Amino-terminal Domain Research Articles

Degradation triggers the alarm

Immunology Inflammasomes are multiprotein complexes that orchestrate proinflammatory cytokine secretion and cell death. Proteases such as anthrax lethal factor can activate an inflammasome known as NLRP1B, but the mechanism for this activation has been unclear. Chui et al. used genome-wide knockout screens to show that proteolysis of NLRP1B by lethal factor induces proteasomal degradation of the amino-terminal domains of NLRP1B and eventual cell death. Sandstrom et al. found that degradation of the amino-terminal domains of NLRP1B resulted in the release of a carboxyl-terminal fragment that activates caspase-1. This process, called “functional degradation,” allows the immune system to detect pathogen-associated activities, much as it recognizes pathogen-associated antigens. Science , this issue p. [82][1], p. [eaau1330][2] [1]: /lookup/volpage/364/82?iss=6435 [2]: /lookup/doi/10.1126/science.aau1330

Prokaryotic primases - structure and function

Primases are responsible for the synthesis of a short oligo RNA, which serves as primer for DNA polymerase. Primases play an essential role in the initiation of DNA replication at the origins, in the synthesis of Okazaki fragments and in the restart of stalled replication forks. Prokaryotic primases based on their structure and sequence alignments are classified as a family of DnaG proteins. Primases from this family contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either interacts with a helicase or is itself a DNA helicase. In this review, we are presenting the comparison of the representative primases from bacteria and bacteriophages, their mode of action and their involvement in DNA replication at the replication fork.

The Natural History of Teneurins: A Billion Years of Evolution in Three Key Steps.

The entire evolutionary history of the animal gene family, Teneurin, can be summed up in three key steps, plus three salient footnotes. In a shared ancestor of all bilaterians, the first step began with gene fusions that created a protein with an amino-terminal intracellular domain bridged via a single transmembrane helix to extracellular EGF-like domains. This first step was completed with a further gene fusion: an additional carboxy-terminal stretch of about 2000 amino acids (aa) was adopted, as-a-whole, from bacteria. The 2000 aa structure in Teneurin was recently solved in three dimensions. The 2000 aa region appears in a number of bacteria, yet was co-opted solely into Teneurin, and into no other eukaryotic proteins. Outside of bilaterian animals, no Teneurins exist, with a “Monosiga brevicollis caveat” brought below, as ‘the third footnote”. Subsequent to the “urTeneurin’s” genesis-by-fusions, all bilaterians bore a single Teneurin gene, always encoding an extraordinarily conserved Type II transmembrane protein with invariant domain content and order. The second key step was a duplication that led to an exception to singleton Teneurin genomes. A pair of Teneurin paralogs, Ten-a and Ten-m, are found in representatives all four Arthropod sub-phyla, in: insects, crustaceans, myriapods, and chelicerates. In contrast, in every other protostome species’ genome, including those of all non-Arthropod ecdysozoan phyla, only a single Teneurin gene occurs. The closest, sister, phylum of arthropods, the Onychophorans (velvet worms), bear a singleton Teneurin. Ten-a and Ten-m therefore arose from a duplication in an urArthropod only after Arthropods split from Onychophorans, but before the splits that led to the four Arthropod sub-phyla. The third key step was a quadruplication of Teneurins at the root of vertebrate radiation. Four Teneurin paralogs (Teneurins 1 through 4) arose first by a duplication of a single chordate gene likely leading to one 1/4–type gene, and one 2/3-type gene: the two copies found in extant jawless vertebrates. Relatively soon thereafter, a second duplication round yielded the 1 through 4 paralog types now found in all jawed vertebrates, from sharks to humans. It is possible to assert that these duplication events correlate well to the Ohno 2R hypothesis.

Direct stimulation of NADP+ synthesis through Akt-mediated phosphorylation of NAD kinase.

Nicotinamide adenine dinucleotide phosphate (NADP+) is essential for producing NADPH, the primary cofactor for reductive metabolism. We find that growth factor signaling through the phosphoinositide 3-kinase (PI3K)-Akt pathway induces acute synthesis of NADP+ and NADPH. Akt phosphorylates NAD kinase (NADK), the sole cytosolic enzyme that catalyzes the synthesis of NADP+ from NAD+ (the oxidized form of NADH), on three serine residues (Ser44, Ser46, and Ser48) within an amino-terminal domain. This phosphorylation stimulates NADK activity both in cells and directly in vitro, thereby increasing NADP+ production. A rare isoform of NADK (isoform 3) lacking this regulatory region exhibits constitutively increased activity. These data indicate that Akt-mediated phosphorylation of NADK stimulates its activity to increase NADP+ production through relief of an autoinhibitory function inherent to its amino terminus.

Classification of the human THAP protein family identifies an evolutionarily conserved coiled coil region

BackgroundThe THAP (Thanatos Associated Proteins) protein family in humans is implicated in various important cellular processes like epigenetic regulation, maintenance of pluripotency, transposition and disorders like cancers and hemophilia. The human THAP protein family which consists of twelve members of different lengths has a well characterized amino terminal, zinc-coordinating, DNA-binding domain called the THAP domain. However, the carboxy terminus of most THAP proteins is yet to be structurally characterized. A coiled coil region is known to help in protein oligomerization in THAP1 and THAP11. It is not known if other human THAP proteins oligomerize. We have used bioinformatic tools to explore the possibility of dimerization of THAP proteins via a coiled coil region.ResultsClassification of human THAP protein into three size based groups led to the identification of an evolutionarily conserved alpha helical region, downstream of the amino terminal THAP domain. Secondary structure predictions, alpha helical wheel plots and protein models demonstrated the strong possibility of coiled coil formation in this conserved, leucine rich region of all THAP proteins except THAP10.ConclusionsThe identification of a predicted oligomerization region in the human THAP protein family opens new directions to investigate the members of this protein family.

The effect of androgen receptor splice variant 7 on the growth of castration-resistant prostate cancer and the efficacy of taxane chemotherapy.

302 Background: Expression of androgen receptor (AR) splice variant 7 (AR-V7) has been identified as a mechanism associated with the development of castration-resistant prostate cancer (CRPC), but a potential link between AR-V7 expression and resistance to taxanes, such as docetaxel or cabazitaxel, has not been unequivocally demonstrated. We clarify the relationship between AR-V7 expression and resistance to taxanes. Furthermore, we assess the inhibitory effect of EPI-002, an antagonist of AR amino-terminal domain (NTD), to an AR-V7 driven CRPC cell line with acquired resistance to docetaxel. Methods: LNCaP95-DR cells, which express AR-V7 and exhibit resistance to enzalutamide and docetaxel, was developed. WST-1 assay or BrdU ELISA assay was performed to evaluate the inhibitory effect of drugs. We examined alterations of AR and AR-V7 signaling using Western blot analyses, real-time RT-qPCR and reporter gene assay. Results: LNCaP95-DR cells showed cross-resistance to cabazitaxel. Although the LNCaP95-DR cells had increased expression of AR-V7 and its target genes (UBE2C, CDC20), knockdown of AR-V7 did not restore sensitivity to docetaxel or cabazitaxel. However, despite resistance to docetaxel and carbazitaxel, EPI-002 had an inhibitory effect on the proliferation of LNCaP95-DR cells that was similar to that achieved with the parental LNCaP95 cells, whereas enzalutamide had no effect on the proliferation of either cell line. Conclusions: Our results suggest that EPI-002 may be an option for the treatment of AR-V7-driven CRPC that is resistant to taxanes.

Physical and functional interaction between nucleoid-associated proteins HU and Lsr2 of Mycobacterium tuberculosis: altered DNA binding and gene regulation.

Nucleoid-associated proteins (NAPs) in bacteria contribute to key activities such as DNA compaction, chromosome organization and regulation of gene expression. HU and Lsr2 are two principal NAPs in Mycobacterium tuberculosis (Mtb). HU is essential for Mtb survival and is one of the most abundant NAPs. It differs from other eubacterial HU proteins in having a long, flexible lysine- and arginine-rich carboxy-terminal domain. Lsr2 of Mtb is the functional analogue of the bacterial NAP commonly called H-NS. Lsr2 binds to and regulates expression of A/T-rich portions of the otherwise G/C-rich mycobacterial chromosome. Here, we demonstrate that HU and Lsr2 interact to form a complex. The interaction occurs primarily through the flexible carboxy-terminal domain of HU and the acidic amino-terminal domain of Lsr2. The resulting complex, upon binding to DNA, forms thick nucleoprotein rods, in contrast to the DNA bridging seen with Lsr2 and the DNA compaction seen with HU. Furthermore, transcription assays indicate that the HU-Lsr2 complex is a regulator of gene expression. This physical and functional interaction between two NAPs, which has not been reported previously, is likely to be important for DNA organization and gene expression in Mtb and perhaps other bacterial species.

Thermodynamics of the NMDA Receptor Amino-Terminal Domain

The N-methyl-D-aspartate receptor (NMDAR) is a ligand-gated ion channel found in post-synaptic neurons that is critically important in learning and memory. This tetrameric membrane protein has two extracellular domains, a ligand-binding domain (LBD) and an amino-terminal domain (ATD), that control the movement of ions through the channel. Specifically, conformational changes in the ATD layer have been linked to the allosteric regulation of channel gating. Previous structural studies of the GluN1/GluN2B NMDAR have identified a ∼20° opening of the GluN2B ATD and a ∼15° rotation of the GluN1 and GluN2B ATD subunits with respect to the dimer interface in the presence of allosteric modulators. In this investigation, we constructed models of both GluN1 and GluN2B ATD monomers and the GluN1/GluN2B dimer with and without allosteric modulators. We then selected order parameters that capture the range of conformational motion adopted by these domains. By performing free energy molecular dynamics simulations, we gain insight into the thermodynamics that drive these observed structural changes.

Mapping Structural Elements to NMDA Receptor Activation Steps

NMDA receptors are heterotetrameric glutamate-activated ionotropic receptors that mediate excitatory synaptic transmission in the brain. Numerous NMDA receptor subunit mutations are associated with epilepsy and several have been identified as causal factors. Single-molecule electrophysiological recordings reveal that NMDA receptors explore many functional configurations. However, our ability to map structural changes to each of these functional configurations has been limited. Here, we integrate single-molecule recordings of GluN1-1a/GluN2A receptors with computational modeling of receptor activation to determine structural elements necessary for receptor activation. We found using single-channel recordings of disease-associated mutation, GluN1-1a I642L (GluN1-1aI642L) reduced receptor open probability within bursts (wild-type: 0.72 ± 0.04; GluN1-1aI642L: 0.04 ± 0.06), mean open duration (wild-type: 4.34 ± 0.11 msec; GluN1-1aI642L: 1.10 ± 0.09 msec), and mean closed duration within bursts (wild-type: 3.21 ± 0.51 msec; GluN1-1aI642L: 11.74 ± 1.20 msec). To determine the structural mechanism by which this disease mutation perturbs channel function, we used GluN1/GluN2B crystal structures to build a homology model of GluN1/GluN2A lacking cytoplasmic and amino terminal domains. Using this model, we performed targeted molecular dynamics simulations of the closed receptor into a putative “open” conformation. We found that residue GluN1 I642 forms additional atomic contacts with L550 on the neighboring GluN2A during this transition. We performed mutant cycle analysis on these pair of residues by recording single-channel currents through GluN1-1a/GluN2A, GluN1-1AI642L/GluN2A, GluN1-1a/GluN2AL550I, or GluN1-1AI642L/GluN2AL550I. Using kinetic modeling of these currents to measure the free energy change associated with each transition step in the receptor activation pathway, we found that the strongest coupling between GluN1 I642 and GluN2A L550 occurs transitions between states C3 to C2 and O1 to O2. Our approach provides a tool by which we can begin to map precise structural elements necessary for each transition step in receptor function.

Signal Peptide Represses Kainate Receptor GluK1 Surface and Synaptic Trafficking through Direct Interaction with Amino-Terminal Domain

Signal Peptide Represses Kainate Receptor GluK1 Surface and Synaptic Trafficking through Direct Interaction with Amino-Terminal Domain

Atomistic Mechanisms Underlying the Activation of G Protein-Coupled Sweet Receptor Heterodimer Mediated by Sugar Alcohol Recognition

The human sweet taste receptor (T1R2-T1R3) plays an important role in recognizing various low-molecular-weight sweet-tasting sugars and proteins, resulting in the release of intracellular heterotrimeric G protein, which leads to a sweet taste perception. Xylitol and sorbitol, which are sugar alcohols naturally found in many fruits and vegetables, exhibit the potential caries-reducing effect and are widely used for diabetic patients as low-calorie sweeteners. In the present study, the computational tools are applied to investigate the structural details of binary complexes formed between sugar alcohols and T1R2-T1R3 heterodimeric receptor. Principle component analysis reveals that the ligand-binding site in T1R2 domain is adapted by the induced-fit mechanism to accommodate the focused sugar alcohols, in which the residues 233-268 are significantly located closer to stabilize ligand. The finding likely suggests that these structural transformations might be the important mechanisms underlying ligand-protein recognitions. The calculated free energies also support the aminoterminal domain of T1R2 monomer as the preferential binding site for such sugar alcohols rather than T1R3 domain, in a correspondence with relatively less water molecules accessible into the T1R2 pocket. The T1R2 E302 residue is found to be the important recognized residue for sugar alcohol binding through a strongly firmed hydrogen bond and electrostatic attraction. Additionally, the binding affinity of xylitol toward T1R2 domain is significantly higher than that of sorbitol, making it a sweeter taste molecule.

Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors

Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases.

Prototype foamy virus intasome aggregation is mediated by outer protein domains and prevented by protocatechuic acid

The integrase (IN) enzyme of retrovirus prototype foamy virus (PFV) consists of four domains: amino terminal extension (NED), amino terminus (NTD), catalytic core (CCD), and carboxyl terminus domains (CTD). A tetramer of PFV IN with two viral DNA ends forms the functional intasome. Two inner monomers are catalytically active while the CCDs of the two outer monomers appear to play only structural roles. The NED, NTD, and CTD of the outer monomers are disordered in intasome structures. Truncation mutants reveal that integration to a supercoiled plasmid increases without the outer monomer CTDs present. Deletion of the outer CTDs enhances the lifetime of the intasome compared to full length (FL) IN or deletion of the outer monomer NTDs. High ionic strength buffer or several additives, particularly protocatechuic acid (PCA), enhance the integration of FL intasomes by preventing aggregation. These data confirm previous studies suggesting the disordered outer domains of PFV intasomes are not required for intasome assembly or integration. Instead, the outer CTDs contribute to aggregation of PFV intasomes which may be inhibited by high ionic strength buffer or the small molecule PCA.

Phosphorylation cycling of Annexin A2 Tyr23 is critical for calcium-regulated exocytosis in neuroendocrine cells

Annexin A2 (AnxA2) is a calcium and lipid binding protein involved in neuroendocrine secretion. We have previously demonstrated that AnxA2 participates in the formation and/or stabilization of lipid microdomains required for structural and spatial organization of the exocytotic machinery in chromaffin cells. However, the regulation of AnxA2 is not fully understood. Numerous phosphorylation sites have been identified in the amino-terminal domain of AnxA2. Phosphorylation of Ser25 and Tyr23 are well established and confirmed to be functionally significant. In particular, phosphorylation of Tyr23 by the tyrosine kinase pp60Src reduces the binding of AnxA2 to both actin filaments and the plasma membrane, two major actors of exocytosis, thus, we examined whether AnxA2 was phosphorylated on Tyr23 during exocytosis. Using immunolabelling and a biochemical approach, we found that nicotine stimulation triggered the phosphorylation of Anx A2 on Tyr23. The expression of two AnxA2 mutants carrying phosphorylation deficient (Y23A) or phosphomimetic (Y23E) mutations reduced the number exocytotic sites. Furthermore, expression of AnxA2-Y23A inhibited the formation of lipid microdomains, whereas the expression of AnxA2-Y23E altered actin filaments associated with docked granules. These results suggest that phosphorylation/dephosphorylation switch at Tyr23 in AnxA2 is critical for calcium-regulated exocytosis in neuroendocrine cells. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

DNA polymerase beta and other gap-filling enzymes in mammalian base excision repair.

DNA polymerase β plays a central role in the base excision DNA repair pathway that cleanses the genome of apurinic/apyrimidinic (AP) sites. AP sites arise in DNA from spontaneous base loss and DNA damage-specific glycosylases that hydrolyze the N-glycosidic bond between the deoxyribose and damaged base. AP sites are deleterious lesions because they can be mutagenic and/or cytotoxic. DNA polymerase β contributes two enzymatic activities, DNA synthesis and lyase, during the repair of AP sites; these activities reside on carboxyl- and amino-terminal domains, respectively. Accordingly, its cellular, structural, and kinetic attributes have been extensively characterized and it serves as model enzyme for the nucleotidyl transferase reaction utilized by other replicative, repair, and trans-lesion DNA polymerases.

The GTPase Domain of MX2 Interacts with HIV-1 Capsid Enabling Its Short Isoform to Moderate Antiviral Restriction

Myxovirus resistance 2 (MX2/MXB) is an interferon (IFN)-induced HIV-1 restriction factor that inhibits viral nuclear DNA accumulation. The amino-terminal domain of MX2 binds the viral capsid and is essential for inhibition. Using in vitro assembled Capsid-Nucleocapsid (CANC) complexes as a surrogate for the HIV-1 capsid lattice, we reveal that the GTPase (G) domain of MX2 contains a second, independent capsid binding site. The importance of this interaction was addressed in a series of competition assays using the naturally occurring non-antiviral short isoform of MX2 that lacks the amino-terminal 25 amino acids. Specifically, we show that the G domain enhances MX2 function, and that the foreshortened isoform acts as a functional suppressor of the full-length protein in a G domain-dependent manner. The interaction of MX2 with its HIV-1 capsid substrate is therefore multi-faceted: there are dual points of contact which, together with protein oligomerization, contribute to the complexity of MX2 regulation.

Methods to Investigate the Roles of β-Arrestin-Dependent RalGDS Activation in GPCR-Stimulated Membrane Blebbing.

G protein-coupled receptors (GPCRs) comprise the largest family of integral membrane proteins, which in addition to signaling via heterotrimeric G proteins can activate small G proteins both directly and indirectly. The activation of a variety of GPCRs leads to the translocation of Ral GDP dissociation stimulator (RalGDS) to the plasma membrane, where it functions as a guanine nucleotide exchange factor of RalA to promote membrane blebbing. The translocation of RalGDS is β-arrestin-dependent and can be inhibited by either the expression of the β-arrestin1 amino-terminal domain or the expression of RalGDS clone 284 (amino acid residues 616-768 of RalGDS). We describe here a methodology for assessing GPCR-dependent stimulation of RalGDS plasma membrane translocation.

Acetylation is the most abundant actin modification in Entamoeba histolytica and modifications of actin's amino-terminal domain change cytoskeleton activities.

Actin is one of the most conserved, abundant, and ubiquitous proteins in all eukaryotes characterised to date. Posttranslation modifications of actin modify the organisation of the actin-rich cytoskeleton. In particular, chemical modifications of actin's amino-terminal region determine how filamentous actin is organised into scaffolds. After assuming that protein modifications account for the multiple functional activities exerted by the single actin in Entamoeba histolytica, we profiled posttranslational modifications of this protein. Acetylation (on 21 different amino acids) was the most abundant modification, followed by phosphorylation. Furthermore, the glycine residue at Position 2 in E. histolytica's actin (Gly2, not found in most other eukaryotic actins) was found to be acetylated. The impact of Gly2 on the amoeba's life cycle and pathogenicity was then assessed in mutagenesis experiments. We found that Gly2 was necessary for cell morphology and division, parasite-host cell adhesion, and host invasion in an in vitro model of amoebic human infection.

Delineating WWOX Protein Interactome by Tandem Affinity Purification-Mass Spectrometry: Identification of Top Interactors and Key Metabolic Pathways Involved.

It has become clear from multiple studies that WWOX (WW domain-containing oxidoreductase) operates as a “non-classical” tumor suppressor of significant relevance in cancer progression. Additionally, WWOX has been recognized for its role in a much wider array of human pathologies including metabolic conditions and central nervous system related syndromes. A myriad of putative functional roles has been attributed to WWOX mostly through the identification of various binding proteins. However, the reality is that much remains to be learned on the key relevant functions of WWOX in the normal cell. Here we employed a Tandem Affinity Purification-Mass Spectrometry (TAP-MS) approach in order to better define direct WWOX protein interactors and by extension interaction with multiprotein complexes under physiological conditions on a proteomic scale. This work led to the identification of both well-known, but more importantly novel high confidence WWOX interactors, suggesting the involvement of WWOX in specific biological and molecular processes while delineating a comprehensive portrait of WWOX protein interactome. Of particular relevance is WWOX interaction with key proteins from the endoplasmic reticulum (ER), Golgi, late endosomes, protein transport, and lysosomes networks such as SEC23IP, SCAMP3, and VOPP1. These binding partners harbor specific PPXY motifs which directly interact with the amino-terminal WW1 domain of WWOX. Pathway analysis of WWOX interactors identified a significant enrichment of metabolic pathways associated with proteins, carbohydrates, and lipids breakdown. Thus, suggesting that WWOX likely plays relevant roles in glycolysis, fatty acid degradation and other pathways that converge primarily in Acetyl-CoA generation, a fundamental molecule not only as the entry point to the tricarboxylic acid (TCA) cycle for energy production, but also as the key building block for de novo synthesis of lipids and amino acids. Our results provide a significant lead on subsets of protein partners and enzymatic complexes with which full-length WWOX protein interacts with in order to carry out its metabolic and other biological functions while also becoming a valuable resource for further mechanistic studies.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of prostate cancer, inhibiting AR activity in a manner that would predict functional tumor selectivity. Small-molecule drug discovery and extensive biological characterization of a lead compound. We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of prostate cancer cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6 hours, and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development.