Protein Signaling System Research Articles

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

Autophagy, molecular chaperones, and unfolded protein response as promoters of tumor recurrence.

Tumor recurrence is a paradoxical function of a machinery, whereby a small proportion of the cancer cell population enters a resistant, dormant state, persists long-term in this condition, and then transitions to proliferation. The dormant phenotype is typical of cancer stem cells, tumor-initiating cells, disseminated tumor cells, and drug-tolerant persisters, which all demonstrate similar or even equivalent properties. Cancer cell dormancy and its conversion to repopulation are regulated by several protein signaling systems that inhibit or induce cell proliferation and provide optimal interrelations between cancer cells and their special niche; these systems act in close connection with tumor microenvironment and immune response mechanisms. During dormancy and reawakening periods, cell proteostasis machineries, autophagy, molecular chaperones, and the unfolded protein response are recruited to protect refractory tumor cells from a wide variety of stressors and therapeutic insults. Proteostasis mechanisms functionally or even physically interfere with the main regulators of tumor relapse, and the significance of these interactions and implications in the tumor recurrence phases are discussed in this review.

Knockdown of GPSM1 Inhibits the Proliferation and Promotes the Apoptosis of B-Cell Acute Lymphoblastic Leukemia Cells by Suppressing the ADCY6-RAPGEF3-JNK Signaling Pathway

B-cell acute lymphoblastic leukemia (B-ALL) is the common type of blood cancer. Although the remission rate has increased, the current treatment options for B-ALL are usually related to adverse reactions and recurrence, so it is necessary to find other treatment options. G protein signaling modulator 1 (GPSM1) is one of several factors that affect the basic activity of the G protein signaling system, but its role in B-ALL has not yet been clarified. In this study, we analyzed the expression of GPSM1 in the Oncomine database and found that the GPSM1 levels were higher in B-ALL cells than in peripheral blood mononuclear cells (PBMCs). Analyses of the Gene Expression Profiling Interactive Analysis (GEPIA) demonstrated that patients with high GPSM1 levels had shorter survival times than those with low levels. Additionally, gene set enrichment analysis (GSEA) suggested that GPSM1 was positively correlated with proliferation, G protein-coupled receptor (GPCR) ligand binding, Gαs signaling and calcium signaling pathways. In further experiments, GPSM1 was found to be highly expressed in Acute lymphoblastic leukemia (ALL) cell lines, and downregulation of GPSM1 inhibited proliferation and promoted cell cycle arrest and apoptosis in BALL-1 and Reh cells. Moreover, knockdown of GPSM1 suppressed ADCY6 and RAPGEF3 expression in BALL-1 and Reh cells. Furthermore, we reported that GPSM1 regulated JNK expression via ADCY6-RAPGEF3. The present study demonstrates that GPSM1 promotes tumor growth in BALL-1 and Reh cells by modulating ADCY6-RAPGEF3-JNK signaling.

Learning Pathway Dynamics from Single-Cell Proteomic Data: A Comparative Study.

Single‐cell platforms provide statistically large samples of snapshot observations capable of resolving intrercellular heterogeneity. Currently, there is a growing literature on algorithms that exploit this attribute in order to infer the trajectory of biological mechanisms, such as cell proliferation and differentiation. Despite the efforts, the trajectory inference methodology has not yet been used for addressing the challenging problem of learning the dynamics of protein signaling systems. In this work, we assess this prospect by testing the performance of this class of algorithms on four proteomic temporal datasets. To evaluate the learning quality, we design new general‐purpose evaluation metrics that are able to quantify performance on (i) the biological meaning of the output, (ii) the consistency of the inferred trajectory, (iii) the algorithm robustness, (iv) the correlation of the learning output with the initial dataset, and (v) the roughness of the cell parameter levels though the inferred trajectory. We show that experimental time alone is insufficient to provide knowledge about the order of proteins during signal transduction. Accordingly, we show that the inferred trajectories provide richer information about the underlying dynamics. We learn that established methods tested on high‐dimensional data with small sample size, slow dynamics, and complex structures (e.g. bifurcations) cannot always work in the signaling setting. Among the methods we evaluate, Scorpius and a newly introduced approach that combines Diffusion Maps and Principal Curves were found to perform adequately in recovering the progression of signal transduction although their performance on some metrics varies from one dataset to another. The novel metrics we devise highlight that it is difficult to conclude, which one method is universally applicable for the task. Arguably, there are still many challenges and open problems to resolve. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Identification of the growth factor–binding sequence in the extracellular matrix protein MAGP-1

Microfibril-associated glycoprotein-1 (MAGP-1) is a component of vertebrate extracellular matrix (ECM) microfibrils that, together with the fibrillins, contributes to microfibril function. Many of the phenotypes associated with MAGP-1 gene inactivation are consistent with dysregulation of the transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling system. We have previously shown that full-length MAGP-1 binds active TGFβ-1 and some BMPs. The work presented here further defines the growth factor-binding domain of MAGP-1. Using recombinant domains and synthetic peptides, along with surface plasmon resonance analysis to measure the kinetics of the MAGP-1-TGFβ-1 interaction, we localized the TGFβ- and BMP-binding site in MAGP-1 to a 19-amino acid-long, highly acidic sequence near the N terminus. This domain was specific for binding active, but not latent, TGFβ-1. Growth factor activity experiments revealed that TGFβ-1 retains signaling activity when complexed with MAGP-1. Furthermore, when bound to fibrillin, MAGP-1 retained the ability to interact with TGFβ-1, and active TGFβ-1 did not bind fibrillin in the absence of MAGP-1. The absence of MAGP was sufficient to raise the amount of total TGFβ stored in the ECM of cultured cells, suggesting that the MAGPs compete with the TGFβ large latent complex for binding to microfibrils. Together, these results indicate that MAGP-1 plays an active role in TGFβ signaling in the ECM.

Multi-functionality of proteins involved in GPCR and G protein signaling: making sense of structure-function continuum with intrinsic disorder-based proteoforms.

GPCR-G protein signaling system recognizes a multitude of extracellular ligands and triggers a variety of intracellular signaling cascades in response. In humans, this system includes more than 800 various GPCRs and a large set of heterotrimeric G proteins. Complexity of this system goes far beyond a multitude of pair-wise ligand-GPCR and GPCR-G protein interactions. In fact, one GPCR can recognize more than one extracellular signal and interact with more than one G protein. Furthermore, one ligand can activate more than one GPCR, and multiple GPCRs can couple to the same G protein. This defines an intricate multifunctionality of this important signaling system. Here, we show that the multifunctionality of GPCR-G protein system represents an illustrative example of the protein structure-function continuum, where structures of the involved proteins represent a complex mosaic of differently folded regions (foldons, non-foldons, unfoldons, semi-foldons, and inducible foldons). The functionality of resulting highly dynamic conformational ensembles is fine-tuned by various post-translational modifications and alternative splicing, and such ensembles can undergo dramatic changes at interaction with their specific partners. In other words, GPCRs and G proteins exist as sets of conformational/basic, inducible/modified, and functioning proteoforms characterized by a broad spectrum of structural features and possessing various functional potentials.

Negative Feedback Phosphorylation of Gγ Subunit Ste18 and the Ste5 Scaffold Synergistically Regulates MAPK Activation in Yeast

SUMMARYHeterotrimeric G proteins (Gαβγ) are essential transducers in G protein signaling systems in all eukaryotes. In yeast, G protein signaling differentially activates mitogen-activated protein kinases (MAPKs)—Fus3 and Kss1—a phenomenon controlled by plasma membrane (PM) association of the scaffold protein Ste5. Here, we show that phosphorylation of the yeast Gγ subunit (Ste18), together with Fus3 docking on Ste5, controls the rate and stability of Ste5/PM association. Disruption of either element alone by point mutation has mild but reciprocal effects on MAPK activation. Disabling both elements results in ultra-fast and stable bulk Ste5/PM localization and Fus3 activation that is 6 times faster and 4 times more amplified compared to wild-type cells. These results further resolve the mechanism by which MAPK negative feedback phosphorylation controls pathway activation and provides compelling evidence that Gγ subunits can serve as intrinsic regulators of G protein signaling.

Combinatorial Phosphorylation-Dependent Synergy Between a G Protein Subunit and the Ste5 Scaffold Regulates Differential MAP-Kinase Activation in Yeast

Heterotrimeric G proteins (Gαβγ) play a critical role as transducers in G protein signaling systems responsible for activating a wide range of physiological processes including neurotransmitter and hormone signaling. In yeast, heterotrimeric G protein signaling results in the distinctly different activation of two mitogen-activated protein kinases (MAPKs) - Fus3 and Kss1 - a phenomenon that requires conformational switching of the scaffold protein Ste5. Here we show that rapid receptor-activated phosphorylation of the intrinsically disordered N-terminal tail of the yeast Gγ subunit (Ste18-Nt) has co-evolved to synergize with MAPK binding on Ste5 to control the kinetics and amplitude of Fus3 and Kss1 signaling. By modulating this synergistic component genetically, we show that the activation kinetics and amplitude of either kinase can be controlled - leading to preferential hyperactivation of Fus3 or Kss1. These results further resolve the mechanism by which scaffolds control pathway activation and provide compelling evidence that the intrinsically disordered N-terminal tails of Gγ subunits can serve as regulators of G protein signaling.

The G Protein Gamma Subunit, Ste18, is a Phosphorylation‐Dependent pH Sensor that Controls MAPK Activation in Yeast

As essential transducers of extracellular stimuli such as hormones and neurotransmitters, G protein signaling (GPS) systems – comprised of seven transmembrane receptors (GPCRs), heterotrimeric G‐proteins (Gαβγ), and regulators of G‐protein signaling (RGS) proteins – are important regulators of cell/environment homeostasis in all eukaryotes. Yeast harbor a single canonical GPS system that regulates a pheromone‐dependent mating response, which has long served as a tool for discovering regulators fundamental for all eukaryotic GPS systems. In wild type yeast, the activation rate and amplitude of pheromone signaling is severely dampened by negative feedback phosphorylation of the canonical Gγ subunit, Ste18, and the MAPK scaffold, Ste5. Here, we show that Ste18 is a pH sensor that undergoes combinatorial phosphorylation/dephosphorylation in response to fluctuations in intracellular pH (pHi). Using a site‐specific phosphorylation‐dependent electrophoretic mobility assay and a pHi probe (pHlourin), we show that Ste18S3 is rapidly phosphorylated in response to intracellular acidification, while Ste18S7 is rapidly phosphorylated in response to mating pheromone. When pHi is lowered, basal phosphorylation on Ste18S7 decreases concomitantly with increasing phosphorylation on Ste18S3. In contrast, pheromone stimulation activates phosphorylation on Ste18S7, but not Ste18S3. We show that phosphomimic mutations on either phosphosite function to prohibit phosphorylation of the other non‐mutated site, demonstrating that phosphorylation is mutually exclusive. Furthermore, phosphorylation at either site, mimicked by glutamate mutations, is sufficient to inhibit ultra‐fast and intense output from the pheromone pathway. Thus, our findings suggest that Ste18Nt acts as a phosphorylation‐dependent pH‐sensor that is capable of restricting pheromone pathway output under acid stress conditions when yeast mating is sub‐optimal. Considering that dysregulation of pHi is linked to chronic and life‐threatening diseases such as cancer, stroke, and Alzheimer's disease, our data reveal the possibility that Gγ subunits serve as pH sensors important for regulating G protein signaling in such diseases.Support or Funding InformationNational Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphorylation of G protein γ subunit Ste18 and the Ste5 scaffold form a braking system that governs pathway activation kinetics and switch‐like signaling in yeast

Heterotrimeric G proteins (Gαβγ) play a critical role as transducers in G protein signaling systems responsible for activating a wide range of physiological processes including neurotransmitter and hormone signaling. In yeast, G protein signaling differentially activates two distinct mitogen‐activated protein kinases (MAPKs) – Fus3 and Kss1 – a phenomenon that requires conformational switching of the scaffold protein Ste5. Here we show that rapid receptor‐activated phosphorylation of the intrinsically disordered N‐terminal tail of the yeast Gγ subunit (Ste18), in combination with MAPK phosphorylation of Ste5, serves as a brake system that controls the rate and stability of scaffold association at the plasma membrane. Disabling the brake by precise point mutation of Ste18 phosphorylation sites or a MAPK docking site on Ste5 differentially governs the kinetics and amplitude of MAPK activation as well as the switch‐like morphological response to the stimulus. Complete elimination of the brake results in a response that is 5 times faster and ~4 times amplified over the response in wild type cells. Finally, we show that phosphorylation of Ste18 appeared late in evolutionary history ‐ at the same time as the MAPK docking site on Ste5, suggesting the possibility that both sides of the brake co‐evolved. Since phosphorylated intrinsically disordered tails are structurally conserved elements found in all Gγ subunits, including mammals, these results provide compelling evidence that such tails may function as intrinsic regulators of G protein signaling by altering the interactions of Gβγ subunits with their binding partners.Support or Funding InformationThis work was supported by funding from NIH grant GM117400 to M.T.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Simulation of the effects of moderate stimulation/inhibition of the β1-adrenergic signaling system and its components in mouse ventricular myocytes.

The β1-adrenergic signaling system is one of the most important protein signaling systems in cardiac cells. It regulates cardiac action potential duration, intracellular Ca(2+) concentration ([Ca(2+)]i) transients, and contraction force. In this paper, a comprehensive experimentally based mathematical model of the β1-adrenergic signaling system for mouse ventricular myocytes is explored to simulate the effects of moderate stimulations of β1-adrenergic receptors (β1-ARs) on the action potential, Ca(2+) and Na(+) dynamics, as well as the effects of inhibition of protein kinase A (PKA) and phosphodiesterase of type 4 (PDE4). Simulation results show that the action potential prolongations reach saturating values at relatively small concentrations of isoproterenol (∼0.01 μM), while the [Ca(2+)]i transient amplitude saturates at significantly larger concentrations (∼0.1-1.0 μM). The differences in the response of Ca(2+) and Na(+) fluxes to moderate stimulation of β1-ARs are also observed. Sensitivity analysis of the mathematical model is performed and the model limitations are discussed. The investigated model reproduces most of the experimentally observed effects of moderate stimulation of β1-ARs, PKA, and PDE4 inhibition on the L-type Ca(2+) current, [Ca(2+)]i transients, and the sarcoplasmic reticulum Ca(2+) load and makes testable predictions for the action potential duration and [Ca(2+)]i transients as functions of isoproterenol concentration.

Functions of thga1 Gene in Trichoderma harzianum Based on Transcriptome Analysis.

Trichoderma spp. are important biocontrol filamentous fungi, which are widely used for their adaptability, broad antimicrobial spectrum, and various antagonistic mechanisms. In our previous studies, we cloned thga1 gene encoding GαI protein from Trichoderma harzianum Th-33. Its knockout mutant showed that the growth rate, conidial yield, cAMP level, antagonistic action, and hydrophobicity decreased. Therefore, Illumina RNA-seq technology (RNA-seq) was used to determine transcriptomic differences between the wild-type strain and thga1 mutant. A total of 888 genes were identified as differentially expressed genes (DEGs), including 427 upregulated and 461 downregulated genes. All DEGs were assigned to KEGG pathway databases, and 318 genes were annotated in 184 individual pathways. KEGG analysis revealed that these unigenes were significantly enriched in metabolism and degradation pathways. GO analysis suggested that the majority of DEGs were associated with catalytic activities and metabolism processes that encode carbohydrate-active enzymes, secondary metabolites, secreted proteins, or transcription factors. According to the functional annotation of these DEGs by KOG, the most abundant group was “secondary metabolite biosynthesis, transport, and catabolism.” Further studies for functional characterization of candidate genes and pathways reported in this paper are necessary to further define the G protein signaling system in T. harzianum.

Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

Meta‐Proteomic Analysis of PTMs in G protein Signaling Systems – New Tools and Unsuspected Discoveries

The collective structural and functional data for prototypical G protein signaling (GPS) systems – defined by 7‐transmembrane receptors (GPCRs), heterotrimeric G proteins (Gαβγ) and Regulators of G‐protein Signaling (RGS) – is arguably the most comprehensive of its kind to date. Comparatively little is known about how G protein structure and function is altered by post‐translational modifications (PTMs). Here we report the first structurally‐resolved, meta‐proteomic map of all empirically validated PTMs for GPS proteins. Using a novel PTM analysis tool called Structural Projection of PTMs (SPoP), a total of 1,910 unique eukaryotic PTMs were coalesced onto available structures within the Protein Data Bank. The resulting dataset has enabled the first‐ever visualization of the PTM topology for each member of a prototypical GPS system. By factoring total PTM observations, protein structure, protein interactions and sequence conservation, we estimated a function potential value for each PTM. Rank order comparison of calculated function potentials with literature‐based evidence of function reveals that SPoP has predictive power. Based on these results, we have identified major PTM hotspots in GPS proteins; predicted and tested the involvement of previously unsuspected PTM regulatory elements; and revealed surprising differences between the PTM topologies of large and small guanine‐nucleotide binding proteins. We propose that meta‐proteomic and structural PTM analysis with SPoP can lead to the discovery of functionally important modification sites that regulate biomedically‐relevant classes of signaling molecules.

Increased expression of AGS3 in rat brain cortex after traumatic brain injury

Receptor-independent activators of G protein signaling (AGS) offer alternative modes of signal processing for the G protein signaling system that has broad mechanistic and functional significance. Previous studies have demonstrated that AGS3, which belongs to the AGS family, is involved in a number of different cellular activities. However, the distribution and function of AGS3 in the central nervous system (CNS) remain unclear. To investigate whether AGS3 is involved in CNS injury and repair, we used an acute traumatic brain injury (TBI) model in adult rats. Western blot analysis and immunohistochemistry showed a significant upregulation of AGS3 in ipsilateral peritrauma cortex. Double immunofluorescence staining showed that AGS3 was coexpressed with NeuN but rarely with glial fibrillary acidic protein. In addition, we detected that active caspase-3 had colocalization with NeuN and AGS3, suggesting that AGS3 might be involved in the neuron apoptosis after TBI. To investigate the potential function of AGS3 further, a neuronal cell line, PC12, was employed to establish a cell apoptosis model. Western blot analysis indicated that AGS3 shared a similar dynamic variation in animal experiments, and phosphorylated cyclic AMP response element-binding protein (CREB) increased in parallel. Additionally, knocking down AGS3 with siRNA partially attenuated the protein level of phosphorylated CREB in PC12 stimulated by H2 O2 , while reinforcing active caspase-3 expression, demonstrating a probable antiapoptotic role through CREB played by AGS3 in neuronal apoptosis. We hypothesize that AGS3 might play an important antiapoptotic role through enhancing phosphorylation of CREB. © 2013 Wiley Periodicals, Inc.

Ric-8 regulation of heterotrimeric G proteins

Resistance to inhibitors of cholinesterase 8 proteins (Ric-8A and Ric-8B) collectively bind the four classes of heterotrimeric G protein α subunits. Ric-8A and Ric-8B act as non-receptor guanine nucleotide exchange factors (GEFs) toward the Gα subunits that each binds in vitro and seemingly regulate diverse G protein signaling systems in cells. Combined evidence from worm, fly and mammalian systems has shown that Ric-8 proteins are required to maintain proper cellular abundances of G proteins. Ric-8 proteins support G protein levels by serving as molecular chaperones that promote Gα subunit biosynthesis. In this review, the evidence that Ric-8 proteins act as non-receptor GEF activators of G proteins in signal transduction contexts will be weighed against the evidence supporting the molecular chaperoning function of Ric-8 in promoting G protein abundance. I will conclude by suggesting that Ric-8 proteins may act in either capacity in specific contexts. The field awaits additional experimentation to delineate the putative multi-functionality of Ric-8 towards G proteins in cells.

Phospholipase C Beta reduces the Binding and Cleavage of Oligonucleotides by Component 3 Promoter of RISC

Phospholipase C beta1 (PLCβ1), a key component in the classical pathway of G protein signaling system, relays information from G-proteins to downstream effectors upon stimulation of G-protein coupled receptors. Recently we found a novel role for PLCβ1 in gene regulation through TRAX, a protein implicated in RNA interference pathway. TRAX in complex with its protein partner translin, a DNA and RNA binding protein, has nuclease activity. We found that in cells, PLCβ1 selectively reverses the siRNA-mediated down-regulation of certain housekeeping genes such as GAPDH and LDH, but not of Cyclophilin A or Hsp90. Over-expression of TRAX inhibited the PLCβ1-mediated rescue of down-regulation of GAPDH and LDH. Our data in HEK and Hela cells indicate that PLCβ1 might play a role in TRAX-mediated RNA-induced silencing complex (RISC) activities of metabolic proteins. We have carried out in-vitro studies to characterize the biochemical properties of TRAX and translin, and the effect of PLCβ1 on binding and rate of hydrolysis of oligonucleotides by TRAX and TRAX-translin octameric complex, Component 3 promoter of RISC (C3PO). From acrylamide as well as agarose native gel electrophoresis and anisotropy measurements, we observed that translin, TRAX and C3PO exist in multiple oligomeric states that bind DNA and RNA. We were able to determine the cleavage rates for ssRNA by TRAX, translin and C3PO. We found that PLCβ1 reduces the rate of hydrolysis by C3PO through decreasing the affinity of a weak binding site of RNA on C3PO complex. The effect of PLCβ1 is seen at higher concentration of C3PO suggesting a role for PLCβ1 at localized elevated levels of C3PO, as in RISC.

Regulation of K Channels by the G Protein Signaling System

Over 25 years ago G protein inwardly rectifying K+ (GIRK) channels were the first effector shown to be activated by the βγ subunits of G proteins (Gβγ). Perfusion of atrial inside-out patches with purified Gβγ subunits stimulated channel activity even when the cells had been treated with pertussis toxin that functionally uncouples endogenous G proteins from receptor signaling. Furthermore, the lack of stimulation of channel activity by the Gα subunits led to the proposal that Gβγ independently activates GIRK channels (Logothetis et al., 1987). A decade later it was appreciated that GIRK channels required the presence of phosphoinositides, such as PIP2, in order to be gated by Gβγ subunits or other gating molecules such as intracellular Na+ or alcohols (Sui et al., 1998). In the following decade through the present times, crystal structures of the GIRK channels have been elucidated revealing the architecture of full-length GIRK channels in the absence or presence of PIP2 (Nishida et al., 1997; Whorton et al., 2011). Although attempts to obtain structures of the channels in complex with Gβγ have not succeeded thus far, insights on the sites of Gβγ and PIP2 action leading to channel gating are beginning to emerge. Seeking to provide a more rigorous test of the hypothesis that Gβγ stimulates GIRK channel activity independently of the remaining components of the system, we have successfully reconstituted the entire G protein signaling complex with the channel into planar lipid bilayers. To our surprise the signaling complex functioned equivalently in the bilayer as it did in excised patches only when the entire complex was reconstituted (i.e. receptor, heterotrimeric G proteins, and GIRK channel). The results obtained in the bilayer system put in question the hypothesis that Gβγ stimulates channel activity independently of the other components of the signaling complex.

Insights into immune structure, recognition, and signaling

In this volume of Immunological Reviews, the articles focus on the structural biology of immunoreceptors and what structure is telling us about function. The issue is unabashedly ‘receptor-centric’ and the proteins covered range from antigen receptors, innate receptors, and cytokine receptors, thereby providing an unusually comprehensive snapshot of the current state of the art in molecular immunology. Of all the major fields in biomedical research, including neurobiology, developmental biology, immunology, microbiology, and genetics, just to name a subset, perhaps structure in the broadest sense has had more of an impact on immunology and played more of a major role in guiding the evolution and maturation of the field than any other. Here, I use the term ‘structure’ to encompass not only direct structural approaches, such as x-ray crystallography and nuclear magnetic resonance spectroscopy (NMR), but also imaging, biophysical, molecular, and mechanistic approaches to interrogate aspects of molecular recognition, oligomerization, and signaling. Perhaps the fact that immunobiology is centered on questions of recognition and discrimination and is largely controlled by receptor-ligand systems has led to an unusually high level of sophistication about structure among immunologists. Personally speaking, having delved into structural questions in neurobiology and developmental biology, I can state without equivocation that the structural and molecular savvy of immunologists has led to greater mechanistic clarity underlying central tenets of the field compared to other disciplines. Dating back to the days of elucidating the first antibody structures and the relation between hypervariable regions and antigen recognition, to the transformative clarity of the first major histocompatibility complex (MHC) structures in explaining MHC restriction, to more recent biophysical and structural investigations of T-cell receptor (TCR) recognition of both classical and non-classical MHC, innate immune receptors such as natural killer (NK) and Toll-like receptors, and cytokine receptors, molecular science has illuminated the path for the experimentalists and assumed a large and highly visible role in immunobiology as a whole.

Whole genome expression profile in neuroblastoma cells exposed to 1-methyl-4-phenylpyridine

Mitochondrial dysfunction and subsequent energy failure is a contributing factor to degeneration of the substantia nigra pars compacta associated with Parkinson's disease (PD). In this study, we investigate molecular events triggered by cell exposure to the mitochondrial toxin 1-methyl-4-phenylpyridine (MPP+) using whole genome-expression microarray, Western Blot and metabolic studies. The data show that MPP+ (500μM) obstructs mitochondrial respiration/oxidative phosphorylation (OXPHOS) in mouse neuroblastoma Neuro-2a cells, juxtaposing accelerated glucose consumption and production of lactic acid. While additional glucose concentrations restored viability in the presence of MPP+ (500μM), the loss of OXPHOS was sustained, suggesting that compensatory anaerobic metabolic systems were fulfilling required energy needs. Under these conditions, MPP+ initiated significant changes to the transcription of 439 genes of which 287 DAVID IDs were identified and subsequent functional annotation clusters identified. Prominent changes were as follows; MPP+ initiated loss of mRNA for mitochondrial encoded 3-hydroxybutyratedehydrogenase, type2(Bdh2), tv1, NADH dehydrogenase 4,5 genes, cytochrome b and NADH dehydrogenase (ubiquinone) flavoprotein 3, concomitant to rise in a mitochondrial fission gene; ganglioside-induced differentiation-associated-protein 1 (GDAP1). The negative changes to OXPHOS components were accompanied by protective forces within the mitochondria espousing elevated ratio of anti/pro-apoptotic processes. These included a loss of apoptotic Bcl-2/adenovirus E1B 19-kDa-interacting protein (BNIP3) and family with sequence similarity 162, member A (FAM162a) and rise of heat shock protein 1 and Lon peptidase 1. There were no changes indicative of free radical damage (e.g. SOD, GSH-Px), rather MPP+ initiated significant elevation in G protein signaling components (which trigger catabolic processes) and anaerobic metabolic systems involving carboxylic acid/transamination reactions (e.g. glutamate oxaloacetate transaminase 1 (GOT1), glutamic pyruvate-alanine transaminase 2 (GPT2), cystathionase and redox proteins such as cytochrome b5 reductase 1 and ferredoxin reductase. Counter-intuitively, the data show reduction of mRNA in glycolytic processes [DAVID enrichment score 9.96 p value 1.90E−19], some corroborated by Western Blot, bringing in to question the sources of lactate observed in the presence of MPP+. Examining this aspect, the data show that diverse carboxylic acids (succinate, oxaloacetate and a-ketoglutarate) are capable of contributing to the lactate pool in addition to phosph(enolpyruvate) or pyruvate in the absence of glucose by this cell line. In conclusion, these findings show that MPP+ negatively affects the transcriptome involved with complex I, but initiated an elevation of G protein signaling and anaerobic metabolic systems involved with nitrogen/carboxylic acid metabolism. Future research will be required to elucidate the survival pathways that drive anaerobic substrate level phosphorylation, and define functional ramification to the loss of mitochondrial FAM162a and BNIP3 proteins.