Scaffolding Complexes Research Articles

In-Situ Structure and Topography of AMPA Receptor Scaffolding Complexes Visualized by CryoET.

Most synapses in the brain transmit information by the presynaptic release of vesicular glutamate, driving postsynaptic depolarization through AMPA-type glutamate receptors (AMPARs). The nanometer-scale topography of synaptic AMPARs regulates response amplitude by controlling the number of receptors activated by synaptic vesicle fusion. The mechanisms controlling AMPAR topography and their interactions with postsynaptic scaffolding proteins are unclear, as is the spatial relationship between AMPARs and synaptic vesicles. Here, we used cryo-electron tomography to map the molecular topography of AMPARs and visualize their in-situ structure. Clustered AMPARs form structured complexes with postsynaptic scaffolding proteins resolved by sub-tomogram averaging. Sub-synaptic topography mapping reveals the presence of AMPAR nanoclusters with exclusion zones beneath synaptic vesicles. Our molecular-resolution maps visualize the predominant information transfer path in the nervous system.

Nanoscale architecture of synaptic vesicles and scaffolding complexes revealed by cryo-electron tomography

The spatial distribution of proteins and their arrangement within the cellular ultrastructure regulates the opening of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in response to glutamate release at the synapse. Fluorescence microscopy imaging revealed that the postsynaptic density (PSD) and scaffolding proteins in the presynaptic active zone (AZ) align across the synapse to form a trans-synaptic "nanocolumn," but the relation to synaptic vesicle release sites is uncertain. Here, we employ focused-ion beam (FIB) milling and cryoelectron tomography to image synapses under near-native conditions. Improved image contrast, enabled by FIB milling, allows simultaneous visualization of supramolecular nanoclusters within the AZ and PSD and synaptic vesicles. Surprisingly, membrane-proximal synaptic vesicles, which fuse to release glutamate, are not preferentially aligned with AZ or PSD nanoclusters. These synaptic vesicles are linked to the membrane by peripheral protein densities, often consistent in size and shape with Munc13, as well as globular densities bridging the synaptic vesicle and plasma membrane, consistent with prefusion complexes of SNAREs, synaptotagmins, and complexin. Monte Carlo simulations of synaptic transmission events using biorealistic models guided by our tomograms predict that clustering AMPARs within PSD nanoclusters increases the variability of the postsynaptic response but not its average amplitude. Together, our data support a model in which synaptic strength is tuned at the level of single vesicles by the spatial relationship between scaffolding nanoclusters and single synaptic vesicle fusion sites.

Regulation of Phosphoinositide Signaling by Scaffolds at Cytoplasmic Membranes.

Cytoplasmic phosphoinositides (PI) are critical regulators of the membrane-cytosol interface that control a myriad of cellular functions despite their low abundance among phospholipids. The metabolic cycle that generates different PI species is crucial to their regulatory role, controlling membrane dynamics, vesicular trafficking, signal transduction, and other key cellular events. The synthesis of phosphatidylinositol (3,4,5)-triphosphate (PI3,4,5P3) in the cytoplamic PI3K/Akt pathway is central to the life and death of a cell. This review will focus on the emerging evidence that scaffold proteins regulate the PI3K/Akt pathway in distinct membrane structures in response to diverse stimuli, challenging the belief that the plasma membrane is the predominant site for PI3k/Akt signaling. In addition, we will discuss how PIs regulate the recruitment of specific scaffolding complexes to membrane structures to coordinate vesicle formation, fusion, and reformation during autophagy as well as a novel lysosome repair pathway.

Deubiquitination complex platform: A plausible mechanism for regulating the substrate specificity of deubiquitinating enzymes.

Deubiquitinating enzymes (DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin‒proteasome degradation and are critical for regulating protein expression levels invivo. Therefore, dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling pathways. To data, extensive studies on the ubiquitin chain specificity of DUBs have been reported, but substrate protein recognition is still not clearly understood. As a breakthrough, the scaffolding role may be significant to substrate protein selectivity. From this perspective, we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate selectivity. Furthermore, we proposed a deubiquitination complex platform (DCP) as a potentially generic mechanism for DUB substrate recognition based on known examples, which might fill the gaps in the understanding of DUB substrate specificity.

Tissue- and cell-specific whole-transcriptome meta-analysis from brain and retina reveals differential expression of dystrophin complexes and new dystrophin spliced isoforms.

The large DMD gene encodes a group of dystrophin proteins in brain and retina, produced from multiple promoters and alternative splicing events. Dystrophins are core components of different scaffolding complexes in distinct cell types. Their absence may thus alter several cellular pathways, which might explain the heterogeneous genotype-phenotype relationships underlying central comorbidities in Duchenne muscular dystrophy (DMD). However, the cell-specific expression of dystrophins and associated proteins (DAPs) is still largely unknown. The present study provides a first RNA-Seq-based reference showing tissue- and cell-specific differential expression of dystrophins, splice variants and DAPs in mouse brain and retina. We report that a cell type may express several dystrophin complexes, perhaps due to expression in separate cell subdomains and/or subpopulations, some of which with differential expression at different maturation stages. We also identified new splicing events in addition to the common exon-skipping events. These include a new exon within intron 51 (E51b) in frame with the flanking exons in retina, as well as inclusions of intronic sequences with stop codons leading to the presence of transcripts with elongated exons 40 and/or 41 (E40e, E41e) in both retina and brain. PCR validations revealed that the new exons may affect several dystrophins. Moreover, immunoblot experiments using a combination of specific antibodies and dystrophin-deficient mice unveiled that the transcripts with stop codons are translated into truncated proteins lacking their C-terminus, which we called N-Dp427 and N-Dp260. This study thus uncovers a range of new findings underlying the complex neurobiology of DMD.

Multilayer regulatory mechanisms of ERK1/2 signals transmitted through the Shoc2 scaffolding complex

Signaling scaffolds guide the flow of information and the spatial organization of the enzymes within the ERK1/2 signaling pathway. However, mechanisms that control assembly and dynamics within scaffolding complexes as well as mechanisms regulating the cellular distribution of these complexes remain largely unknown. Our studies unravel a novel, multi‐level paradigm in which allosteric modifications alter the ability of the scaffold protein Shoc2 to actively accelerate transmission of ERK1/2 signals. Shoc2 accelerates ERK1/2 signaling by tethering to close proximity essential proteins of the pathway, Ras and RAF‐1. Germ‐line mutations in Shoc2 affect spatial distribution of Shoc2 or its ability to assemble the signaling complex and cause Noonan‐like syndrome, a congenital disorder that presents with a wide spectrum of developmental abnormalities.Our lab studies mechanisms that fine‐tune ERK1/2 signals transmitted through the Shoc2 complex. We found that Shoc2 assembles an elegant multi‐protein complex that incorporates a number of proteins of the ubiquitin system. To fine‐tune amplitude of ERK1/2 signal transmitted via the complex, Shoc2 tethers the E3 ligase HUWE1, the (AAA+) ATPases, PSMC5 and VCP/p97 as well as the deubiquitinating enzyme, USP7. All of these enzymes are integral to the intricate feedback mechanism by which ubiquitination controls amplitude of the Shoc2‐ERK1/2 signals.Our studies show that HUWE1 ubiquitinates Shoc2 and RAF‐1, while ATPases modulate ubiquitination of Shoc2 and RAF‐1 through remodeling of the complex. We used fluorescence microscopy and biochemical analysis to demonstrate that PSMC5 and VCP/p97 are involved in recruitment of the Shoc2 scaffolding complexes to endosomes for complex remodeling. Importantly, we found that in the context of the Shoc2 complex, USP7 functions as a “molecular switch” that uses its enzymatic activity to trigger the “turn‐off” of the ERK1/2 signals transmitted via the scaffolding complex. We found that congenital Shoc2 mutations affect mechanisms controlling Shoc2 ubiquitination leading to aberrant signal transmission.In summary, our studies are the first to demonstrate that the Shoc2 scaffold employs multi‐protein enzymatic machinery to govern the amplitude of Shoc2‐ERK1/2 signals. We also show that mislocalization of the Shoc2 complexes affects remodeling of the scaffolding module and may lead to the developmental pathology. Overall, these studies significantly advance our understanding of the mechanisms by which non‐enzymatic scaffolds regulate specificity and dynamics of the ERK1/2 signaling networks.Support or Funding InformationNational Cancer Institute (R00CA126161 to EG), National Institute of General Medical Sciences (GM113087 to EG), American Cancer Society (RSG‐14‐172‐01‐CSM to EG)

Homo- and Heterotypic Association Regulates Signaling by the SgK269/PEAK1 and SgK223 Pseudokinases

SgK269/PEAK1 is a pseudokinase and scaffolding protein that plays a critical role in regulating growth factor receptor signal output and is implicated in the progression of several cancers, including those of the breast, colon, and pancreas. SgK269 is structurally related to SgK223, a human pseudokinase that also functions as a scaffold but recruits a distinct repertoire of signaling proteins compared with SgK269. Structural similarities between SgK269 and SgK223 include a predicted α-helical region (designated CH) immediately preceding the conserved C-terminal pseudokinase (PK) domain. Structure-function analyses of SgK269 in MCF-10A mammary epithelial cells demonstrated a critical role for the CH and PK regions in promoting cell migration and Stat3 activation. Characterization of the SgK269 "interactome" by mass spectrometry-based proteomics identified SgK223 as a novel binding partner, and association of SgK269 with SgK223 in cells was dependent on the presence of the CH and PK domains of both pseudokinases. Homotypic association of SgK269 and SgK223 was also demonstrated and exhibited the same structural requirements. Further analysis using pulldowns and size-exclusion chromatography underscored the critical role of the CH region in SgK269/SgK223 association. Importantly, although SgK269 bridged SgK223 to Grb2, it was unable to activate Stat3 or efficiently enhance migration in SgK223 knock-out cells generated by CRISPR/Cas9. These results reveal previously unrecognized interplay between two oncogenic scaffolds and demonstrate a novel signaling mechanism for pseudokinases whereby homotypic and heterotypic association is used to assemble scaffolding complexes with distinct binding properties and hence qualitatively regulate signal output.

Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

The surprising dynamics of scaffolding proteins.

The function of scaffolding proteins is to bring together two or more proteins in a relatively stable configuration, hence their name. Numerous scaffolding proteins are found in nature, many having multiple protein–protein interaction modules. Over the past decade, examples of scaffolding complexes long thought to be stable have instead been found to be surprisingly dynamic. These studies are scattered among different biological systems, and so the concept that scaffolding complexes might not always represent stable entities and that their dynamics can be regulated has not garnered general attention. We became aware of this issue in our studies of a scaffolding protein in microvilli, which forced us to reevaluate its contribution to their structure. The purpose of this Perspective is to draw attention to this phenomenon and discuss why complexes might show regulated dynamics. We also wish to encourage more studies on the dynamics of “stable” complexes and to provide a word of caution about how functionally important dynamic associations may be missed in biochemical and proteomic studies.

Differences in β‐arrestin dependent signaling by PAR2 and NK1R are directed by their C terminal tails

PAR2 and NK1R are 7 transmembrane receptors (7TMRs), which signal by Gαq leading to Ca2+ release and protein kinase C (PKC) activation. Both receptors are desensitized by β‐arrestin binding to their C termini. They can also activate ERK1/2 through β‐arrestin scaffolding complexes. They differ in ERK1/2 mediated physiological outcomes: cell migration versus proliferation. Using β‐arrestins, PAR2 activates cofilin, to promote chemotaxis. We hypothesized that the differences in β‐arrestin dependent signaling by 7TMRs can be attributed to how the molecular scaffolds bind the C‐tail of the receptors. Using wild type and C‐tail chimeras, we showed that the rate of desensitization, internalization, subcellular localization post endocytosis and ERK1/2 mediated responses depend on the nature of interaction of β‐arrestins with the C‐termini. BRET assays showed that PAR2 recruits both β‐arrestin 1/2 faster and with greater affinity than NK1R. We further show that initial Gαq signaling is necessary for β‐arrestin 1/2 recruitment to NK1R. PAR2 recruits β‐arrestin 1/2 even when Gαq pathway is blocked. Assays with C terminal phosphorylation mutants of PAR2 indicated that, phosphorylation of certain residues are necessary for β‐arrestin 1/2 recruitment. Phosphorylation at putative PKC sites determine stability of PAR2‐β‐arrestin 1/2 complex. This in turn ensures downstream cofilin activation and cell migration

Investigating inactive conformations of protein kinases

Protein kinases are found in countless signal transduction pathways and play key roles in the regulation of intracellular signaling events. Kinases exist in equilibria between catalytically active and inactive states. While evolutionary pressure limits kinases to one active state conformation, several inactive conformations have been described. The MAP kinase p38alpha has been shown to adopt a specific inactive conformation that appears to be inaccessible to other MAP kinases despite extensive structural homology. Seeking to understand the reason for this disparity, we found two residue positions that sensitize MAP kinases to small molecule inhibitors that are selective for specific inactive conformations. These ligands inhibit the activities of engineered mutants of p38delta and Erk2 more potently than their wild‐type counterparts by several orders of magnitude. We have determined a crystal structure of inhibitor‐sensitive Erk2 bound to one of these ligands in the active site and found that the inhibitor disrupts a key salt bridge necessary for catalysis, rendering the kinase inactive. The ability to manipulate kinases in a conformationally‐dependent manner could allow the targeted study of kinases in scaffolding complexes and other noncatalytic roles.Supported by NIH (R01GM086858) and American Heart Association (11PRE7260025)

Matchmaking: SK channels, C‐boutons and motor units

Matchmaking: SK channels, C‐boutons and motor units

Mechanisms of disease: inflammasome activation and the development of type 2 diabetes

Over the recent past, the importance of aberrant immune cell activation as one of the contributing mechanisms to the development of insulin-resistance and type 2 diabetes (T2D) has been recognized. Among the panoply of pro-inflammatory cytokines that are linked to chronic metabolic diseases, new data suggests that interleukin-1β (IL-1β) may play an important role in initiating and sustaining inflammation-induced organ dysfunction in T2D. Therefore, factors that control secretion of bioactive IL-1β have therapeutic implications. In this regard, the identification of multiprotein scaffolding complexes, “inflammasomes,” has been a great advance in our understanding of this process. The secretion of bioactive IL-1β is predominantly controlled by activation of caspase-1 through assembly of a multiprotein scaffold, “inflammasome” that is composed of NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) ASC (apoptosis associated speck-like protein containing a CARD) and procaspase-1. The NLRP3 inflammasome appears to be an important sensor of metabolic dysregulation and controls obesity-associated insulin resistance and pancreatic beta cell dysfunction. Initial clinical “proof of concept” studies suggest that blocking IL-1β may favorably modulate factors related to development and treatment of T2D. However, this potential therapeutic approach remains to be fully substantiated through phase-II clinical studies. Here, we outline the new immunological mechanisms that link metabolic dysfunction to the emergence of chronic inflammation and discuss the opportunities and challenges of future therapeutic approaches to dampen NLRP3 inflammasome activation or IL-1β signaling for controlling type 2 diabetes.

The role of lipids in defining membrane protein interactions: insights from mass spectrometry

Cellular membranes comprise hundreds of lipids in which protein complexes, such as ion channels, receptors, and scaffolding complexes, are embedded. These protein assemblies act as signalling and trafficking platforms for processes fundamental to life. Much effort in recent years has focused on identifying the protein components of these complexes after their extraction from the lipid membrane in detergent micelles. Spectacular advances have been made using X-ray crystallography, providing in some cases detailed information about the mechanism of pumping and channel gating. These structural studies are leading to a growing realisation that, to understand their function, it is not only the structures of the protein components that are important but also knowledge of the protein-lipid interactions. This review highlights recent insights gained from this knowledge, surveys methods being developed for probing these interactions, and focuses specifically on the potential of mass spectrometry in this growing area of research.

Scaffolding of the cofilin pathway by β‐arrestins

β‐arrestin‐1 (β1) is a key mediator of G‐protein‐coupled receptor (GPCR) signal termination and G‐protein‐independent signaling. Our lab uncovered the role for β‐arrestins in the control of actin dynamics downstream of the GPCR, protease‐activated receptor‐2 (PAR2), via regulation of cofilin activity. Here, we demonstrate that β1 can form at least 2 scaffolding complexes that regulate cofilin activity, one with cofilin and its phosphatase CIN, and another with its upstream negative regulator, LIM kinase (LIMK). Previous studies have demonstrated that β1 scaffolding of cofilin with CIN facilitates cofilin dephosphorylation by bringing substrate and phosphatase together. The studies presented here demonstrated that β1 also directly inhibits LIMK1 activity which further contributes to cofilin dephosphorylation. Combining the results from sandwich immunoassay, spot peptide array, bioluminescence resonance energy transfer (BRET) and in vitro kinase assay, we prove that β1 facilitates cofilin activation by directly binding both LIMK1 and cofilin. We have identified putative sites of interaction for both LIMK and cofilin on β1 between residues 146–182. These data elucidate suggesting a novel cofilin activation mechanism by which β1 enhances cofilin dephosphorylation by specifically binding to LIMK and preventing phosphorylation and inactivation of cofilin.

Structural biology of MAPK (p38/ERK) regulation by phosphatases and scaffolding proteins

~70% of all eukaryotic cellular proteins are regulated by phosphorylation. While structural data for kinases and phosphatases are readily available, only a handful of protein:protein complex structures, which are necessary to understand the biological function of the underlying regulation, have been described. Here we report our efforts to close this gap by describing our novel, structural insights into the regulation of MAPKs (p38, ERK2) by the kinase interaction motif family KIM‐PTPs (HePTP, STEP, PTP‐SL). In order to obtain a molecular picture of these large MAPK protein:protein complexes, we are integrating high resolution techniques (NMR/X‐ray) for the analysis of basic components with lower resolution techniques (SAXS) for constraints on the higher order, dynamic, multi‐protein assemblies. This has allowed us to determine the first detailed structures of MAPK:MAPK regulatory protein complexes, which, in combination with a thorough ITC study, has enabled us to identify the molecular determinants that drive MAPK specificity and fidelity. Finally, we are also providing new structural insights into the MAPK spatial control by elucidating the structures of MAPK scaffolding complexes.Supported by RSG‐08‐067‐01‐LIB from the American Cancer Society.

Emerging methodologies to investigate lipid–protein interactions

Cellular membranes are composed of hundreds of different lipids, ion channels, receptors and scaffolding complexes that act as signalling and trafficking platforms for processes fundamental to life. Cellular signalling and membrane trafficking are often regulated by peripheral proteins, which reversibly interact with lipid molecules in highly regulated spatial and temporal fashions. In most cases, one or more modular lipid-binding domain(s) mediate recruitment of peripheral proteins to specific cellular membranes. These domains, of which more than 10 have been identified since 1989, harbour structurally selective lipid-binding sites. Traditional in vitro and in vivo studies have elucidated how these domains coordinate their cognate lipids and thus how the parent proteins associate with membranes. Cellular activities of peripheral proteins and subsequent physiological processes depend upon lipid binding affinities and selectivity. Thus, the development of novel sensitive and quantitative tools is essential in furthering our understanding of the function and regulation of these proteins. As this field expands into new areas such as computational biology, cellular lipid mapping, single molecule imaging, and lipidomics, there is an urgent need to integrate technologies to detail the molecular architecture and mechanisms of lipid signalling. This review surveys emerging cellular and in vitro approaches for studying protein-lipid interactions and provides perspective on how integration of methodologies directs the future development of the field.

Erratum of “Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies”

Erratum of “Dystrophins, Utrophins, and Associated Scaffolding Complexes: Role in Mammalian Brain and Implications for Therapeutic Strategies”

Rescue of a Dystrophin-like Protein by Exon Skipping In Vivo Restores GABAA-receptor Clustering in the Hippocampus of the mdx Mouse

Dystrophin, the cytoskeletal protein whose defect is responsible for Duchenne muscular dystrophy (DMD), is normally expressed in both muscles and brain. Genetic loss of brain dystrophin in the mdx mouse model of DMD reduces the capacity for type A gamma-aminobutyric acid (GABA(A))-receptor clustering in central inhibitory synapses, which is thought to be a main molecular defect leading to brain and cognitive alterations in this syndrome. U7 small nuclear RNAs modified to encode antisense sequences and expressed from recombinant adeno-associated viral (rAAV) vectors have proven efficient after intramuscular injection to induce skipping of the mutated exon 23 and rescue expression of a functional dystrophin-like product in muscle tissues of mdx mice in vivo. Here, we report that intrahippocampal injection of a single dose of rAAV2/1-U7 can rescue substantial levels of brain dystrophin expression (15-25%) in mdx mice for months. This is sufficient to completely restore GABA(A)-receptor clustering in pyramidal and dendritic layers of CA1 hippocampus, suggesting exon-skipping strategies offer the prospect to investigate and correct both brain and muscle alterations in DMD. This provides new evidence that in the adult brain dystrophin is critical for the control of GABA(A)-receptor clustering, which may have an important role in activity-dependent synaptic plasticity in hippocampal circuits.

Undergraduate Laboratory: Increasing Awareness of the Role of Lipids in Biochemistry

Cellular membranes harbor receptors, ion channels, lipid domains, lipid signals, and scaffolding complexes, which function to maintain cellular growth, metabolism, and homeostasis. Moreover, abnormalities in lipid metabolism attributed to genetic changes among other causes are often associated with diseases such as cancer. Thus, there is a need to comprehensively understand molecular events occurring within and on membranes as a means of grasping disease etiology and identifying viable targets for drug development. The lipid bilayer has a highly polarized structure that consists of a central hydrocarbon core and two flanking interfacial regions that are highly dynamic and may contain >10,000 different lipids. This dynamic variety of glycerolipids, sphingolipids, and sterols in the membrane organelles provide spatial and temporal architecture to direct signaling processes through target proteins. The central lipid bilayer viewed as a hub for biological interactions can serve an integral role in biochemistry education. To increase what has traditionally been an underserved area of biochemical education, we have designed a lipid‐based curriculum for the undergraduate classroom and laboratory. An overview of the curriculum and laboratory experiments will be given.