Biotin tagging coupled with amino acid‐coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade‐offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid‐coded mass tagging, for highly sensitive and accurate screening of mammalian protein–protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin‐tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled‐down complex amino acid‐coded mass tagging serves as “in‐spectra” quantitative markers to distinguish those bait‐specific interactors from non‐specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid‐coded mass tagging approach, we first biotin‐tagged in vivo a multi‐functional protein family member, 14‐3‐3ε, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14‐3‐3ε were identified in high confidence.     

Proteomic analysis of the 14-3-3 family in Arabidopsis

In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.     

Effect of pressure on helix‐coil transition of an alanine‐based peptide: An FTIR study

Protein–protein interactions are mediated by complementary amino acids defining complementary surfaces. Typically not all members of a family of related proteins interact equally well with all members of a partner family; thus analysis of the sequence record can reveal the complementary amino acid partners that confer interaction specificity. This article develops methods for learning and using probabilistic graphical models of such residue “cross‐coupling” constraints between interacting protein families, based on multiple sequence alignments and information about which pairs of proteins are known to interact. Our models generalize traditional consensus sequence binding motifs, and provide a probabilistic semantics enabling sound evaluation of the plausibility of new possible interactions. Furthermore, predictions made by the models can be explained in terms of the underlying residue interactions. Our approach supports different levels of prior knowledge regarding interactions, including both one‐to‐one (e.g., pairs of proteins from the same organism) and many‐to‐many (e.g., experimentally identified interactions), and we present a technique to account for possible bias in the represented interactions. We apply our approach in studies of PDZ domains and their ligands, fundamental building blocks in a number of protein assemblies. Our algorithms are able to identify biologically interesting cross‐coupling constraints, to successfully identify known interactions, and to make explainable predictions about novel interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Cyclic strain modulates migration and proliferation of vascular smooth muscle cells via Rho‐GDIα, Rac1, and p38 pathway

Cyclic strain is an important inducer of proliferation and migration of vascular smooth muscle cells (VSMCs) which are involved in vascular remodeling during hypertension. However, its mechanism remains to be elucidated. VSMCs of rat aorta were exposed to cyclic strains in vitro with defined parameters, the static, 5%‐strain (physiological) and 15%‐strain (pathological), at 1.25 Hz for 24 h respectively. Then the possible signaling molecules participated in strain‐induced VSMC migration and proliferation were investigated. The results showed that 15%‐strain significantly increased VSMC migration and proliferation in comparison with 5%‐strain. Expression of Rho GDP dissociation inhibitor alpha (Rho‐GDIα) was repressed by 15%‐strain, but expressions of phospho‐Rac1 and phospho‐p38 were increased. Expressions of phospho‐Akt and phospho‐ERK1/2 were similar between the static, 5%‐strain and 15%‐strain groups. Rho‐GDIα “knock‐down” by target siRNA transfection increased migration and proliferation of VSMCs, and up‐regulated phosphorylation of Rac1 and p38 in all groups. Rac1 “knock‐down” repressed migration and proliferation of VSMCs, down‐regulated phosphorylation of p38, but had no effect on Rho‐GDIα expression. When siRNAs of Rho‐GDIα and Rac1 were co‐transfected to VSMCs, the expressions of Rho‐GDIα and phospho‐Rac1 were both decreased, and the effects of Rho‐GDIα “knock‐down” were blocked. Rho‐GDIα “knock‐down” promoted while Rac1 “knock‐down” postponed the assembly of stress fibers and focal adhesions in static. The results demonstrate that the pathological cyclic strain might induce migration and proliferation of VSMCs via repressing expression of Rho‐GDIα, which subsequently verified phosphorylations of Rac1 and p38. J. Cell. Biochem. 109: 906–914, 2010. © 2010 Wiley‐Liss, Inc.     

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti‐aging and metabolic effects of sirtuins

Sirtuins are a family of NAD⁺‐dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti‐aging effects of human homologues of sirtuins, SIRT1‐7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti‐aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI‐TOF/TOF‐MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co‐immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti‐aging and metabolic regulatory activities of sirtuins.     

14‐3‐3ζ binds to and stabilizes phospho‐beclin 1S295 and induces autophagy in hepatocellular carcinoma cells

Data from The Cancer Genome Atlas (TCGA) indicate that the expression levels of 14‐3‐3ζ and beclin 1 (a key molecule involved in cellular autophagy) are up‐regulated and positively correlated with each other (R = .5, P < .05) in HCC tissues. Chemoresistance developed in hepatoma cancer cells is associated with autophagy initiation. This study aimed to explore 14‐3‐3ζ’s role in regulating autophagy in HCC cells, with a focus on beclin 1. The co‐localization of 14‐3‐3ζ and beclin 1 was detectable in primary HCC tissues. To simulate in vivo tumour microenvironment (hypoxia), CSQT‐2 and HCC‐LM3 cells were exposed to 2% oxygen for 24 hours. The protein levels of 14‐3‐3ζ and phospho‐beclin 1^S295 peaked at 12 hours following hypoxia. Meanwhile, the strongest autophagy flux occurred: LC3II was increased, and p62 was decreased significantly. By sequencing the coding area of BECN 1 gene of CSQT‐2 and HCC‐LM3 cells, we found that the predicted translational products of BECN 1 gene contained RLPS²⁹⁵VP (R, arginine; L, leucine; P, proline; S, serine; V, valine), a classic 14‐3‐3ζ binding motif. CO‐IP results confirmed that 14‐3‐3ζ bound to beclin 1, and this connection was markedly weakened when S²⁹⁵ was mutated into A²⁹⁵ (alanine). Further, 14‐3‐3ζ overexpression prevented phospho‐beclin 1^S295 from degradation and enhanced its binding to VPS34, whilst its knockdown accelerated the degradation. Additionally, 14‐3‐3ζ enhanced the chemoresistance of HCC cells to cis‐diammined dichloridoplatium by activating autophagy. Our work reveals that 14‐3‐3ζ binds to and stabilizes phospho‐beclin 1^S295 and induces autophagy in HCC cells to resist chemotherapy.     

Association of Multiple Phosphorylated Proteins with the 14-3-3 Regulatory Hubs: Problems and Perspectives

14-3-3 proteins are well-known universal regulators binding a vast number of partners by recognizing their phosphorylated motifs, typically located within the intrinsically disordered regions. The abundance of such phosphomotifs ensures the involvement of 14-3-3 proteins in sophisticated protein–protein interaction networks that govern vital cellular processes. Thousands of 14-3-3 partners have been either experimentally identified or predicted, but the spatiotemporal hierarchy of the processes based on 14-3-3 interactions is not clearly understood. This is exacerbated by the lack of available structural information on full regulatory complexes involving 14-3-3, which resist high-resolution structural studies due to the presence of intrinsically disordered regions. Although deducing three-dimensional structures is of particular urgency, structural advances are lagging behind the rate at which novel 14-3-3 partners are discovered. Here I attempted to critically review the current state of the field and in particular to dissect the unknowns, focusing on questions that could help in moving the frontiers forward.     

Constraints imposed by non‐functional protein–protein interactions on gene expression and proteome size

Crowded intracellular environments present a challenge for proteins to form functional specific complexes while reducing non‐functional interactions with promiscuous non‐functional partners. Here we show how the need to minimize the waste of resources to non‐functional interactions limits the proteome diversity and the average concentration of co‐expressed and co‐localized proteins. Using the results of high‐throughput Yeast 2‐Hybrid experiments, we estimate the characteristic strength of non‐functional protein–protein interactions. By combining these data with the strengths of specific interactions, we assess the fraction of time proteins spend tied up in non‐functional interactions as a function of their overall concentration. This allows us to sketch the phase diagram for baker's yeast cells using the experimentally measured concentrations and subcellular localization of their proteins. The positions of yeast compartments on the phase diagram are consistent with our hypothesis that the yeast proteome has evolved to operate closely to the upper limit of its size, whereas keeping individual protein concentrations sufficiently low to reduce non‐functional interactions. These findings have implication for conceptual understanding of intracellular compartmentalization, multicellularity and differentiation.     

The Integration of Proteome-Wide PTM Data with Protein Structural and Sequence Features Identifies Phosphorylations that Mediate 14-3-3 Interactions

14-3-3s are abundant proteins that regulate essentially all aspects of cell biology, including cell cycle, motility, metabolism, and cell death. 14-3-3s work by docking to phosphorylated Ser/Thr residues on a large network of client proteins and modulating client protein function in a variety of ways. In recent years, aided by improvements in proteomics, the discovery of 14-3-3 client proteins has far outpaced our ability to understand the biological impact of individual 14-3-3 interactions. The rate-limiting step in this process is often the identification of the individual phospho-serines/threonines that mediate 14-3-3 binding, which are difficult to distinguish from other phospho-sites by sequence alone. Furthermore, trial-and-error molecular approaches to identify these phosphorylations are costly and can take months or years to identify even a single 14-3-3 docking site phosphorylation. To help overcome this challenge, we used machine learning to analyze predictive features of 14-3-3 binding sites. We found that accounting for intrinsic protein disorder and the unbiased mass spectrometry identification rate of a given phosphorylation significantly improves the identification of 14-3-3 docking site phosphorylations across the proteome. We incorporated these features, coupled with consensus sequence prediction, into a publicly available web app, called “14-3-3 site-finder”. We demonstrate the strength of this approach through its ability to identify 14-3-3 binding sites that do not conform to the loose consensus sequence of 14-3-3 docking phosphorylations, which we validate with 14-3-3 client proteins, including TNK1, CHEK1, MAPK7, and others. In addition, by using this approach, we identify a phosphorylation on A-kinase anchor protein-13 (AKAP13) at Ser2467 that dominantly controls its interaction with 14-3-3.     

Multifunctional 14-3-3 proteins of plant cell

The 14-3-3 proteins are a family of highly conserved proteins found in all eukaryotes - from the yeasts to mammals. They regulate several cellular processes recognizing unique conservative, mostly phosphorylated motif of partner proteins. Binding of the 14-3-3 proteins regulates their partners through a variety of mechanisms, such as altering their catalytic activity, subcellular localization, stability or altering their interactions with other protein molecules. The native 14-3-3 proteins are present in form of homo- and hetero-dimers. The most structurally variable N-and C-termini are responsible for isoform specific protein-protein interactions, and cellular localization. In plant cell, 14-3-3 proteins appear to play an important role in regulation of key enzymes of carbon and nitrogen metabolism, modulation ion pumps and channels. They are also involved in signal transduction pathways and even in gene expression.     

14-3-3 proteins in neurological disorders

14-3-3 proteins were originally discovered as a family of proteins that are highly expressed in the brain. Through interactions with a multitude of binding partners, 14-3-3 proteins impact many aspects of brain function including neural signaling, neuronal development and neuroprotection. Although much remains to be learned and understood, 14-3-3 proteins have been implicated in a variety of neurological disorders based on evidence from both clinical and laboratory studies. Here we will review previous and more recent research that has helped us understand the roles of 14-3-3 proteins in both neurodegenerative and neuropsychiatric diseases.     

Grb7 and Hax1 may colocalize partially to mitochondria in EGF‐treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1

Growth factor receptor bound protein 7 (Grb7) is a signal‐transducing adaptor protein that mediates specific protein–protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein‐binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras‐GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti‐apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor‐treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time‐dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.     

Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false‐positive control

Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface‐exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital‐acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface “shaving” technique with either trypsin or proteinase‐K combined with LC‐MS/MS. Trypsin‐derived data were controlled using a “false‐positive” strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin‐shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface‐exposed peptides. Trypsin and proteinase‐K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase‐K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false‐positive strategy improved enrichment of surface‐exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance–surface protein SACOL0050 (pls) and penicillin‐binding protein 2′ (mecA), as well as bifunctional autolysin and the extracellular matrix‐binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface‐exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.     

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β‐turns in peptide immunogenicity

Earlier immunological experiments with a synthetic 36‐residue peptide (75‐110) from Influenza hemagglutinin have been shown to elicit anti‐peptide antibodies (Ab) which could cross‐react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide (⁹⁸YPYDVPDYASLRS¹¹⁰) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS‐AA/L all‐atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β‐turn formed by “DYAS” sequence) with greater preference at 310 K while, it samples a native‐like conformation with relatively greater propensity at 293 K. The sequence “DYAS” samples β‐turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β‐turn involving “DYAS” sequence and in addition it also samples a β‐turn formed by the sequence “YPYD” at its N‐terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag–Ab recognition are discussed. Proteins 2015; 83:1352–1367. © 2015 Wiley Periodicals, Inc.