Facilitates Complex Formation Research Articles

Differential reliance of CTD-nuclear envelope phosphatase 1 on its regulatory subunit in ER lipid synthesis and storage.

Lipin 1 is an ER enzyme that produces diacylglycerol, the lipid intermediate that feeds into the synthesis of glycerophospholipids for membrane expansion or triacylglycerol for storage into lipid droplets. CTD-Nuclear Envelope Phosphatase 1 (CTDNEP1) regulates lipin 1 to restrict ER membrane synthesis, but a role for CTDNEP1 in lipid storage in mammalian cells is not known. Furthermore, how NEP1R1, the regulatory subunit of CTDNEP1, contributes to these functions in mammalian cells is not fully understood. Here, we show that CTDNEP1 is reliant on NEP1R1 for its stability and function in limiting ER expansion. CTDNEP1 contains an amphipathic helix at its N-terminus that targets to the ER, nuclear envelope and lipid droplets. We identify key residues at the binding interface of CTDNEP1 and NEP1R1 and show that they facilitate complex formation in vivo and in vitro. We demonstrate that NEP1R1 binding to CTDNEP1 shields CTDNEP1 from proteasomal degradation to regulate lipin 1 and restrict ER size. Unexpectedly, NEP1R1 was not required for CTDNEP1's role in restricting lipid droplet biogenesis. Thus, the reliance of CTDNEP1 function on NEP1R1 depends on cellular demands for membrane production versus lipid storage. Together, our work provides a framework into understanding how the ER regulates lipid synthesis under different metabolic conditions.

Liquid Dynamics Determine Transition Metal-N-Heterocyclic Carbene Complex Formation.

The mechanism of metal-N-heterocyclic carbene (NHC) complex formation from imidazolium salts in the presence of weak bases was investigated through theoretical methods. Quantum chemical calculations revealed that the two bases considered here, sodium acetate and trimethylamine, both facilitate complex formation. In contrast to previous experiments, these calculations indicated a slightly lower barrier with the amine. Molecular dynamics simulations showed that the ionic nature of the [AuCl2 ]- and imidazolium ions, as well as the sodium acetate base keep these species associated in the reaction mixture through ion pairing. This pre-association of the components produces those clusters that are essential for the metal complex formation reaction. The neutral amine, however, remains mostly separated from the other reaction partners, making it a significantly less effective base.

Novel Interactors of the SH2 Domain of the Signaling Adaptors CRK and CRKL Identified in Neuro2A Cells

CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of signaling adaptor proteins that serve important roles in the regulation of fundamental cellular processes, including cell motility and proliferation, in a variety of cell types. The Src Homology 2 (SH2) domain of CRK and CRKL interacts with proteins containing phosphorylated tyrosine-X-X-proline (pYXXP) motifs, facilitating complex formation during signaling events. A handful of CRK/CRKL-SH2-specific interactors have been identified to date, although in silico analyses suggest that many additional interactors remain to be found. To identify CRK/CRKL-SH2 interactors with potential involvement in neuronal development, we conducted a mass spectrometry-based proteomics screen using a neuronal cell line (Neuro2A, or N2A). This resulted in the identification of 132 (6 known and 126 novel) YXXP-containing CRK/CRKL-SH2 interactors, of which 77 were stimulated to bind to the CRK/CRKL-SH2 domain following tyrosine phosphatase inhibition. Approximately half of the proteins identified were common interactors of both the CRK- and CRKL-SH2 domains. However, both CRK family member SH2 domains exhibited unique binding partners across experimental replicates. These findings reveal an abundance of novel neuronal CRK/CRKL-SH2 domain binding partners and suggest that CRK family SH2 domains possess undescribed docking preferences beyond the canonical pYXXP motif. KEYWORDS: CRK; CRKL; SH2; LC-MS/MS; Proteomics; Neurodevelopment; Signal Transduction

IQ Motif Containing GTPase Activating Proteins (IQGAPs), A-Kinase Anchoring Proteins (AKAPs) and Kinase Suppressor of Ras Proteins (KSRs) in Scaffolding Oncogenic Pathways and Their Therapeutic Potential.

Scaffolding proteins colocalize interacting partners on their surface and facilitate complex formation. They have multiple domains and motifs, which provide binding sites for various molecules. This property of scaffolding proteins helps in the orderly transduction of signals. Abnormal signal transduction is frequently observed in cancers, which can also be attributed to the altered functionality of scaffolding proteins. IQ motif containing GTPase activating proteins (IQGAPs), kinase suppressor of Ras (KSR), and A-kinase anchoring proteins (AKAPs) tether oncogenic pathways RAS/RAF/MEK/ERK, PI3K/AKT, Hippo, Wnt, and CDC42/RAC to them. Scaffolding proteins are attractive drug targets as they are the controlling hub for multiple pathways and regulate crosstalk between them. The first part of this review describes the human scaffolding proteins known to play a role in oncogenesis, pathways altered by them, and the impact on oncogenic processes. The second part provides information on the therapeutic potential of scaffolding proteins and future possibilities. The information on the explored and unexplored areas of the therapeutic potential of scaffolding proteins will be equally helpful for biologists and chemists.

Molecular convergence by differential domain acquisition is a hallmark of chromosomal passenger complex evolution

The chromosomal passenger complex (CPC) is a heterotetrameric regulator of eukaryotic cell division, consisting of an Aurora-type kinase and a scaffold built of INCENP, Borealin, and Survivin. While most CPC components are conserved across eukaryotes, orthologs of the chromatin reader Survivin have previously only been found in animals and fungi, raising the question of how its essential role is carried out in other eukaryotes. By characterizing proteins that bind to the Arabidopsis Borealin ortholog, we identified BOREALIN RELATED INTERACTOR 1 and 2 (BORI1 and BORI2) as redundant Survivin-like proteins in the context of the CPC in plants. Loss of BORI function is lethal and a reduced expression of BORIs causes severe developmental defects. Similar to Survivin, we find that the BORIs bind to phosphorylated histone H3, relevant for correct CPC association with chromatin. However, this interaction is not mediated by a BIR domain as in previously recognized Survivin orthologs but by an FHA domain, a widely conserved phosphate-binding module. We find that the unifying criterion of Survivin-type proteins is a helix that facilitates complex formation with the other two scaffold components and that the addition of a phosphate-binding domain, necessary for concentration at the inner centromere, evolved in parallel in different eukaryotic groups. Using sensitive similarity searches, we find conservation of this helical domain between animals and plants and identify the missing CPC component in most eukaryotic supergroups. Interestingly, we also detect Survivin orthologs without a defined phosphate-binding domain, likely reflecting the situation in the last eukaryotic common ancestor.

Unraveling the phase behavior of cricket protein isolate and alginate in aqueous solution

The associative phase behavior of cricket protein isolate (CPI) and sodium alginate (AL) in aqueous solutions was explored using turbidimetry, methylene blue spectroscopy, zeta potentiometry, dynamic light scattering, and confocal microscopy as a function of pH, biopolymer ratio, total biopolymer concentration (CT), and ionic strength. When both biopolymers had net-negative charges, soluble complexes formed between pH 6.0 and 8.0, however when both biopolymers had opposing net charges, insoluble complexes formed as complex coacervates below pH 5.5, defined as pHφ1, followed by precipitates below another critical pH 3.0 (pHp). Increasing the CPI:AL weight ratio or CT facilitated complex formation, and the addition of salts (NaCl/KCl) had a salt-enhancement and salt-reduction impact at low and high salt concentrations, respectively. Ionic interactions between oppositely charged CPI and AL were mainly responsible for the formation of their insoluble complexes, while hydrogen bonding and hydrophobic interactions also played significant roles.

EIF3H Orchestrates Hippo Pathway-Mediated Oncogenesis via Catalytic Control of YAP Stability.

EIF3H is presumed to be a critical translational initiation factor. Here, our unbiased screening for tumor invasion factors has identified an unexpected role for EIF3H as a deubiquitylating enzyme that dictates breast tumor invasion and metastasis by modulating the Hippo-YAP pathway. EIF3H catalyzed YAP for deubiquitylation, resulting in its stabilization. Structure-based molecular modeling and simulations coupled with biochemical characterization unveiled a unique catalytic mechanism for EIF3H in dissociating polyubiquitin chains from YAP through a catalytic triad consisting of Asp90, Asp91, and Gln121. Trp119 and Tyr 140 on EIF3H directly interacted with the N-terminal region of YAP1, facilitating complex formation of EIF3H and YAP1 for YAP1 deubiquitylation. Stabilization of YAP via elevated EIF3H promoted tumor invasion and metastasis. Interference of EIF3H-mediated YAP deubiquitylation blocked YAP-induced tumor progression and metastasis in breast cancer models. These findings point to a critical role for YAP regulation by EIF3H in tumor invasion and metastasis. SIGNIFICANCE: This work demonstrates that EIF3H is a novel bona fide deubiquitinase that counteracts YAP ubiquitylation and proteolysis, and stabilization of YAP by EIF3H promotes tumor invasion and metastasis.

Evidence that Primary Microrna Bends in the Presence of DGCR8 Seen using Both Saxs and FRET Measurements

RNA's structural dynamics are critically important in the processing of microRNAs (miRNAs). We and others have demonstrated that non-A-form structural elements and the conformational flexibility they support are necessary for efficient RNA binding and cleavage by the proteins associated with the two major stages of miRNA processing [Roth, 2013; Quarles, 2013; Quarles, 2015]. However, it is unclear whether the flexibility of primary microRNA (pri-miRNA) is necessary to facilitate complex formation with protein partners. Here, we study the flexibility and bending of a primary microRNA (pri-miR-16) in the presence of a small recombinant construct of the DGCR8 protein that binds double stranded RNA (dsRNA) in vitro. This so-called DGCR8-core contains two dsRNA-binding domains (dsRBD) and a C-terminal tail. We use Small Angle X-ray Scattering (SAXS) to measure the conformations of pri-miR-16 in the presence and absence of the DGCR8-core. SAXS provides global structural insights about freely diffusing macromolecules, including complexes, in solution. With contrast variation, SAXS detects only the nucleic acid (NA) component of a protein-NA complex, by masking out the protein contribution to the scattering profile. Using contrast variation SAXS, we find strong evidence that pri-miR-16 bends in the presence of the DGCR8-core. Complementary studies using single molecule FRET (smFRET) experiments confirm this model.

Is Graphene Oxide a Chemoattractant?

The use of nanomaterials to regulate cell surface receptors is considered a novel strategy to manipulate cell behaviors. However, recognition is important to drive nanoparticle-cell complex formation. Here, we report a novel approach that uses graphene oxide (GO) as a chemoattractant to lure bacteria to nanosurface, facilitating complex formation. The amount of Escherichia coli (E. coli) cells attracted into capillaries containing 20 mg/L GO was more than 8.6-fold higher than that attracted into capillaries containing 20 mg/L glucose. The inherent mechanism involved interference with transmembrane chemoreceptors and activation of the chemotactic system via GO attachment and a subsequent increase in cell aggregation and migration via self-secreted quorum sensing molecules. The key feature of this strategy is the potential to improve the efficiency of the nanoparticle-cell recognition pattern and to expedite the development of surface-contact-related nanotechnology.

Exploring the Limits of Bivalency by DNA-Based Spatial Screening.

Multivalency can facilitate complex formation when monovalent receptor-ligand interactions are weak. However, enhanced binding of two multivalent binding partners should be avoidable, for example when bivalent receptors ought to utilize multimolecular interactions to cross-link binding partners. We herein report the first systematic study to assess the criteria deciding whether a bivalent system engages in bivalency-enhanced interactions or cross-linking. We used DNA-instructed self-assembly to arrange the cucurbit[7]uril-adamantane host-guest system in 70-360 Å distance. Measurements and statistical mechanics analyses revealed that the affinity gain is controlled by 1) the distance between recognition modules, 2) the scaffold flexibility, and, importantly, 3) the strength of the monovalent interaction. We show that the bivalency effect can extend beyond 150 Å and discuss how, on the contrary, weak monovalent interactions reduce the concentration threshold for cross-linking. The findings are of interest for inhibitor design.

A Golgi-Released Subpopulation of the Trans-Golgi Network Mediates Protein Secretion in Arabidopsis.

Spatiotemporal coordination of protein trafficking among organelles is essential for eukaryotic cells. The post-Golgi interface, including the trans-Golgi network (TGN), is a pivotal hub for multiple trafficking pathways. The Golgi-released independent TGN (GI-TGN) is a compartment described only in plant cells, and its cellular and physiological roles remain elusive. In Arabidopsis (Arabidopsis thaliana), the SYNTAXIN OF PLANTS (SYP) 4 group Qa-SNARE (soluble N-ethylmaleimide) membrane fusion proteins are shared components of TGN and GI-TGN and regulate secretory and vacuolar transport. Here we reveal that GI-TGNs mediate the transport of the R-SNARE VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 to the plasma membrane. In interactions with a nonadapted powdery mildew pathogen, the SYP4 group of SNAREs is required for the dynamic relocation of VAMP721 to plant-fungus contact sites via GI-TGNs, thereby facilitating complex formation with its cognate SNARE partner PENETRATION1 to restrict pathogen entry. Furthermore, quantitative proteomic analysis of leaf apoplastic fluid revealed constitutive and pathogen-inducible secretion of cell wall-modification enzymes in a SYP4- and VAMP721-dependent manner. Hence, the GI-TGN acts as a transit compartment between the Golgi apparatus and the plasma membrane. We propose a model in which the GA-TGN matures into the GI-TGN and then into secretory vesicles by increasing the abundance of VAMP721-dependent secretory pathway components.

Dynamic Hydrogels from Host-Guest Supramolecular Interactions.

Hydrogel biomaterials are pervasive in biomedical use. Applications of these soft materials range from contact lenses to drug depots to scaffolds for transplanted cells. A subset of hydrogels is prepared from physical cross-linking mediated by host-guest interactions. Host macrocycles, the most recognizable supramolecular motif, facilitate complex formation with an array of guests by inclusion in their portal. Commonly, an appended macrocycle forms a complex with appended guests on another polymer chain. The formation of poly(pseudo)rotaxanes is also demonstrated, wherein macrocycles are threaded by a polymer chain to give rise to physical cross-linking by secondary non-covalent interactions or polymer jamming. Host-guest supramolecular hydrogels lend themselves to a variety of applications resulting from their dynamic properties that arise from non-covalent supramolecular interactions, as well as engineered responsiveness to external stimuli. These are thus an exciting new class of materials.

Microfluidic isoform sequencing shows widespread splicing coordination in the human transcriptome.

Understanding transcriptome complexity is crucial for understanding human biology and disease. Technologies such as Synthetic long-read RNA sequencing (SLR-RNA-seq) delivered 5 million isoforms and allowed assessing splicing coordination. Pacific Biosciences and Oxford Nanopore increase throughput also but require high input amounts or amplification. Our new droplet-based method, sparse isoform sequencing (spISO-seq), sequences 100k–200k partitions of 10–200 molecules at a time, enabling analysis of 10–100 million RNA molecules. SpISO-seq requires less than 1 ng of input cDNA, limiting or removing the need for prior amplification with its associated biases. Adjusting the number of reads devoted to each molecule reduces sequencing lanes and cost, with little loss in detection power. The increased number of molecules expands our understanding of isoform complexity. In addition to confirming our previously published cases of splicing coordination (e.g., BIN1), the greater depth reveals many new cases, such as MAPT. Coordination of internal exons is found to be extensive among protein coding genes: 23.5%–59.3% (95% confidence interval) of highly expressed genes with distant alternative exons exhibit coordination, showcasing the need for long-read transcriptomics. However, coordination is less frequent for noncoding sequences, suggesting a larger role of splicing coordination in shaping proteins. Groups of genes with coordination are involved in protein–protein interactions with each other, raising the possibility that coordination facilitates complex formation and/or function. We also find new splicing coordination types, involving initial and terminal exons. Our results provide a more comprehensive understanding of the human transcriptome and a general, cost-effective method to analyze it.

Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans.

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, composed of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of the yeast Shu complex member Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs rfs-1 and rip-1 suggest that they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast two-hybrid assay that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1 Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting that RIP-1 facilitates complex formation with SWS-1 and RFS-1 We propose that SWS-1, RIP-1, and RFS-1 compose a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.

Separation of Binary Mixtures of Histidine, Proline, and Methionine in Extraction Systems Based on Water-Soluble Polymers of Vinyl Series

Results of the extraction separation of binary mixtures of histidine, proline, and methionine using water-soluble homo- and copolymers with different molecular weights are presented. The recoveries and separation factors in the systems methionine–proline and histidine–proline are calculated. Amino acids were quantitatively determined by spectrophotometry using their intrinsic absorption in the UV spectral region. The efficiency of amino acid separation was increased using systems additionally containing chloride ions, which facilitate complex formation.

Bone and Calcium Research Update 2015. The regulation of proliferation and differentiation by the ER stress transducer in chondrocytes

BBF2H7 is an ER-resident transmembrane transcription factor which is structurally similar to ATF6, one of the canonical ER stress transducers, and preferentially expresses in chondrocytes. This molecule is processed at the transmembrane region in response to ER stress or differentiation stimuli. The cleaved amino (N) -terminal cytoplasmic domain promotes transcription of the ER-Golgi trafficking protein Sec23a, and plays a crucial role in secretion of extracellular matrix proteins from chondrocytes during cartilage formation. On the contrary, the cleaved C-terminal luminal domain of BBF2H7 is secreted from chondrocytes. The secreted BBF2H7 C-terminus directly binds to both Indian Hedgehog and its receptor Patched-1, and facilitates complex formation of the ligand-receptor followed by activation of Hedgehog signaling to promote proliferation in neighbouring chondrocytes. The dual N- and C-terminal functions of BBF2H7 triggered by physiological ER stress could allow chondrocytes to promote simultaneously distinct cellular events for differentiation and proliferation in developing cartilage.

A SIRT7-Dependent Acetylation Switch of GABPβ1 Controls Mitochondrial Function

Mitochondrial activity is controlled by proteins encoded by both nuclear and mitochondrial DNA. Here, we identify Sirt7 as a crucial regulator of mitochondrial homeostasis. Sirt7 deficiency in mice induces multisystemic mitochondrial dysfunction, which is reflected by increased blood lactate levels, reduced exercise performance, cardiac dysfunction, hepatic microvesicular steatosis, and age-related hearing loss. This link between SIRT7 and mitochondrial function is translatable in humans, where SIRT7 overexpression rescues the mitochondrial functional defect in fibroblasts with a mutation in NDUFSI. These wide-ranging effects of SIRT7 on mitochondrial homeostasis are the consequence of the deacetylation of distinct lysine residues located in the hetero- and homodimerization domains of GABPβ1, a master regulator of nuclear-encoded mitochondrial genes. SIRT7-mediated deacetylation of GABPβ1 facilitates complex formation with GABPα and the transcriptional activation of the GABPα/GABPβ heterotetramer. Altogether, these data suggest that SIRT7 is a dynamic nuclear regulator of mitochondrial function through its impact on GABPβ1 function.

Phosphorylation of Grb14 BPS domain by GSK-3 correlates with complex forming of Grb14 and insulin receptor

Growth factor receptor-bound protein 14 (Grb14) interacts with insulin receptor (IR) through the between PH and SH2 (BPS) domain. Grb14-IR complex formation is initiated by insulin stimulation, and the binding event results in the inhibition of insulin signalling. Thus, Grb14 is regarded as an endogenous suppressor of insulin signal transduction; however, there are no studies describing the mechanism whereby Grb14-IR complex formation is suppressed in the absence of insulin stimulation. In the present study, multiple phosphorylation motifs for glycogen synthase kinase 3 (GSK-3) were identified within the Grb14 BPS domain (Ser(358), Ser(362) and Ser(366) of human Grb14). Pharmacological inhibition as well as knockdown of GSK-3 facilitated complex formation between Grb14 and IR, implicating GSK-3 activity in regulating Grb14-IR binding. In situ proximity ligation assay and in vitro kinase assays of phosphopeptides suggested that serine residues in the BPS domain would be substrates for GSK-3. The kinase assays also indicated phosphoserine 370 (in human Grb14) was required for the phosphorylation of Ser(358), Ser(362) and Ser(366) by GSK-3. Grb14-IR binding was also facilitated by replacement of the serines with Ala. We also observed that Ser(366) of endogenous Grb14 in Hep G2 cell was phosphorylated and the phosphorylation was influenced by treatments with insulin, as well as the GSK-3 inhibitor.

Hyperoxidized peroxiredoxin 2 interacts with the protein disulfide- isomerase ERp46

Prx (peroxiredoxin) 2 protects cells from deleterious oxidative damage. It catalyses the breakdown of hydroperoxides through a highly reactive cysteine residue and has been linked to chaperone activity that promotes cell survival under conditions of oxidative stress. It may also be involved in redox signalling by binding to other proteins. In the present study we have searched for binding partners of Prx2in H2O2-treated Jurkat and human umbilical vein endothelial cells and discovered that the hyperoxidized form selectively co-precipitated with the protein disulfide-isomerase ERp46. Mutant analyses revealed that loss of the peroxidative cysteine residue of Prx2 also facilitated complex formation with ERp46, even without H2O2 treatment, whereas the resolving cysteine residue of Prx2 was indispensible for the interaction to occur. The complex involved a stable non-covalent interaction that was disassociated by the reduction of intramolecular disulfides in ERp46, or by disruption of the decameric structure of hyperoxidized Prx2. This is the first example of a protein interaction dependent on the hyperoxidized status of a Prx.

Tetraspanin CD151 Stimulates Adhesion-dependent Activation of Ras, Rac, and Cdc42 by Facilitating Molecular Association between β1 Integrins and Small GTPases

Tetraspanin CD151 associates with laminin-binding α(3)β(1)/α(6)β(1) integrins in epithelial cells and regulates adhesion-dependent signaling events. We found here that CD151 plays a role in recruiting Ras, Rac1, and Cdc42, but not Rho, to the cell membrane region, leading to the formation of α(3)β(1)/α(6)β(1) integrin-CD151-GTPases complexes. Furthermore, cell adhesion to laminin enhanced CD151 association with β(1) integrin and, thereby, increased complex formation between the β(1) family of integrins and small GTPases, Ras, Rac1, and Cdc42. Adhesion receptor complex-associated small GTPases were activated by CD151-β(1) integrin complex-stimulating adhesion events, such as α(3)β(1)/α(6)β(1) integrin-activating cell-to-laminin adhesion and homophilic CD151 interaction-generating cell-to-cell adhesion. Additionally, FAK and Src appeared to participate in this adhesion-dependent activation of small GTPases. However, engagement of laminin-binding integrins in CD151-deficient cells or CD151-specific siRNA-transfected cells did not activate these GTPases to the level of cells expressing CD151. Small GTPases activated by engagement of CD151-β(1) integrin complexes contributed to CD151-induced cell motility and MMP-9 expression in human melanoma cells. Importantly, among the four tetraspanin proteins that associate with β(1) integrin, only CD151 exhibited the ability to facilitate complex formation between the β(1) family of integrins and small GTPases and stimulate β(1) integrin-dependent activation of small GTPases. These results suggest that CD151 links α(3)β(1)/α(6)β(1) integrins to Ras, Rac1, and Cdc42 by promoting the formation of multimolecular complexes in the membrane, which leads to the up-regulation of adhesion-dependent small GTPase activation.