Tandem Affinity Research Articles

CRIP1 involves the pathogenesis of multiple myeloma via dual-regulation of proteasome and autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of the plasma cells. The maintenance of protein homeostasis is critical for MM cell survival. Elevated levels of paraproteins in MM cells are cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as a backbone agent and successfully improved the outcome of patients; however, the increasing activity of autophagy suppresses the sensitivity to PIs treatment. The transcription levels of CRIP1 were explored in plasma cells obtained from healthy donors, patients with newly diagnosed multiple myeloma (NDMM), and relapsed/refractory multiple myeloma (RRMM) using Gene expression omnibus datasets. Doxycycline-inducible CRIP1-shRNA and CRIP1 overexpressed MM cell lines were constructed to explore the role of CRIP1 in MM pathogenesis. Proliferation, invasion, migration, proteasome activity and autophagy were examined in MM cells with different CRIP1 levels. Co-immunoprecipitation (Co-IP) with Tandem affinity purification/Mass spectrum (TAP/MS) was performed to identify the binding proteins of CRIP1. The mouse xenograft model was used to determine the role of CRIP1 in the proliferation and drug-resistance of MM cells. High CRIP1 expression was associated with unfavorable clinical outcomes in patients with MM and served as a biomarker for RRMM with shorter overall survival. Invitro and invivo studies showed that CRIP1 plays a critical role in protein homeostasis via the dual regulation of the activities of proteasome and autophagy in MM cells. A combined analysis of RNA-seq, Co-IP and TAP/MS demonstrated that CRIP1 promotes proteasome inhibitors resistance in MM cells by simultaneously binding to de-ubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promoted proteasome activity and autophagosome maturation by facilitating the dequbiquitination and stabilization of PA200. Our findings clarified the pivotal roles of the CRIP1/USP7/PA200 complex in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the pathogenesis of MM. A full list of funding sources can be found in the acknowledgements section.

CRIP1 Involves the Pathogenesis of Multiple Myeloma Via Dual-Regulation of Proteasome and Autophagy

Multiple myeloma (MM) is an incurable hematological malignancy of plasma cells. The maintenance of protein homeostasis is critical for the survival of MM cells. Elevated paraprotein in MM cells would be cleared by proteasomes or lysosomes, which are independent but inter-connected with each other. Proteasome inhibitors (PIs) work as the backbone agent and successfully improved the outcome of patients, but the increasing activity of autophagy suppresses the sensitivity to PIs treatment. In the present study, we elucidated that CRIP1 was a critical player in protein homeostasis via the dual regulation of proteasome and autophagy in MM. A combination analysis of RNA-seq, Co-IP and Tandem affinity purification/mass spectrometry identified that high-level CRIP1 represents a biomarker for relapsed/refractory MM with shorter overall-survival of patients. CRIP1 plays a critical role in MM progression and PIs resistance by binding to deubiquitinase USP7 and proteasome coactivator PA200. CRIP1 promotes proteasome activity and autophagosome maturation in MM by stabilizing PA200. Therefore, our findings clarified the pivotal roles of CRIP1/USP7/PA200 complex formation in ubiquitin-dependent proteasome degradation and autophagy maturation involved in the progression of MM.

CRIP1 INDUCES PIS RESISTANCE BY FORMING A COMPLEX WITH USP7 /PA200 AND ENHANCING PROTEASOME ACTIVITY AND AUTOPHAGY IN MULTIPLE MYELOMA

Introduction: Multiple myeloma (MM) is a plasma malignancy characterized by an accumulation of misfolded immunoglobulins in the cell. Although proteasome inhibitor (PI) can treat MM by inhibiting ubiquitin-proteasome system (UPS), autophagy as a compensatory protein clearance mechanism, it still leads to drug resistance. The discovery of new targets that can simultaneously inhibit UPS and autophagy will provide great potential for the treatment of MM. Methods: The proliferation, invasion, migration, proteasome activity and autophagy were detected in CRIP1- knockdown /overexpression and control MM cells. Co-IP (Co-Immunoprecipitation) with TAP/MS (Tandem affinity purification/Mass spectrum) was performed to detect the binding of USP7, CRIP1 and PA200. Myeloma xenograft model was used to determine the role of CRIP1 to promote proliferation of MM cells and induce BTZ resistance in vivo. Results: The levels of CRIP1 were significantly increased in plasma cells from NDMM (newly diagnosed multiple myeloma) compared to healthy donors, and further increased in patients with RRMM (relapsed/refractory multiple myeloma). Moreover, the high expression of CRIP1 is significantly related to the poor prognosis of MM patients, especially those treated with BTZ. Our results in vitro and in vivo showed that CRIP1 knockdown noteworthy inhibited the cell growth, invasion, migration. MM cells were more sensitive to PIs-induced cell growth arrest when CRIP1 knockdown. The proteasome activity and the protein level of LC3B-Ⅱ of the MM cells were significantly reduced with CRIP1 knockdown. Co-IP analysis showed that CRIP1 formed a complex with USP7/PA200. The protein level of CRIP1 decreased and the ubiquitination CRIP1 increased with USP7 inhibition. These data suggested that CRIP1 is the substrate of USP7.CRIP1 promotes the de-ubiquitination and stabilization of PA200 mediated by USP7 by interacting with USP7 and PA200, which in turn promotes MM cell survival and drug resistant. Functional inhibition of CRIP1 by knocking down of USP7 or PSME4 in CRIP1 OE MM cell line led to a decrease of the activity of proteasome and autophagy, thus enhancing the sensitivity to PIs in intro and in vivo. Conclusion: CRIP1 plays a critical role in MM progression and PIs resistance by formation of CRIP1/USP7/PA200 complex. High level of CRIP1 is a biomarker for high-risk MM patients. CRIP1 is a potential target for MM therapy, can simultaneously inhibit proteasome activity and autophagy. Encore Abstract - previously submitted to EHA 2023 The research was funded by the following foundations:(a) CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-040 and 2022-I2M-1-022), (b) National NaturalScience Foundation of China (82170194, 81920108006, 82270175) and Natural Science Foundation of Fujian Province of China (2021J02040). Keywords: Diagnostic and Prognostic Biomarkers, Multiple Myeloma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

Tumor protein D52 (TPD52) affects cancer cell metabolism by negatively regulating AMPK.

The AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, with deregulation leading to cancer and other diseases. However, how this pathway is dysregulated in cancer has not been well clarified. Using a tandem affinity purification/mass-spec technique and biochemical analyses, we identified tumor protein D52 (TPD52) as an AMPKα-interacting molecule. To explore the biological effects of TPD52 in cancers, we conducted biochemical and metabolic assays in vitro and in vivo with cancer cells and TPD52 transgenic mice. Finally, we assessed the clinical significance of TPD52 expression in breast cancer patients using bioinformatics techniques. TPD52, initially identified to be overexpressed in many human cancers, was found to form a stable complex with AMPK in cancer cells. TPD52 directly interacts with AMPKα and inhibits AMPKα kinase activity in vitro and in vivo. In TPD52 transgenic mice, overexpression of TPD52 leads to AMPK inhibition and multiple metabolic defects. Clinically, high TPD52 expression predicts poor survival of breast cancer patients. The findings revealed that TPD52 is a novel regulator of energy stress-induced AMPK activation and cell metabolism. These results shed new light on AMPK regulation and understanding of the etiology of cancers with TPD52 overexpression.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in invivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.

Induction of autophagy and suppression of type I IFN secretion by CSFV

ABSTRACT Macroautophagy/autophagy plays an essential role in cellular responses to pathogens. However, the precise mechanisms and signaling pathways that modulate cellular autophagy in classical swine fever virus (CSFV)-infected host cells have not been confirmed. In this study, we showed that CSFV infection inhibits the phosphorylation of MTOR (mechanistic target of rapamycin kinase), subsequently leading to autophagy initiation. We also show that MAPK/ERK (mitogen-activated protein kinase) signaling is involved in CSFV-induced autophagy. The CSFV-induced inhibition of AKT/PKB (AKT serine/threonine kinase)-MTOR was observed to be partially responsible for the MTOR inactivation and subsequent autophagy initiation. Moreover, the CAMKK2/CaMKKβ (calcium/calmodulin dependent protein kinase kinase 2)-PRKAA/AMPK (protein kinase AMP-activated catalytic subunit alpha) axis was found to be involved in CSFV-induced autophagy. Meanwhile, CSFV non-structural protein NS5A induced autophagy via the CAMKK2-PRKAA-MTOR signaling pathway but not the AKT-MTOR or MAPK1/ERK2-MAPK3/ERK1-MTOR pathway. Although the AKT-MTOR pathway also plays an important role in the induction of autophagy by CSFV. We also found the interaction between HSP90AB1/HSPCB and NS5A by tandem affinity purification/liquid chromatography-mass spectrometry (LC-MS) and immunoprecipitation. Furthermore, the CSFV-induced [Ca2+]cyto increase potently induced autophagy through CAMKK2 and PRKAA. Moreover, we isolated and identified the BECN1/Beclin 1 protein complexes by tandem affinity purification/LC-MS and immunoprecipitation, the interaction between BECN1 and MAVS was confirmed by immunoprecipitation, laser scanning confocal microscope technology, and GST affinity-isolation experiments. Furthermore, CSFV-mediated autophagy suppressing type I IFN production is related to the interaction between MAVS and BECN1. Finally, the modulation of autophagy induction pathways by different autophagy regulatory factors significantly affected the replication of CSFV. Abbreviations: AKT: AKT serine/threonine kinase; AMPK: Adenosine monophosphate-activated protein kinase; CAMKK2: Calcium/calmodulin dependent protein kinase kinase 2; CSFV: Classical swine fever virus; HRP: Horseradish peroxidase; HSP90AB1: Heat shock protein 90 alpha family class B member 1; IFN: Interferon; ISGs: IFN-stimulated genes; LC-MS: Liquid chromatography-mass spectrometry; MAP1LC3/LC3: Microtubule associated protein 1 light chain 3; MAPK: Mitogen-activated protein kinase; MAVS: Mitochondrial antiviral signaling protein; MOI: Multiplicity of infection; MTOR: Mechanistic target of rapamycin kinase; PBS: Phosphate-buffered saline; PRKAA: Protein kinase AMP-activated catalytic subunit alpha; shRNA: short hairpin RNA.

The Indispensable Role of Histone Methyltransferase PoDot1 in Extracellular Glycoside Hydrolase Biosynthesis of Penicillium oxalicum.

Histone methylation is associated with transcription regulation, but its role for glycoside hydrolase (GH) biosynthesis is still poorly understood. We identified the histone H3 lysine 79 (H3K79)-specific methyltransferase PoDot1 in Penicillium oxalicum. PoDot1 affects conidiation by regulating the transcription of key regulators (BrlA, FlbC, and StuA) of asexual development and is required in normal hyphae septum and branch formation by regulating the transcription of five septin-encoding genes, namely, aspA, aspB, aspC, aspD, and aspE. Tandem affinity purification/mass spectrometry showed that PoDot1 has no direct interaction with transcription machinery, but it affects the expressions of extracellular GH genes extensively. The expression of genes (amy15A, amy13A, cel7A/cbh1, cel61A, chi18A, cel3A/bgl1, xyn10A, cel7B/eg1, cel5B/eg2, and cel6A/cbh2) that encode the top 10 GHs was remarkably downregulated by Podot1 deletion (ΔPodot1). Consistent with the decrease in gene transcription level, the activities of amylases and cellulases were significantly decreased in ΔPodot1 mutants in agar (solid) and fermentation (liquid) media. The repression of GH gene expressions caused by PoDot1 deletion was not mediated by key transcription factors, such as AmyR, ClrB, CreA, and XlnR, but was accompanied by defects in global demethylated H3K79 (H3K79me2) and trimethylated H3K79 (H3K79me3). The impairment of H3K79me2 on specific GH gene loci was observed due to PoDot1 deletion. The results implies that defects of H3K79 methylation is the key reason of the downregulated transcription level of GH-encoding genes and reveals the indispensable role of PoDot1 in extracellular GH biosynthesis.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

Abstract 1349: Functional and mechanistic characterization of PRMT6 regulated autophagy in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is a major health burden worldwide. Tumor recurrence and therapy resistance represent major obstacles in the treatment of the disease, with their mechanism of action largely uncharacterized. Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis including degradation of damaged organelles and unwanted proteins as well as the support of cellular biosynthesis in response to environmental stress, preventing cells from undergoing apoptosis. Our group has recently reported on the regulatory role of protein arginine methyltransferase 6 (PRMT6) down-regulation in maintenance of HCC, particularly potentiating resistance to sorafenib and chemotherapy. By tandem affinity purification/mass spectrometry analysis of cells with PRMT6 stably overexpressed, we identified a number of autophagy related proteins as potential interacting partners of PRMT6, suggesting a possible role of PRMT6 in modulating autophagy in HCC. Treatment of HCC cells with stress induced conditions, including sorafenib, nutrient deprivation and hypoxic condition, resulted in a marked reduction of PRMT6 expression, concomitant with elevated levels of LC3BII. Further, under these various stimuli, knockdown of PRMT6 in HCC cells would also result in enhanced expression of LC3BII, accumulation of GFP-LC3B positive puncta staining as well as an increase in autophagosome formation. Consistently, treatment of HCC cells with PRMT6 overexpressed would leading to an opposing effect. Further studies by immunoprecipitation analysis validated PRMT6 to physically interact with AMBRA1 (activating molecule in BECN1-regulated autophagy protein 1), a well-known autophagy initiative component, which takes part in autophagy vesicle nucleation, a critical step for autophagic initiation. Our findings suggest PRMT6 down-regulation in HCC tumors to promote tumorigenicity through autophagic flux de-regulation. Targeting the mechanisms of stress response may provide novel therapeutic insights for the disease. Citation Format: Noélia Che, Man Tong, Kai Yu Ng, Phillis WF Kau, Xin Yuan Guan, Michael SY Huen, Stephanie Ma. Functional and mechanistic characterization of PRMT6 regulated autophagy in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1349.

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.

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.

Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

Biophysical Characterisation and Quantification of Nucleic Acid-Protein Interactions: EMSA, MST and SPR.

Cell viability is only possible due to a dynamic range of essential nucleic acid-protein complex formation. DNA replication and repair, gene expression, transcription and protein synthesis are well-known processes mediated by nucleic acids (DNA and RNA) - protein interactions. Novel nucleic acid- protein complexes have been identified in the past few years aided by the development of numerous new techniques such as RNA capture or Tandem RNA Affinity Purification (TRAP). However, the biophysical and biochemical details of these interactions are mostly unknown. Here, we present three techniques (Electrophoretic Mobility Shift Assays, Microscale Thermophoresis and Surface Plasmon Resonance) that are commonly used to quantify and characterize DNA-protein and RNA-protein interactions and discuss their main advantages and limitations.

Isolation of Selenoprotein-P and Determination of Se Concentration Incorporated in Proteins in Human and Mouse Plasma by Tandem Heparin Affinity and Size-exclusion Column HPLC-ICPMS

Sel-P is considered to be the most important selenoprotein for evaluating the selenium (Se) status in the body. To isolate and determine Sel-P in plasma, we have developed an analytical method combining heparin affinity (AF) and size-exclusion column (SEC) high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). We used this method to validate the adsorption efficiency of selenoproteins on a heparin AF column, and to then determine the Se concentrations incorporated in proteins in human and mouse plasma. The adsorption efficiency of Sel-P on a heparin column was more than 90% for both human and mouse plasma. Tandem AF and SEC separation proved to be useful for determining the Se concentrations incorporated in Sel-P in mouse plasma, but not in human plasma, because of nonspecific adsorption of plasma-extracellular glutathione peroxidase (eGPx) and albumin on the heparin AF column. Ultimately, we used the tandem AF and SEC separation method for mouse plasma and SEC separation alone for human plasma. The Se concentration incorporated in selenoproteins determined by our method showed good agreements with the total Se concentration determined following acid digestion.

Proteomic Insights into Inflammatory Airway Diseases

Airways disease presenting as viral bronchiolitis, asthma and chronic obstructive pulmonary disease (COPD) is an increasingly important source of morbidity in Western Countries. Although these diseases affect distinct age groups, have different initiators and manifest variable amounts of parenchymal remodeling, each process is driven by a common fundamental process involving cellular inflammation. Inflammation is a coordinated multi-cellular response to the presence of pathogens or allergens culminating in altering resident leukocyte populations and remodeling structural tissues in the lung. In this review, we will discuss the contributions of innate and chronic adaptive immunity underlying inflammation seen in bronchiolitis, asthma, both mild and severe, and COPD. We will illustrate how applications of proteomics have begun to provide new insights into critical questions regarding the cellular and innate response to inflammation, role of reactive oxygen stress, identification of phenotypic subgroups using molecular profiling, and how proteomics provides insights into therapeutic responses. We will discuss the available sampling strategies for airway biofluids ( bronchoalveolar lavage, induced sputum, exhaled bronchial breath condensates, and nasopharyngeal aspiration) and the advantages and limitations of each. Selectively applied and properly designed proteomics studies provide an added dimension of information to reduce the impact and burden of these common inflammatory airway diseases. Keywords: Asthma, innate immunity, inflammation, RIG-I, tandem affinity purification/molecular profiling, Reactive oxygen species (ROS), leukocyte, cytokines

Abstract 2357: Defining the Ezrin-CLIC4 interaction in osteosarcoma

Abstract Ezrin, a linker protein between the cytoskeleton and plasma membrane, plays an important role in the metastatic phenotype of cancers that includes osteosarcoma and rhabdomyosarcoma. To better understand the role of ezrin in metastasis, we have identified proteins that bind the N-terminus of ezrin using tandem mass spectrometry affinity chromatography. One of the mass spectrometry identified proteins is the p53, TNFα, and c-Myc regulated stress-response protein Chloride Intracellular Channel 4 (CLIC4). Under conditions of cell stress, CLIC4 has been shown to translocate to the nucleus and subsequently cause cell-cycle arrest and/or accelerate apoptosis in cancer cells including osteosarcoma. Emerging data from our lab suggests that ezrin may provide metastatic cells with an early survival advantage by helping to resist the stresses of metastasis and resultant apoptosis. Combining these two lines of reasoning, we hypothesized that ezrin prevents CLIC4 induced apoptosis during times of stress by maintaining CLIC4 out of the nucleus. Using cell free conditions we have demonstrated a direct relationship between ezrin and CLIC4 recombinant proteins and through co-immunoprecipitation in osteosarcoma cells. In addition, experiments to define the minimal binding determinants of the ezrin-CLIC4 interaction are currently underway. We have confirmed co-expression of ezrin and CLIC4 in multiple human osteosarcoma cell lines and human sarcomas using both immunohistochemistry and expression microarray. We have recently developed assays and reagents to monitor and manipulate CLIC4 nuclear localization under conditions of stress. The outcome of this work will be to define the nature and biological significance of the ezrin-CLIC4 interaction in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2357.

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.

Structural Organization of Essential Iron-Sulfur Clusters in the Evolutionarily Highly Conserved ATP-binding Cassette Protein ABCE1

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.