MultiBac: expanding the research toolbox for multiprotein complexes

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms

Methods for protein complex prediction and their contributions towards understanding the organization, function and dynamics of complexes

Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organization of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight challenges faced by these methods, in particular detection of sparse and small or sub- complexes and discerning of overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area.Comment: 1 Tabl

ReCLIP (Reversible Cross-Link Immuno-Precipitation): An Efficient Method for Interrogation of Labile Protein Complexes

The difficulty of maintaining intact protein complexes while minimizing non-specific background remains a significant limitation in proteomic studies. Labile interactions, such as the interaction between p120-catenin and the E-cadherin complex, are particularly challenging. Using the cadherin complex as a model-system, we have developed a procedure for efficient recovery of otherwise labile protein-protein interactions. We have named the procedure “ReCLIP” (Reversible Cross-Link Immuno-Precipitation) to reflect the primary elements of the method. Using cell-permeable, thiol-cleavable crosslinkers, normally labile interactions (i.e. p120 and E-cadherin) are stabilized in situ prior to isolation. After immunoprecipitation, crosslinked binding partners are selectively released and all other components of the procedure (i.e. beads, antibody, and p120 itself) are discarded. The end result is extremely efficient recovery with exceptionally low background. ReCLIP therefore appears to provide an excellent alternative to currently available affinity-purification approaches, particularly for studies of labile complexes

Biochemical And Structural Characterization Of The Core Subunits Of Gpi Transamidase

BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF THE CORE SUBUNITS OF GPI TRANSAMIDASE by DILANI G GAMAGE May 2015 Advisor: Prof. Tamara L. Hendrickson Major: Chemistry (Biochemistry) Degree: Doctor of Philosophy Glycosylphosphatidylinositol transamidase (GPI-T) is a complicated, membrane-bound, multi-subunit enzyme that catalyzes an essential post-translational modification. This enzyme attaches GPI anchors to the C-termini of various proteins that contain a proper GPI-T signal sequence. Gpi8, Gaa1, Gpi16, Gpi17 and Gab1 are the five known subunits that may encompass the fungal GPI-T; Gpi8 is the catalytic subunit, but the functions of the other subunits remain essentially unknown. In humans, different GPI-T subunits are upregulated in different cancers, making GPI-T a target for cancer research. However, in spite of the importance of this enzyme, little is known about how it assembles into an active enzyme complex, the stoichiometry of this complex, or the roles of the different components. Here we use soluble domains of the three core subunits (Gpi8, Gpi16 and Gaa1) to investigate the stoichiometry of the enzyme as well as to study the functions of each subunit in vitro. Additionally, overexpression of the full-length core subunits was used to study the enzyme\u27s behavior on transamidation in vivo. Due to the complex nature of this protein and the fact that it is membrane associated, we set out to simply this enzyme into a more tractable system. In chapters 2 and 3 of this thesis, we focused on the soluble domains of the core subunits, Gpi81-306, Gaa150-343 and Gpi161-551. These soluble domains were overexpressed and their interactions and stoichiometry were characterized. Gpi8, the catalytic subunit, has weak sequence similarity to caspase-1 and assembles into a homodimer. Also, N-linked glycosylation of one asparagine in this subunit is not essential for dimerization. Co-immunoprecipitation of the soluble domains of Gpi81-306:Gaa150-343, Gpi81-306:Gpi161-551 and Gpi161-551:Gaa150-343 demonstrated that these subunits interact with each other at least in heterodimeric complexes. Initial characterization of the Gpi823-306:Gaa150-343 complex is consistent with the formation of an heterotetramer. Also, these three subunits Gpi81-306:Gaa150-343:Gpi161-551 can be co-purified as an intact complex. Preliminary results show that this core heterotrimer has nucleophile-independent activity. Our results will help to elucidate the function and resolve the complexity of GPI-T. Efforts are underway to determine the stoichiometry of each subunit and the contribution of each subunit towards transamidase activity. To better understand how changes in expression affect GPI-T activity, and as a model for this enzyme in cancer, we have developed an in vivo strategy to monitor and quantify the effect of subunit overexpression on cell surface presentation of GPI-anchored proteins in Saccharomyces cerevisiae. Here we used Invertase as a reporter enzyme. Three GPI-T signal sequences were appended to the C-terminus of invertase and the amount of cell surfaced, GPI anchored invertase was measured. Overexpression of Gpi8, the catalytic subunit had little effect on GPI anchoring of invertase with two of these three signal sequences; however, the amount of cell surface invertase was nearly doubled when the weakest signal sequence was used. Compared to Gpi8, overexpression of either Gpi16 or Gaa1 downregulated GPI-T activity with all three signal sequences. To our knowledge, these results represent the first direct examination of the impact of subunit overexpression directly on GPI-T activity. Our results suggest that overexpression of a single GPI-T subunit either disrupts assembly of active GPI-T or frees these subunits to participate different cellular functions. The results presented in this dissertation represent the beginning of a new era aimed at understanding GPI-T and provide new tools and approaches to achieve this important goal

Analysis of High-Throughput Data - Protein-Protein Interactions, Protein Complexes and RNA Half-life

The development of high-throughput techniques has lead to a paradigm change in biology from the small-scale analysis of individual genes and proteins to a genome-scale analysis of biological systems. Proteins and genes can now be studied in their interaction with each other and the cooperation within multi-subunit protein complexes can be investigated. Moreover, time-dependent dynamics and regulation of these processes and associations can now be explored by monitoring mRNA changes and turnover. The in-depth analysis of these large and complex data sets would not be possible without sophisticated algorithms for integrating different data sources, identifying interesting patterns in the data and addressing the high variability and error rates in biological measurements. In this thesis, we developed such methods for the investigation of protein interactions and complexes and the corresponding regulatory processes. In the first part, we analyze networks of physical protein-protein interactions measured in large-scale experiments. We show that the topology of the complete interactomes can be confidently extrapolated despite high numbers of missing and wrong interactions from only partial measurements of interaction networks. Furthermore, we find that the structure and stability of protein interaction networks is not only influenced by the degree distribution of the network but also considerably by the suppression or propagation of interactions between highly connected proteins. As analysis of network topology is generally focused on large eukaryotic networks, we developed new methods to analyze smaller networks of intraviral and virus-host interactions. By comparing interactomes of related herpesviral species, we could detect a conserved core of protein interactions and could address the low coverage of the yeast two-hybrid system. In addition, common strategies in the interaction of the viruses with the host cell were identified. New affinity purification methods now make it possible to directly study associations of proteins in complexes. Due to experimental errors the individual protein complexes have to be predicted with computational methods from these purification results. As previously published methods relied more or less heavily on existing knowledge on complexes, we developed an unsupervised prediction algorithm which is independent from such additional data. Using this approach, high-quality protein complexes can be identified from the raw purification data alone for any species purification experiments are performed. To identify the direct, physical interactions within these predicted complexes and their subcomponent structure, we describe a new approach to extract the highest scoring subnetwork connecting the complex and interactions not explained by alternative paths of indirect interactions. In this way, important interactions within the complexes can be identified and their substructure can be resolved in a straightforward way. To explore the regulation of proteins and complexes, we analyzed microarray measurements of mRNA abundance, de novo transcription and decay. Based on the relationship between newly transcribed, pre-existing and total RNA, transcript half-life can be estimated for individual genes using a new microarray normalization method and a quality control can be applied. We show that precise measurements of RNA half-life can be obtained from de novo transcription which are of superior accuracy to previously published results from RNA decay. Using such precise measurements, we studied RNA half-lives in human B-cells and mouse fibroblasts to identify conserved patterns governing RNA turnover. Our results show that transcript half-lives are strongly conserved and specifically correlated to gene function. Although transcript half-life is highly similar in protein complexes and \mbox{families}, individual proteins may deviate significantly from the remaining complex subunits or family members to efficiently support the regulation of protein complexes or to create non-redundant roles of functionally similar proteins. These results illustrate several of the many ways in which high-throughput measurements lead to a better understanding of biological systems. By studying large-scale measure\-ments in this thesis, the structure of protein interaction networks and protein complexes could be better characterized, important interactions and conserved strategies for herpes\-viral infection could be identified and interesting insights could be gained into the regulation of important biological processes and protein complexes. This was made possible by the development of novel algorithms and analysis approaches which will also be valuable for further research on these topics

Cryo-EM structures of eukaryotic translation termination and ribosome recycling complexes containing eRF1, eRF3 and ABCE1

Translation of an mRNA template into a polypeptide chain is terminated on a stop codon. The stop is recognized in the ribosomal A site by release factors, that subsequently release the polypeptide. This event is followed by ribosome recycling leading to a dissociation of the ribosome into subunits. Eukaryotic eRF1 recognizes all three stop codons and is delivered to the ribosomal A site in a ternary complex with GTP-bound eRF3. ABCE1, a highly conserved ATPase, stimulates peptide release and splits the ribosome in concert with eRF1. The first goal of this work was to study the structural rearrangements of eRF1, eRF3 and ABCE1 on the ribosome during translation termination and ribosome recycling. Two cryo-EM structures were obtained at sub-nanometer resolution: the pre-termination complex containing eRF1 and eRF3, and a termination/pre-recycling complex containing eRF1 and ABCE1. The pre-termination stage showed eRF1 packed against eRF3, unable to catalyze peptide release. In the termination/pre-recycling complex, eRF1 assumed an extended conformation which is further stabilized by ABCE1, with the central domain of eRF1 swung out toward the CCA end of the P-site tRNA. ABCE1 adopted a half-closed conformation of its two nucleotide-binding domains in the termination/pre-recycling complex. According to a model based of these results, splitting the ribosome would require the closing of the two nucleotide-binding domains and a rotation of the iron-sulfur cluster domain of ABCE1, which would in turn push eRF1 into the intersubunit space. Supporting this idea, ABCE1 was shown to remain bound to the small ribosomal subunit after in vitro splitting. 40S-bound ABCE1 adopted a fully closed conformation and in which re-association of the large ribosomal subunit is prevented. As a second goal of this work, native S. cerevisiae ABCE1-bound small ribosomal subunits were purified to complement the in vitro studies and explore the supposed involvement of ABCE1 in translation initiation. Cryo-EM of native 40S-ABCE1 complexes indeed confirmed the closed conformation of ABCE1. Moreover, these complexes were associated with initiator tRNA and eIF1A, an initiation factor which binds the ribosomal A site and is involved in multiple processes in initiation including subunit joining. These results are clearly hinting at an active role of ABCE1 during translation initiation. Yet, the exact role of ABCE1 will be subject of further studies

Investigation Of The Saccharomyces Cerevisiae Gpi Transamidase: Insights Into Its Activity And Subunit-Subunit Interactions

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a eukaryotic, posttranslational modification catalyzed by GPI transamidase (GPI-T). The Saccharomyces cerevisiae GPI-T is composed of five membrane-bound subunits: Gaa1, Gpi8, Gpi16, Gpi17, and Gab1. Structural and functional studies have been hindered by the complexity of this enzyme. Conditions to purify the Gpi8:Gaa1:Gpi16 GPI-T heterotrimer from yeast have been reported, but an understanding of the subunit functions, interactions, and stoichiometry remain unclear. Furthermore, a reliable, quantitative, in vitro assay for this important post-translational modification has remained elusive for nearly three decades. Our laboratory has developed an in vitro peptide cleavage assay that correlates changes in fluorescence to GPI-T activity. Using this peptide cleavage assay, it was demonstrated that the purified, full-length GPI-T retains activity, providing the first method to measure GPI-T activity in a quantitative, time-dependent manner. This dissertation research presents the characterization of the soluble domains of the GPI-T heterotrimeric complex, composed of Gpi823-306, Gaa150-343, and Gpi1620-551. Each soluble domain interacts with one another without the need for the third subunit. This soluble GPI-T heterotrimer can be purified as one complex without its transmembrane domains. Most importantly, this simplified heterotrimer retains transamidase activity, demonstrating that these three subunits comprise the functional core of GPI-T. These results contribute to our understanding of how this enzyme is structurally organized, provide a method to screen potential GPI-T inhibitors, and open the door to further understand how GPI-T is involved in normal cellular function and pathogenesis

An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

Contains fulltext : 158967.pdf (publisher's version ) (Open Access)Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine

Proteomic Investigation of the HIV Receptors CD4 and DC-Sign/CD209

HIV infection and disease is a multistage process that involves a variety of cell types as the virus spreads through the body. Initially, dendritic cells (DCs) present at the mucosal site of infection bind and internalise HIV for degradation and presentation to T cells. As the DCs migrate to lymph nodes and mature, part of the internalised virions remains infective inside endosomal compartments. During formation of the immunological synapse between CD4 T cells and DCs, infective virions from dendritic cells are transferred to CD4 T cells leading to a strong infection of those cells allowing rapid virus dissemination throughout the body and establishment of the typical HIV infection. Various membrane receptors are involved in this process. Initial HIV binding to DCs is mediated by C-type lectin receptors such as the mannose receptor or DC-SIGN (DC specific intracellular adhesion molecule 3 grabbing non integrin) which is followed by virus internalisation and lysis albeit virus induced changes in endocytic routing prevents a proportion from degradation. Productive infection of DCs has also been observed allowing trans infection of CD4 T cells through a different mechanism. HIV infection of CD4 T cells, DCs and other cells is a multistep process initiated by binding of HIV envelope gp120 to the CD4 receptor, a 55 kDa transmembrane glycoprotein. Subsequent conformational changes in gp120 allow binding to a chemokine receptor, either CCR5 or CXCR4, followed by membrane fusion and infection. The aim of this thesis was to investigate protein associations with the HIV receptors DC-SIGN and CD4 in order to elucidate the mechanism of complex formation, virus entry and/or defining target sites for antiretroviral drugs. This thesis used a proteomic approach for studying the receptors with mass spectrometry-based protein identification as its core technology. A range of different approaches were developed and compared for identification of protein interactions and characterisation of the identified protein associations. An affinity purification of the CD4 receptor complex from lymphoid cells was used as the basis for detecting novel CD4-binding proteins. For this approach a strategy based on mass spectrometry identification of CD4 associating proteins using affinity chromatography and affinity-tag mediated purification of tryptic peptides was developed. This method proved successful for the identification of CD4 interacting proteins such as the strongly associated kinase p56lck, however a limited number of non-specifically bound proteins were also identified along the receptor complex. Using one-dimensional SDS-polyacrylamide gel electrophoresis followed by in-gel digests and mass spectrometry analysis, a large number of non-specifically binding proteins were identified along the CD4/lck complex. Evaluation of different lysis buffers in several independent experiments demonstrated that there was a large and inconsistent array of proteins that were obviously non-specifically bound to the receptor. No further specific binding partners were detected. These data suggested that protein interactions of CD4 on this cell type are of weak and/or transient nature. It also demonstrated a need for careful interpretation of proteomic data in the light of the propensity of non-specific binding under these conditions. To overcome dissociation of weak protein interactions, a method was developed using chemical cross-linking to preserve weak protein interactions on lymphoid cells. Affinity purification was used to purify CD4 along with cross-linked associated proteins and mass spectrometry analysis identified an interaction with the transferrin receptor CD71 and the tyrosine phosphatase CD45. The CD45-CD4 interaction is well known. The CD4-CD71 interaction was demonstrated to be a result from colocalization of the two molecules during formation of endocytic vesicles. Flow cytometry-based fluorescence resonance energy transfer (FRET) measurements were applied to confirm colocalization. A similar interaction was suspected for CD4 and DC-SIGN on the plasma membrane of DCs as cis infection of DCs has been demonstrated i.e. initial binding to DC-SIGN then to CD4/CCR5 on the same cell. Therefore, protein associations of DC-SIGN were investigated using the developed techniques. Using cross-linking, DC-SIGN was shown to assemble in large complexes on the surface of immature monocyte-derived DCs. Mass spectrometry analysis of the purified complexes identified them as homo-oligomers of DC-SIGN. The absence of CD4 suggested that the fraction interacting with CD4 at any one time must be small. The complexes of DC-SIGN were further characterised to be tetramers and successfully co-immunoprecipitated with HIV gp120 and mannan. DC-SIGN monomers were not evident demonstrating that the assembly of DC-SIGN into tetramers is required for high affinity binding of its natural and viral ligands. Thus potential antiviral agents aimed at blocking the early stage of HIV binding to DCs must simulate tetramers in order to neutralise the virus efficiently. Overall the thesis provides new information on protein interactions of CD4 and DC-SIGN, a careful investigation of "proteomics" techniques for identifying the proteins in affinity-purified samples and demonstrates the need for multifaceted analytical approaches to probe complex cellular systems