Multiprotein Assemblies Research Articles

Self-Assembly of the RZZ Complex into Filaments Drives Kinetochore Expansion in the Absence of Microtubule Attachment

SummaryThe kinetochore is a dynamic multi-protein assembly that forms on each sister chromatid and interacts with microtubules of the mitotic spindle to drive chromosome segregation. In animals, kinetochores without attached microtubules expand their outermost layer into crescent and ring shapes to promote microtubule capture and spindle assembly checkpoint (SAC) signaling. Kinetochore expansion is an example of protein co-polymerization, but the mechanism is not understood. Here, we present evidence that kinetochore expansion is driven by oligomerization of the Rod-Zw10-Zwilch (RZZ) complex, an outer kinetochore component that recruits the motor dynein and the SAC proteins Mad1-Mad2. Depletion of ROD in human cells suppresses kinetochore expansion, as does depletion of Spindly, the adaptor that connects RZZ to dynein, although dynein itself is dispensable. Expansion is also suppressed by mutating ZWILCH residues implicated in Spindly binding. Conversely, supplying cells with excess ROD facilitates kinetochore expansion under otherwise prohibitive conditions. Using the C. elegans early embryo, we demonstrate that ROD-1 has a concentration-dependent propensity for oligomerizing into micrometer-scale filaments, and we identify the ROD-1 β-propeller as a key regulator of self-assembly. Finally, we show that a minimal ROD-1-Zw10 complex efficiently oligomerizes into filaments in vitro. Our results suggest that RZZ’s capacity for oligomerization is harnessed by kinetochores to assemble the expanded outermost domain, in which RZZ filaments serve as recruitment platforms for SAC components and microtubule-binding proteins. Thus, we propose that reversible RZZ self-assembly into filaments underlies the adaptive change in kinetochore size that contributes to chromosome segregation fidelity.

Two-Dimensional Biochemical Purification for Global Proteomic Analysis of Macromolecular Protein Complexes.

A high-resolution two-dimensional (2-D) proteomic fractionation technique for the systematic purification and subsequent mass spectrometry-based identification of endogenous protein macromolecular complexes is described. The method hyphenates preparative isoelectric focusing (IEF) with mixed-bed ion exchange chromatography (IEX) to efficiently separate cell- or tissue- derived soluble protein mixtures, allowing for more effective and less biased physiochemical characterization of stable multiprotein assemblies. After comprehensive 2D fractionation of cell-free lysates, each fraction is subjected to quantitative tandem mass spectrometry (MS/MS) and subsequent computational analysis to map high-confidence protein-protein interactions (PPIs). Herein, the experimental component (workflow protocols) for this global "interactome" network mapping platform is described.

VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

Biochemical and Functional Interplay Between Ion Channels and the Components of the Dystrophin-Associated Glycoprotein Complex

Dystrophin is a cytoskeleton-linked membrane protein that binds to a larger multiprotein assembly called the dystrophin-associated glycoprotein complex (DGC). The deficiency of dystrophin or the components of the DGC results in the loss of connection between the cytoskeleton and the extracellular matrix with significant pathophysiological implications in skeletal and cardiac muscle as well as in the nervous system. Although the DGC plays an important role in maintaining membrane stability, it can also be considered as a versatile and flexible molecular complex that contribute to the cellular organization and dynamics of a variety of proteins at specific locations in the plasma membrane. This review deals with the role of the DGC in transmembrane signaling by forming supramolecular assemblies for regulating ion channel localization and activity. These interactions are relevant for cell homeostasis, and its alterations may play a significant role in the etiology and pathogenesis of various disorders affecting muscle and nerve function.

Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force-dependence.

Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two-color and three-color super-resolution stimulated emission depletion microscopy to determine the spatial distributions and co-localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi-protein assemblies. The loss of contractile force reduced the nanoscale co-localization between different types of proteins, while it increased this co-localization between markers of the same type. This suggests that actomyosin-dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell-matrix adhesions. These observations are consistent with the possibility that proteins in cell-matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein-specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.

Phase Transitions in the Assembly and Function of Human miRISC

miRISC is a multi-protein assembly that uses microRNAs (miRNAs) to identify mRNAs targeted for repression. Dozens of miRISC-associated proteins have been identified, and interactions between many factors have been examined in detail. However, the physical nature of the complex remains unknown. Here, we show that two core protein components of human miRISC, Argonaute2 (Ago2) and TNRC6B, condense into phase-separated droplets invitro and in live cells. Phase separation is promoted by multivalent interactions between the glycine/tryptophan (GW)-rich domain of TNRC6B and three evenly spaced tryptophan-binding pockets in the Ago2 PIWI domain. miRISC droplets formed invitro recruit deadenylation factors and sequester target RNAs from the bulk solution. The condensation of miRISC is accompanied by accelerated deadenylation of target RNAs bound to Ago2. The combined results may explain how miRISC silences mRNAs of varying size and structure and provide experimental evidence that protein-mediated phase separation can facilitate an RNA processing reaction.

Stoichiometric balance of protein copy numbers is measurable and functionally significant in a protein-protein interaction network for yeast endocytosis.

Stoichiometric balance, or dosage balance, implies that proteins that are subunits of obligate complexes (e.g. the ribosome) should have copy numbers expressed to match their stoichiometry in that complex. Establishing balance (or imbalance) is an important tool for inferring subunit function and assembly bottlenecks. We show here that these correlations in protein copy numbers can extend beyond complex subunits to larger protein-protein interactions networks (PPIN) involving a range of reversible binding interactions. We develop a simple method for quantifying balance in any interface-resolved PPINs based on network structure and experimentally observed protein copy numbers. By analyzing such a network for the clathrin-mediated endocytosis (CME) system in yeast, we found that the real protein copy numbers were significantly more balanced in relation to their binding partners compared to randomly sampled sets of yeast copy numbers. The observed balance is not perfect, highlighting both under and overexpressed proteins. We evaluate the potential cost and benefits of imbalance using two criteria. First, a potential cost to imbalance is that ‘leftover’ proteins without remaining functional partners are free to misinteract. We systematically quantify how this misinteraction cost is most dangerous for strong-binding protein interactions and for network topologies observed in biological PPINs. Second, a more direct consequence of imbalance is that the formation of specific functional complexes depends on relative copy numbers. We therefore construct simple kinetic models of two sub-networks in the CME network to assess multi-protein assembly of the ARP2/3 complex and a minimal, nine-protein clathrin-coated vesicle forming module. We find that the observed, imperfectly balanced copy numbers are less effective than balanced copy numbers in producing fast and complete multi-protein assemblies. However, we speculate that strategic imbalance in the vesicle forming module allows cells to tune where endocytosis occurs, providing sensitive control over cargo uptake via clathrin-coated vesicles.

Stabilizing the Retromer Complex in a Human Stem Cell Model of Alzheimer's Disease Reduces TAU Phosphorylation Independently of Amyloid Precursor Protein.

SummaryDeveloping effective therapeutics for complex diseases such as late-onset, sporadic Alzheimer’s disease (SAD) is difficult due to genetic and environmental heterogeneity in the human population and the limitations of existing animal models. Here, we used hiPSC-derived neurons to test a compound that stabilizes the retromer, a highly conserved multiprotein assembly that plays a pivotal role in trafficking molecules through the endosomal network. Using this human-specific system, we have confirmed previous data generated in murine models and show that retromer stabilization has a potentially beneficial effect on amyloid beta generation from human stem cell-derived neurons. We further demonstrate that manipulation of retromer complex levels within neurons affects pathogenic TAU phosphorylation in an amyloid-independent manner. Taken together, our work demonstrates that retromer stabilization is a promising candidate for therapeutic development in AD and highlights the advantages of testing novel compounds in a human-specific, neuronal system.

Nanodiscs: A Controlled Bilayer Surface for the Study of Membrane Proteins.

The study of membrane proteins and receptors presents many challenges to researchers wishing to perform biophysical measurements to determine the structure, function, and mechanism of action of such components. In most cases, to be fully functional, proteins and receptors require the presence of a native phospholipid bilayer. In addition, many complex multiprotein assemblies involved in cellular communication require an integral membrane protein as well as a membrane surface for assembly and information transfer to soluble partners in a signaling cascade. Incorporation of membrane proteins into Nanodiscs renders the target soluble and provides a native bilayer environment with precisely controlled composition of lipids, cholesterol, and other components. Likewise, Nanodiscs provide a surface of defined area useful in revealing lipid specificity and affinities for the assembly of signaling complexes. In this review, we highlight several biophysical techniques made possible through the use of Nanodiscs.

Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module.

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners. Among the MAGUKs, Dlg1 proteins exist as homo-oligomers, and the oligomerization is solely dependent on the L27 domain. Here we provide biochemical and structural evidence of homodimerization via the L27 domain of Dlg1 from Drosophila melanogaster. The structure reveals that the core of the dimer is formed by a distinctive six-helix assembly, involving all three conserved helices from each subunit (monomer). The homodimer interface is extended by the C-terminal tail of the L27 domain of Dlg1, which forms a two-stranded antiparallel β-sheet. The structure reconciles and provides a structural context for a large body of available mutational data. From our analyses, we conclude that the observed L27 homodimerization is most likely a feature unique to the Dlg1 orthologs within the MAGUK family.

Co-translational control of protein complex formation: a fundamental pathway of cellular organization?

Analyses of proteomes from a large number of organisms throughout the domains of life highlight the key role played by multiprotein complexes for the implementation of cellular function. While the occurrence of multiprotein assemblies is ubiquitous, the understanding of pathways that dictate the formation of quaternary structure remains enigmatic. Interestingly, there are now well-established examples of protein complexes that are assembled co-translationally in both prokaryotes and eukaryotes, and indications are that the phenomenon is widespread in cells. Here, we review complex assembly with an emphasis on co-translational pathways, which involve interactions of nascent chains with other nascent or mature partner proteins, respectively. In prokaryotes, such interactions are promoted by the polycistronic arrangement of mRNA and the associated co-translation of functionally related cell constituents in order to enhance otherwise diffusion-dependent processes. Beyond merely stochastic events, however, co-translational complex formation may be sensitive to subunit availability and allow for overall regulation of the assembly process. We speculate how co-translational pathways may constitute integral components of quality control systems to ensure the correct and complete formation of hundreds of heterogeneous assemblies in a single cell. Coupling of folding of intrinsically disordered domains with co-translational interaction of binding partners may furthermore enhance the efficiency and fidelity with which correct conformation is attained. Co-translational complex formation may constitute a fundamental pathway of cellular organization, with profound importance for health and disease.

Antigen Processing at the Atomic Level: MD Simulations of MHCI and its Peptide-Loading Complex

Antigens exposed at the cell surface by major histocompatibility complex class I (MHCI) proteins enable self/non-self recognition by cytotoxic T cells, protecting the organism against viral infections and cancer-causing mutations. To perform their role, MHCI must first be loaded with an antigenic peptide inside the endoplasmic reticulum (ER), a process controlled by a multi-protein assembly called the peptide-loading complex (PLC). In the absence of any experimental structure of the PLC, we used molecular dynamics (MD) simulations to study its individual components, their assembly and their function. We have predicted the structure of the tapasin • MHCI interface, and shown how tapasin both protects empty MHCI from unfolding and catalyses the selection of high-affinity peptides through a molecular tug-of-war mechanism: tapasin pulls on a region of the MHCI peptide binding groove to open it, while the peptide simultaneously tries to close the groove. Low-affinity contenders “lose” this challenge and are exchanged until a high-affinity one binds to and closes the groove, thereby initiating complex break-down. We have also shown how tapasin recruits the transporter associated with antigen processing (TAP) into the PLC via transmembrane interactions. In addition, by truncating antigens or removing them from the MHCI binding groove, we made a spatially resolved map of MHCI plasticity, revealing how peptide loading status affects key structural regions contacting tapasin. Finally, we have integrated the previous elements to build a computational model of the full PLC. Our MD simulations explain experimental kinetics and mutagenesis data, and represent the first in-depth, atomic-level study of the mechanism underlying the PLC, an important step towards a better understanding of adaptive immunity.

MS Western, a Method of Multiplexed Absolute Protein Quantification is a Practical Alternative to Western Blotting

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.

Automated Computational Inference of Multi-protein Assemblies from Biochemical Co-purification Data.

Biology has amassed a wealth of information about the function of a multitude of protein-coding genes across species. The challenge now is to understand how all these proteins work together to form a living organism, and a crucial step for gaining this knowledge is a complete description of the molecular "wiring circuits" that underlie cellular processes. In this chapter, we describe a general computational framework for predicting multi-protein assemblies from biochemical co-fractionation data.

The Dengue Virus Replication Complex: From RNA Replication to Protein-Protein Interactions to Evasion of Innate Immunity.

Viruses from the Flavivirus family are the causative agents of dengue fever, Zika, Japanese encephalitis, West Nile encephalitis or Yellow fever and constitute major or emerging public health problems. A better understanding of the flavivirus replication cycle is likely to offer new opportunities for the design of antiviral therapies to treat severe conditions provoked by these viruses, but it should also help reveal fundamental biological mechanisms of the host cell. During virus replication, RNA synthesis is mediated by a dynamic and membrane-bound multi-protein assembly, named the replication complex (RC). The RC is composed of both viral and host-cell proteins that assemble within vesicles composed of the endoplasmic reticulum membrane, near the nucleus. At the heart of the flavivirus RC lies NS4B, a viral integral membrane protein that plays a role in virulence and in down-regulating the innate immune response. NS4B binds to the NS2B-NS3 protease-helicase, which itself interacts with the NS5 methyl-transferase polymerase. We present an overview of recent structural and functional data that augment our understanding of how viral RNA is replicated by dengue virus. We focus on structural data that illuminate the various roles played by proteins NS2B-NS3, NS4B and NS5. By participating in viral RNA cap methylation, the NS5 methyltransferase enables the virus to escape the host cell innate immune response. We present the molecular basis for this activity. We summarize what we know about the network of interactions established by NS2B-NS3, NS4B and NS5 (their "interactome"). This leads to a working model that is captured in the form of a rather naïve "cartoon", which we hope will be refined towards an atomic model in the near future.

Dual-Color and 3D Super-Resolution Microscopy of Multi-protein Assemblies.

Breaking the resolution limit of conventional microscopy by super-resolution microscopy (SRM) led to many new biological insights into protein assemblies at the nanoscale. Here we provide detailed protocols for single-molecule localization microscopy (SMLM) to image the structure of a protein complex. As examples, we show how to acquire single- and dual-color super-resolution images of the nuclear pore complex (NPC) and dual-color 3D data on actin and paxillin in focal adhesions.

The human CTF4-orthologue AND-1 interacts with DNA polymerase α/primase via its unique C-terminal HMG box.

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.

Native DIGE: Efficient Tool to Elucidate Protein Interactomes.

Protein-protein interactions and multi-protein assemblies are inherent features of proteomes, involving soluble and membrane proteins. This imparts structural and functional heterogeneity to the proteome. One needs to consider this aspect while studying changes in abundance or activities of proteins in response to any physiological stimulus. Abundance changes in components of a given proteome can be best visualized and quantified using electrophoresis-based approaches. Here, we describe the method of Blue Native Difference Gel Electrophoresis (BN DIGE) to quantify abundance changes in proteins in the context of protein-protein interactions. This method confers an additional advantage to monitor quantitative changes in membrane proteins, which otherwise is a difficult task.

Molecular regulation and pharmacological targeting of the β-catenin destruction complex.

The β‐catenin destruction complex is a dynamic cytosolic multiprotein assembly that provides a key node in Wnt signalling regulation. The core components of the destruction complex comprise the scaffold proteins axin and adenomatous polyposis coli and the Ser/Thr kinases casein kinase 1 and glycogen synthase kinase 3. In unstimulated cells, the destruction complex efficiently drives degradation of the transcriptional coactivator β‐catenin, thereby preventing the activation of the Wnt/β‐catenin pathway. Mutational inactivation of the destruction complex is a major pathway in the pathogenesis of cancer. Here, we review recent insights in the regulation of the β‐catenin destruction complex, including newly identified interaction interfaces, regulatory elements and post‐translationally controlled mechanisms. In addition, we discuss how mutations in core destruction complex components deregulate Wnt signalling via distinct mechanisms and how these findings open up potential therapeutic approaches to restore destruction complex activity in cancer cells.Linked ArticlesThis article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc

GSK3\u03b2 and ERK regulate the expression of 78\u2009kDa SG2NA and ectopic modulation of its level affects phases of cell cycle

Striatin and SG2NA are essential constituents of the multi-protein STRIPAK assembly harbouring protein phosphatase PP2A and several kinases. SG2NA has several isoforms generated by mRNA splicing and editing. While the expression of striatin is largely restricted to the striatum in brain, that of SG2NAs is ubiquitous. In NIH3T3 cells, only the 78 kDa isoform is expressed. When cells enter into the S phase, the level of SG2NA increases; reaches maximum at the G2/M phase and declines thereafter. Downregulation of SG2NA extends G1 phase and its overexpression extends G2. Ectopic expression of the 35 kDa has no effects on the cell cycle. Relative abundance of phospho-SG2NA is high in the microsome and cytosol and the nucleus but low in the mitochondria. Okadoic acid, an inhibitor of PP2A, increases the level of SG2NA which is further enhanced upon inhibition of proteasomal activity. Phospho-SG2NA is thus more stable than the dephosphorylated form. Inhibition of GSK3β by LiCl reduces its level, but the inhibition of ERK by PD98059 increases it. Thus, ERK decreases the level of phospho-SG2NA by inhibiting GSK3β. In cells depleted from SG2NA by shRNA, the levels of pGSK3β and pERK are reduced, suggesting that these kinases and SG2NA regulate each other’s expression.