Tandem Affinity Purification-mass Spectrometry Research Articles

USP7 interacts with and destabilizes oncoprotein SET

Oncoprotein SE translocation (SET) is frequently overexpressed in different types of tumors and correlated with poor prognosis of cancer patients. Targeting SET has been considered a promising strategy for cancer intervention. However, the mechanisms by which SET is regulated under cellular conditions are largely unknown. Here, by performing a tandem affinity purification-mass spectrometry (TAP-MS), we identify that the ubiquitin-specific protease 7 (USP7) forms a stable protein complex with SET in cancer cells. Further analyses reveal that the acidic domain of SET directly binds USP7 while both catalytic domain and ubiquitin-like (UBL) domains of USP7 are required for SET binding. Knockdown of USP7 has no effect on the mRNA level of SET. However, we surprisingly find that USP7 depletion leads to a dramatic elevation of SET protein levels, suggesting that USP7 plays a key role in destabilizing oncoprotein SET, possibly through an indirect mechanism. To our knowledge, our data report the first deubiquitinase (DUB) that physically associates with oncoprotein SET and imply an unexpected regulatory effect of USP7 on SET stability.

LRRC59 promotes the progression of oral squamous cell carcinoma by interacting with SRP pathway components and enhancing the secretion of CKAP4-containing exosomes

BackgroundAs a ribosome receptor, LRRC59 was thought to regulate mRNA translation on the ER membrane. Evidence suggests that LRRC59 is overexpressed in a number of human malignancies and is associated with poor prognoses, but its primary biological function in the development of oral squamous cell carcinoma (OSCC) remains obscure. ObjectiveThe purpose of this study is to investigate at the expression changes and functional role of LRRC59 in OSCC. MethodsLRRC59 gene expression and correlation with prognosis of OSCC patients were first examined using the data from The Cancer Genome Atlas (TCGA) databases. Following that, a series of functional experiments, including cell counting kit-8, cell cycle analysis, wound healing assays, and transwell assays, were carried out to analyze the biological roles of LRRC59 in tumor cells. Mechanistically, we employed Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach to isolate and identify protein complexes of LRRC59. Downstream regulatory proteins of LRRC59 were verified through immunoprecipitation and immunofluorescence experiments. Furthermore, we isolated exosomes from OSCC cell supernatant and conducted co-culture experiments to examine the effect of LRRC59 knockdown on OSCC cells. ResultsIn samples from OSCC patients, LRRC59 was highly expressed and correlated with poor prognoses. Moreover, the gene sets analysis based on TCGA RNA-seq data indicated that LRRC59 seemed to be strongly related with protein secretory and OSCC migration. Upregulated levels of LRRC59 are more prone to lymph node metastasis in OSCC patients. LRRC59 knockdown impaired the ability of OSCC cell proliferation, migration, and invasion invitro. Mechanistically, our TAP-MS data situate LRRC59 in a functional nexus for mRNA translation regulation via interactions with SRP pathway components, translational initiation factors, CRD-mediated mRNA stabilization factors. More importantly, we found that LRRC59 interacted with cytoskeleton-associated protein 4 (CKAP4) and promoted the formation of CKAP4-containing exosomes. We also revealed that the LRRC59-CKAP4 axis was a crucial regulator of CKAP4-containing exosome secretion in OSCC cells for migration and invasion. ConclusionsTherefore, based on our findings, LRRC59 may serve as a potential biomarker for OSCC patients, and LRRC59-induced exosome secretion via the CKAP4 axis may serve as a potential therapeutic target for OSCC.

Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

Identification and Validation of a PPP1R12A-Related Five-Gene Signature Associated With Metabolism to Predict the Prognosis of Patients With Prostate Cancer.

BackgroundProstate cancer (PCa) is the most common malignant male neoplasm in the American male population. Our prior studies have demonstrated that protein phosphatase 1 regulatory subunit 12A (PPP1R12A) could be an efficient prognostic factor in patients with PCa, promoting further investigation. The present study attempted to construct a gene signature based on PPP1R12A and metabolism-related genes to predict the prognosis of PCa patients.MethodsThe mRNA expression profiles of 499 tumor and 52 normal tissues were extracted from The Cancer Genome Atlas (TCGA) database. We selected differentially expressed PPP1R12A-related genes among these mRNAs. Tandem affinity purification-mass spectrometry was used to identify the proteins that directly interact with PPP1R12A. Gene set enrichment analysis (GSEA) was used to extract metabolism-related genes. Univariate Cox regression analysis and a random survival forest algorithm were used to confirm optimal genes to build a prognostic risk model.ResultsWe identified a five-gene signature (PPP1R12A, PTGS2, GGCT, AOX1, and NT5E) that was associated with PPP1R12A and metabolism in PCa, which effectively predicted disease-free survival (DFS) and biochemical relapse-free survival (BRFS). Moreover, the signature was validated by two internal datasets from TCGA and one external dataset from the Gene Expression Omnibus (GEO).ConclusionThe five-gene signature is an effective potential factor to predict the prognosis of PCa, classifying PCa patients into high- and low-risk groups, which might provide potential novel treatment strategies for these patients.

The Interaction of NEK2 with USP7 Causes Resistance to Proteasome Inhibitor in Multiple Myeloma

Background: Drug resistance is the major reason for treatment failure in multiple myeloma (MM). Our previous work has demonstrated that high expression of chromosomal instability (CIN) genes results in increased cell survival and drug resistance and is associated with poor outcome in MM and other cancers. NEK2 was the most significant CIN gene to induce drug resistance and poor outcome in MM. Though we have demonstrated that NEK2 induces drug resistance through activation of efflux drug pumps, the detail mechanisms remain unknown.Materials and Methods: Multiple MM cell lines, xenograft MM NOD-Rag1nullmice with both subcutaneous and by tail vein injection, and primary MM samples were included in this study to test NEK2 inhibitors and gene expression profiles (GEP). The tandem affinity purification - mass spectrometry (TAP-MS) and co-immunoprecipitation (Co-IP) were applied for the identification of NEK2 interacting partners. The dual-luciferase reporter assay and GEPs were performed to determine whether the interaction of NEK2 with USP7 activates the canonical NF-kB signaling pathway. Chromatin immunoprecipitation (ChIP)-qPCR and western blots were carried out to determine the direct target genes of the axis NEK2/USP7/NF-kB.Results: We demonstrate that NEK2 is stabilized by the de-ubiquitinating enzyme USP7, leading to accumulation of NEK2 within the cancer cells. Accumulation of NEK2 activates the canonical NF-kB through the AKT/IKK/IKB-a/p65 axis and, using this pathway as framework, we uncovered a novel bona fide NF-kB target, heparanase, a protein that is known to promote bone destruction in MM patients. A NF-kB or USP7 inhibitor could overcome NEK2-induced bortezomib resistance. The NEK2 inhibitor, INH1, showed great therapeutic effects on MM in vitro and in vivo . To corroborate our findings, we subcutaneously injected NEK2-OE ARP1 MM cells into NOD.Cg-Rag1 mice and targeted NEK2 with the small molecule drugs INH1 or P5091 (USP7 inhibitor) alone or in combination with bortezomib. As expected bortezomib had very little effect in reducing tumor burden since NEK2-OE cells are highly resistant to this drug. Inhibition of NEK2 with P5091 or INH1 alone had a strong anti-tumor effect and a synergistic effect was observed when combined with bortezomib.Conclusion: Our data present a novel mechanism for the interaction of NEK2 with USP7 driving drug resistance in MM. We conclude that (1) USP7 in MM cells leads to the de-ubiquitination and escape of proteasomal degradation of NEK2, leading to its accumulation in cancer cells. (2) Accumulated NEK2 binds and phosphorylates PP1a, suppressing its phosphatase activity thus promoting AKT overactivation. (3) AKT then phosphorylates the IKKa/b complex, triggering the canonical NF-kB cascade which phosphorylates and targets IKBa for proteasomal degradation, releasing the sequestered p65 to be phosphorylated at S536 and translocation to the nucleus where it drives bortezomib resistance by activating its target genes. Our data also show that INH1 and P5091 can deplete NEK2 protein in vitro and in vivo and suppress NEK2 downstream targets resulting in overcoming bortezomib resistance. Our findings should facilitate novel targeted approaches to the therapy and prevention of myeloma progression and relapse. DisclosuresNo relevant conflicts of interest to declare.

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition

Persistent R-loops (RNA–DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5′-3′-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2’s association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.

Abstract B21: Proteomic profiling of tandem affinity purified MAP4K family kinases

Abstract MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases and recently have been reported to regulate Hippo pathway via LATS1/2 activation. However, the diverse roles of MAP4Ks in biologic process are still unclear. To identify the MAP4K family kinase interaction network, we employed a tandem affinity purification-mass spectrometry (TAP-MS) approach using SFB tag-conjugated MAP4K1-7. Through a proteomic analysis, we could guess that MAP4K family kinases, especially MAP4K2, may regulate autophagy or interact with striatin-interacting phosphatase and kinase (STRIPAK) components. These results offer a framework for further studies on Hippo pathway and biologic processes involving MAP4Ks. Note: This abstract was not presented at the conference. Citation Format: Gayoung Seo, Han Han, Bing Yang, Rebecca Vargas, Yuxuan Chen, Wenqi Wang. Proteomic profiling of tandem affinity purified MAP4K family kinases [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B21.

Delineating WWOX protein interactome by tandem affinity purification-mass spectrometry

Delineating WWOX protein interactome by tandem affinity purification-mass spectrometry

The autophagy protein ATG9A promotes HIV-1 infectivity

BackgroundNef is a multifunctional accessory protein encoded by HIV-1, HIV-2 and SIV that plays critical roles in viral pathogenesis, contributing to viral replication, assembly, budding, infectivity and immune evasion, through engagement of various host cell pathways.ResultsTo gain a better understanding of the role of host proteins in the functions of Nef, we carried out tandem affinity purification-mass spectrometry analysis, and identified over 70 HIV-1 Nef-interacting proteins, including the autophagy-related 9A (ATG9A) protein. ATG9A is a transmembrane component of the machinery for autophagy, a catabolic process in which cytoplasmic components are degraded in lysosomal compartments. Pulldown experiments demonstrated that ATG9A interacts with Nef from not only HIV-1 and but also SIV (cpz, smm and mac). However, expression of HIV-1 Nef had no effect on the levels and localization of ATG9A, and on autophagy, in the host cells. To investigate a possible role for ATG9A in virus replication, we knocked out ATG9A in HeLa cervical carcinoma and Jurkat T cells, and analyzed virus release and infectivity. We observed that ATG9A knockout (KO) had no effect on the release of wild-type (WT) or Nef-defective HIV-1 in these cells. However, the infectivity of WT virus produced from ATG9A-KO HeLa and Jurkat cells was reduced by ~ fourfold and eightfold, respectively, relative to virus produced from WT cells. This reduction in infectivity was independent of the interaction of Nef with ATG9A, and was not due to reduced incorporation of the viral envelope (Env) glycoprotein into the virus. The loss of HIV-1 infectivity was rescued by pseudotyping HIV-1 virions with the vesicular stomatitis virus G glycoprotein.ConclusionsThese studies indicate that ATG9A promotes HIV-1 infectivity in an Env-dependent manner. The interaction of Nef with ATG9A, however, is not required for Nef to enhance HIV-1 infectivity. We speculate that ATG9A could promote infectivity by participating in either the removal of a factor that inhibits infectivity or the incorporation of a factor that enhances infectivity of the viral particles. These studies thus identify a novel host cell factor implicated in HIV-1 infectivity, which may be amenable to pharmacologic manipulation for treatment of HIV-1 infection.

XRN2 Depletion is Synthetic Lethal with PARP1 Inhibition

Persistent R‐loops (RNA‐DNA hybrids with a displaced single‐stranded DNA) create DNA damage and lead to genomic instability. The 5’‐3’‐exoribonuclease 2 (XRN2) degrades RNA to resolve R‐loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. XRN2 function in DSB repair is not fully understood. We hypothesize that XRN2 plays a critical role in multiple DNA repair pathways. Here, using tandem affinity purification‐mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70‐Ku80, DNA‐PKcs, PARP1, MCM2‐7, PCNA, RPA1), and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non‐homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2‐deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2‐deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. The XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 loss and PARP1 inhibition.Support or Funding InformationThis work was largely supported by the National Institutes of Health/National Cancer Institute (NCI/NIH) [R01 CA139217] to D.A.B, a minority supplement [R01 CA139217‐05S1] and the Cancer Biology Training Grant T32CA124334‐06 to E.A.M. (PI: Dr. Jerry Shay), Simmons Comprehensive Cancer Center, UT Southwestern. This work was also supported by grant 5P30CA142543, NCI/NIH to the UT Southwestern Proteomics Core. The UT Southwestern Proteomics Core was also supported, in part, by a grant from the Cancer Prevention and Research Institute of Texas [CPRIT, RP1206130] to Dr. Hamid Mirzaei. Funding for open access charges was from NCI/NIH CA139217 to D.A.B. Research reported in this study was also supported by the UT Southwestern CCSG grant 5P30CA142543 from the NCI/NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P1-06-10: Combined tandem affinity purification mass-spectrometry technique with genome-editing CRISPR-Cas9 knockout screening to identify potential subunits in the BRCA1 complex

Abstract Background: Genomic instability is one of tumor characteristics. Enhancing the DNA damage load or impairing the ability of DNA damage repair has been therapeutics of cancer. Breast cancer type 1 susceptibility gene (BRCA1) has been studied a lot about the DNA damage and repair in breast cancer and ovarian cancer. BRCA1 mutation increases the risk of developing breast cancer by an eighth to tenth. The C terminal domain of BRCA1 can associates with three proteins Abraxas, Bach1 and CtIP in a phosphorylation-dependent manner forming mutually exclusive complexes, namely BRCA1-A, B, and C complexes. The BRCA1-A complex is necessary in DNA damage repair, and study implicates the complex play roles in chemotherapy resistance. Methods: We explored tandem affinity purification mass-spectrometry (TAP-MAS) technique to identify potential subunits associated with NBA1(one component of BRCA1-A complex). Then we made the genome-editing CRISPR-Cas9 sgRNA library into lentivirus to infect U2OS cells. And 5 Gy dose of Ionizing radiation (IR) was used to induce DNA damage on the cell. After 14 days cultivation, we extracted the DNA from the cells, performed polymerase chain reaction (PCR) and analyzed the correlation between potential genes and DNA damage-repair passage by bioinformatics methods. We generated 200 breast cancer patient tissue samples in our cancer center and performed immunostaining assay. Results: By the TAP-MAS technique of NBA1 tagged with HA and Flag, we found 93 potential subunits except those known subunits in BRCA1-A complex. Combined with CRISPR-Cas9 sgRNA library screening, we scored the relativity of DNA damage and repair passageway of identified 93 potential subunits. We found that nucleoside-triphosphatase, cancer-related (NTPCR) as a new potential subunit of BRCA1-A complex (P value=0.0034) had the highest score. Endogenous and exogenous co-IP validated NTPCR physically associates with subunits within BRCA1-A complex. Besides, we found that NTPCR associated with the same domain of NBA1 as the other subunits in BRCA1-A complex. Immunohistochemistry of patient tissue samples indicated that high levels of NTPCR expression was correlated with poor prognosis in multivariate analysis (HR: 4.990; 95%CI: 1.433-17.378; p value: 0.012). Conclusion: NTPCR is a new subunit in BRCA1-A complex. And high expression of NTPCR is a negative prognostic factor in breast cancer patients. Citation Format: Zhao H, Jiang H, Sun W, Shao Z, Hu X. Combined tandem affinity purification mass-spectrometry technique with genome-editing CRISPR-Cas9 knockout screening to identify potential subunits in the BRCA1 complex [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-10.

Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved.

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a “non-classical” tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies.

Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma.

Drug resistance remains the key problem in cancer treatment. It is now accepted that each myeloma patient harbors multiple subclones and subclone dominance may change over time. The coexistence of multiple subclones with high or low chromosomal instability (CIN) signature causes heterogeneity and drug resistance with consequent disease relapse. In this study, using a tandem affinity purification-mass spectrometry (TAP-MS) technique, we found that NEK2, a CIN gene, was bound to the deubiquitinase USP7. Binding to USP7 prevented NEK2 ubiquitination resulting in NEK2 stabilization. Increased NEK2 kinase levels activated the canonical NF-κB signaling pathway through the PP1α/AKT axis. Newly diagnosed myeloma patients with activated NF-κB signaling through increased NEK2 activity had poorer event-free and overall survivals based on multiple independent clinical cohorts. We also found that NEK2 activated heparanase, a secreted enzyme, responsible for bone destruction in an NF-κB-dependent manner. Intriguingly, both NEK2 and USP7 inhibitors showed great efficacy in inhibiting myeloma cell growth and overcoming NEK2-induced and -acquired drug resistance in xenograft myeloma mouse models.

Nek2 Stabilization By Usp7 Leads to Activation of NF-Kb in Multiple Myeloma

NIMA (Never In Mitosis Gene A)-Related Kinase 2 (Nek2), a centrosomal Serine/Threonine kinase, is a key player in numerous malignancies. Overexpression of Nek2 has been related to many cancers including Multiple Myeloma (MM). In MM, Nek2 is one of the chromosomal instability genes associated with drug resistance and disease relapse. However, very little is known about the mechanisms that lead to these Nek2-driven disparities. Here, we show that the Ubiquitin Specific Peptidase 7 (USP7) stabilizes Nek2 leading to activation of NF-kb pathway.Using gene expression profile (GEP) data from patients and cell lines we discovered that Nek2 overexpression leads to increases of several targets of the NF-kb pathway. We, thus, hypothesize that Nek2 is activating NF-kb. To address this, we overexpressed Nek2 and tested the classic canonical NF-kb hallmarks proteins by western blotting. Nek2 overexpression led to an increase in phosphorylation of IKK, activator of NF-kb, and to decrease levels of IKb-alpha, a negative regulator of the pathway. Nek2 overexpression also increased nuclear and phosphorylated p65 on residue S536, known as active transcriptional site. To further confirm that Nek2 is activating canonical NF-kb luciferase assay was performed. The luciferase reporter is driven by a p65 promoter and in cells overexpressing Nek2 luciferase levels were increased.To characterize Nek2 interacting partners a tandem affinity purification/mass spectrometry (TAP/MS) approach was performed. We found that Nek2 binds to Usp7, a deubiquitinase overexpressed in numerous cancers. This led to hypothesize that that Nek2, a known target of the ubiquitin proteasome system, is being stabilized by the Usp7 contributing to its overexpression and the increased activation of the NF-kb pathway. To test our hypothesis, we treated different cancer cell lines with the commercially available Usp7 inhibitor, P5091, or silenced the protein using shRNA. In both case, we found a reduction in Nek2 protein level. Additionally, we overexpressed Usp7 and Nek2 increased confirming that Usp7 stabilizes Nek2. To further show that Usp7 stabilizes Nek2 by de-ubiquitination, we overexpressed Usp7 and analyzed Nek2 ubiquitination after immunoprecipitation. When Usp7 was overexpressed no ubiquitination of Nek2 was detected.Finally, by using GEP data from MM patients, we found that individuals who overexpressed Nek2 along with an active NF-kb signature have worst event free survival as well as overall survival, indicating Nek2 overexpression leading to increased NF-kb signature has clinical significance. DisclosuresNo relevant conflicts of interest to declare.

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client

Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

A two-layer integration framework for protein complex detection.

BackgroundProtein complexes carry out nearly all signaling and functional processes within cells. The study of protein complexes is an effective strategy to analyze cellular functions and biological processes. With the increasing availability of proteomics data, various computational methods have recently been developed to predict protein complexes. However, different computational methods are based on their own assumptions and designed to work on different data sources, and various biological screening methods have their unique experiment conditions, and are often different in scale and noise level. Therefore, a single computational method on a specific data source is generally not able to generate comprehensive and reliable prediction results.ResultsIn this paper, we develop a novel Two-layer INtegrative Complex Detection (TINCD) model to detect protein complexes, leveraging the information from both clustering results and raw data sources. In particular, we first integrate various clustering results to construct consensus matrices for proteins to measure their overall co-complex propensity. Second, we combine these consensus matrices with the co-complex score matrix derived from Tandem Affinity Purification/Mass Spectrometry (TAP) data and obtain an integrated co-complex similarity network via an unsupervised metric fusion method. Finally, a novel graph regularized doubly stochastic matrix decomposition model is proposed to detect overlapping protein complexes from the integrated similarity network.ConclusionsExtensive experimental results demonstrate that TINCD performs much better than 21 state-of-the-art complex detection techniques, including ensemble clustering and data integration techniques.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-0939-3) contains supplementary material, which is available to authorized users.

The Kub5-Hera/RPRD1B interactome: a novel role in preserving genetic stability by regulating DNA mismatch repair.

Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, co-immunoprecipitation and gel-filtration chromatography to define higher-order protein complexes containing K-H scaffolding protein to gain insight into its cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, p15RS) and discovered several novel associated proteins that function in RNA metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes (PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein metabolic processes, including translation. Notably, this approach directed us to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) where K-H depletion led to concomitant MMR deficiency and compromised global microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor treatment restored MMR protein loss. These findings represent a novel mechanism to acquire MMR deficiency/microsatellite alterations. A significant proportion of colon, endometrial and ovarian cancers exhibit k-h expression/copy number loss and may have severe mutator phenotypes with enhanced malignancies that are currently overlooked based on sporadic MSI+ screening.

SLC27A4 regulate ATG4B activity and control reactions to chemotherapeutics-induced autophagy in human lung cancer cells.

Autophagy is a highly conserved self-digestion process to promote cell survival in response to nutrient starvation and other metabolic stresses in eukaryotic cells. Dysregulation of this system is linked with numerous human diseases, including cancers. ATG4B, a cysteine protease required for autophagy, cleaves the C-terminal amino acid of ATG8 family proteins to reveal a C-terminal glycine which is necessary for ATG8 proteins conjugation to phosphatidylethanolamine (PE) and insertion to autophagosome precursor membranes. However, the mechanism governing the protein stability of ATG4B in human cancer cells is not fully understood. In this study, tandem affinity purification/mass spectrometry (TAP/MS) were applied to the investigation of the interaction between ATG4B and potential candidate proteins. Then, co-immunoprecipitation (Co-IP) and GST-pull down assays indicated that the candidate protein-SLC27A4 directly interacts with ATG4B in lung cancer cell lines. Intriguingly, we also found that ATG4B protein expression was increased in parallel with SLC27A4 in lung cancer cell lines as well as lung tumor tissues. However, relevant functional research of SLC27A4 in autophagy or oncotherapy has not been investigated before. In this study, we hypothesized that SLC27A4 might act as a mediator of ATG4B, in some respects, through the protein binding directly. Further, we found that the high expression level of SLC7A4 increased the ATG4B stability and was conducive to rapid reaction to everolimus (RAD001)-induced autophagy in human lung cancer cells. As expected, the results showed that SLC27A4 could help to maintain the protein stability and intracellular concentration of ATG4B, thereby triggering rapid autophagy through releasing ATG4B to cytoplasm under conditions of reduced nutrient availability or during stress of chemotherapy in lung cancer cells. Reduced SLC27A4 by si-RNA also showed the enhanced therapeutic efficiency of everolimus, doxorubicin, and cisplatin in human lung cancer cell lines. Collectively, this study may help researchers better understand the mechanism of autophagy vitality in human cancers and SLC27A4/ATG4B complex might act as a new potential therapeutic target of lung tumor chemotherapy.

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.