Intermediate Domains Research Articles

Identification of 21 novel immune-type receptors in miiuy croaker and expression pattern of three typical inhibitory members

Novel immune-type receptor (NITR) genes belong to the immunoglobulin superfamily and are encoded by clusters of multigene families. NITRs encode type I transmembrane proteins and are only found in teleosts. In the current study, total 21 NITR genes are identified from miiuy croaker (Miichthys miiuy) and named as MmNITR1 to MmNITR21. Miiuy croaker NITR genes that encoded one or two extracellular immunoglobulin (Ig) domains, a transmembrane (TM) region, an immunoreceptor tyrosine-based inhibitor motif (ITIM) in the cytoplasmic (Cyt) region. The majority of MmNITRs possess cytoplasmic ITIM that can be classified as inhibitory receptors. However, a smaller number of NITRs (MmNITR8, MmNITR15 and MmNITR16) can be classified as activating receptors by the lack of cytoplasmic ITIMs and presence of a positively charged residue within their transmembrane domain. As typical inhibitory receptors, MmNITR1, MmNITR2 and MmNITR3 have different characteristics of the structure. In MmNITR1 gene, variable (V) and intermediate (I) domains are encoded by two separate exons. In contrast to MmNITR1, MmNITR3 gene encode V and I domains in a single exon. And MmNITR2 gene is characterized by the presence of only one Ig-like (V-type) extracellular domain and lack of J or J-like motifs. Also MmNITR2 gene displays an additional exon which is 48bp long between the V domain and the TM region. Two and four potential N-link giycosylation sites (N-X-S/T) are present in the extracellular Ig domains. Real-time RT-PCR results showed that upon induction with Vibrio anguillarum, NITR gene expressions were induced by bacteria in kidney, liver and spleen. Meanwhile, NITRs are also primarily detected in different tissues. Phylogenetic analyses of NITR V domains indicate that MmNITR1 and MmNITR2 are more similar than MmNITR3.

90K, an interferon-stimulated gene product, reduces the infectivity of HIV-1

BackgroundIn response to viral infections, interferons induce the transcription of several hundred genes in mammalian cells. Specific antiviral functions, however, have only been attributed to a few of them. 90K/LGALS3BP has been reported to be an interferon-stimulated gene that is upregulated in individuals with cancer or HIV-1 infection.ResultsHere, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K’s antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4+ T-cells and PBMCs. 90K’s susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1.ConclusionsThus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions.

Crystal structure of a GroEL-ADP complex in the relaxed allosteric state at 2.7 Å resolution

The chaperonin proteins GroEL and GroES are cellular nanomachines driven by the hydrolysis of ATP that facilitate the folding of structurally diverse substrate proteins. In response to ligand binding, the subunits of a ring cycle in a concerted manner through a series of allosteric states (T, R, and R″), enabling work to be performed on the substrate protein. Removing two salt bridges that ordinarily break during the allosteric transitions of the WT permitted the structure of GroEL-ADP in the R state to be solved to 2.7 Å resolution. Whereas the equatorial domain displays almost perfect sevenfold symmetry, the apical domains, to which substrate proteins bind, and to a lesser extent, the intermediate domains display a remarkable asymmetry. Freed of intersubunit contacts, the apical domain of each subunit adopts a different conformation, suggesting a flexibility that permits interaction with diverse substrate proteins. This result contrasts with a previous cryo-EM study of a related allosteric ATP-bound state at lower resolution. After artificially imposing sevenfold symmetry it was concluded that a GroEL ring in the R-ATP state existed in six homogeneous but slightly different states. By imposing sevenfold symmetry on each of the subunits of the crystal structure of GroEL-ADP, we showed that the synthetic rings of (X-ray) GroEL-ADP and (cryo-EM) GroEL-ATP are structurally closely related. A deterministic model, the click stop mechanism, that implied temporal transitions between these states was proposed. Here, however, these conformational states are shown to exist as a structurally heterogeneous ensemble within a single ring.

Flux pattern transitions in the intermediate state of a type-I superconductor driven by an ac field

The intermediate state of a type-I superconductor Pb film is studied by a scanning Hall probe and scanning ac-susceptibility microscopies under static and oscillating applied magnetic fields. The structure of the typical flux patterns during magnetic field penetration/expulsion shows a strong hysteresis. Under the action of an ac field, the multiply quantized flux tubes in a type-I superconductor reveal a dynamical reordering similar to what is observed in the Campbell regime for vortices in a type-II superconductor. Most strikingly, after shaking, higher density flux tube patterns demonstrate a reorganization from a superheated metastable tubular pattern to a stable stripe pattern. We provide direct experimental evidence that the flux reorganization behavior is a dynamical transition. The local distribution of the potentials providing pinning to intermediate state patterns is mapped out, which is, as far as we know, the first direct visualization of confinement of intermediate state domains by pinning centers.

Characterization of Pore Geometry of Indiana Limestone in Relation to Mechanical Compaction

We studied the pore structure in intact and inelastically compacted Indiana limestone using X-ray microtomography imaging. Guided by detailed microstructural observations and using Otsu’s global thresholding method, the 3D images acquired at voxel side length of 4 μm were segmented into three domains: solid grains, macropores and an intermediate zone dominated by microporosity. The macropores were individually identified by morphological processing and their shape quantified by their sphericity and equivalent diameter. Our new data revealed a significant reduction of the number of macropores in hydrostatically and triaxially compressed samples with respect to the intact material, in agreement with previous microstructural analysis. The intermediate (microporosity) domains remained interconnected in compacted samples. Our data suggest that the inelastic compaction in Indiana limestone is manifested as not only a decrease in the volume fraction of the microporosity backbone but also a corresponding decrease in its thickness.

Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch

Morphogenesis of the vertebrate head relies on proper dorsal–ventral (D–V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.

Deformations of strongly pseudoconvex domains

We show that two smoothly bounded, strongly pseudoconvex domains which are diffeomorphic may be smoothly deformed into each other, with all intermediate domains being strongly pseudoconvex. This result relates to Lempert’s ideas about Kobayashi extremal discs, and also has intrinsic interest.

The Canalicular System and the Osteoblast Domain in Human Secondary Osteons

The lacunar-canalicular system in human secondary osteons was examined by two complementary techniques: light microscopy analysis of undecalcified thick sections and the SEM cortex-fractured surface technique. Unlike the earlier definitions of 'osteoblastic domain' presented as the matrix volume produced by osteoblasts in the process of osteon infilling, this study measured the domain by the length of osteoblast dendritic processes. The domain extension was defined along radial vectors advancing from the reversal line towards the central canal. According to their lengths, domains were divided into three classes: peripheral, intermediate and internal. The mean length of peripheral domains was significantly shorter than those of the intermediate and internal domains. This suggests that the infilling process is modulated by an initial preparatory phase characterised by osteoblast adhesion to the wall of the cutting cone, and a limited matrix synthesis, followed by a regular matrix volume apposition organised in concentric layers. In addition to the radial canaliculae arranged along converging vectors in planes perpendicular to the central canal, we distinguished a further class of canaliculae, the equatorial canaliculae originating from the major perimeter of the lacuna and spreading out radially in the plane of the same lacuna (therefore, perpendicularly to the radial canaliculae). The whole lacunar-canalicular network was structured as a closed system around the vascular axis of the central canal with very few canaliculae crossing the reversal line and connecting the neighbouring osteons. These anatomical observations contribute to our knowledge of lacunar-canalicular system development.

The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel-shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I-domain loops increase K(M) and decrease V(max) for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine.

Involvement of residues of the ϕ29 terminal protein intermediate and priming domains in the formation of a stable and functional heterodimer with the replicative DNA polymerase

Bacteriophage ϕ29 genome consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5′ end (TP-DNA) that together with a specific sequence constitutes the replication origins. To initiate replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the origins and initiates replication using as primer the hydroxyl group of TP residue Ser232. The 3D structure of the DNA polymerase/TP heterodimer allowed the identification of TP residues that could be responsible for interaction with the DNA polymerase. Here, we examined the role of TP residues Arg158, Arg169, Glu191, Asp198, Tyr250, Glu252, Gln253 and Arg256 by in vitro analyses of mutant derivatives. The results showed that substitution of these residues had an effect on either the stability of the TP/DNA polymerase complex (R158A) or in the functional interaction of the TP at the polymerization active site (R169A, E191A, Y250A, E252A, Q253A and R256A), affecting the first steps of ϕ29 TP-DNA replication. These results allow us to propose a role for these residues in the maintenance of the equilibrium between TP-priming domain stabilization and its gradual exit from the polymerization active site of the DNA polymerase as new DNA is being synthesized.

Gremlin 2 regulates distinct roles of BMP and Endothelin 1 signaling in dorsoventral patterning of the facial skeleton

Patterning of the upper versus lower face involves generating distinct pre-skeletal identities along the dorsoventral (DV) axes of the pharyngeal arches. Whereas previous studies have shown roles for BMPs, Endothelin 1 (Edn1) and Jagged1b-Notch2 in DV patterning of the facial skeleton, how these pathways are integrated to generate different skeletal fates has remained unclear. Here, we show that BMP and Edn1 signaling have distinct roles in development of the ventral and intermediate skeletons, respectively, of the zebrafish face. Using transgenic gain-of-function approaches and cell-autonomy experiments, we find that BMPs strongly promote hand2 and msxe expression in ventral skeletal precursors, while Edn1 promotes the expression of nkx3.2 and three Dlx genes (dlx3b, dlx5a and dlx6a) in intermediate precursors. Furthermore, Edn1 and Jagged1b pattern the intermediate and dorsal facial skeletons in part by inducing the BMP antagonist Gremlin 2 (Grem2), which restricts BMP activity to the ventral-most face. We therefore propose a model in which later cross-inhibitory interactions between BMP and Edn1 signaling, in part mediated by Grem2, separate an initially homogenous ventral region into distinct ventral and intermediate skeletal precursor domains.

Pairs of Domains Where All Intermediate Domains are Treed

For a ring extension \({R \subseteq S, (R, S)}\) is called a treed pair if every ring \({T, R \subseteq T \subseteq S,}\) is treed. When R is a field it is shown that the only treed pairs are those satisfying tr.deg[S : R] ≤ 1. For dim(R) ≥ 1 several satisfactory results are given. We establish a relationship between treed pairs, GD pairs and residually algebraic pairs.

Variabilidade das anomalias de temperatura da superfície do mar no oceano atlântico sudoeste e sua relação com o fenomeno El Niño-Oscilação Sul

Este trabalho apresenta um estudo sobre a variabilidade dos campos de anomalia da temperatura da superfície do mar (ATSM), obtidos através imagens de satélites em regiões sob a influência dos diferentes regimes oceanográficos e/ou biofísicos no Oceano Atlântico Sudoeste (ATS). Para tanto, séries temporais de ATSM de 1985 a 2006 foram investigadas em nove sub-áreas no ATS, divididas nos domínios subtropical, intermediário e subantártico. Análise espectral aplicada às séries de ATSM revelou ciclos no período interanual entre 1,6 e 3,7 anos. Análise de correlação cruzada foi usada para estabelecer a relação entre os ciclos presentes na ATSM e os eventos do fenômeno El Niño Oscilação Sul (ENOS), representados aqui a partir do Índice Multivariado do ENOS (MEI). Os resultados evidenciam uma correlação estatisticamente significante, defasada no tempo, entre o ENOS e anomalias termais no ATS, porém sem uma associação direta entre os episódios quentes (frios) de El Niño (La Niña) e ATSMs positivas (negativas). Dentre as diversas áreas de estudo, a correlação é máxima sobre a região da Confluência Brasil-Malvinas (-0,78), com defasagem de tempo de 9 meses após a La Niña de maio de 1988 a maio de 1989.

Molecular Microdomains in a Sensory Terminal, the Vestibular Calyx Ending

Many primary vestibular afferents form large cup-shaped postsynaptic terminals (calyces) that envelope the basolateral surfaces of type I hair cells. The calyceal terminals both respond to glutamate released from ribbon synapses in the type I cells and initiate spikes that propagate to the afferent's central terminals in the brainstem. The combination of synaptic and spike initiation functions in these unique sensory endings distinguishes them from the axonal nodes of central neurons and peripheral nerves, such as the sciatic nerve, which have provided most of our information about nodal specializations. We show that rat vestibular calyces express an unusual mix of voltage-gated Na and K channels and scaffolding, cell adhesion, and extracellular matrix proteins, which may hold the ion channels in place. Protein expression patterns form several microdomains within the calyx membrane: a synaptic domain facing the hair cell, the heminode abutting the first myelinated internode, and one or two intermediate domains. Differences in the expression and localization of proteins between afferent types and zones may contribute to known variations in afferent physiology.

Crystal structure of the novel PaiB transcriptional regulator from Geobacillus stearothermophilus

The pai operon is transcriptionally regulated by PaiB and has been found to be essential for cell growth and involved in the negative control of sporulation and degradative enzyme production. The pai operon was initially cloned as a fragment of the Bacillus subtilis chromosome consisting of two genes paiA and paiB.1 The presence of both of these genes on a multicopy vector revealed that both decrease the levels of extracellular degradative enzymes such as subtilisin, neutral protease, levansucrase, α-amylase, and alkaline phosphatases and also greatly reduce the frequency of sporulation.2 Based on the study by Strauch (1993), paiB appears to encode a regulator belonging to the same family as the AbrB, Hpr, and Sin regulators. AbrB, Hpr, Sin, and PaiB control the synthesis of degradative enzymes that occurs during the transition between vegetative growth and the onset of the stationary phase and sporulation in B. subtilis.2 Known sporulation genes have been identified as targets for direct control by AbrB (spo0E, spo0H, and spoVG) and Sin (spoIIA, spoIID, spoIIE, and spoIIG).3 Hpr and PaiB may also regulate spo genes directly as evidenced by the Spo phenotype of strains carrying multicopy plasmids that overexpress these two genes.1 Preliminary bioinformatic data suggested that PaiB might be a split barrel flavin-binding regulator. Herein, we report the first crystal structure of the Pai transcriptional regulator: PaiB from Geobacillus stearothermophilus. The structure of PaiB demonstrates that it is a homodimer and that each monomer consists of three domains: large, intermediate, and C-terminal. The large and intermediate domains fold into a structure similar to pyridoxine 5′-phosphate oxidase (PNPO) and form an analogous flavin mononucleotide (FMN)-binding cavity. The small C-terminal domain of PaiB possesses a helix-turn-helix (HTH) DNA-binding fold. The comparisons of sequence and structure show that PaiB is a representative of a new sequence family of transcriptional regulators. Detailed analysis of the protein structure provides insights into the potential biological function.

Combinatorial double-sided auctions for network bandwidth allocation: a budget-balanced and decentralized approach

Telecommunication networks are now an interconnection of competitive operators that need to cooperate to ensure end-to-end delivery of traffic. Inter-domain agreements have to be performed, and pricing is seen as a relevant way to reward intermediate domains for forwarding the traffic of others. In previous works, Vickrey–Clark–Groves (VCG) double-sided auctions have been applied because they provide proper incentives, lead to an efficient use of the network, and verify other relevant characteristics. However, it has been highlighted that the resource allocation schemes applying VCG auction are neither budget-balanced nor solvable in a decentralized way. In this paper, we apply combinatorial double-sided auction to allocate the bandwidth resources over nodes. While previous works were using a centralized algorithm, we use here a new pricing rule, leading to a new budget-balanced pricing scheme for which allocations and charges can be computed in a decentralized way. We also analyze the impact of this scheme on the game over declared costs of nodes.

Cryo-EM Structure of a Group II Chaperonin in the Prehydrolysis ATP-Bound State Leading to Lid Closure

Chaperonins are large ATP-driven molecular machines that mediate cellular protein folding. Group II chaperonins use their "built-in lid" to close their central folding chamber. Here we report the structure of an archaeal group II chaperonin in its prehydrolysis ATP-bound state at subnanometer resolution using single particle cryo-electron microscopy (cryo-EM). Structural comparison of Mm-cpn in ATP-free, ATP-bound, and ATP-hydrolysis states reveals that ATP binding alone causes the chaperonin to close slightly with a ∼45° counterclockwise rotation of the apical domain. The subsequent ATP hydrolysis drives each subunit to rock toward the folding chamber and to close the lid completely. These motions are attributable to the local interactions of specific active site residues with the nucleotide, the tight couplings between the apical and intermediate domains within the subunit, and the aligned interactions between two subunits across the rings. This mechanism of structural changes in response to ATP is entirely different from those found in group I chaperonins.

Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer

Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins.

Chaperonin-Mediated Protein Folding

Chaperonins are remarkable ring assemblies that provide kinetic assistance to protein folding to the native state. They function through the action of the central cavities of their rings. The best studied such system is bacterial GroEL and its cooperating co-chaperonin GroES, which mediate productive folding of a large number of proteins in the presence of ATP. I will recount the early functional studies of chaperonins in mitochondria, carried out collaboratively with Ulrich Hartl, and describe our subsequent X-ray, EM, and biochemical studies of GroEL leading to an understanding of the structure and reaction cycle, studies carried out with Paul Sigler and Helen Saibil. The structure and mechanism studies revealed that an open ring of GroEL exposes hydrophobic surfaces that capture non-native proteins and prevent them from aggregation. In the presence of ATP, which binds cooperatively in the seven equatorial sites of an open ring, the cochaperonin ring GroES associates, producing large rigid body movements of the apical and intermediate domains of the bound, so-called cis ring, releasing substrate protein into a now-encapsulated central cavity that has hydrophilic walls. Folding occurs in this space in what is the longest-lived state of the reaction cycle. ATP hydrolysis gates rapid entry of ATP into the opposite ring, which both allosterically ejects the cis ligands and sets up a new folding active ring. I will present recent cryoEM studies of the allosteric movements driven by ATP binding, which both facilitate substrate protein binding and enable initial contact of GroES with GroEL, carried out with Helen Saibil, as well as discuss cis folding studies, carried out in part with Kurt Wüthrich.

Dual Action of ATP Hydrolysis Couples Lid Closure to Substrate Release into the Group II Chaperonin Chamber

Group II chaperonins are ATP-dependent ring-shaped complexes that bind nonnative polypeptides and facilitate protein folding in archaea and eukaryotes. A built-in lid encapsulates substrate proteins within the central chaperonin chamber. Here, we describe the fate of the substrate during the nucleotide cycle of group II chaperonins. The chaperonin substrate-binding sites are exposed, and the lid is open in both the ATP-free and ATP-bound prehydrolysis states. ATP hydrolysis has a dual function in the folding cycle, triggering both lid closure and substrate release into the central chamber. Notably, substrate release can occur in the absence of a lid, and lid closure can occur without substrate release. However, productive folding requires both events, so that the polypeptide is released into the confined space of the closed chamber where it folds. Our results show that ATP hydrolysis coordinates the structural and functional determinants that trigger productive folding.