S1 of the Spike glycoprotein contains the Receptor Binding Domain (RBD) and it is responsible for the interaction with the angiotensin-converting enzyme 2 (ACE2). Although ACE2 is the primary Spike host receptor currently studied, it has been demonstrated that SARS-CoV-2 is also able to infect cells expressing low levels of ACE2, indicating that the virus may have alternative receptors on the host cells. The identification of the alternative receptors can better elucidate the pathogenicity and the tropism of SARS-CoV-2. Therefore, we investigated the Spike S1 interactomes, starting from host membrane proteins of non-pulmonary cell lines, such as human kidney (HK-2), normal colon (NCM460D), and colorectal adenocarcinoma (Caco-2). We employed an affinity purification-mass spectrometry (AP-MS) to pull down, from the membrane protein extracts of all cell lines, the protein partners of the recombinant form of the Spike S1 domain. The purified interactors were identified by a shotgun proteomics approach. The lists of S1 potential interacting proteins were then clusterized according to cellular localization, biological processes, and pathways, highlighting new possible S1 intracellular functions, crucial not only for the entrance mechanisms but also for viral replication and propagation processes.

OS10.4.A A macrophage-based drug delivery platform for glioma treatment

Abstract Background There is an urgent need for more effective treatment strategies against gliomas. At present, even though various drugs have potent anti-tumor activity in vitro, their application in vivo is limited by ineffective delivery and systemic toxicity. Therefore, novel strategies are needed to deliver these drugs effectively and safely to the tumor site. Here, we developed an adoptive transfer strategy against malignant brain tumors utilizing macrophages that are loaded with ferritin-protein cages containing drugs or other proteins and transfer these nanocarriers to cancer cells in vitro and in vivo. Material and Methods Live-time imaging, microscopy and flow cytometry were utilized to investigate the transfer of ferritin cages from loaded macrophages to human or mouse glioma cells. Co-cultures of glioma cells and macrophages loaded with ferritin-drug cages were used to study the anti-glioma activity in vitro and orthotopic immunocompetent mouse glioma models were used to study the anti-glioma activity in vivo. Affinity purification-mass spectrometry (AP-MS) was used to elucidate the mechanisms of transfer by characterizing the interactome of ferritin cages within macrophages and cancer cells. Results We observed a high transfer efficiency of ferritin-cages from loaded macrophages into human and mouse glioma cells in vitro in co-culture assays and confirmed the transfer from macrophages to glioma cells also in vivo upon intravenous or intratumoral treatment of GL-261 or CT-2A glioma-bearing mice. To study the anti-glioma activity with therapeutically active payloads, we loaded murine/human macrophages with ferritin cages carrying cytotoxic payloads. Co-culture of these loaded macrophages with murine or human glioma cells in vitro revealed a time- and concentration-dependent cytotoxicity to glioma cells. In vivo, intravenous or intratumoral administration of ferritin-drug protein cages was tolerated without toxicities and conferred a survival benefit in two orthotopic murine glioma models (GL-261 and CT-2A). Interactome studies of ferritin-cage-binding proteins revealed phagocytic and cytoskeleton re-arrangement pathways to be involved in uptake and transfer of ferritin-cages from macrophages to glioma cells. Conclusion This ‘Trojan Horse’ approach constitutes a promising platform to deliver cytotoxic drugs effectively and safely to gliomas and provides a rationale for clinical translation.

Abstract PO017: HBV alters YAP regulation in liver cancer by remodeling PP2A complexes

Abstract Viral hepatitis promotes hepatocellular carcinoma (HCC) initiation by inducing inflammation, cellular stress and cell death. However, the cell-intrinsic effects of viral infection in advanced HCC remain unclear. We used affinity purification mass spectrometry to develop a complete map of 145 interactions among Hepatitis B Virus (HBV) and host proteins in the HUH7 HCC tumor cell line, identifying known and novel HBV/host protein-protein interactions. We integrated this map with HCC genomes, identifying 61 proteins with preferential mutation in non-HBV HCC, suggesting that their interaction with HBV plays a role in cancer. Focusing on proteins that directly interact with the HBV oncoprotein X (HBx), we found that HBx rewires the effect of the PP2A phosphatase on HCC signaling. HBx binding excluded striatin-family regulatory subunits from the PP2A complex, causing Hippo kinase activation. This effect creates a requirement for integrin signaling to mTOR complex 2 to maintain expression of the YAP oncoprotein, critical for HCC growth. Thus, HBV rewires HCC signaling and may promote targetable dependencies. Citation Format: John Gordan, Adriana Pitea, Rigney E Turnham, Manon Eckhardt, Gwendolyn M Jang, Huat Lim, Alex L Choi, Sourav Bandyopadhyay, Danielle Swaney, Kevan Shokat, Trey Ideker, Nevan Krogan. HBV alters YAP regulation in liver cancer by remodeling PP2A complexes [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO017.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and β-cell function. Human islets were treated with interleukin-1β and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate β-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

Abstract P1007: Cardiac Fibroblast BAG3 Controls TGFBR2 Signaling And Contributes To Engineered Cardiac Tissue Function.

Introduction: Mutations in BAG3 result in dilated cardiomyopathy with cardiac fibrosis. Currently, the role of BAG3 in non-myocyte cell types is not known. We tested the hypothesis that BAG3 controls cardiac fibroblast (CF) function and that the loss of fibroblast-specific BAG3 is deleterious, using cells and engineered cardiac tissues (ECT) derived from pluripotent stem cells (iPSC). Methods: Isogenic iPSCs containing homozygous knockout (KO) of BAG3 were differentiated into CFs. Mass spectrometry measured the effect of BAG3 loss on CF proteome. EdU staining and flow cytometry were used to assess CF proliferation. Transforming growth factor beta (TGF) signaling pathway activity was measured using a luciferase reporter and Western blotting. Affinity purification mass spectrometry (AP-MS) identified high-confidence interactors of BAG3 in CFs. To assess the role of fibroblast-specific BAG3 in cardiac function, conditional KO ECTs were generated using wild-type cardiomyocytes mixed with either wild-type CFs or KO CFs. ECTs were functionally assessed using video microscopy and immunofluorescence. The effects of CF BAG3 loss on tissue gene expression are under study by single nuclear RNA sequencing. Results: Analysis of proteomics data revealed enrichment of TGF and proliferation pathways in KO cells. KO-CFs had increased proliferation measured by EdU staining and increased SMAD luciferase reporter activity. Western blotting showed that KO CFs have higher expression of TGF beta receptor 2 (TGFBR2) and activation of canonical TGF signaling. AP-MS identified TGFBR2 as an interacting partner of BAG3, suggesting BAG3 mediated TGFBR2 turnover. In ECT, the loss of CF BAG3 caused a reduction in cardiac tissue contractile force (50% reduction, n = 10-12 tissues per group). Immunofluorescence revealed significantly increased tissue fibrosis in CF-KO cardiac tissues quantified by vimentin staining. Conclusions: BAG3 loss results in increased TGFBR2 signaling and proliferation in cardiac fibroblasts. Conditional KO ECTs demonstrate that CF BAG3 affects tissue function. These data suggest that fibrosis related to BAG3 pathology may be a result of a primary deficiency in CFs rather than a secondary response to cardiomyocyte death.

Abstract 3906: Characterization of mTORC2 interactome reveals association with microtubules in glioblastoma and neuroblastoma cells

Abstract The Mechanistic Target of Rapamycin Complex 2 (mTORC2) is a multiprotein complex representing one of the key players in the mTOR signaling pathway. This signal transduction is essential for the regulation of various cellular activities, including proper cell growth, proliferation, differentiation, survival, cell motility, and metabolism. Aberration of the mTOR cascade can be found in several types of disorders, including cancers and neurodegenerative diseases. It has been reported to correlate with acquired aggressive characteristics promoting metastasis, drug resistance, and recurrence in cancer cells and more progressive phenotypes in other diseased cells. More specifically, mTORC2 signaling promotes actin cytoskeleton reorganization, cell motility, cell survival, and has been recently reported to be responsible for DNA damage control. Unlike mTORC1 that has been more thoroughly studied, molecular mechanisms of mTORC2 are yet to be elaborated. Therefore, characterization of the mTORC2 signaling pathway will provide further information needed for better understandings of the biology of diseases. Previously, we have identified multiple cytoskeleton regulators, including actin-binding and microtubule-associated proteins as binding partners of mTORC2 in glioblastoma cells by affinity purification-mass spectrometry (AP-MS). In this work, we provided extensive characterization of mTORC2 interactome in both neuronal and non-neuronal cells focusing on microtubule-associated proteins. Interestingly, apart from many MAP proteins found in non-neuronal cells (glioblastoma), we identified Tau isoforms and their regulatory proteins such as Tau kinases from affinity-purified mTORC2 in neuroblastoma cells. The quantitative proteomic analyses of mTORC2-interacting proteins in various cellular conditions were performed to help determine the dynamics of the mTORC2-associated microtubule network. These novel protein-protein interactions acquired from this study suggested additional mTORC2 functions in different types of brain cells, which might also be beneficial to the understandings of neurodegenerative diseases in addition to cancers. Citation Format: Narawit Pacharakullanon, Piriya Wongkongkathep, Fuyuhiko Tamanoi, Joseph A. Loo, Trairak Pisitkun, Naphat Chantaravisoot. Characterization of mTORC2 interactome reveals association with microtubules in glioblastoma and neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3906.

Proteomic Characterization of a Candidate Polygenic Driver of Metabolism in Non-small Cell Lung Cancer

Proteome analysis revealed signatures of co-expressed upregulated metabolism proteins highly conserved between primary and non-small cell lung cancer (NSCLC) patient-derived xenograft tumors (Li et al. 2014, Nat. Communications 5:5469). The C10 signature is encoded by seven genes (ADSS, ATP2A2, CTPS1, IMPDH2, PKM2, PTGES3, SGPL1) and DNA alterations in C10-encoding genes are associated with longer survival in a subset of NSCLC. To explore the C10 signature as an oncogenic driver and address potential mechanisms of action, C10 protein expression and protein–protein interactions were determined. In independent NSCLC cohorts, the coordinated expression of C10 proteins was significant and mutations in C10 genes were associated with better outcome. Affinity purification-mass spectrometry and in vivo proximity-based biotin identification defined a C10 interactome involving 667 proteins including candidate drug targets and clusters associated with glycolysis, calcium homeostasis, and nucleotide and sphingolipid metabolism. DNA alterations in genes encoding C10 interactome components were also found to be associated with better survival. These data support the notion that the coordinated upregulation of the C10 signature impinges metabolic processes that collectively function as an oncogenic driver in NSCLC.

Human transcription factor protein interaction networks

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 identifiesmore 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.

Abstract 529: The Intra-molecular Pathway Of Nck1 In Atherogenic Endothelial Activation

Rational: Atherosclerosis, a chronic progressive disease, is the underlying disease process in ischemic heart disease (IHD). Despite our advances, IHD is still the leading cause of disease mortality worldwide. Endothelial Activation due to disturbed blood flow (d-flow) is one of the earliest changes in atherosclerosis. However, the molecular pathway whereby d-flow induces endothelial activation is unclear. Methodology & Results: Herein we identified the signaling protein Nck1 as a key regulator to atherogenic endothelial activation. Nck1 is an adaptor molecule with a multi-domain structure, and in response to d-flow, we found that the Nck1 SH2 (phosophotyrosine binder) and the Nck1 SH3.1 (with a high affinity to bind to proline rich peptides) are critical domains of Nck1 to regulate the prolinflammatory signal activation. In addition, the small molecule inhibitor, AX-024 (50 nM), which occupies the DY pocket of the Nck1 SH3.1, prevents d-flow induced endothelial activation, suggesting downstream mediator that binds to the Nck1 SH3.1 and mediates the activation. In order to determine the Nck1 specific signaling partners, we used affinity purification-mass spectrometry and we identified 6 signaling proteins that significantly interact with Nck1 in response to d-flow. In vivo, our endothelial-specific Nck1 knock out mice show significantly less atherosclerosis lesion burden compared to their littermate controls. Conclusions: A recent GWAS analysis linked Nck1 to IHD, underscoring its importance as a potential therapeutic target. AX-024 is a first-in-class a small molecule inhibitor to Nck1, and is currently being tested in clinical trials. Our data confirms a role for AX-024 in preventing endothelial activation after d-flow exposure and sheds light on a previously uncharacterized function of Nck1 adaptor protein in atherogenic endothelial activation.

Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation.

Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cyclic adenosine monophosphate (cAMP)-dependent signaling pathway and downstream mediators. To investigate protein–protein interaction partners of FSHR at resting state and upon FSH stimulation, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. Interestingly, while only WT FSHR interacted with FSH, insulin-like growth factor 1 receptor (IGF1R), for example, interacted with both WT and A189V FSHR upon FSH stimulation. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR.

The cargo adapter protein CLINT1 is phosphorylated by the Numb-associated kinase BIKE and mediates dengue virus infection

The signaling pathways and cellular functions regulated by the four Numb-associated kinases are largely unknown. We reported that AAK1 and GAK control intracellular trafficking of RNA viruses and revealed a requirement for BIKE in early and late stages of dengue virus (DENV) infection. However, the downstream targets phosphorylated by BIKE have not yet been identified. Here, to identify BIKE substrates, we conducted a barcode fusion genetics-yeast two-hybrid screen and retrieved publicly available data generated via affinity-purification mass spectrometry. We subsequently validated 19 of 47 putative BIKE interactors using mammalian cell–based protein–protein interaction assays. We found that CLINT1, a cargo-specific adapter implicated in bidirectional Golgi-to-endosome trafficking, emerged as a predominant hit in both screens. Our experiments indicated that BIKE catalyzes phosphorylation of a threonine 294 CLINT1 residue both in vitro and in cell culture. Our findings revealed that CLINT1 phosphorylation mediates its binding to the DENV nonstructural 3 protein and subsequently promotes DENV assembly and egress. Additionally, using live-cell imaging we revealed that CLINT1 cotraffics with DENV particles and is involved in mediating BIKE’s role in DENV infection. Finally, our data suggest that additional cellular BIKE interactors implicated in the host immune and stress responses and the ubiquitin proteasome system might also be candidate phosphorylation substrates of BIKE. In conclusion, these findings reveal cellular substrates and pathways regulated by the understudied Numb-associated kinase enzyme BIKE, a mechanism for CLINT1 regulation, and control of DENV infection via BIKE signaling, with potential implications for cell biology, virology, and host-targeted antiviral design.

A network biology approach to identify crucial host targets for COVID-19.

The ongoing global pandemic of COVID-19, caused by SARS-CoV-2 has killed more than 5.9 million individuals out of ∼43 million confirmed infections. At present, several parts of the world are encountering the 3rd wave. Mass vaccination has been started in several countries but they are less likely to be broadly available for the current pandemic, repurposing of the existing drugs has drawn highest attention for an immediate solution. A recent publication has mapped the physical interactions of SARS-CoV-2 and human proteins by affinity-purification mass spectrometry (AP-MS) and identified 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Here, we taken a network biology approach and constructed a human protein-protein interaction network (PPIN) with the above SARS-CoV-2 targeted proteins. We utilized a combination of essential network centrality measures and functional properties of the human proteins to identify the critical human targets of SARS-CoV-2. Four human proteins, namely PRKACA, RHOA, CDK5RAP2, and CEP250 have emerged as the best therapeutic targets, of which PRKACA and CEP250 were also found by another group as potential candidates for drug targets in COVID-19. We further found candidate drugs/compounds, such as guanosine triphosphate, remdesivir, adenosine monophosphate, MgATP, and H-89 dihydrochloride that bind the target human proteins. The urgency to prevent the spread of infection and the death of diseased individuals has prompted the search for agents from the pool of approved drugs to repurpose them for COVID-19. Our results indicate that host targeting therapy with the repurposed drugs may be a useful strategy for the treatment of SARS-CoV-2 infection.

SARS-CoV-2 helicase NSP13 hijacks the host protein EWSR1 to promote viral replication by enhancing RNA unwinding activity

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. ResultsIn this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. ConclusionsOur results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.

Human transcription factor protein interaction networks

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.

Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration

SUMMARYTau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau’s interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau’s role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.