Truncation Constructs Research Articles

Overexpression of Diacylglycerol KinaseηEnhances Gαq-Coupled G Protein–Coupled Receptor Signaling

Multiple genome-wide association studies have linked diacylglycerol kinase η (DGKη) to bipolar disorder (BPD). Moreover, DGKη expression is increased in tissue from patients with BPD. How increased levels of this lipid kinase might affect cellular functions is currently unclear. Here, we overexpressed mouse DGKη in human embryonic kidney 293 cells to examine substrate specificity and signaling downstream of endogenous G protein-coupled receptors (GPCRs). We found that DGKη can phosphorylate diacylglycerol (DAG) with different acyl side chains (8:0, 12:0, 18:1). In addition, overexpression of DGKη enhanced calcium mobilization after stimulating muscarinic receptors with carbachol and after stimulating purinergic receptors with ATP. This effect required DGKη catalytic activity, as assessed using a kinase-dead (G389D) mutant and multiple truncation constructs. DGKη was localized throughout the cytosol and did not translocate to the plasma membrane after stimulation with carbachol. Since protein kinase C (PKC) can be activated by DAG and promotes receptor desensitization, we also examined functional interactions between PKC and DGKη. We found that acute activation of PKC with phorbol 12-myristate 13-acetate shortened carbachol-evoked calcium responses and occluded the effect of overexpressed DGKη. Moreover, inhibition of PKC activity with bisindolylmaleimide I (BIM I) produced the same enhancing effect on carbachol-evoked calcium mobilization as overexpressed DGKη, and overexpression of DGKη produced no additional effect on calcium mobilization in the presence of BIM I. Taken together, our data suggest that DGKη enhances GPCR signaling by reducing PKC activation.

Neto1 associates with the NMDA receptor/amyloid precursor protein complex

Neuropilin tolloid-like 1 (Neto1), is a CUB domain-containing transmembrane protein that was recently identified as a novel component of the NMDA receptor complex. Here, we have investigated the possible association of Neto1 with the amyloid precursor protein (APP)695/GluN1/GluN2A and APP695/GluN1/GluN2B NMDA receptor trafficking complexes that we have previously identified. Neto1(HA) was shown to co-immunoprecipitate with assembled NMDA receptors via GluN2A or GluN2B subunits; Neto1(HA) did not co-immunoprecipitate APP695(FLAG) . Co-immunoprecipitations from mammalian cells co-transfected with APP695(FLAG) , Neto1(HA) and GluN1/GluN2A or GluN1/GluN2B revealed that all four proteins co-exist within one macromolecular complex. Immunoprecipitations from native brain tissue similarly revealed the existence of a GluN1/GluN2A or GluN2B/APP/Neto1 complex. Neto1(HA) caused a reduction in the surface expression of both NMDA receptor subtypes, but had no effect on APP695(FLAG) - or PSD-95α(c-Myc) enhanced surface receptor expression. The Neto1 binding domain of GluN2A was mapped using GluN1/GluN2A chimeras and GluN2A truncation constructs. The extracellular GluN2A domain does not contribute to association with Neto1(HA) but deletion of the intracellular tail resulted in a loss of Neto-1(HA) co-immunoprecipitation which was paralleled by a loss of association between GluN2A and SAP102. Thus, Neto1 is concluded to be a component of APP/NMDA receptor trafficking complexes.

Functional analysis of PEX13 mutation in a Zellweger syndrome spectrum patient reveals novel homooligomerization of PEX13 and its role in human peroxisome biogenesis

In humans, the concerted action of at least 13 different peroxisomal PEX proteins is needed for proper peroxisome biogenesis. Mutations in any of these PEX genes can lead to lethal neurometabolic disorders of the Zellweger syndrome spectrum (ZSS). Previously, we identified the W313G mutation located within the SH3 domain of the peroxisomal protein, PEX13. As this tryptophan residue is highly conserved in almost all known SH3 proteins, we investigated the pathogenic mechanism of the W313G mutation and its role in PEX13 interactions and functions in peroxisome biogenesis. Here, we report for the first time that human PEX13 interacts with itself in peroxisomes in living cells. We demonstrate that the import of PTS1 (peroxisomal targeting signal 1) proteins is specifically disrupted when homooligomerization of PEX13 is interrupted. Live cell FRET microscopy in living cells as well as co-immunoprecipitation experiments reveal that the highly conserved W313 residue is important for self-association of PEX13 but is not required for interaction with PEX14, a well-established interaction partner at the peroxisomal membrane. Experiments with truncated constructs indicate that although the W313G mutation resides in the C-terminal SH3 domain, the N-terminal half is necessary for peroxisomal localization, which in turn appears to be crucial for homooligomerization. Furthermore, rescue of homooligomerization in the W313G mutant cells through complementation with truncation constructs restores import of peroxisomal matrix proteins. Taken together, the thorough analyses of a ZSS patient mutation unraveled the general cell biological function of PEX13 and its mechanism in the import of peroxisomal matrix PTS1 proteins.

The tetraspanin network modulates MT1-MMP cell surface trafficking

The membrane-type 1 matrix metalloproteinase (MT1-MMP) drives fundamental physiological and pathophysiological processes. Among other substrates, MT1-MMP cleaves components of the extracellular matrix and activates other matrix-cleaving proteases such as MMP-2. Trafficking is a highly effective means to modulate MT1-MMP cell surface expression, and hence regulate its function. Here, we describe the complex interaction of MT1-MMP with tetraspanins, their effects on MT1-MMP intracellular trafficking and proteolytic function. Tetraspanins are credited as membrane organizers that form a network within the membrane to regulate the trafficking of associated proteins. In short, we found MT1-MMP to interact with the tetraspanin-associated EWI-2 protein by a yeast two-hybrid screen. Immunoprecipitation analysis confirmed this interaction and further revealed that MT1-MMP also stably interacts with distinct tetraspanins (CD9, CD37, CD53, CD63, CD81, and CD82) and the tetraspanin-like MAL protein. By using different MT1-MMP truncation constructs and mutants, we observed that all tetraspanins and MAL associated with the hemopexin domain of MT1-MMP. Moreover, this interaction was independent of O-glycosylation of MT1-MMP and exclusively occurred in the endoplasmic reticulum. Here, the respective subcellular compartment was identified by fitting the MT1-MMP interaction pattern to a model for post-translational processing of MT1-MMP. In addition, tetraspanins differentially affected the cell surface localization of MT1-MMP, its capacity to activate pro-MMP-2, and the collagen invasion capacity. Interestingly, the degree of tetraspanin-MT1-MMP association did not correlate with its impact on MT1-MMP function. Tetraspanins thus distinctly affect MT1-MMP subcellular localization and function, and may constitute an effective mechanism to control MT1-MMP-dependent proteolysis at the cell surface.

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

Characterization of the Intramolecular Interactions in the Circularly Permuted GTPase Nucleostemin

Nucleostemin is a unique protein that plays a role in cell cycle regulation, cellular stress sensing, telomere maintenance, and tumor suppression. Similar to other nucleolar proteins, compartmentalization plays a large role in regulating the activity of NS. The mechanism behind this redistribution is poorly defined but it is linked to the GTPase properties of the protein and an uncharacterized inhibitory region that directs complex formation between NS and various cellular partners. We have been studying the structural and biochemical properties of drosophilia NS1 to better understand the molecular basis of this dynamic localization process. The canonical GTPase core contains a six-stranded β sheet surrounded by 5 α helices. The nucleotide-binding site of the protein is defined by four conserved amino acid sequence motifs, the G-1 through G-4 boxes. cpGTPases are characterized by a reordering of the characteristic G boxes specifically to a G4-G1-G3 pattern, thus a circular permutation of the classic GTPase arrangement. There is little known about how altering the positioning of the G-boxes affects the nucleotide binding and regulatory properties of the protein. Work from our group and other laboratories demonstrated that cpGTPases have broad substrate specificity and can hydrolyze GTP and ATP. We have also identified several structural domains in dNS1 using limited proteolysis. Truncation constructs have been made to elucidate the role of each domain and how the physical interactions between these domains contribute to the biological properties of the protein. Taken together, these data point to a mechanism of action that differs from the larger GTPase family.

Sequence Specific pausing of RecQ Helicase

Escherichia coli RecQ helicase is a Super-Family 2 helicase that plays an essential role in the maintenance of genome stability via its participation in the repair and homologous recombination of DNA. The strand separation (unwinding) mechanism of RecQ has not been well characterized to date. Here, we report the study of RecQ unwinding using magnetic tweezers and focus on understanding the sequence specific pausing behavior observed in both dsDNA unwinding and ssDNA translocation. We compare the unwinding and pausing behavior of RecQ on different DNA sequences and in different pulling and unwinding geometries, which allow us to examine how the key unwinding properties (i.e., unwinding rate, processivity, pause location, pause duration, and their distributions) depend on DNA sequence, DNA geometry, and applied tension on the DNA. DNA unwinding by wild-type RecQ helicase is interrupted by strong sequence-dependent pauses. Pausing is significantly reduced for thrombin-cleaved RecQ and truncation constructs lacking the HRDC domain, and for point mutants that disrupt the single-stranded binding affinity of the HRDC domain. We propose a model for the sequence dependent pausing by RecQ and speculate on the in vivo ramifications of this behavior.

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

BackgroundThe envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.Methodology/Principal FindingsIn this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.Conclusions/SignificanceA C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.

PI3Kβ downstream of GPCRs - crucial partners in oncogenesis

Of the class IA PI3Ks, p110β is the only member to simultaneously signal downstream of both Receptor Tyrosine Kinases (RTKs) and G Protein-Coupled Receptors (GPCRs). While the mechanism of RTK-mediated activation of p85/p110 heterodimers has been characterized [1, 2], defining the mechanism of Gβγ-mediated activation of p110β downstream of GPCRs has been challenging due to the transient nature of the interaction. The binding of p110β to Gβγ is weak, but is reinforced by interactions with lipid membranes, where Gβγ resides. A combination of sequence analysis and hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) experiments has enabled us to map the regions of interaction on both p110β and Gβγ [3]. Furthermore, HDX-MS data analysis provided a unique insight into the mechanism of p110β activation by Gβγ, showing that p110β kinase domain makes stronger interaction with membranes in the presence of Gβγ. The regulatory p85 subunit inhibits basal activity of p110β, largely by the inhibitory contacts that the nSH2 and the cSH2 domains of p85 make with the p110β subunit [1, 2]. The nSH2 contacts the helical domain of p110β close to the residues essential for Gβγ binding. Whether the nSH2 has to disengage from p110β to allow Gβγ binding is still uncertain. In vitro, p110β/p85 truncation constructs lacking either the nSH2 or both SH2 domains are still activated by Gγ. In contrast, activation of p110β/p85 constructs by tyrosyl phosphorylated phosphopeptides require the presence of at least one p85 SH2 domain. Thus, Gβγ and phosphopeptides activate the enzyme via distinct mechanisms, resulting in synergistic activation when both activators are present. Whether there is an allosteric component of activation in addition to membrane recruitment mediated by Gβγ remains an open question. Based on the current results and on the structure of a membrane interacting heterotrimeric G-protein, we can begin to model how the activated p110β would interact with membranes. In this model, Gβγ makes no direct contact with the kinase domain of p110β, suggesting a mechanism where Gβγ stimulates p110β activity by increasing its membrane residence time. A similar mechanism has been proposed for the other Gβγ-sensitive PI3K, p110γ [4]. The crystal structure of p110β in a complex with Gβγ will answer many remaining questions. Wild-type p110β is transforming when over-expressed in fibroblasts [5], and it is the major p110 isoform required for driving PTEN−/− tumors [6]. However, it has not been previously possible to determine whether GPCR activation of p110β/p85 was required in these cases. Using a mutant p110β or a cell-permeable peptide, both of which block GPCR- mediated but not RTK-mediated activation of p110β/p85, we showed that GPCR inputs to p110β/p85 are required for transformation, proliferation, chemotaxis, and invasion driven by either p110β over-expression or stimulation with GPCR ligands [3]. Furthermore, a requirement for GPCR activation of p110β/p85 was seen in the growth of PTEN−/− cell lines, but not PTEN+/+ cells. Surprisingly, the peptide inhibitor of p110β/Gβγ binding blocked the growth of PTEN−/− cells, whereas the p110β-specific kinase inhibitor, TGX-221, did not. This suggests a role for a Gβγ-mediated scaffolding function of p110β in proliferation. This is consistent with previous studies showing that some functions of p110β are kinase independent [7, 8]. Our work suggests that in some tumors, inhibitors specifically targeting the Gβγ-p110β interaction might be more potent than inhibitors targeting p110β catalytic activity. Our study also suggests that the identification of the GPCRs that drive PTEN−/− tumors could provide an important alternative therapeutic approach for the treatment of these tumors. Understanding the regulation of p110β catalytic activity, as well as defining its scaffolding functions, will be important in developing drugs that target its functions in human disease.

Phosphorylation of Tyrosine 340 in the Pleckstrin Homology Domain of RIAM Is Required for Inside-Out Activation of LFA-1 and LFA-1: ICAM-1-Mediated Adhesion

Abstract 837Adhesion of lymphocytes to antigen presenting cells (APCs) is a critical event linking innate and adaptive immunity and a mandatory step for the initiation of T cell immune responses. Control of lymphocyte adhesion to APC is accomplished through the regulation of the principle adhesion molecule on the lymphocyte surface, the β2 integrin designated lymphocyte functional antigen 1 (LFA-1), which binds to intercellular adhesion molecule 1 (ICAM-1) on the surface of APCs. In order to mediate its adhesive function LFA-1 must be activated via a process referred to as inside-out signaling, which results in conformation changes of the receptor that extends the ectodomains of the α and β chains leading to a high affinity state. Because LFA-1-mediated adhesion is a central link between innate and adaptive immunity, understanding the mechanisms implicated in inside-out-mediated LFA-1 activation is a subject of intense investigation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate inside-out activation of LFA-1. Among the few signaling molecules that have been implicated in this process in hematopoietic cells are the small GTPase Rap1 and its downstream effector RIAM. RIAM is a multidomain protein that includes a talin binding region, two coiled-coiled regions, a small N-terminus proline-rich region, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, via which interacts with Ena/VASP family proteins and profilin and is recruited to the sites of actin turnover. Through its C-terminus RIAM also interacts with the SH3-domain of PLC-γ1. The RA and PH domains of RIAM function as an integral unit and as a proximity detector since both are required for translocation of RIAM to the plasma membrane. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. We determined that the integral RA-PH structural unit of RIAM has also an integral functional role because both the RA and PH regions are required for LFA-1-mediated adhesion. Here we sought to determine how TCR proximal signaling events regulate RIAM function leading to inside-out activation of LFA-1 and LFA-1:ICAM-1 mediated adhesion. Using primary human T lymphocytes and Jurkat T cells we determined that upon TCR/CD3-mediated stimulation, activated Src family kinases Fyn and Lck associate with and induce tyrosine phosphorylation of RIAM. To identify the precise domain(s) of RIAM, which undergo phosphorylation by these kinases, we co-expressed individual truncation constructs of RIAM N-terminus, RA-PH, and C-terminus regions along with the active or inactive form of Fyn or Lck in COS cells. Immunoprecipitations and immunoblot assays revealed that active Fyn and Lck mediated robust and selective tyrosine phosphorylation of the RA-PH structural unit of RIAM. Detailed analysis by mass spectrometry identified that tyrosine 340 (Y340) within the PH domain of RIAM was the specific target. The PH domain of RIAM has specificity for PI(4,5)P2 and is an integral component of the RA-PH proximity detector. We hypothesized that phosphorylation of tyrosine 340 in the PH domain might have a regulatory role in the function of the RA-PH structural unit in mediating LFA-1 activation. To address this issue we used site directed mutagenesis and we introduced a tyrosine to phenylalanine mutation in site 340 (Y34F) of RIAM, rendering this residue resistant to phosphorylation mediated by Fyn and Lck. Expression of RIAM Y340F in Jurkat T cells abrogated LFA-1 activation and LFA-1-mediated adhesion in response to TCR/CD3 stimulation. Under these conditions, activation of LFA-1 in response to PMA, which bypasses TCR-mediated signaling, remained intact. Thus, besides recruitment of RIAM to the plasma membrane, TCR-mediated activation of Src family kinases and phosphorylation of RIAM PH domain is a mandatory requirement for the function of the RA-PH proximity detector as a regulator of LFA-1 activation. Our results provide a biochemical, molecular and mechanistic link between TCR-mediated signaling and inside-out activation of LFA-1 thereby initiating LFA-1: ICAM-1-mediated adhesion and cross-talk between T cells and APC. Disclosures:No relevant conflicts of interest to declare.

Striatal-enriched Protein-tyrosine Phosphatase (STEP) Regulates Pyk2 Kinase Activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.

Sigma-1 receptor alters the kinetics of Kv1.3 voltage gated potassium channels but not the sensitivity to receptor ligands

Sigma1 receptors (Sigma1R) are intracellular chaperone proteins that bind psychotropic drugs and also clinically used drugs such as ketamine and haloperidol. Co-expression of the Sigma1R has been reported to enhance the sensitivity of several voltage-gated ion channels to Sigma1R ligands. Kv1.3 is the predominant voltage-gated potassium channel expressed in T lymphocytes with a documented role in immune activation. To gain a better understanding of Sigma1R modulation of Kv ion channels, we investigated the effects of Sigma1R co-expression on Kv1.3 physiology and pharmacology in ion channels expressed in Xenopus oocytes. We also explored the protein domains of Kv1.3 necessary for protein:protein interaction between Kv1.3 and Sigma1R through co-immunoprecipitation studies. Slowly inactivating outward-going currents consistent with Kv1.3 expression were elicited on step depolarizations. The current characterized by Erev, V1/2, and slope factor remained unchanged when co-expressed with Sigma1R. Analysis of inactivation time constant revealed a faster Kv1.3 current decay when co-expressed with Sigma1R. However the sensitivity to Sigma1R ligands remained unaltered when co-expressed with the Sigma1R in contrast to the previously reported modulation of ligand sensitivity in closely related Kv1.4 and Kv1.5 voltage gated potassium channels. Co-immunoprecipitation assays of various Kv1.3 truncation constructs indicated that the transmembrane domain of the Kv1.3 protein was responsible for the protein:protein interaction with the Sigma1R. Sigma1R likely interacts with different domains of Kv ion channel family proteins resulting in distinct modulation of different channels.

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

C-Jun and c-Fos regulate the complement factor H promoter in murine astrocytes

The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimer's disease, Parkinson's disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241base pair (bp) region at −416bp to −175bp that showed the strongest activity. Through supershift analysis, it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation.

Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway

SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway.

The Ca2+ Channel β4c Subunit Interacts with Heterochromatin Protein 1 via a PXVXL Binding Motif

The β subunits of voltage-gated Ca(2+) channels are best known for their roles in regulating surface expression and gating of voltage-gated Ca(2+) channel α(1) subunits. Recent evidence, however, indicates that these proteins have a variety of Ca(2+) channel-independent functions. For example, on the molecular level, they regulate gene expression, and on the whole animal level, they regulate early cell movements in zebrafish development. In the present study, an alternatively spliced, truncated β4 subunit (β4c) is identified in the human brain and shown to be highly expressed in nuclei of vestibular neurons. Pull-down assays, nuclear magnetic resonance, and isothermal titration calorimetry demonstrate that the protein interacts with the chromo shadow domain (CSD) of heterochromatin protein 1γ. Site-directed mutagenesis reveals that the primary CSD interaction occurs through a β4c C-terminal PXVXL consensus motif, adding the β4c subunit to a growing PXVXL protein family with epigenetic responsibilities. These proteins have multiple nuclear functions, including transcription regulation (TIF1α) and nucleosome assembly (CAF1). An NMR-based two-site docking model of β4c in complex with dimerized CSD is presented. Possible roles for the interaction are discussed.

The amino acid tryptophan prevents the biosynthesis of dermatan sulfate

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-216 to leucine (W216L) in the chimera S222A increased the amount of l-IdoA to 12-16%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W216L) in HEK293 cells. Co-staining of S222A-HA and S222A(W216L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W216L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W216L). However, the binding of fibroblasts growth factor-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.

Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [3H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing ‘intrinsically disordered region (IDR)’ (∼85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.

Rod/Zw10 Complex Is Required for PIASy-dependent Centromeric SUMOylation

SUMO conjugation of cellular proteins is essential for proper progression of mitosis. PIASy, a SUMO E3 ligase, is required for mitotic SUMOylation of chromosomal proteins, yet the regulatory mechanism behind the PIASy-dependent SUMOylation during mitosis has not been determined. Using a series of truncated PIASy proteins, we have found that the N terminus of PIASy is not required for SUMO modification in vitro but is essential for mitotic SUMOylation in Xenopus egg extracts. We demonstrate that swapping the N terminus of PIASy protein with the corresponding region of other PIAS family members abolishes chromosomal binding and mitotic SUMOylation. We further show that the N-terminal domain of PIASy is sufficient for centromeric localization. We identified that the N-terminal domain of PIASy interacts with the Rod/Zw10 complex, and immunofluorescence further reveals that PIASy colocalizes with Rod/Zw10 in the centromeric region. We show that the Rod/Zw10 complex interacts with the first 47 residues of PIASy which were particularly important for mitotic SUMOylation. Finally, we show that depletion of Rod compromises the centromeric localization of PIASy and SUMO2/3 in mitosis. Together, we demonstrate a fundamental mechanism of PIASy to localize in the centromeric region of chromosome to execute centromeric SUMOylation during mitosis.

Crystallization and preliminary X-ray crystallographic studies of human FAIM protein.

Fas apoptosis inhibitory molecule (FAIM), an antagonist of Fas-induced cell death, is highly conserved and is broadly expressed in many tissues. It has been found that FAIM can stimulate neurite outgrowth in PC12 cells and primary neurons. However, the molecular mechanisms of action of FAIM are not understood in detail. Here, full-length human FAIM and two truncation constructs have successfully been cloned, expressed and purified in Escherichia coli. FAIM (1-90) was crystallized and diffracted to a resolution of 2.5 A; the crystal belonged to space group P3(1), with unit-cell parameters a=b=58.02, c=71.11 A, alpha=beta=90, gamma=120 degrees. There were two molecules in the asymmetric unit.