Dynamic Subunit Exchange and the Regulation of Microtubule Assembly by the Stress Response Protein Human αB Crystallin

Background

The small heat shock protein (sHSP), human αB crystallin, forms large, polydisperse complexes that modulate the tubulin-microtubule equilibrium using a dynamic mechanism that is poorly understood. The interactive sequences in αB crystallin for tubulin are surface exposed, and correspond to interactive sites for the formation of αB crystallin complexes.

Methodology/Principal Findings

There is sequence homology between tubulin and the interactive domains in the β8-strand of the core domain and the C-terminal extension of αB crystallin. This study investigated the hypothesis that the formation of tubulin and αB crystallin quaternary structures was regulated through common interactive domains that alter the dynamics of their assembly. Size exclusion chromatography (SEC), SDS-PAGE, microtubule assembly assays, aggregation assays, multiple sequence alignment, and molecular modeling characterized the dynamic response of tubulin assembly to increasing concentrations of αB crystallin. Low molar ratios of αB crystallin∶tubulin were favorable for microtubule assembly and high molar ratios of αB crystallin∶tubulin were unfavorable for microtubule assembly. Interactions between αB crystallin and unassembled tubulin were observed using SEC and SDS-PAGE.

Conclusions/Significance

Subunits of αB crystallin that exchange dynamically with the αB crystallin complex can interact with tubulin subunits to regulate the equilibrium between tubulin and microtubules.     

Surveillance-Activated Defenses Block the ROS–Induced Mitochondrial Unfolded Protein Response

Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPR^mt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPR^mt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPR^mt. Activation of the UPR^mt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPR^mt, suggesting that surveillance-activated defenses specifically inhibit the UPR^mt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection

Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated.     

Structural Basis of Protein Kinase Cα Regulation by the C-Terminal Tail

Protein kinase C (PKC) isoenzymes are multi-modular proteins activated at the membrane surface to regulate signal transduction processes. When activated by second messengers, PKC undergoes a drastic conformational and spatial transition from the inactive cytosolic state to the activated membrane-bound state. The complete structure of either state of PKC remains elusive. We demonstrate, using NMR spectroscopy, that the isolated Ca²⁺-sensing membrane-binding C2 domain of the conventional PKCα interacts with a conserved hydrophobic motif of the kinase C-terminal region, and we report a structural model of the complex. Our data suggest that the C-terminal region plays a dual role in regulating the PKC activity: activating, through sensitization of PKC to intracellular Ca²⁺ oscillations; and auto-inhibitory, through its interaction with a conserved positively charged region of the C2 domain.     

Archaeal aIF2B Interacts with Eukaryotic Translation Initiation Factors eIF2α and eIF2Bα: Implications for aIF2B Function and eIF2B Regulation

Translation initiation is down-regulated in eukaryotes by phosphorylation of the α-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2α interacts with a subcomplex of eIF2B formed by the three regulatory subunits α/GCN3, β/GCD7, and δ/GCD2, blocking the GDP-GTP exchange activity of the catalytic ?-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO₂ fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the α, β, and δ subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the α-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the α-subunit of yeast eIF2 in vitro and to interact with eIF2Bα/GCN3 in vivo in yeast. The aIF2B-eIF2α interaction was however independent of eIF2α phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2α, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2B?, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2α.     

The Unfolded Protein Response is Triggered in Rat Neurons of the Dorsal Raphe Nucleus After Single-Prolonged Stress

The dorsal raphe nucleus (DRN) has been suggested playing an important role in the pathophysiology of post-traumatic stress disorder (PTSD), however the underlying cellular mechanisms are not fully understood. The endoplasmic reticulum (ER) is a critical organelle for synthesis of membrane and secretory proteins, and perturbations in ER lead to the unfolded protein response (UPR). In the present experiment, we hypothesized UPR may be associated with the PTSD, and there is an induction of UPR in the DRN neurons of the PTSD-like rats. We first observed the morphological changes of ER in the DRN neurons of the rats exposed to single-prolonged stress (SPS), a model of PTSD, and then we also detected the expression of ER chaperones glucose regulated protein 78 (GRP78) and glucose regulated protein (GRP94) which are two key sensors and mediators of the UPR and are considered an ER stress-specific inducible proteins using methods of western blot and immunohistochemical analysis. Our results demonstrated there were abnormal expansion of ER and up-regulation expression of GRP78 and GRP94 after SPS, which indicated that the UPR was triggered in the DRN neurons of the PTSD-like rats. These results are consistent with our speculation that UPR may be associated with the PTSD, and suggest us the UPR may be a new critical cellular mechanisms of PTSD.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.     

Protein synthesis attenuation by phosphorylation of eIF2α is required for the differentiation of Trypanosoma cruzi into infective forms

Chagas' disease is a potentially life-threatening illness caused by the unicellular protozoan parasite Trypanosoma cruzi. It is transmitted to humans by triatomine bugs where T. cruzi multiplies and differentiates in the digestive tract. The differentiation of proliferative and non-infective epimastigotes into infective metacyclic trypomastigotes (metacyclogenesis) can be correlated to nutrient exhaustion in the gut of the insect vector. In vitro, metacyclic-trypomastigotes can be obtained when epimastigotes are submitted to nutritional stress suggesting that metacyclogenesis is triggered by nutrient starvation. The molecular mechanism underlying such event is not understood. Here, we investigated the role of one of the key signaling responses elicited by nutritional stress in all other eukaryotes, the inhibition of translation initiation by the phosphorylation of the eukaryotic initiation factor 2α (eIF2α), during the in vitro differentiation of T. cruzi. Monospecific antibodies that recognize the phosphorylated Tc-eIF2α form were generated and used to demonstrate that parasites subjected to nutritional stress show increased levels of Tc-eIF2α phosphorylation. This was accompanied by a drastic inhibition of global translation initiation, as determined by polysomal profiles. A strain of T. cruzi overexpressing a mutant Tc-eIF2α, incapable of being phosphorylated, showed a block on translation initiation, indicating that such a nutritional stress in trypanosomatids induces the conserved translation inhibition response. In addition, Tc-eIF2α phosphorylation is critical for parasite differentiation since the overexpression of the mutant eIF2α in epimastigotes abolished metacyclogenesis. This work defines the role of eIF2α phosphorylation as a key step in T. cruzi differentiation.     

Interactome Screening Identifies the ER Luminal Chaperone Hsp47 as a Regulator of the Unfolded Protein Response Transducer IRE1α

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