Up-regulation of OLR1 expression by TBC1D3 through activation of TNFα/NF-κB pathway promotes the migration of human breast cancer cells.

PubMed

Wang, Bei; Zhao, Huzi; Zhao, Lei; Zhang, Yongchen; Wan, Qing; Shen, Yong; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-11-01

Metastatic spread of cancer cells is the most life-threatening aspect of breast cancer and involves multiple steps including cell migration. We recently found that the TBC1D3 oncogene promotes the migration of breast cancer cells, and its interaction with CaM enhances the effects of TBC1D3. However, little is known regarding the mechanism by which TBC1D3 induces the migration of cancer cells. Here, we demonstrated that TBC1D3 stimulated the expression of oxidized low density lipoprotein receptor 1 (OLR1), a stimulator of cell migration, in breast cancer cells at the transcriptional level. Depletion of OLR1 by siRNAs or down-regulation of OLR1 expression using pomalidomide, a TNFα inhibitor, significantly decreased TBC1D3-induced migration of these cells. Notably, TBC1D3 overexpression activated NF-κB, a major effector of TNFα signaling, while inhibition of TNFα signaling suppressed the effects of TBC1D3. Consistent with this, NF-κB inhibition using its specific inhibitor caffeic acid phenethyl ester decreased both TBC1D3-induced OLR1 expression and cell migration, suggesting a critical role for TNFα/NF-κB signaling in TBC1D3-induced migration of breast cancer cells. Mechanistically, TBC1D3 induced activation of this signaling pathway on multiple levels, including by increasing the release of TNFα, elevating the transcription of TNFR1, TRAF1, TRAF5 and TRAF6, and decreasing the degradation of TNFR1. In summary, these studies identify the TBC1D3 oncogene as a novel regulator of TNFα/NF-κB signaling that mediates this oncogene-induced migration of human breast cancer cells by up-regulating OLR1. Copyright © 2017 Elsevier B.V. All rights reserved.

Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

PubMed

Zhao, Huzi; Zhang, Lina; Zhang, Yongchen; Zhao, Lei; Wan, Qing; Wang, Bei; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-05-30

The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

PVD TBC experience on GE aircraft engines

NASA Technical Reports Server (NTRS)

Maricocchi, Antonio; Bartz, Andi; Wortman, David

1995-01-01

The higher performance levels of modern gas turbine engines present significant challenges in the reliability of materials in the turbine. The increased engine temperatures required to achieve the higher performance levels reduce the strength of the materials used in the turbine sections of the engine. Various forms of thermal barrier coatings (TBC's) have been used for many years to increase the reliability of gas turbine engine components. Recent experience with the physical vapor deposition (PVD) process using ceramic material has demonstrated success in extending the service life of turbine blades and nozzles. Engine test results of turbine components with a 125 micron (0.005 in) PVD TBC have demonstrated component operating temperatures of 56-83 C (100-150 F) lower than non-PVD TBC components. Engine testing has also revealed the TBC is susceptible to high angle particle impact damage. Sand particles and other engine debris impact the TBC surface at the leading edge of airfoils and fracture the PVD columns. As the impacting continues, the TBC erodes away in local areas. Analysis of the eroded areas has shown a slight increase in temperature over a fully coated area, however a significant temperature reduction was realized over an airfoil without TBC.

Toward Precision Medicine: TBC1D4 Disruption Is Common Among the Inuit and Leads to Underdiagnosis of Type 2 Diabetes.

PubMed

Manousaki, Despoina; Kent, Jack W; Haack, Karin; Zhou, Sirui; Xie, Pingxing; Greenwood, Celia M; Brassard, Paul; Newman, Deborah E; Cole, Shelley; Umans, Jason G; Rouleau, Guy; Comuzzie, Anthony G; Richards, J Brent

2016-11-01

A common nonsense mutation in TBC1D4 was recently found to substantially increase the odds of type 2 diabetes in Greenlandic Inuit, leading to exclusively increased postprandial glucose. We investigated the frequency and effect of the TBC1D4 mutation on glucose metabolism and type 2 diabetes diagnosis among Canadian and Alaskan Inuit. Exome sequencing of the TBC1D4 variant was performed in 114 Inuit from Nunavik, Canada, and Sanger sequencing was undertaken in 1,027 Alaskan Inuit from the Genetics of Coronary Artery Disease in Alaskan Natives (GOCADAN) Study. Association testing evaluated the effect of the TBC1D4 variant on diabetes-related metabolic traits and diagnosis. The TBC1D4 mutation was present in 27% of Canadian and Alaskan Inuit. It was strongly associated with higher glucose (effect size +3.3 mmol/L; P = 2.5 x 10 -6 ) and insulin (effect size +175 pmol/L; P = 0.04) 2 h after an oral glucose load in homozygote carriers. TBC1D4 carriers with prediabetes and type 2 diabetes had an increased risk of remaining undiagnosed unless postprandial glucose values were tested (odds ratio 5.4 [95% CI 2.5-12]) compared with noncarriers. Of carriers with prediabetes or type 2 diabetes, 32% would remain undiagnosed without an oral glucose tolerance test (OGTT). Disruption of TBC1D4 is common among North American Inuit, resulting in exclusively elevated postprandial glucose. This leads to underdiagnosis of type 2 diabetes, unless an OGTT is performed. Accounting for genetic factors in the care of Inuit with diabetes provides an opportunity to implement precision medicine in this population. © 2016 by the American Diabetes Association.

PVD TBC experience on GE aircraft engines

NASA Technical Reports Server (NTRS)

Bartz, A.; Mariocchi, A.; Wortman, D. J.

1995-01-01

The higher performance levels of modern gas turbine engines present significant challenges in the reliability of materials in the turbine. The increased engine temperatures required to achieve the higher performance levels reduce the strength of the materials used in the turbine sections of the engine. Various forms of Thermal Barrier Coatings (TBC's) have been used for many years to increase the reliability of gas turbine engine components. Recent experience with the Physical Vapor Deposition (PVD) process using ceramic material has demonstrated success in extending the service life of turbine blades and nozzles. Engine test results of turbine components with a 125 micrometer (0.005 in) PVD TBC have demonstrated component operating temperatures of 56-83 C (100-150 F) lower than uncoated components. Engine testing has also revealed the TBC is susceptible to high angle particle impact damage. Sand particles and other engine debris impact the TBC surface at the leading edge of airfoils and fracture the PVD columns. As the impacting continues the TBC erodes away in local areas. Analysis of the eroded areas has shown a slight increase in temperature over a fully coated area, however, a significant temperature reduction was realized over an airfoil without any TBC.

The hominoid-specific gene TBC1D3 promotes generation of basal neural progenitors and induces cortical folding in mice

PubMed Central

Ju, Xiang-Chun; Hou, Qiong-Qiong; Sheng, Ai-Li; Wu, Kong-Yan; Zhou, Yang; Jin, Ying; Wen, Tieqiao; Yang, Zhengang; Wang, Xiaoqun; Luo, Zhen-Ge

2016-01-01

Cortical expansion and folding are often linked to the evolution of higher intelligence, but molecular and cellular mechanisms underlying cortical folding remain poorly understood. The hominoid-specific gene TBC1D3 undergoes segmental duplications during hominoid evolution, but its role in brain development has not been explored. Here, we found that expression of TBC1D3 in ventricular cortical progenitors of mice via in utero electroporation caused delamination of ventricular radial glia cells (vRGs) and promoted generation of self-renewing basal progenitors with typical morphology of outer radial glia (oRG), which are most abundant in primates. Furthermore, down-regulation of TBC1D3 in cultured human brain slices decreased generation of oRGs. Interestingly, localized oRG proliferation resulting from either in utero electroporation or transgenic expression of TBC1D3, was often found to underlie cortical regions exhibiting folding. Thus, we have identified a hominoid gene that is required for oRG generation in regulating the cortical expansion and folding. DOI: http://dx.doi.org/10.7554/eLife.18197.001 PMID:27504805

PVD TBC experience on GE aircraft engines

NASA Astrophysics Data System (ADS)

Maricocchi, A.; Bartz, A.; Wortman, D.

1997-06-01

The higher performance levels of modern gas turbine engines present significant challenges in the reli-ability of materials in the turbine. The increased engine temperatures required to achieve the higher per-formance levels reduce the strength of the materials used in the turbine sections of the engine. Various forms of thermal barrier coatings have been used for many years to increase the reliability of gas turbine engine components. Recent experience with the physical vapor deposition process using ceramic material has demonstrated success in extending the service life of turbine blades and nozzles. Engine test results of turbine components with a 125 μm (0.005 in.) PVD TBC have demonstrated component operating tem-peratures of 56 to 83 °C (100 to 150 °F) lower than non-PVD TBC components. Engine testing has also revealed that TBCs are susceptible to high angle particle impact damage. Sand particles and other engine debris impact the TBC surface at the leading edge of airfoils and fracture the PVD columns. As the impacting continues, the TBC erodes in local areas. Analysis of the eroded areas has shown a slight increase in temperature over a fully coated area ; however, a significant temperature reduc-tion was realized over an airfoil without TBC.

[Family of ribosomal proteins S1 contains unique conservative domain].

PubMed

Deriusheva, E I; Machulin, A V; Selivanova, O M; Serdiuk, I N

2010-01-01

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

PubMed

Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

A myosin II ATPase inhibitor reduces force production, glucose transport, and phosphorylation of AMPK and TBC1D1 in electrically stimulated rat skeletal muscle.

PubMed

Blair, David R; Funai, Katsuhiko; Schweitzer, George G; Cartee, Gregory D

2009-05-01

Contraction-stimulated glucose transport by skeletal muscle appears to be caused by the cumulative effects of multiple inputs [potentially including AMP-activated protein kinase (AMPK), Ca(2+) flux, and force production], making it challenging to isolate the roles of these putative regulatory factors. To distinguish the effects of force production from the direct consequences of Ca(2+) flux, the predominantly type II rat epitrochlearis muscle was incubated without (vehicle) or with N-benzyl-p-toluenesulfonamide (BTS), a highly specific myosin II ATPase inhibitor that prevents force production by electrically stimulated (ES) type II fibers without altering cytosolic Ca(2+). In ES muscles, BTS vs. vehicle had an 84% reduction in force production and a 57% decrement in contraction-stimulated 3-O-methylglucose transport (3MGT). BTS did not alter the ES increase in phosphorylation of CaMKII (indicative of cytosolic Ca(2+)) or the amount of glycogen depletion. ES caused significant reductions in ATP (48%) and phosphocreatine (67%) concentrations for vehicle-treated muscles. For BTS-treated muscles, ES did not reduce ATP and caused only a 42% decrease in phosphocreatine. There was an ES increase in phosphorylation of AMPK, acetyl-CoA carboxylase (an AMPK substrate), and TBC1D1 for vehicle-treated muscles but not for BTS-treated muscles. These results point toward an essential role for tension-related events, including AMPK activation, in the 57% contraction-stimulated increase in 3MGT that was inhibited by BTS and further suggest a possible role for TBC1D1 phosphorylation. Non-tension-related events (e.g., increased cytosolic Ca(2+) rather than increased AMPK and TBC1D1 phosphorylation) are implicated in the contraction-stimulated increase in 3MGT that persisted in the presence of BTS.

Characterization of a Linked Jumonji Domain of the KDM5/JARID1 Family of Histone H3 Lysine 4 Demethylases.

PubMed

Horton, John R; Engstrom, Amanda; Zoeller, Elizabeth L; Liu, Xu; Shanks, John R; Zhang, Xing; Johns, Margaret A; Vertino, Paula M; Fu, Haian; Cheng, Xiaodong

2016-02-05

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the

TBC2health: a database of experimentally validated health-beneficial effects of tea bioactive compounds.

PubMed

Zhang, Shihua; Xuan, Hongdong; Zhang, Liang; Fu, Sicong; Wang, Yijun; Yang, Hua; Tai, Yuling; Song, Youhong; Zhang, Jinsong; Ho, Chi-Tang; Li, Shaowen; Wan, Xiaochun

2017-09-01

Tea is one of the most consumed beverages in the world. Considerable studies show the exceptional health benefits (e.g. antioxidation, cancer prevention) of tea owing to its various bioactive components. However, data from these extensively published papers had not been made available in a central database. To lay a foundation in improving the understanding of healthy tea functions, we established a TBC2health database that currently documents 1338 relationships between 497 tea bioactive compounds and 206 diseases (or phenotypes) manually culled from over 300 published articles. Each entry in TBC2health contains comprehensive information about a bioactive relationship that can be accessed in three aspects: (i) compound information, (ii) disease (or phenotype) information and (iii) evidence and reference. Using the curated bioactive relationships, a bipartite network was reconstructed and the corresponding network (or sub-network) visualization and topological analyses are provided for users. This database has a user-friendly interface for entry browse, search and download. In addition, TBC2health provides a submission page and several useful tools (e.g. BLAST, molecular docking) to facilitate use of the database. Consequently, TBC2health can serve as a valuable bioinformatics platform for the exploration of beneficial effects of tea on human health. TBC2health is freely available at http://camellia.ahau.edu.cn/TBC2health. © The Author 2016. Published by Oxford University Press.

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms.

PubMed

Fulcher, Luke J; Bozatzi, Polyxeni; Tachie-Menson, Theresa; Wu, Kevin Z L; Cummins, Timothy D; Bufton, Joshua C; Pinkas, Daniel M; Dunbar, Karen; Shrestha, Sabin; Wood, Nicola T; Weidlich, Simone; Macartney, Thomas J; Varghese, Joby; Gourlay, Robert; Campbell, David G; Dingwell, Kevin S; Smith, James C; Bullock, Alex N; Sapkota, Gopal P

2018-05-22

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Dynamics of the Tec‐family tyrosine kinase SH3 domains

PubMed Central

Roberts, Justin M.; Tarafdar, Sreya; Joseph, Raji E.; Andreotti, Amy H.; Smithgall, Thomas E.; Engen, John R.

2016-01-01

Abstract The Src Homology 3 (SH3) domain is an important regulatory domain found in many signaling proteins. X‐ray crystallography and NMR structures of SH3 domains are generally conserved but other studies indicate that protein flexibility and dynamics are not. We previously reported that based on hydrogen exchange mass spectrometry (HX MS) studies, there is variable flexibility and dynamics among the SH3 domains of the Src‐family tyrosine kinases and related proteins. Here we have extended our studies to the SH3 domains of the Tec family tyrosine kinases (Itk, Btk, Tec, Txk, Bmx). The SH3 domains of members of this family augment the variety in dynamics observed in previous SH3 domains. Txk and Bmx SH3 were found to be highly dynamic in solution by HX MS and Bmx was unstructured by NMR. Itk and Btk SH3 underwent a clear EX1 cooperative unfolding event, which was localized using pepsin digestion and mass spectrometry after hydrogen exchange labeling. The unfolding was localized to peptide regions that had been previously identified in the Src‐family and related protein SH3 domains, yet the kinetics of unfolding were not. Sequence alignment does not provide an easy explanation for the observed dynamics behavior, yet the similarity of location of EX1 unfolding suggests that higher‐order structural properties may play a role. While the exact reason for such dynamics is not clear, such motions can be exploited in intra‐ and intermolecular binding assays of proteins containing the domains. PMID:26808198

A structural portrait of the PDZ domain family.

PubMed

Ernst, Andreas; Appleton, Brent A; Ivarsson, Ylva; Zhang, Yingnan; Gfeller, David; Wiesmann, Christian; Sidhu, Sachdev S

2014-10-23

PDZ (PSD-95/Discs-large/ZO1) domains are interaction modules that typically bind to specific C-terminal sequences of partner proteins and assemble signaling complexes in multicellular organisms. We have analyzed the existing database of PDZ domain structures in the context of a specificity tree based on binding specificities defined by peptide-phage binding selections. We have identified 16 structures of PDZ domains in complex with high-affinity ligands and have elucidated four additional structures to assemble a structural database that covers most of the branches of the PDZ specificity tree. A detailed comparison of the structures reveals features that are responsible for the diverse specificities across the PDZ domain family. Specificity differences can be explained by differences in PDZ residues that are in contact with the peptide ligands, but these contacts involve both side-chain and main-chain interactions. Most PDZ domains bind peptides in a canonical conformation in which the ligand main chain adopts an extended β-strand conformation by interacting in an antiparallel fashion with a PDZ β-strand. However, a subset of PDZ domains bind peptides with a bent main-chain conformation and the specificities of these non-canonical domains could not be explained based on canonical structures. Our analysis provides a structural portrait of the PDZ domain family, which serves as a guide in understanding the structural basis for the diverse specificities across the family. Copyright © 2014 Elsevier Ltd. All rights reserved.

Degradation Mechanisms of an Advanced Jet Engine Service-Retired TBC Component

NASA Astrophysics Data System (ADS)

Wu, Rudder T.; Osawa, Makoto; Yokokawa, Tadaharu; Kawagishi, Kyoko; Harada, Hiroshi

Current use of TBCs is subjected to premature spallation failure mainly due to the formation of thermally grown oxides (TGOs). Although extensive research has been carried out to gain better understanding of the thermo - mechanical and -chemical characteristics of TBCs, laboratory-scale studies and simulation tests are often carried out in conditions significantly differed from the complex and extreme environment typically of a modern gas-turbine engine, thus, failed to truly model service conditions. In particular, the difference in oxygen partial pressure and the effects of contaminants present in the engine compartment have often been neglected. In this respect, an investigation is carried out to study the in-service degradation of an EB-PVD TBC coated nozzle-guide vane. Several modes of degradation were observed due to three factors: 1) presence of residual stresses induced by the thermal-expansion mismatches, 2) evolution of bond coat microstructure and subsequent formation of oxide spinels, 3) deposition of CMAS on the surface of TBC.

ELMO Domains, Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases*

PubMed Central

East, Michael P.; Bowzard, J. Bradford; Dacks, Joel B.; Kahn, Richard A.

2012-01-01

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases. PMID:23014990

Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

PubMed

Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

2017-04-01

Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Evaluation of the hyplex® TBC PCR test for detection of Mycobacterium tuberculosis complex in clinical samples

PubMed Central

2010-01-01

Background Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples. Results A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively. Conclusions The hyplex® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens. PMID:20356361

Structural Characterization of the Predominant Family of Histidine Kinase Sensor Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Z.; Hendrickson, W

2010-01-01

Histidine kinase (HK) receptors are used ubiquitously by bacteria to monitor environmental changes, and they are also prevalent in plants, fungi, and other protists. Typical HK receptors have an extracellular sensor portion that detects a signal, usually a chemical ligand, and an intracellular transmitter portion that includes both the kinase domain itself and the site for histidine phosphorylation. While kinase domains are highly conserved, sensor domains are diverse. HK receptors function as dimers, but the molecular mechanism for signal transduction across cell membranes remains obscure. In this study, eight crystal structures were determined from five sensor domains representative of themore » most populated family, family HK1, found in a bioinformatic analysis of predicted sensor domains from transmembrane HKs. Each structure contains an inserted repeat of PhoQ/DcuS/CitA (PDC) domains, and similarity between sequence and structure is correlated across these and other double-PDC sensor proteins. Three of the five sensors crystallize as dimers that appear to be physiologically relevant, and comparisons between ligated structures and apo-state structures provide insights into signal transmission. Some HK1 family proteins prove to be sensors for chemotaxis proteins or diguanylate cyclase receptors, implying a combinatorial molecular evolution.« less

Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex.

PubMed

Kandhakumari, Gandhi; Stephen, Selvaraj

2017-01-01

At present, three rapid kits are available globally for the confirmation of Mycobacterium tuberculosis complex (MTBC) in cultures by MPT64 antigen (MPT64 Ag) detection. These include Capilia TB, SD Bioline, and BD MGIT TBc Identification (TBcID). The third kit is yet to be validated in India. We have tested this kit and compared with SD Bioline using conventional tests as gold standard. Seventy-one MTBC (70 M. tuberculosis and one Mycobacterium bovis) and four nontuberculous mycobacteria (NTM) were isolated from 649 clinical specimens in MGIT 960 and/or Lowenstein-Jensen slants (LJ). MPT64 Ag was detected by both TBcID and SD Bioline kits in all the 71 clinical isolates and the reference strain M. tuberculosis H37Rv. All NTM species tested were negative by the two different kits. Thus, TBcID kit showed 100% concordance in terms of sensitivity and specificity. Rapid kits confirm MTBC cultures within 15 min in contrast to several weeks' time required by conventional techniques.

CORAL: aligning conserved core regions across domain families.

PubMed

Fong, Jessica H; Marchler-Bauer, Aron

2009-08-01

Homologous protein families share highly conserved sequence and structure regions that are frequent targets for comparative analysis of related proteins and families. Many protein families, such as the curated domain families in the Conserved Domain Database (CDD), exhibit similar structural cores. To improve accuracy in aligning such protein families, we propose a profile-profile method CORAL that aligns individual core regions as gap-free units. CORAL computes optimal local alignment of two profiles with heuristics to preserve continuity within core regions. We benchmarked its performance on curated domains in CDD, which have pre-defined core regions, against COMPASS, HHalign and PSI-BLAST, using structure superpositions and comprehensive curator-optimized alignments as standards of truth. CORAL improves alignment accuracy on core regions over general profile methods, returning a balanced score of 0.57 for over 80% of all domain families in CDD, compared with the highest balanced score of 0.45 from other methods. Further, CORAL provides E-values to aid in detecting homologous protein families and, by respecting block boundaries, produces alignments with improved 'readability' that facilitate manual refinement. CORAL will be included in future versions of the NCBI Cn3D/CDTree software, which can be downloaded at http://www.ncbi.nlm.nih.gov/Structure/cdtree/cdtree.shtml. Supplementary data are available at Bioinformatics online.

Generation of calibrated tungsten target x-ray spectra: modified TBC model.

PubMed

Costa, Paulo R; Nersissian, Denise Y; Salvador, Fernanda C; Rio, Patrícia B; Caldas, Linda V E

2007-01-01

In spite of the recent advances in the experimental detection of x-ray spectra, theoretical or semi-empirical approaches for determining realistic x-ray spectra in the range of diagnostic energies are important tools for planning experiments, estimating radiation doses in patients, and formulating radiation shielding models. The TBC model is one of the most useful approaches since it allows for straightforward computer implementation, and it is able to accurately reproduce the spectra generated by tungsten target x-ray tubes. However, as originally presented, the TBC model fails in situations where the determination of x-ray spectra produced by an arbitrary waveform or the calculation of realistic values of air kerma for a specific x-ray system is desired. In the present work, the authors revisited the assumptions used in the original paper published by . They proposed a complementary formulation for taking into account the waveform and the representation of the calculated spectra in a dosimetric quantity. The performance of the proposed model was evaluated by comparing values of air kerma and first and second half value layers from calculated and measured spectra by using different voltages and filtrations. For the output, the difference between experimental and calculated data was better then 5.2%. First and second half value layers presented differences of 23.8% and 25.5% in the worst case. The performance of the model in accurately calculating these data was better for lower voltage values. Comparisons were also performed with spectral data measured using a CZT detector. Another test was performed by the evaluation of the model when considering a waveform distinct of a constant potential. In all cases the model results can be considered as a good representation of the measured data. The results from the modifications to the TBC model introduced in the present work reinforce the value of the TBC model for application of quantitative evaluations in radiation

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

PubMed Central

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-01-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

The MB2 gene family of Plasmodium species has a unique combination of S1 and GTP-binding domains

PubMed Central

Romero, Lisa C; Nguyen, Thanh V; Deville, Benoit; Ogunjumo, Oluwasanmi; James, Anthony A

2004-01-01

Background Identification and characterization of novel Plasmodium gene families is necessary for developing new anti-malarial therapeutics. The products of the Plasmodium falciparum gene, MB2, were shown previously to have a stage-specific pattern of subcellular localization and proteolytic processing. Results Genes homologous to MB2 were identified in five additional parasite species, P. knowlesi, P. gallinaceum, P. berghei, P. yoelii, and P. chabaudi. Sequence comparisons among the MB2 gene products reveal amino acid conservation of structural features, including putative S1 and GTP-binding domains, and putative signal peptides and nuclear localization signals. Conclusions The combination of domains is unique to this gene family and indicates that MB2 genes comprise a novel family and therefore may be a good target for drug development. PMID:15222903

Degradation of TBC Systems in Environments Relevant to Advanced Gas Turbines for IGCC Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gleeson, Brian

2014-09-30

Air plasma sprayed (APS) thermal barrier coatings (TBCs) are used to provide thermal insulation for the hottest components in gas turbines. Zirconia stabilized with 7wt% yttria (7YSZ) is the most common ceramic top coat used for turbine blades. The 7YSZ coating can be degraded from the buildup of fly-ash deposits created in the power-generation process. Fly ash from an integrated gasification combined cycle (IGCC) system can result from coal-based syngas. TBCs are also exposed to harsh gas environments containing CO 2, SO 2, and steam. Degradation from the combined effects of fly ash and harsh gas atmospheres has the potentialmore » to severely limit TBC lifetimes. The main objective of this study was to use lab-scale testing to systematically elucidate the interplay between prototypical deposit chemistries (i.e., ash and its constituents, K 2SO 4, and FeS) and environmental oxidants (i.e., O 2, H 2O and CO 2) on the degradation behavior of advanced TBC systems. Several mechanisms of early TBC failure were identified, as were the specific fly-ash constituents responsible for degradation. The reactivity of MCrAlY bondcoats used in TBC systems was also investigated. The specific roles of oxide and sulfate components were assessed, together with the complex interplay between gas composition, deposit chemistry and alloy reactivity. Bondcoat composition design strategies to mitigate corrosion were established, particularly with regard to controlling phase constitution and the amount of reactive elements the bondcoat contains in order to achieve optimal corrosion resistance.« less

SH2-catalytic domain linker heterogeneity influences allosteric coupling across the SFK family.

PubMed

Register, A C; Leonard, Stephen E; Maly, Dustin J

2014-11-11

Src-family kinases (SFKs) make up a family of nine homologous multidomain tyrosine kinases whose misregulation is responsible for human disease (cancer, diabetes, inflammation, etc.). Despite overall sequence homology and identical domain architecture, differences in SH3 and SH2 regulatory domain accessibility and ability to allosterically autoinhibit the ATP-binding site have been observed for the prototypical SFKs Src and Hck. Biochemical and structural studies indicate that the SH2-catalytic domain (SH2-CD) linker, the intramolecular binding epitope for SFK SH3 domains, is responsible for allosterically coupling SH3 domain engagement to autoinhibition of the ATP-binding site through the conformation of the αC helix. As a relatively unconserved region between SFK family members, SH2-CD linker sequence variability across the SFK family is likely a source of nonredundant cellular functions between individual SFKs via its effect on the availability of SH3 and SH2 domains for intermolecular interactions and post-translational modification. Using a combination of SFKs engineered with enhanced or weakened regulatory domain intramolecular interactions and conformation-selective inhibitors that report αC helix conformation, this study explores how SH2-CD sequence heterogeneity affects allosteric coupling across the SFK family by examining Lyn, Fyn1, and Fyn2. Analyses of Fyn1 and Fyn2, isoforms that are identical but for a 50-residue sequence spanning the SH2-CD linker, demonstrate that SH2-CD linker sequence differences can have profound effects on allosteric coupling between otherwise identical kinases. Most notably, a dampened allosteric connection between the SH3 domain and αC helix leads to greater autoinhibitory phosphorylation by Csk, illustrating the complex effects of SH2-CD linker sequence on cellular function.

TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

PubMed

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

miR-17-5p Regulates Endocytic Trafficking through Targeting TBC1D2/Armus

PubMed Central

Serva, Andrius; Knapp, Bettina; Tsai, Yueh-Tso; Claas, Christoph; Lisauskas, Tautvydas; Matula, Petr; Harder, Nathalie; Kaderali, Lars; Rohr, Karl; Erfle, Holger; Eils, Roland; Braga, Vania; Starkuviene, Vytaute

2012-01-01

miRNA cluster miR-17-92 is known as oncomir-1 due to its potent oncogenic function. miR-17-92 is a polycistronic cluster that encodes 6 miRNAs, and can both facilitate and inhibit cell proliferation. Known targets of miRNAs encoded by this cluster are largely regulators of cell cycle progression and apoptosis. Here, we show that miRNAs encoded by this cluster and sharing the seed sequence of miR-17 exert their influence on one of the most essential cellular processes – endocytic trafficking. By mRNA expression analysis we identified that regulation of endocytic trafficking by miR-17 can potentially be achieved by targeting of a number of trafficking regulators. We have thoroughly validated TBC1D2/Armus, a GAP of Rab7 GTPase, as a novel target of miR-17. Our study reveals regulation of endocytic trafficking as a novel function of miR-17, which might act cooperatively with other functions of miR-17 and related miRNAs in health and disease. PMID:23285084

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

PubMed Central

2012-01-01

Background Proteins convey the majority of biochemical and cellular activities in organisms. Over the course of evolution, proteins undergo normal sequence mutations as well as large scale mutations involving domain duplication and/or domain shuffling. These events result in the generation of new proteins and protein families. Processes that affect proteome evolution drive species diversity and adaptation. Herein, change over the course of metazoan evolution, as defined by birth/death and duplication/deletion events within protein families and domains, was examined using the proteomes of 9 metazoan and two outgroup species. Results In studying members of the three major metazoan groups, the vertebrates, arthropods, and nematodes, we found that the number of protein families increased at the majority of lineages over the course of metazoan evolution where the magnitude of these increases was greatest at the lineages leading to mammals. In contrast, the number of protein domains decreased at most lineages and at all terminal lineages. This resulted in a weak correlation between protein family birth and domain birth; however, the correlation between domain birth and domain member duplication was quite strong. These data suggest that domain birth and protein family birth occur via different mechanisms, and that domain shuffling plays a role in the formation of protein families. The ratio of protein family birth to protein domain birth (domain shuffling index) suggests that shuffling had a more demonstrable effect on protein families in nematodes and arthropods than in vertebrates. Through the contrast of high and low domain shuffling indices at the lineages of Trichinella spiralis and Gallus gallus, we propose a link between protein redundancy and evolutionary changes controlled by domain shuffling; however, the speed of adaptation among the different lineages was relatively invariant. Evaluating the functions of protein families that appeared or disappeared at the

TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

PubMed Central

Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

2012-01-01

Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

A Protein Domain and Family Based Approach to Rare Variant Association Analysis.

PubMed

Richardson, Tom G; Shihab, Hashem A; Rivas, Manuel A; McCarthy, Mark I; Campbell, Colin; Timpson, Nicholas J; Gaunt, Tom R

2016-01-01

It has become common practice to analyse large scale sequencing data with statistical approaches based around the aggregation of rare variants within the same gene. We applied a novel approach to rare variant analysis by collapsing variants together using protein domain and family coordinates, regarded to be a more discrete definition of a biologically functional unit. Using Pfam definitions, we collapsed rare variants (Minor Allele Frequency ≤ 1%) together in three different ways 1) variants within single genomic regions which map to individual protein domains 2) variants within two individual protein domain regions which are predicted to be responsible for a protein-protein interaction 3) all variants within combined regions from multiple genes responsible for coding the same protein domain (i.e. protein families). A conventional collapsing analysis using gene coordinates was also undertaken for comparison. We used UK10K sequence data and investigated associations between regions of variants and lipid traits using the sequence kernel association test (SKAT). We observed no strong evidence of association between regions of variants based on Pfam domain definitions and lipid traits. Quantile-Quantile plots illustrated that the overall distributions of p-values from the protein domain analyses were comparable to that of a conventional gene-based approach. Deviations from this distribution suggested that collapsing by either protein domain or gene definitions may be favourable depending on the trait analysed. We have collapsed rare variants together using protein domain and family coordinates to present an alternative approach over collapsing across conventionally used gene-based regions. Although no strong evidence of association was detected in these analyses, future studies may still find value in adopting these approaches to detect previously unidentified association signals.

The MPS1 family of protein kinases.

PubMed

Liu, Xuedong; Winey, Mark

2012-01-01

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs.

The MPS1 Family of Protein Kinases

PubMed Central

Liu, Xuedong; Winey, Mark

2014-01-01

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs. PMID:22482908

Satisfaction Domains Differ between the Patient and Their Family in Adult Intensive Care Units

PubMed Central

Song, Ge; Sim, Pei Zhen; Ting, Kit Cheng; Yoo, Jeffrey Kwang Sui; Wang, Qing Li; Mascuri, Raudhah Binte Haji Mohamad; Ong, Venetia Hui Ling; Phua, Jason; Kowitlawakul, Yanika

2016-01-01

Background. Patients' and family's satisfaction data from the Asian intensive care units (ICUs) is lacking. Objective. Domains between patient and family satisfaction and contribution of each domain to the general satisfaction were studied. Method. Over 3 months, adult patients across 4 ICUs staying for more than 48 hours with abbreviated mental test score of 7 or above and able to understand English and immediate family members were surveyed by separate validated satisfaction questionnaires. Results. Two hundred patients and 194 families were included in the final analysis. Significant difference in the satisfaction scores was observed between the ICUs. Patients were most and least satisfied in the communication (4.2 out of 5) and decision-making (2.9 out of 5) domains, respectively. Families were most and least satisfied in the relationship with doctors (3.9 out of 5) and family's involvement domains (3.3 out of 5), respectively. Domains contributing most to the general satisfaction were the illness management domain for patients (β coefficient = 0.44) and characteristics of doctors and nurses domain for family (β coefficient = 0.45). Discussion. In an Asian ICU community, patients and families differ in their expectations and valuations of health care processes. Health care providers have difficult tasks in attending to these different domains. PMID:28044138

A meta-analysis of work-family conflict and various outcomes with a special emphasis on cross-domain versus matching-domain relations.

PubMed

Amstad, Fabienne T; Meier, Laurenz L; Fasel, Ursula; Elfering, Achim; Semmer, Norbert K

2011-04-01

A literature review of studies analyzing work-family conflict and its consequences was conducted, and 427 effect sizes were analyzed meta-analytically. Work-family conflict was analyzed bidirectionally in terms of work interference with family (WIF) and family interference with work (FIW). We assessed 3 categories of potential outcomes: work-related outcomes, family-related outcomes, and domain-unspecific outcomes. Results show that WIF and FIW are consistently related to all 3 types of outcomes. Both types of interrole conflict showed stronger relationships to same-domain outcomes than to cross-domain outcomes. Thus, WIF was more strongly associated with work-related than with family-related outcomes, and FIW was more strongly associated with family-related than with work-related outcomes. In moderator analyses, parenthood could not explain variability in effect sizes. However, time spent at work did moderate the relationships between WIF and family-related outcomes, as well as FIW and domain-unspecific outcomes.

A Novel Interaction between the SH2 Domain of Signaling Adaptor Protein Nck-1 and the Upstream Regulator of the Rho Family GTPase Rac1 Engulfment and Cell Motility 1 (ELMO1) Promotes Rac1 Activation and Cell Motility*

PubMed Central

Zhang, Guo; Chen, Xia; Qiu, Fanghua; Zhu, Fengxin; Lei, Wenjing; Nie, Jing

2014-01-01

Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoGV12A), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity. PMID:24928514

Development and Optimization of Tailored Composite TBC Design Architectures for Improved Erosion Durability

NASA Astrophysics Data System (ADS)

Schmitt, Michael P.; Schreiber, Jeremy M.; Rai, Amarendra K.; Eden, Timothy J.; Wolfe, Douglas E.

2017-08-01

Rare-earth pyrochlores, RE2Zr2O7, have been identified as potential thermal barrier coating (TBC) materials due to their attractive thermal properties and CMAS resistance. However, they possess a low fracture toughness which results in poor erosion durability/foreign object damage resistance. This research focuses on the development of tailored composite air plasma spray (APS) TBC design architectures utilizing a t' Low-k secondary toughening phase (ZrO2-2Y2O3-1Gd2O3-1Yb2O3; mol.%) to enhance the erosion durability of a hyper-stoichiometric pyrochlore, NZO (ZrO2-25Nd2O3-5Y2O3-5Yb2O3; mol.%). In this study, composite coatings have been deposited with 30, 50, and 70% (wt.%) t' Low-k toughening phase in a horizontally aligned lamellar morphology which enhances the toughening response of the coating. The coatings were characterized via SEM and XRD and were tested for erosion durability before and after isothermal heat treatment at 1100 °C. Analysis with mixing laws indicated improved erosion performance; however, a lack of long-term thermal stability was shown via isothermal heat treatments at 1316 °C. An impact stress analysis was performed using finite element analysis of a coating cross section, representing the first microstructurally realistic study of mechanical properties of TBCs with the results correlating well with observed behavior.

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non-syndromic mental retardation.

PubMed

Basel-Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-03-01

The molecular basis of autosomal recessive non-syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I-kappaB kinase/NFkappaB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. A previously unknown signal transduction pathway is important in human cognitive development.

A novel in-frame deletion affecting the BAR domain of OPHN1 in a family with intellectual disability and hippocampal alterations

PubMed Central

Santos-Rebouças, Cíntia Barros; Belet, Stefanie; Guedes de Almeida, Luciana; Ribeiro, Márcia Gonçalves; Medina-Acosta, Enrique; Bahia, Paulo Roberto Valle; Alves da Silva, Antônio Francisco; Lima dos Santos, Flávia; Borges de Lacerda, Glenda Corrêa; Pimentel, Márcia Mattos Gonçalves; Froyen, Guy

2014-01-01

Oligophrenin-1 (OPHN1) is one of at least seven genes located on chromosome X that take part in Rho GTPase-dependent signaling pathways involved in X-linked intellectual disability (XLID). Mutations in OPHN1 were primarily described as an exclusive cause of non-syndromic XLID, but the re-evaluation of the affected individuals using brain imaging displayed fronto-temporal atrophy and cerebellar hypoplasia as neuroanatomical marks. In this study, we describe clinical, genetic and neuroimaging data of a three generation Brazilian XLID family co-segregating a novel intragenic deletion in OPHN1. This deletion results in an in-frame loss of exon 7 at transcription level (c.781_891del; r.487_597del), which is predicted to abolish 37 amino acids from the highly conserved N-terminal BAR domain of OPHN1. cDNA expression analysis demonstrated that the mutant OPHN1 transcript is stable and no abnormal splicing was observed. Features shared by the affected males of this family include neonatal hypotonia, strabismus, prominent root of the nose, deep set eyes, hyperactivity and instability/intolerance to frustration. Cranial MRI scans showed large lateral ventricles, vermis hypoplasia and cystic dilatation of the cisterna magna in all affected males. Interestingly, hippocampal alterations that have not been reported in patients with loss-of-function OPHN1 mutations were found in three affected individuals, suggesting an important function for the BAR domain in the hippocampus. This is the first description of an in-frame deletion within the BAR domain of OPHN1 and could provide new insights into the role of this domain in relation to brain and cognitive development or function. PMID:24105372

The Identification and Structure of an N-Terminal PR Domain Show that FOG1 Is a Member of the PRDM Family of Proteins

PubMed Central

Clifton, Molly K.; Westman, Belinda J.; Thong, Sock Yue; O’Connell, Mitchell R.; Webster, Michael W.; Shepherd, Nicholas E.; Quinlan, Kate G.; Crossley, Merlin; Blobel, Gerd A.; Mackay, Joel P.

2014-01-01

FOG1 is a transcriptional regulator that acts in concert with the hematopoietic master regulator GATA1 to coordinate the differentiation of platelets and erythrocytes. Despite considerable effort, however, the mechanisms through which FOG1 regulates gene expression are only partially understood. Here we report the discovery of a previously unrecognized domain in FOG1: a PR (PRD-BF1 and RIZ) domain that is distantly related in sequence to the SET domains that are found in many histone methyltransferases. We have used NMR spectroscopy to determine the solution structure of this domain, revealing that the domain shares close structural similarity with SET domains. Titration with S-adenosyl-L-homocysteine, the cofactor product synonymous with SET domain methyltransferase activity, indicated that the FOG PR domain is not, however, likely to function as a methyltransferase in the same fashion. We also sought to define the function of this domain using both pulldown experiments and gel shift assays. However, neither pulldowns from mammalian nuclear extracts nor yeast two-hybrid assays reproducibly revealed binding partners, and we were unable to detect nucleic-acid-binding activity in this domain using our high-diversity Pentaprobe oligonucleotides. Overall, our data demonstrate that FOG1 is a member of the PRDM (PR domain containing proteins, with zinc fingers) family of transcriptional regulators. The function of many PR domains, however, remains somewhat enigmatic for the time being. PMID:25162672

Moisture-Induced TBC Spallation on Turbine Blade Samples

NASA Technical Reports Server (NTRS)

Smialek, James L.

2011-01-01

Delayed failure of TBCs is a widely observed laboratory phenomenon, although many of the early observations went unreported. "The weekend effect" or "DeskTop Spallation" (DTS) is characterized by initial survival of a TBC after accelerated laboratory thermal cycling, then failure by exposure to ambient humidity or water. Once initiated, failure can occur quite dramatically in less than a second. To this end, the water drop test and digital video recordings have become useful techniques in studies at NASA (Smialek, Zhu, Cuy), DECHMA (Rudolphi, Renusch, Schuetze), and CNRS Toulouse/SNECMA (Deneux, Cadoret, Hervier, Monceau). In the present study the results for a commercial turbine blade, with a standard EB-PVD 7YSZ TBC top coat and Pt-aluminide diffusion bond monitored by weight change and visual appearance. Failures were distributed widely over a 5-100 hr time range, depending on temperature. At some opportune times, failure was captured by video recording, documenting the appearance and speed of the moisture-induced spallation process. Failure interfaces exhibited alumina scale grains, decorated with Ta-rich oxide particles, and alumina inclusions as islands and streamers. The phenomenon is thus rooted in moisture-induced delayed spallation (MIDS) of the alumina scale formed on the bond coat. In that regard, many studies show the susceptibility of alumina scales to moisture, as long as high strain energy and a partially exposed interface exist. The latter conditions result from severe cyclic oxidation conditions, which produce a highly stressed and partially damaged scale. In one model, it has been proposed that moisture reacts with aluminum in the bond coat to release hydrogen atoms that 'embrittle' the interface. A negative synergistic effect with interfacial sulfur is also invoked.

Diversification of HP1-like Chromo Domain Proteins in Tetrahymena thermophila.

PubMed

Wiley, Emily A; Horrell, Scott; Yoshino, Alyssa; Schornak, Cara C; Bagnani, Claire; Chalker, Douglas L

2018-01-01

Proteins that possess a chromo domain are well-known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy-terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi-directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates. © 2017 The Authors. Journal of Eukaryotic Microbiology published by Wiley Periodicals, Inc. on behalf of International Society of Protistologists.

The CC2D1A, a member of a new gene family with C2 domains, is involved in autosomal recessive non‐syndromic mental retardation

PubMed Central

Basel‐Vanagaite, L; Attia, R; Yahav, M; Ferland, R J; Anteki, L; Walsh, C A; Olender, T; Straussberg, R; Magal, N; Taub, E; Drasinover, V; Alkelai, A; Bercovich, D; Rechavi, G; Simon, A J; Shohat, M

2006-01-01

Background The molecular basis of autosomal recessive non‐syndromic mental retardation (NSMR) is poorly understood, mostly owing to heterogeneity and absence of clinical criteria for grouping families for linkage analysis. Only two autosomal genes, the PRSS12 gene on chromosome 4q26 and the CRBN on chromosome 3p26, have been shown to cause autosomal recessive NSMR, each gene in only one family. Objective To identify the gene causing autosomal recessive NSMR on chromosome 19p13.12. Results The candidate region established by homozygosity mapping was narrowed down from 2.4 Mb to 0.9 Mb on chromosome 19p13.12. A protein truncating mutation was identified in the gene CC2D1A in nine consanguineous families with severe autosomal recessive NSMR. The absence of the wild type protein in the lymphoblastoid cells of the patients was confirmed. CC2D1A is a member of a previously uncharacterised gene family that carries two conserved motifs, a C2 domain and a DM14 domain. The C2 domain is found in proteins which function in calcium dependent phospholipid binding; the DM14 domain is unique to the CC2D1A protein family and its role is unknown. CC2D1A is a putative signal transducer participating in positive regulation of I‐κB kinase/NFκB cascade. Expression of CC2D1A mRNA was shown in the embryonic ventricular zone and developing cortical plate in staged mouse embryos, persisting into adulthood, with highest expression in the cerebral cortex and hippocampus. Conclusions A previously unknown signal transduction pathway is important in human cognitive development. PMID:16033914

CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome.

PubMed

Brioude, F; Oliver-Petit, I; Blaise, A; Praz, F; Rossignol, S; Le Jule, M; Thibaud, N; Faussat, A-M; Tauber, M; Le Bouc, Y; Netchine, I

2013-12-01

Russell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a cell proliferation inhibitor encoded by an imprinted gene in the 11p15 ICR2 domain. CDKN1C mutations lead to Beckwith Wiedemann syndrome (BWS, overgrowth syndrome) and in IMAGe syndrome which associates growth retardation and adrenal insufficiency. We searched for CDKN1C mutations in a cohort of clinically diagnosed RSS patients with no molecular anomaly. The coding sequence and intron-exon boundaries of CDKN1C were analysed in 97 RSS patients. The impact of CDKN1C variants on the cell cycle in vitro were determined by flow cytometry. Stability of CDKN1C was studied by western immunoblotting after inhibition of translation with cycloheximide. We identified the novel c.836G>[G;T] (p.Arg279Leu) mutation in a familial case of intrauterine growth retardation (IUGR) with RSS phenotype and no evidence of IMAGe. All the RSS patients inherited this mutation from their mothers (consistent with monoallelic expression from the maternal allele of the gene). A mutation of this amino acid (p.Arg279Pro) has been reported in cases of IMAGe. Functional analysis showed that Arg279Leu (RSS) did not affect the cell cycle, whereas the Arg279Pro mutation (IMAGe) led to a gain of function. Arg279Leu (RSS) led to an increased stability which could explain an increased activity of CDKN1C. CDKN1C mutations cause dominant maternally transmitted RSS, completing the molecular mirror with BWS. CDKN1C should be investigated in cases with family history of RSS.

Moisture-Induced TBC Spallation on Turbine Blade Samples

NASA Technical Reports Server (NTRS)

Smialek, James

2011-01-01

Delayed failure of TBCs is a widely observed laboratory phenomenon, although many of the early observations went unreported. The weekend effect or DeskTop Spallation (DTS) is characterized by initial survival of a TBC after accelerated laboratory thermal cycling, then failure by exposure to ambient humidity or water. Once initiated, failure can occur quite dramatically in less than a second. To this end, the water drop test and digital video recordings have become useful techniques in studies at NASA (Smialek, Zhu, Cuy), DECHMA (Rudolphi, Renusch, Schuetze), and CNRS Toulouse/SNECMA (Deneux, Cadoret, Hervier, Monceau). In the present study the results for a commercial turbine blade, with a standard EB-PVD 7YSZ TBC top coat and Pt-aluminide diffusion bond coat are reported. Cut sections were intermittently oxidized at 1100, 1150, and 1200 C and monitored by weight change and visual appearance. Failures were distributed widely over a 5-100 hr time range, depending on temperature. At some opportune times, failure was captured by video recording, documenting the appearance and speed of the moisture-induced spallation process. Failure interfaces exhibited alumina scale grains, decorated with Ta-rich oxide particles, and alumina inclusions as islands and streamers. The phenomenon is thus rooted in moisture-induced delayed spallation (MIDS) of the alumina scale formed on the bond coat. In that regard, many studies show the susceptibility of alumina scales to moisture, as long as high strain energy and a partially exposed interface exist. The latter conditions result from severe cyclic oxidation conditions, which produce a highly stressed and partially damaged scale. In one model, it has been proposed that moisture reacts with aluminum in the bond coat to release hydrogen atoms that embrittle the interface. A negative synergistic effect with interfacial sulfur is also invoked.

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Sulfur and Moisture Effects on Alumina Scale and TBC Spallation

NASA Technical Reports Server (NTRS)

Smialek, James L.

2007-01-01

cause is related to a hydrogen embrittlement reaction: Al alloy + 3 H2O = Al(OH)3 + 3H(+) + 3e(-). This mechanism is derived from an analogous moisture-induced hydrogen embrittlement mechanism originally shown for Ni3Al and FeAl intermetallics. Consequently, a cathodic hydrogen charging technique was used to demonstrate that electrolytic de-scaling occurs for these otherwise adherent alumina scales formed on Y-doped Rene'N5, in support of hydrogen effects. Finally, some TBC observations are discussed in light of all of the above. Plasma sprayed 8YSZ coatings, produced on PWA1484 without a bond coat, were found to survive more than 1000 1-hr cycles at 1100 C when desulfurized to below 0.1 ppmw. At higher sulfur (1.2 ppmw) levels, moisture sensitivity and delayed TBC failure, referred to as Desk Top Spallation, occurred at just 200 hr. Despite a large degree of scatter, a factor of 5 in life improvement is indicated for desulfurized samples in cyclic furnace tests, confirming the beneficial effect of low sulfur alloys on model TBC systems. (DTS and moisture effects are also observed on commercially applied PVD 7YSZ coatings on Rene'N5+Y with Pt-aluminide bond coats). These types of catastrophic failure were subverted on the model system by segmenting the substrate into a network of 0.010 high ribs, spaced in. apart, prior to plasma spraying. No failures occurred after 1000 cycles at 1150 C or after 2000 cycles at 1100 C, even after water immersion. The benefit is described in terms of elasticity models and a critical buckling stress.

Discovery and Characterization of Novel Cyclotides Originated from Chimeric Precursors Consisting of Albumin-1 Chain a and Cyclotide Domains in the Fabaceae Family*

PubMed Central

Nguyen, Giang Kien Truc; Zhang, Sen; Nguyen, Ngan Thi Kim; Nguyen, Phuong Quoc Thuc; Chiu, Ming Sheau; Hardjojo, Antony; Tam, James P.

2011-01-01

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1–T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants. PMID:21596752

Involvement of family members in life with type 2 diabetes: Six interconnected problem domains of significance for family health identity and healthcare authenticity

PubMed Central

Grabowski, Dan; Andersen, Tue Helms; Varming, Annemarie; Ommundsen, Christine; Willaing, Ingrid

2017-01-01

Objectives: Family involvement plays a key role in diabetes management. Problems and challenges related to type 2-diabetes often affect the whole family, and relatives are at increased risk of developing diabetes themselves. We highlight these issues in our objectives: (1) to uncover specific family problems associated with mutual involvement in life with type 2-diabetes and (2) to analytically look at ways of approaching these problems in healthcare settings. Methods: Qualitative data were gathered in participatory problem assessment workshops. The data were analysed in three rounds using radical hermeneutics. Results: Problems were categorized in six domains: knowledge, communication, support, everyday life, roles and worries. The final cross-analysis focusing on the link between family identity and healthcare authenticity provided information on how the six domains can be approached in healthcare settings. Conclusion: The study generated important knowledge about problems associated with family involvement in life with type 2 diabetes and about how family involvement can be supported in healthcare practice. PMID:28839943

Scale Adhesion, Sulfur Content, and TBC Failure on Single Crystal Superalloys

NASA Technical Reports Server (NTRS)

Smialek, James L.

2002-01-01

This paper summarizes the main effects of sulfur impurity content on the cyclic oxidation resistance of single crystal superalloys, with emphasis on scale and TBC adhesion. Eleven hundred degrees C cyclic oxidation of PWA 1480 produces scale spallation leading to a weight loss of more than 30 Mg/sq cm after 500 one-hr cycles for a sulfur content of 6 ppmw. The sulfur content was reduced to levels below 0.1 ppmw by hydrogen annealing, resulting in weight gains of only 0.5 to 1.0 Mg/sq cm after 1000 one-hr cycles. Samples were produced with various sulfur contents by adjusting the annealing temperature, time, and sample thickness (i.e., diffusion product Dt/L(exp 2)). The subsequent cyclic oxidation behavior, mapped over a sulfur content/thickness diagram, shows a transition to adherent behavior at sulfur levels equivalent to 1 monolayer of total segregation. Additional information is contained in the original extended abstract.

Chlamydomonas reinhardtii LFO1 Is an IsdG Family Heme Oxygenase

DOE PAGES

Lojek, Lisa J.; Farrand, Allison J.; Wisecaver, Jennifer H.; ...

2017-08-16

Heme is essential for respiration across all domains of life. However, heme accumulation can lead to toxicity if cells are unable to either degrade or export heme or its toxic by-products. Under aerobic conditions, heme degradation is performed by heme oxygenases, enzymes which utilize oxygen to cleave the tetrapyrrole ring of heme. The HO-1 family of heme oxygenases has been identified in both bacterial and eukaryotic cells, whereas the IsdG family has thus far been described only in bacteria. We identified a hypothetical protein in the eukaryotic green alga Chlamydomonas reinhardtii, which encodes a protein containing an antibiotic biosynthesis monooxygenasemore » (ABM) domain consistent with those associated with IsdG family members. This protein, which we have named LFO1, degrades heme, contains similarities in predicted secondary structures to IsdG family members, and retains the functionally conserved catalytic residues found in all IsdG family heme oxygenases. These data establish LFO1 as an IsdG family member and extend our knowledge of the distribution of IsdG family members beyond bacteria. To gain further insight into the distribution of the IsdG family, we used the LFO1 sequence to identify 866 IsdG family members, including representatives from all domains of life. These results indicate that the distribution of IsdG family heme oxygenases is more expansive than previously appreciated, underscoring the broad relevance of this enzyme family. This work establishes a protein in the freshwater alga Chlamydomonas reinhardtii as an IsdG family heme oxygenase. This protein, LFO1, exhibits predicted secondary structure and catalytic residues conserved in IsdG family members, in addition to a chloroplast localization sequence. Additionally, the catabolite that results from the degradation of heme by LFO1 is distinct from that of other heme degradation products. Using LFO1 as a seed, we performed phylogenetic analysis, revealing that the IsdG family is

Chlamydomonas reinhardtii LFO1 Is an IsdG Family Heme Oxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lojek, Lisa J.; Farrand, Allison J.; Wisecaver, Jennifer H.

Heme is essential for respiration across all domains of life. However, heme accumulation can lead to toxicity if cells are unable to either degrade or export heme or its toxic by-products. Under aerobic conditions, heme degradation is performed by heme oxygenases, enzymes which utilize oxygen to cleave the tetrapyrrole ring of heme. The HO-1 family of heme oxygenases has been identified in both bacterial and eukaryotic cells, whereas the IsdG family has thus far been described only in bacteria. We identified a hypothetical protein in the eukaryotic green alga Chlamydomonas reinhardtii, which encodes a protein containing an antibiotic biosynthesis monooxygenasemore » (ABM) domain consistent with those associated with IsdG family members. This protein, which we have named LFO1, degrades heme, contains similarities in predicted secondary structures to IsdG family members, and retains the functionally conserved catalytic residues found in all IsdG family heme oxygenases. These data establish LFO1 as an IsdG family member and extend our knowledge of the distribution of IsdG family members beyond bacteria. To gain further insight into the distribution of the IsdG family, we used the LFO1 sequence to identify 866 IsdG family members, including representatives from all domains of life. These results indicate that the distribution of IsdG family heme oxygenases is more expansive than previously appreciated, underscoring the broad relevance of this enzyme family. This work establishes a protein in the freshwater alga Chlamydomonas reinhardtii as an IsdG family heme oxygenase. This protein, LFO1, exhibits predicted secondary structure and catalytic residues conserved in IsdG family members, in addition to a chloroplast localization sequence. Additionally, the catabolite that results from the degradation of heme by LFO1 is distinct from that of other heme degradation products. Using LFO1 as a seed, we performed phylogenetic analysis, revealing that the IsdG family is

A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505

Assessment of Cyclic Lifetime of NiCoCrAlY/ZrO2-Based EB-PVD TBC Systems via Reactive Element Enrichment in the Mixed Zone of the TGO Scale

NASA Astrophysics Data System (ADS)

Fritscher, Klaus; Braue, Wolfgang; Schulz, Uwe

2013-05-01

The chemical composition of the alumina-zirconia mixed zone (MZ) of an electron beam physical vapor deposited thermal barrier coating (EB-PVD TBC) system is affected by service conditions and by the interdiffusion of elements from the substrate alloy below and the zirconia top coat. Three NiCoCrAlY bond-coated Ni-base substrates with YPSZ or CeSZ EB-PVD TBCs were subjected to a cyclic furnace oxidation test (FCT) at 1373 K (1100 °C) in order to provide experimental evidence of a link between chemistry of the MZ, the substrate alloy, the ceramic top coat, and the time in the FCT. Energy dispersive spectroscopy of the MZ revealed preferred accumulation of Cr, Zr, Y, and Ce. The concentration of the reactive elements (RE = Ce + Y + Zr) was related to the respective average lifetimes of the TBC systems at 1373 K (1100 °C). The RE content in the MZ turned out to be a life-limiting parameter for YPSZ and CeSZ TBC systems which can be utilized to predict their relative lifetimes on the individual substrates. Conversely, the TBC failure mechanisms of YPSZ and CeSZ TBC systems are dissimilar.

Domain analysis of Ras-association domain family member 6 upon interaction with MDM2.

PubMed

Sarkar, Aradhan; Iwasa, Hiroaki; Hossain, Shakhawoat; Xu, Xiaoyin; Sawada, Takeru; Shimizu, Takanobu; Maruyama, Junichi; Arimoto-Matsuzaki, Kyoko; Hata, Yutaka

2017-01-01

The tumor suppressor Ras-association domain family member 6 (RASSF6) has Ras-association domain (RA) and Salvador/RASSF/Hippo domain (SARAH). RASSF6 antagonizes MDM2, stabilizes p53, and induces apoptosis and cell cycle arrest. We previously demonstrated the interaction between RASSF6 and MDM2, but did not determine how both proteins interact with each other. We have shown here that N-terminal, RA, and SARAH domains of RASSF6 interact with MDM2 at distinct regions. RA binds to the RING-finger region of MDM2 and stabilizes p53. SARAH binds RA and blocks the interaction between RA and MDM2. RA overexpression induces p53-dependent apoptosis and senescence. In the presence of active KRas, the interaction between RA and MDM2 is recovered. In this way, RA and SARAH play an important role in Ras-mediated regulation of p53. © 2017 Federation of European Biochemical Societies.

DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia.

PubMed

Markham, Nicholas O; Doll, Caleb A; Dohn, Michael R; Miller, Rachel K; Yu, Huapeng; Coffey, Robert J; McCrea, Pierre D; Gamse, Joshua T; Reynolds, Albert B

2014-09-01

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101-amino acid "head domain" comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain-specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin-mediated development. © 2014 Markham et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

TOPSIS based parametric optimization of laser micro-drilling of TBC coated nickel based superalloy

NASA Astrophysics Data System (ADS)

Parthiban, K.; Duraiselvam, Muthukannan; Manivannan, R.

2018-06-01

The technique for order of preference by similarity ideal solution (TOPSIS) approach was used for optimizing the process parameters of laser micro-drilling of nickel superalloy C263 with Thermal Barrier Coating (TBC). Plasma spraying was used to deposit the TBC and a pico-second Nd:YAG pulsed laser was used to drill the specimens. Drilling angle, laser scan speed and number of passes were considered as input parameters. Based on the machining conditions, Taguchi L8 orthogonal array was used for conducting the experimental runs. The surface roughness and surface crack density (SCD) were considered as the output measures. The surface roughness was measured using 3D White Light Interferometer (WLI) and the crack density was measured using Scanning Electron Microscope (SEM). The optimized result achieved from this approach suggests reduced surface roughness and surface crack density. The holes drilled at an inclination angle of 45°, laser scan speed of 3 mm/s and 400 number of passes found to be optimum. From the Analysis of variance (ANOVA), inclination angle and number of passes were identified as the major influencing parameter. The optimized parameter combination exhibited a 19% improvement in surface finish and 12% reduction in SCD.

Multiplex families with epilepsy

PubMed Central

Afawi, Zaid; Oliver, Karen L.; Kivity, Sara; Mazarib, Aziz; Blatt, Ilan; Neufeld, Miriam Y.; Helbig, Katherine L.; Goldberg-Stern, Hadassa; Misk, Adel J.; Straussberg, Rachel; Walid, Simri; Mahajnah, Muhammad; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Kahana, Esther; Masalha, Rafik; Kramer, Uri; Ekstein, Dana; Shorer, Zamir; Wallace, Robyn H.; Mangelsdorf, Marie; MacPherson, James N.; Carvill, Gemma L.; Mefford, Heather C.; Jackson, Graeme D.; Scheffer, Ingrid E.; Bahlo, Melanie; Gecz, Jozef; Heron, Sarah E.; Corbett, Mark; Mulley, John C.; Dibbens, Leanne M.; Korczyn, Amos D.

2016-01-01

Objective: To analyze the clinical syndromes and inheritance patterns of multiplex families with epilepsy toward the ultimate aim of uncovering the underlying molecular genetic basis. Methods: Following the referral of families with 2 or more relatives with epilepsy, individuals were classified into epilepsy syndromes. Families were classified into syndromes where at least 2 family members had a specific diagnosis. Pedigrees were analyzed and molecular genetic studies were performed as appropriate. Results: A total of 211 families were ascertained over an 11-year period in Israel. A total of 169 were classified into broad familial epilepsy syndrome groups: 61 generalized, 22 focal, 24 febrile seizure syndromes, 33 special syndromes, and 29 mixed. A total of 42 families remained unclassified. Pathogenic variants were identified in 49/211 families (23%). The majority were found in established epilepsy genes (e.g., SCN1A, KCNQ2, CSTB), but in 11 families, this cohort contributed to the initial discovery (e.g., KCNT1, PCDH19, TBC1D24). We expand the phenotypic spectrum of established epilepsy genes by reporting a familial LAMC3 homozygous variant, where the predominant phenotype was epilepsy with myoclonic-atonic seizures, and a pathogenic SCN1A variant in a family where in 5 siblings the phenotype was broadly consistent with Dravet syndrome, a disorder that usually occurs sporadically. Conclusion: A total of 80% of families were successfully classified, with pathogenic variants identified in 23%. The successful characterization of familial electroclinical and inheritance patterns has highlighted the value of studying multiplex families and their contribution towards uncovering the genetic basis of the epilepsies. PMID:26802095

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family.

PubMed

Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C; Dénervaud-Tendon, Valérie; Vermeer, Joop E M; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

2014-08-01

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. © 2014 American Society of Plant Biologists. All Rights Reserved.

Genome dynamics explain the evolution of flowering time CCT domain gene families in the Poaceae.

PubMed

Cockram, James; Thiel, Thomas; Steuernagel, Burkhard; Stein, Nils; Taudien, Stefan; Bailey, Paul C; O'Sullivan, Donal M

2012-01-01

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica.

PubMed

Ackermann, Nikolaus; Tiller, Maximilian; Anding, Gisela; Roggenkamp, Andreas; Heesemann, Jürgen

2008-07-01

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.

Development and initial evaluation of an enhanced measure of boundary flexibility for the work and family domains.

PubMed

Matthews, Russell A; Barnes-Farrell, Janet L

2010-07-01

This manuscript reports the development of a measure of work and family domain boundary flexibility. Building on previous research, we propose an expanded definition of boundary flexibility that includes two components-flexibility-ability and flexibility-willingness-and we develop a measure designed to capture this more comprehensive definition of boundary flexibility. Flexibility-ability is conceptualized as an individual's perception of personal and situational constraints that affect boundary management, and flexibility-willingness is conceptualized as an individual difference variable that captures the motivation to engage in boundary flexing. An additional feature of domain boundaries, permeability, is also examined. Data are presented from two studies. Study 1 (N = 244) describes the development of a multiscale measure that extends current conceptual definitions of boundary flexibility. Study 2 (N = 225) describes the refinement and evaluation of this measure. Confirmatory factor analysis, reliability evidence, interscale correlations, and correlations with important work-family constructs (e.g., domain centrality, work-family conflict) provide initial construct validity evidence for the measure.

Cancer Research Advance in CKLF-like MARVEL Transmembrane Domain Containing Member Family (Review).

PubMed

Lu, Jia; Wu, Qian-Qian; Zhou, Ya-Bo; Zhang, Kai-Hua; Pang, Bing-Xin; Li, Liang; Sun, Nan; Wang, Heng-Shu; Zhang, Song; Li, Wen-Jian; Zheng, Wei; Liu, Wei

2016-01-01

CKLF-like MARVEL transmembrane domain-containing family (CMTM) is a novel family of genes first reported at international level by Peking University Human Disease Gene Research Center. The gene products are between chemokines and the transmembrane-4 superfamily. Loaceted in several human chromosomes, CMTMs, which are unregulated in kinds of tumors, are potential tumor suppressor genes consisting of CKLF and CMTM1 to CMTM8. CMTMs play important roles in immune, male reproductive and hematopoietic systems. Also, it has been approved that CMTM family has strong connection with diseases of autoimmunity, haematopoietic system and haematopoietic system. The in-depth study in recent years found the close relation between CMTMs and umorigenesis, tumor development and metastasis. CMTM family has a significant clinical value in diagnosis and treatment to the diseases linking to tumor and immune system.

Chemical Shift Assignments of the C-terminal Eps15 Homology Domain-3 EH Domain*

PubMed Central

Caplan, Steve; Sorgen, Paul L.

2013-01-01

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation. PMID:23754701

Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun

2014-09-12

Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domainmore » of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.« less

APS TBC performance on directionally-solidified superalloy substrates with HVOF NiCoCrAlYHfSi bond coatings

DOE PAGES

Lance, Michael J.; Unocic, Kinga A.; Haynes, James A.; ...

2015-09-04

Directionally-solidified (DS) superalloy components with advanced thermal barrier coatings (TBC) to lower the metal operating temperature have the potential to replace more expensive single crystal superalloys for large land-based turbines. In order to assess relative TBC performance, furnace cyclic testing was used with superalloys 1483, X4 and Hf-rich DS 247 substrates and high velocity oxygen fuel (HVOF)-NiCoCrAlYHfSi bond coatings at 1100 °C with 1-h cycles in air with 10% H 2O. With these coating and test conditions, there was no statistically-significant effect of substrate alloy on the average lifetime of the air plasma sprayed (APS) yttria-stabilized zirconia (YSZ) top coatingsmore » on small coupons. Using photo-stimulated luminescence piezospectroscopy maps at regular cycling intervals, the residual compressive stress in the α-Al 2O 3 scale underneath the YSZ top coating and on a bare bond coating was similar for all three substrates and delaminations occurred at roughly the same rate and frequency. As a result, x-ray fluorescence (XRF) measurements collected from the bare bond coating surface revealed higher Ti interdiffusion occurring with the 1483 substrate, which contained the highest Ti content.« less

Genome Dynamics Explain the Evolution of Flowering Time CCT Domain Gene Families in the Poaceae

PubMed Central

Cockram, James; Thiel, Thomas; Steuernagel, Burkhard; Stein, Nils; Taudien, Stefan; Bailey, Paul C.; O'Sullivan, Donal M.

2012-01-01

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken. PMID:23028921

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1: FUNCTIONAL INTERACTIONS BETWEEN THE KUNITZ-TYPE INHIBITOR DOMAIN-1 AND THE NEIGHBORING POLYCYSTIC KIDNEY DISEASE-LIKE DOMAIN.

PubMed

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J Preben; Andreasen, Peter A; Jensen, Jan K

2016-07-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Probing the electrostatics and pharmacologic modulation of sequence-specific binding by the DNA-binding domain of the ETS-family transcription factor PU.1: a binding affinity and kinetics investigation

PubMed Central

Munde, Manoj; Poon, Gregory M. K.; Wilson, W. David

2013-01-01

Members of the ETS family of transcription factors regulate a functionally diverse array of genes. All ETS proteins share a structurally-conserved but sequence-divergent DNA-binding domain, known as the ETS domain. Although the structure and thermodynamics of the ETS-DNA complexes are well known, little is known about the kinetics of sequence recognition, a facet that offers potential insight into its molecular mechanism. We have characterized DNA binding by the ETS domain of PU.1 by biosensor-surface plasmon resonance (SPR). SPR analysis revealed a striking kinetic profile for DNA binding by the PU.1 ETS domain. At low salt concentrations, it binds high-affinity cognate DNA with a very slow association rate constant (≤105 M−1 s−1), compensated by a correspondingly small dissociation rate constant. The kinetics are strongly salt-dependent but mutually balance to produce a relatively weak dependence in the equilibrium constant. This profile contrasts sharply with reported data for other ETS domains (e.g., Ets-1, TEL) for which high-affinity binding is driven by rapid association (>107 M−1 s−1). We interpret this difference in terms of the hydration properties of ETS-DNA binding and propose that at least two mechanisms of sequence recognition are employed by this family of DNA-binding domain. Additionally, we use SPR to demonstrate the potential for pharmacological inhibition of sequence-specific ETS-DNA binding, using the minor groove-binding distamycin as a model compound. Our work establishes SPR as a valuable technique for extending our understanding of the molecular mechanisms of ETS-DNA interactions as well as developing potential small-molecule agents for biotechnological and therapeutic purposes. PMID:23416556

Family resources study: part 1: family resources, family function and caregiver strain in childhood cancer

PubMed Central

2011-01-01

Background Severe illness can disrupt family life, cause family dysfunction, strain resources, and cause caregiver burden. The family's ability to cope with crises depends on their resources. This study sought to assess families of children with cancer in terms of family function-dysfunction, family caregiver strain and the adequacy of family resources using a new family resources assessment instrument. Methods This is a cross-sectional study involving 90 Filipino family caregivers of children undergoing cancer treatment. This used a self-administered questionnaire composed of a new 12-item family resources questionnaire (SCREEM-RES) based on the SCREEM method of analysis, Family APGAR to assess family function-dysfunction; and Modified Caregiver Strain Index to assess strain in caring for the patient. Results More than half of families were either moderately or severely dysfunctional. Close to half of caregivers were either predisposed to strain or experienced severe strain, majority disclosed that their families have inadequate economic resources; many also report inaccessibility to medical help in the community and insufficient educational resources to understand and care for their patients. Resources most often reported as adequate were: family's faith and religion; help from within the family and from health providers. SCREEM-RES showed to be reliable with Cronbach's alpha of 0.80. There is good inter-item correlation between items in each domain: 0.24-0.70. Internal consistency reliability for each domain was also good: 0.40-0.92. Using 2-point scoring system, Cronbach's alpha were slightly lower: full scale (0.70) and for each domain 0.26-.82. Results showed evidence of association between family resources and family function based on the family APGAR but none between family resources and caregiver strain and between family function and caregiver strain. Conclusion Many Filipino families of children with cancer have inadequate resources, especially economic

Family resources study: part 1: family resources, family function and caregiver strain in childhood cancer.

PubMed

Panganiban-Corales, Avegeille T; Medina, Manuel F

2011-10-31

Severe illness can disrupt family life, cause family dysfunction, strain resources, and cause caregiver burden. The family's ability to cope with crises depends on their resources. This study sought to assess families of children with cancer in terms of family function-dysfunction, family caregiver strain and the adequacy of family resources using a new family resources assessment instrument. This is a cross-sectional study involving 90 Filipino family caregivers of children undergoing cancer treatment. This used a self-administered questionnaire composed of a new 12-item family resources questionnaire (SCREEM-RES) based on the SCREEM method of analysis, Family APGAR to assess family function-dysfunction; and Modified Caregiver Strain Index to assess strain in caring for the patient. More than half of families were either moderately or severely dysfunctional. Close to half of caregivers were either predisposed to strain or experienced severe strain, majority disclosed that their families have inadequate economic resources; many also report inaccessibility to medical help in the community and insufficient educational resources to understand and care for their patients. Resources most often reported as adequate were: family's faith and religion; help from within the family and from health providers. SCREEM-RES showed to be reliable with Cronbach's alpha of 0.80. There is good inter-item correlation between items in each domain: 0.24-0.70. Internal consistency reliability for each domain was also good: 0.40-0.92. Using 2-point scoring system, Cronbach's alpha were slightly lower: full scale (0.70) and for each domain 0.26-.82. Results showed evidence of association between family resources and family function based on the family APGAR but none between family resources and caregiver strain and between family function and caregiver strain. Many Filipino families of children with cancer have inadequate resources, especially economic; and are moderately or severely

Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

PubMed

Bainbridge, Travis W; DeAlmeida, Venita I; Izrael-Tomasevic, Anita; Chalouni, Cécile; Pan, Borlan; Goldsmith, Joshua; Schoen, Alia P; Quiñones, Gabriel A; Kelly, Ryan; Lill, Jennie R; Sandoval, Wendy; Costa, Mike; Polakis, Paul; Arnott, David; Rubinfeld, Bonnee; Ernst, James A

2014-01-01

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

Functional Pathways of Social Support for Mental Health in Work and Family Domains Among Chinese Scientific and Technological Professionals.

PubMed

Gan, Yiqun; Gan, Tingting; Chen, Zhiyan; Miao, Miao; Zhang, Kan

2015-10-01

This study investigated the role of social support in the complex pattern of associations among stressors, work-family interferences and depression in the domains of work and family. A questionnaire was administered to a nationwide sample of 11,419 Chinese science and technology professionals. Several structural equation models were specified to determine whether social support functioned as a predictor or a mediator. Using Mplus 5.0, we compared the moderation model, the independence model, the antecedent model and the mediation model. The results revealed that the relationship between work-family interference and social support was domain specific. The independence model fit the data best in the work domain. Both the moderation model and the antecedent model fit the family domain data equally well. The current study was conducted to answer the need for comprehensive investigations of cultural uniqueness in the antecedents of work-family interference. The domain specificity, i.e. the multiple channels of the functions of support in the family domain and not in the work domain, ensures that this study is unique and culturally specific. Copyright © 2014 John Wiley & Sons, Ltd.

SUPERFAMILY 1.75 including a domain-centric gene ontology method.

PubMed

de Lima Morais, David A; Fang, Hai; Rackham, Owen J L; Wilson, Derek; Pethica, Ralph; Chothia, Cyrus; Gough, Julian

2011-01-01

The SUPERFAMILY resource provides protein domain assignments at the structural classification of protein (SCOP) superfamily level for over 1400 completely sequenced genomes, over 120 metagenomes and other gene collections such as UniProt. All models and assignments are available to browse and download at http://supfam.org. A new hidden Markov model library based on SCOP 1.75 has been created and a previously ignored class of SCOP, coiled coils, is now included. Our scoring component now uses HMMER3, which is in orders of magnitude faster and produces superior results. A cloud-based pipeline was implemented and is publicly available at Amazon web services elastic computer cloud. The SUPERFAMILY reference tree of life has been improved allowing the user to highlight a chosen superfamily, family or domain architecture on the tree of life. The most significant advance in SUPERFAMILY is that now it contains a domain-based gene ontology (GO) at the superfamily and family levels. A new methodology was developed to ensure a high quality GO annotation. The new methodology is general purpose and has been used to produce domain-based phenotypic ontologies in addition to GO.

Degradation of TBC Systems in Environments Relevant to Advanced Gas Turbines for IGCC Systems

NASA Astrophysics Data System (ADS)

Bohna, Nathaniel Allan

Plasma sprayed (PS) thermal barrier coatings (TBCs) are used to provide thermal insulation for the hottest components in gas turbines. Zirconia stabilized with 7wt% yttria (7YSZ) is the most common ceramic top coat used for turbine blades. The 7YSZ coating can be degraded by the buildup of fly-ash deposits which can arise from the fuel source (coal/biomass) used in the combustion process in gas turbines. Fly-ash from the integrated gasification combined cycle (IGCC) process can result from coal-based syngas and also from ambient air which passes through the system. TBCs are also exposed to harsh gas environments containing CO2, SO2, and steam. As presented in this thesis, degradation from the combined effects of fly-ash and harsh gas atmosphere can severely limit TBC lifetimes. It is well established that degradation at very high temperatures (≥1250°C) from deposits consisting of the oxides CaO-MgO-Al2O3-SiO 2 results from extensive liquid silicate infiltration into the porous top coat of the YSZ. This infiltration causes early failure resulting from chemical and/or mechanical damage to the ceramic layer. Damage resulting from liquid infiltration, however, is not typically considered at relatively lower temperatures around 1100°C because liquid silicates would not be expected to form from the oxides in the deposit. A key focus of this study is to assess the mode and extent of TBC degradation at 1100°C in cases when some amount of liquid forms owing to the presence of K2SO4 as a minor ash constituent. Two types of liquid infiltrations are observed depending on the principal oxide (i.e., CaO or SiO2) in the deposit. The degradation is primarily the result of mechanical damage, which results from infiltration caused by the interaction of liquid K2SO4 with either the CaO or SiO2. The TBCs used in this work are representative of commonly used coatings used in the hottest sections of land-based gas turbines. The specimens consist of 7YSZ top coats deposited on

Structural and functional properties of the N transcriptional activation domain of thyroid transcription factor-1: similarities with the acidic activation domains.

PubMed Central

Tell, G; Perrone, L; Fabbro, D; Pellizzari, L; Pucillo, C; De Felice, M; Acquaviva, R; Formisano, S; Damante, G

1998-01-01

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development. PMID:9425125

Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor).

PubMed

Okumura, Masayo; Yamakawa, Hisashi; Ohara, Osamu; Owaribe, Katsushi

2002-02-22

BPAG1 (bullous pemphigoid antigen 1) was originally identified as a 230-kDa hemidesmosomal protein and belongs to the plakin family, because it consists of a plakin domain, a coiled-coil rod domain and a COOH-terminal intermediate filament binding domain. To date, alternatively spliced products of BPAG1, BPAG1e, and BPAG1n are known. BPAG1e is expressed in epithelial tissues and localized to hemidesmosomes, on the other hand, BPAG1n is expressed in neural tissues and muscles and has an actin binding domain at the NH(2)-terminal of BPAG1e. BPAG1 is also known as a gene responsible for Dystonia musculorum (dt) neurodegeneration syndrome of the mouse. Another plakin family protein MACF (microtubule actin cross-linking factor) has also an actin binding domain and the plakin domain at the NH(2)-terminal. However, in contrast to its high homology with BPAG1 at the NH(2)-terminal, the COOH-terminal structure of MACF, including a microtubule binding domain, resembles dystrophin rather than plakins. Here, we investigated RNAs and proteins expressed from the BPAG1 locus and suggest novel alternative splicing variants, which include one consisting of the COOH-terminal domain structure homologous to MACF. The results indicate that BPAG1 has three kinds of cytoskeletal binding domains and seems to play an important role in linking the different types of cytoskeletons.

Hetero-oligomerization among the TIF family of RBCC/TRIM domain-containing nuclear cofactors: a potential mechanism for regulating the switch between coactivation and corepression.

PubMed

Peng, Hongzhuang; Feldman, Irina; Rauscher, Frank J

2002-07-12

The RING-B box-coiled-coil (RBCC) motif (also re-named recently as the tripartite motif (TRIM)) is a widely distributed motif that is hypothesized to be a protein-protein interface. The RBCC/TRIM domain of the corepressor KAP-1 is both necessary and sufficient to interact directly with the transcription repressor KRAB domain. Each subdomain of the KAP-1-RBCC contributes directly to the oligomerization and/or ligand recognition. Little is known about the function or the natural binding ligands for the RBCC/TRIM domain of the other TIF family members. In order to investigate whether hetero-oligomerization might be a biological regulatory mechanism, we have evaluated the hetero-oligomerization potential of the TIF family members including KAP-1, TIF1alpha, TIF1gamma, and the RBCC/TRIM family members including PML1, and MID1. We have reconstituted and characterized the oligomerization for these proteins using baculovirus and mammalian expression systems by biochemical approaches. Our data indicate that the RBCC/TRIM domains of KAP-1, TIF1alpha and TIF1gamma exist in a homo-oligomeric state. However, there is little cross-talk between KAP-1 and other TIF family members, suggesting that a high degree of specificity for oligomerization interface and ligand recognition is intrinsically built into the RBCC/TRIM domain of KAP-1. Finally, we demonstrate that TIF1alpha interacts with TIF1gamma and the coiled-coil region of TIF1gamma is necessary for this interaction. The hetero-oligomerization between TIF1alpha and TIF1gamma implies a potential regulatory mechanism for transcriptional regulation. (c) 2002 Elsevier Science Ltd.

Endophilin-A1 BAR domain interaction with arachidonyl CoA.

PubMed

Petoukhov, Maxim V; Weissenhorn, Winfried; Svergun, Dmitri I

2014-01-01

Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

Biochemical and structural characterization of a novel family of cystathionine beta-synthase domain proteins fused to a Zn ribbon-like domain.

PubMed

Proudfoot, Michael; Sanders, Stephen A; Singer, Alex; Zhang, Rongguang; Brown, Greg; Binkowski, Andrew; Xu, Linda; Lukin, Jonathan A; Murzin, Alexey G; Joachimiak, Andrzej; Arrowsmith, Cheryl H; Edwards, Aled M; Savchenko, Alexei V; Yakunin, Alexander F

2008-01-04

We have identified a novel family of proteins, in which the N-terminal cystathionine beta-synthase (CBS) domain is fused to the C-terminal Zn ribbon domain. Four proteins were overexpressed in Escherichia coli and purified: TA0289 from Thermoplasma acidophilum, TV1335 from Thermoplasma volcanium, PF1953 from Pyrococcus furiosus, and PH0267 from Pyrococcus horikoshii. The purified proteins had a red/purple color in solution and an absorption spectrum typical of rubredoxins (Rds). Metal analysis of purified proteins revealed the presence of several metals, with iron and zinc being the most abundant metals (2-67% of iron and 12-74% of zinc). Crystal structures of both mercury- and iron-bound TA0289 (1.5-2.0 A resolution) revealed a dimeric protein whose intersubunit contacts are formed exclusively by the alpha-helices of two cystathionine beta-synthase subdomains, whereas the C-terminal domain has a classical Zn ribbon planar architecture. All proteins were reversibly reduced by chemical reductants (ascorbate or dithionite) or by the general Rd reductase NorW from E. coli in the presence of NADH. Reduced TA0289 was found to be capable of transferring electrons to cytochrome C from horse heart. Likewise, the purified Zn ribbon protein KTI11 from Saccharomyces cerevisiae had a purple color in solution and an Rd-like absorption spectrum, contained both iron and zinc, and was reduced by the Rd reductase NorW from E. coli. Thus, recombinant Zn ribbon domains from archaea and yeast demonstrate an Rd-like electron carrier activity in vitro. We suggest that, in vivo, some Zn ribbon domains might also bind iron and therefore possess an electron carrier activity, adding another physiological role to this large family of important proteins.

Desmin myopathy with severe cardiomyopathy in a Uruguayan family due to a codon deletion in a new location within the desmin 1A rod domain.

PubMed

Vernengo, Luis; Chourbagi, Oussama; Panuncio, Ana; Lilienbaum, Alain; Batonnet-Pichon, Sabrina; Bruston, Francine; Rodrigues-Lima, Fernando; Mesa, Rosario; Pizzarossa, Carlos; Demay, Laurence; Richard, Pascale; Vicart, Patrick; Rodriguez, Maria-Mirta

2010-03-01

Desmin myopathy is a heterogeneous neuromuscular disorder characterized by skeletal myopathy and cardiomyopathy, inherited mostly in an autosomal dominant pattern. We report a five generation Uruguayan family with severe cardiomyopathy and skeletal myopathy. Its most striking features are: atrial dilation, arrhythmia, conduction block and sudden death due to conduction impairment. Affected skeletal muscle shows alteration of mitochondria with paracrystallin inclusions and granulofilamentous material scattered in the muscle fibres. This family carries an unusual deletion p.E114del within the 1A rod domain of desmin. Transfected cells expressing the mutated desmin show punctuated and speckled cytoplasmic aggregates. The mutation causes a local conformational change in heptads a/d residues and charge positions. These findings lead to the hypothesis that coiled-coil interactions may be impaired, resulting in severe alterations in the desmin network. This is the first time that a mutation affecting this domain in the desmin molecule is described in a desminopathy. Copyright 2010. Published by Elsevier B.V.

Domain organization, genomic structure, evolution, and regulation of expression of the aggrecan gene family.

PubMed

Schwartz, N B; Pirok, E W; Mensch, J R; Domowicz, M S

1999-01-01

Proteoglycans are complex macromolecules, consisting of a polypeptide backbone to which are covalently attached one or more glycosaminoglycan chains. Molecular cloning has allowed identification of the genes encoding the core proteins of various proteoglycans, leading to a better understanding of the diversity of proteoglycan structure and function, as well as to the evolution of a classification of proteoglycans on the basis of emerging gene families that encode the different core proteins. One such family includes several proteoglycans that have been grouped with aggrecan, the large aggregating chondroitin sulfate proteoglycan of cartilage, based on a high number of sequence similarities within the N- and C-terminal domains. Thus far these proteoglycans include versican, neurocan, and brevican. It is now apparent that these proteins, as a group, are truly a gene family with shared structural motifs on the protein and nucleotide (mRNA) levels, and with nearly identical genomic organizations. Clearly a common ancestral origin is indicated for the members of the aggrecan family of proteoglycans. However, differing patterns of amplification and divergence have also occurred within certain exons across species and family members, leading to the class-characteristic protein motifs in the central carbohydrate-rich region exclusively. Thus the overall domain organization strongly suggests that sequence conservation in the terminal globular domains underlies common functions, whereas differences in the central portions of the genes account for functional specialization among the members of this gene family.

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

PubMed

Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

Characterization of the molecular basis of group II intron RNA recognition by CRS1-CRM domains.

PubMed

Keren, Ido; Klipcan, Liron; Bezawork-Geleta, Ayenachew; Kolton, Max; Shaya, Felix; Ostersetzer-Biran, Oren

2008-08-22

CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain.

Deletion of RUNX1 exons 1 and 2 associated with familial platelet disorder with propensity to acute myeloid leukemia.

PubMed

Cavalcante de Andrade Silva, Marcela; Krepischi, Ana Cristina Victorino; Kulikowski, Leslie Domenici; Zanardo, Evelin Aline; Nardinelli, Luciana; Leal, Aline Medeiros; Costa, Silvia Souza; Muto, Nair Hideki; Rocha, Vanderson; Velloso, Elvira Deolinda Rodrigues Pereira

2018-04-01

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) associated with RUNX1 mutations is an autosomal dominant disorder included in the group of the myeloid neoplasms with germ line predisposition. We describe two brothers who were diagnosed with hematological malignancies (one with AML and the other with T-cell lymphoblastic lymphoma). There was a history of leukemia in the paternal family and two of their siblings presented with low platelet counts and no history of significant bleeding. A microdeletion encompassing exons 1-2 of RUNX1 (outside the cluster region of the Runt Homology domain and the transactivation domain) was detected in six family members using array-CGH and MLPA validation. A low platelet count was not present in all deletion carriers and, therefore, it should not be used as an indication for screening in suspected families and family members. Copyright © 2018 Elsevier Inc. All rights reserved.

The threshold bootstrap clustering: a new approach to find families or transmission clusters within molecular quasispecies.

PubMed

Prosperi, Mattia C F; De Luca, Andrea; Di Giambenedetto, Simona; Bracciale, Laura; Fabbiani, Massimiliano; Cauda, Roberto; Salemi, Marco

2010-10-25

Phylogenetic methods produce hierarchies of molecular species, inferring knowledge about taxonomy and evolution. However, there is not yet a consensus methodology that provides a crisp partition of taxa, desirable when considering the problem of intra/inter-patient quasispecies classification or infection transmission event identification. We introduce the threshold bootstrap clustering (TBC), a new methodology for partitioning molecular sequences, that does not require a phylogenetic tree estimation. The TBC is an incremental partition algorithm, inspired by the stochastic Chinese restaurant process, and takes advantage of resampling techniques and models of sequence evolution. TBC uses as input a multiple alignment of molecular sequences and its output is a crisp partition of the taxa into an automatically determined number of clusters. By varying initial conditions, the algorithm can produce different partitions. We describe a procedure that selects a prime partition among a set of candidate ones and calculates a measure of cluster reliability. TBC was successfully tested for the identification of type-1 human immunodeficiency and hepatitis C virus subtypes, and compared with previously established methodologies. It was also evaluated in the problem of HIV-1 intra-patient quasispecies clustering, and for transmission cluster identification, using a set of sequences from patients with known transmission event histories. TBC has been shown to be effective for the subtyping of HIV and HCV, and for identifying intra-patient quasispecies. To some extent, the algorithm was able also to infer clusters corresponding to events of infection transmission. The computational complexity of TBC is quadratic in the number of taxa, lower than other established methods; in addition, TBC has been enhanced with a measure of cluster reliability. The TBC can be useful to characterise molecular quasipecies in a broad context.

Structure and DNA-Binding Sites of the SWI1 AT-rich Interaction Domain (ARID) Suggest Determinants for Sequence-Specific DNA Recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Suhkmann; Zhang, Ziming; Upchurch, Sean

2004-04-16

2 ARID is a homologous family of DNA-binding domains that occur in DNA binding proteins from a wide variety of species, ranging from yeast to nematodes, insects, mammals and plants. SWI1, a member of the SWI/SNF protein complex that is involved in chromatin remodeling during transcription, contains the ARID motif. The ARID domain of human SWI1 (also known as p270) does not select for a specific DNA sequence from a random sequence pool. The lack of sequence specificity shown by the SWI1 ARID domain stands in contrast to the other characterized ARID domains, which recognize specific AT-rich sequences. We havemore » solved the three-dimensional structure of human SWI1 ARID using solution NMR methods. In addition, we have characterized non-specific DNA-binding by the SWI1 ARID domain. Results from this study indicate that a flexible long internal loop in ARID motif is likely to be important for sequence specific DNA-recognition. The structure of human SWI1 ARID domain also represents a distinct structural subfamily. Studies of ARID indicate that boundary of the DNA binding structural and functional domains can extend beyond the sequence homologous region in a homologous family of proteins. Structural studies of homologous domains such as ARID family of DNA-binding domains should provide information to better predict the boundary of structural and functional domains in structural genomic studies. Key Words: ARID, SWI1, NMR, structural genomics, protein-DNA interaction.« less

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

PubMed

Konno, Kotaro; Shimura, Sachiko; Ueno, Chihiro; Arakawa, Toru; Nakamura, Masatoshi

2018-03-01

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants. Copyright © 2018 Elsevier Ltd. All rights reserved.

Dimerization of the docking/adaptor protein HEF1 via a carboxy-terminal helix-loop-helix domain.

PubMed

Law, S F; Zhang, Y Z; Fashena, S J; Toby, G; Estojak, J; Golemis, E A

1999-10-10

HEF1, p130(Cas), and Efs define a family of multidomain docking proteins which plays a central coordinating role for tyrosine-kinase-based signaling related to cell adhesion. HEF1 function has been specifically implicated in signaling pathways important for cell adhesion and differentiation in lymphoid and epithelial cells. While the SH3 domains and SH2-binding site domains (substrate domains) of HEF1 family proteins are well characterized and binding partners known, to date the highly conserved carboxy-terminal domains of the three proteins have lacked functional definition. In this study, we have determined that the carboxy-terminal domain of HEF1 contains a divergent helix-loop-helix (HLH) motif. This motif mediates HEF1 homodimerization and HEF1 heterodimerization with a recognition specificity similar to that of the transcriptional regulatory HLH proteins Id2, E12, and E47. We had previously demonstrated that the HEF1 carboxy-terminus expressed as a separate domain in yeast reprograms cell division patterns, inducing constitutive pseudohyphal growth. Here we show that pseudohyphal induction by HEF1 requires an intact HLH, further supporting the idea that this motif has an effector activity for HEF1, and implying that HEF1 pseudohyphal activity derives in part from interactions with yeast helix-loop-helix proteins. These combined results provide initial insight into the mode of function of the HEF1 carboxy-terminal domain and suggest that the HEF1 protein may interact with cellular proteins which control differentiation. Copyright 1999 Academic Press.

Characterization of the Materials Synthesized by High Pressure-High Temperature Treatment of a Polymer Derived t-BC2N Ceramic

PubMed Central

Matizamhuka, Wallace R.; Sigalas, Iakovos; Herrmann, Mathias; Dubronvinsky, Leonid; Dubrovinskaia, Natalia; Miyajima, Nobuyoshi; Mera, Gabriela; Riedel, Ralf

2011-01-01

Bulk B-C-N materials were synthesized under static high thermobaric conditions (20 GPa and 2,000 °C) in a multianvil apparatus from a polymer derived t-BC1.97N ceramic. The bulk samples were characterised using X-ray synchrotron radiation and analytical transmission electron microscopy in combination with electron energy loss spectroscopy. Polycrystalline B-C-N materials with a cubic type structure were formed under the applied reaction conditions, but the formation of a ternary cubic diamond-like c-BC2N compound, could not be unambiguously confirmed. PMID:28824124

The SAM domains of Anks family proteins are critically involved in modulating the degradation of EphA receptors.

PubMed

Kim, Jieun; Lee, Haeryung; Kim, Yujin; Yoo, Sooyeon; Park, Eunjeong; Park, Soochul

2010-04-01

We recently reported that the phosphotyrosine-binding (PTB) domain of Anks family proteins binds to EphA8, thereby positively regulating EphA8-mediated signaling pathways. In the current study, we identified a potential role for the SAM domains of Anks family proteins in EphA signaling. We found that SAM domains of Anks family proteins directly bind to ubiquitin, suggesting that Anks proteins regulate the degradation of ubiquitinated EphA receptors. Consistent with the role of Cbl ubiquitin ligases in the degradation of Eph receptors, our results revealed that the ubiquitin ligase c-Cbl induced the ubiquitination and degradation of EphA8 upon ligand binding. Ubiquitinated EphA8 also bound to the SAM domains of Odin, a member of the Anks family proteins. More importantly, the overexpression of wild-type Odin protected EphA8 and EphA2 from undergoing degradation following ligand stimulation and promoted EphA-mediated inhibition of cell migration. In contrast, a SAM domain deletion mutant of Odin strongly impaired the function of endogenous Odin, suggesting that the mutant functions in a dominant-negative manner. An analysis of Odin-deficient primary embryonic fibroblasts indicated that Odin levels play a critical role in regulating the stability of EphA2 in response to ligand stimulation. Taken together, our studies suggest that the SAM domains of Anks family proteins play a pivotal role in enhancing the stability of EphA receptors by modulating the ubiquitination process.

1H, 13C, and 15N resonance assignments for Escherichia coli ytfP, a member of the broadly conserved UPF0131 protein domain family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aramini, James M.; Swapna, G.V.T.; Huang, Yuanpeng

2005-11-01

Protein ytfP from Escherichia coli (Swiss-Prot ID: YTFP-ECOLI; NESG target ID: ER111; Wunderlich et al., 2004) is a 113-residue member of the UPF0131 protein family (Pfam ID: PF03674) of unknown function. This domain family is found in organisms from all three kingdoms, archaea, eubacteria and eukaryotes. Using triple resonance NMR techniques, we have determined 97% of backbone and 91% of side chain 1H, 13C, and 15N resonance assignments. The chemical shift and 3J(HN?Ha) scalar coupling data reveal a mixed a/b topology,????????. BMRB deposit with Accession No. 6448. Reference: Wunderlich et al. (2004) Proteins, 56, 181?187.

The family B1 GPCR: structural aspects and interaction with accessory proteins.

PubMed

Couvineau, Alain; Laburthe, Marc

2012-01-01

G protein coupled receptors (GPCRs) play a crucial role in physiology and pathophysiology in humans. Beside the large family A (rhodopsin-like receptors) and family C GPCR (metabotropic glutamate receptors), the small family B1 GPCR (secretin-like receptors) includes important receptors such as vasoactive intestinal peptide receptors (VPAC), pituitary adenylyl cyclase activating peptide receptor (PAC1R), secretin receptor (SECR), growth hormone releasing factor receptor (GRFR), glucagon receptor (GCGR), glucagon like-peptide 1 and 2 receptors (GLPR), gastric inhibitory peptide receptor (GIPR), parathyroid hormone receptors (PTHR), calcitonin receptors (CTR) and corticotropin-releasing factor receptors (CRFR). They represent very promising targets for the development of drugs having therapeutical impact on many diseases such as chronic inflammation, neurodegeneration, diabetes, stress and osteoporosis. Over the past decade, structure-function relationship studies have demonstrated that the N-terminal ectodomain (N-ted) of family B1 receptors plays a pivotal role in natural ligand recognition. Structural analysis of some family B1 GPCR N-teds revealed the existence of a Sushi domain fold consisting of two antiparallel β sheets stabilized by three disulfide bonds and a salt bridge. The family B1 GPCRs promote cellular responses through a signaling pathway including predominantly the Gsadenylyl cyclase-cAMP pathway activation. Family B1 GPCRs also interact with a few accessory proteins which play a role in cell signaling, receptor expression and/or pharmacological profiles of receptors. These accessory proteins may represent new targets for the design of new drugs. Here, we review the current knowledge regarding: i) the structure of family B1 GPCR binding domain for natural ligands and ii) the interaction of family B1 GPCRs with accessory proteins.

Interrole conflict and self-efficacy to manage work and family demands mediate the relationships of job and family demands with stress in the job and family domains.

PubMed

Smoktunowicz, Ewelina; Cieslak, Roman; Demerouti, Evangelia

2017-09-01

This study derives from Work-Home Resources model (ten Brummelhuis, L. L., & Bakker, A. B. (2012). A resource perspective on the work-home interface: The work-home resources model. American Psychologist, 67(7), 545-556. doi: 10.1037/a0027974 ) and Social Cognitive Theory (Bandura, A. (1986). Social foundations of thought and action: A social cognitive theory. Englewood Cliffs, NJ, US: Prentice-Hall, Inc.) to investigate mechanisms responsible for the effect of job and family demands on work- and family-related perceived stress. We hypothesized that interrole conflict and self-efficacy to manage work and family demands operate either independently or sequentially transmitting the effects of demands on perceived stress. A sample of 100 employees of various occupations participated in the study conducted online in two waves: Time 1 (T1) and Time 2 (T2) with a three-month interval. Regression analysis with bootstrapping was applied. Interrole conflict (T1) did not mediate the relationships between demands (T1) and perceived stress (T2), whereas self-efficacy (T1) mediated only those between family demands (T1) and stress (T2). However, data supported the sequential mediation hypotheses: Demands (T1) were associated with increased interrole conflict (T1) which in turn decreased self-efficacy (T1) and ultimately resulted in the elevated perceived stress at work and in the family (T2). Demands originating in one domain can impact stress both in the same and other life areas through the sequence of interrole conflict and context-specific self-efficacy.

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

1H, 15N and 13C resonance assignments of the J-domain of co-chaperone Sis1 from Saccharomyces cerevisiae.

PubMed

Pinheiro, Glaucia M S; Amorim, Gisele C; Iqbal, Anwar; Ramos, C H I; Almeida, Fabio C L

2018-04-30

Protein folding in the cell is usually aided by molecular chaperones, from which the Hsp70 (Hsp = heat shock protein) family has many important roles, such as aiding nascent folding and participating in translocation. Hsp70 has ATPase activity which is stimulated by binding to the J-domain present in co-chaperones from the Hsp40 family. Hsp40s have many functions, as for instance the binding to partially folded proteins to be delivered to Hsp70. However, the presence of the J-domain characterizes Hsp40s or, by this reason, as J-proteins. The J-domain alone can stimulate Hsp70 ATPase activity. Apparently, it also maintains the same conformation as in the whole protein although structural information on full J-proteins is still missing. This work reports the 1 H, 15 N and 13 C resonance assignments of the J-domain of a Hsp40 from Saccharomyces cerevisiae, named Sis1. Secondary structure and order parameter prediction from chemical shifts are also reported. Altogether, the data show that Sis1 J-domain is highly structured and predominantly formed by α-helices, results that are in very good agreement with those previously reported for the crystallographic structure.

Status of Tamil Language in Singapore: An Analysis of Family Domain

ERIC Educational Resources Information Center

Kadakara, Shanmugam

2015-01-01

This paper addresses the phenomenon of Language Maintenance and Language Shift through a qualitative study of Tamil language in the family domain in Singapore. The influence of Singapore's bilingual policy and the institutional support offered for maintenance of Tamil language provide the context in which the central research problem of the status…

ATP Binding to p97/VCP D1 Domain Regulates Selective Recruitment of Adaptors to Its Proximal N-Domain

PubMed Central

Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

2012-01-01

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell. PMID:23226521

ATP binding to p97/VCP D1 domain regulates selective recruitment of adaptors to its proximal N-domain.

PubMed

Chia, Wei Sheng; Chia, Diana Xueqi; Rao, Feng; Bar Nun, Shoshana; Geifman Shochat, Susana

2012-01-01

p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD-related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.

Regulation of mTORC1 by PI3K signaling.

PubMed

Dibble, Christian C; Cantley, Lewis C

2015-09-01

The class I phosphoinositide 3-kinase (PI3K)-mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling network directs cellular metabolism and growth. Activation of mTORC1 [composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8(mLST8), 40-kDa proline-rich Akt substrate (PRAS40), and DEP domain-containing mTOR-interacting protein (DEPTOR)] depends on the Ras-related GTPases (Rags) and Ras homolog enriched in brain (Rheb) GTPase and requires signals from amino acids, glucose, oxygen, energy (ATP), and growth factors (including cytokines and hormones such as insulin). Here we discuss the signal transduction mechanisms through which growth factor-responsive PI3K signaling activates mTORC1. We focus on how PI3K-dependent activation of Akt and spatial regulation of the tuberous sclerosis complex (TSC) complex (TSC complex) [composed of TSC1, TSC2, and Tre2-Bub2-Cdc16-1 domain family member 7 (TBC1D7)] switches on Rheb at the lysosome, where mTORC1 is activated. Integration of PI3K- and amino acid-dependent signals upstream of mTORC1 at the lysosome is detailed in a working model. A coherent understanding of the PI3K-mTORC1 network is imperative as its dysregulation has been implicated in diverse pathologies including cancer, diabetes, autism, and aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cold Shock Domain Family Members YB-1 and MSY4 Share Essential Functions during Murine Embryogenesis▿ †

PubMed Central

Lu, Zhi Hong; Books, Jason T.; Ley, Timothy J.

2006-01-01

Three cold shock domain (CSD) family members (YB-1, MSY2, and MSY4) exist in vertebrate species ranging from frogs to humans. YB-1 is expressed throughout embryogenesis and is ubiquitously expressed in adult animals; it protects cells from senescence during periods of proliferative stress. YB-1-deficient embryos die unexpectedly late in embryogenesis (embryonic day 18.5 [E18.5] to postnatal day 1) with a runting phenotype. We have now determined that MSY4, but not MSY2, is also expressed during embryogenesis; its abundance declines substantially from E9.5 to E17.5 and is undetectable on postnatal day 1(adult mice express MSY4 in testes only). Whole-mount analysis revealed similar patterns of YB-1 and MSY4 RNA expression in E11.5 embryos. To determine whether MSY4 delays the death of YB-1-deficient embryos, we created and analyzed MSY4-deficient mice and then generated YB-1 and MSY4 double-knockout embryos. MSY4 is dispensable for normal development and survival, but the testes of adult mice have excessive spermatocyte apoptosis and seminiferous tubule degeneration. Embryos doubly deficient for YB-1 and MSY4 are severely runted and die much earlier (E8.5 to E11.5) than YB-1-deficient embryos, suggesting that MSY4 indeed shares critical cellular functions with YB-1 in the embryonic tissues where they are coexpressed. PMID:16954378

Conservation and divergence of C-terminal domain structure in the retinoblastoma protein family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liban, Tyler J.; Medina, Edgar M.; Tripathi, Sarvind

The retinoblastoma protein (Rb) and the homologous pocket proteins p107 and p130 negatively regulate cell proliferation by binding and inhibiting members of the E2F transcription factor family. The structural features that distinguish Rb from other pocket proteins have been unclear but are critical for understanding their functional diversity and determining why Rb has unique tumor suppressor activities. We describe here important differences in how the Rb and p107 C-terminal domains (CTDs) associate with the coiled-coil and marked-box domains (CMs) of E2Fs. We find that although CTD–CM binding is conserved across protein families, Rb and p107 CTDs show clear preferences formore » different E2Fs. A crystal structure of the p107 CTD bound to E2F5 and its dimer partner DP1 reveals the molecular basis for pocket protein–E2F binding specificity and how cyclin-dependent kinases differentially regulate pocket proteins through CTD phosphorylation. Our structural and biochemical data together with phylogenetic analyses of Rb and E2F proteins support the conclusion that Rb evolved specific structural motifs that confer its unique capacity to bind with high affinity those E2Fs that are the most potent activators of the cell cycle.« less

The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

PubMed Central

Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

2016-01-01

One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain*

PubMed Central

Yates, Luke A.; Lumb, Craig N.; Brahme, Nina N.; Zalyte, Ruta; Bird, Louise E.; De Colibus, Luigi; Owens, Raymond J.; Calderwood, David A.; Sansom, Mark S. P.; Gilbert, Robert J. C.

2012-01-01

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species. PMID:23132860

Assignment of protein sequences to existing domain and family classification systems: Pfam and the PDB.

PubMed

Xu, Qifang; Dunbrack, Roland L

2012-11-01

Automating the assignment of existing domain and protein family classifications to new sets of sequences is an important task. Current methods often miss assignments because remote relationships fail to achieve statistical significance. Some assignments are not as long as the actual domain definitions because local alignment methods often cut alignments short. Long insertions in query sequences often erroneously result in two copies of the domain assigned to the query. Divergent repeat sequences in proteins are often missed. We have developed a multilevel procedure to produce nearly complete assignments of protein families of an existing classification system to a large set of sequences. We apply this to the task of assigning Pfam domains to sequences and structures in the Protein Data Bank (PDB). We found that HHsearch alignments frequently scored more remotely related Pfams in Pfam clans higher than closely related Pfams, thus, leading to erroneous assignment at the Pfam family level. A greedy algorithm allowing for partial overlaps was, thus, applied first to sequence/HMM alignments, then HMM-HMM alignments and then structure alignments, taking care to join partial alignments split by large insertions into single-domain assignments. Additional assignment of repeat Pfams with weaker E-values was allowed after stronger assignments of the repeat HMM. Our database of assignments, presented in a database called PDBfam, contains Pfams for 99.4% of chains >50 residues. The Pfam assignment data in PDBfam are available at http://dunbrack2.fccc.edu/ProtCid/PDBfam, which can be searched by PDB codes and Pfam identifiers. They will be updated regularly.

Backbone assignment of the little finger domain of a Y-family DNA polymerase.

PubMed

Ma, Dejian; Fowler, Jason D; Suo, Zucai

2011-10-01

Sulfolobus solfataricus DNA polymerase IV (Dpo4), a prototype Y-family DNA polymerase, contains a unique little finger domain besides a catalytic core. Here, we report the chemical shift assignments for the backbone nitrogens, α and β carbons, and amide protons of the little finger domain of Dpo4. This work and our published backbone assignment for the catalytic core provide the basis for investigating the conformational dynamics of Dpo4 during catalysis using solution NMR spectroscopy.

Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

PubMed

Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

2013-08-01

Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Identification of YTH Domain-Containing Proteins as the Readers for N1-Methyladenosine in RNA.

PubMed

Dai, Xiaoxia; Wang, Tianlu; Gonzalez, Gwendolyn; Wang, Yinsheng

2018-06-05

N1-methyladenosine (m 1 A) is an important post-transcriptional modification in RNA; however, the exact biological role of m 1 A remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of m 1 A in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to m 1 A in RNA. We also found that Trp 432 in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N 6 -methyladenosine (m 6 A), is required for its recognition of m 1 A. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and m 1 A sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to m 1 A in cells. Together, our results uncovered YTH domain-containing proteins as readers for m 1 A in RNA and provided new insight into the functions of m 1 A in RNA biology.

Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins

PubMed Central

Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

2016-01-01

Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells. PMID:26826387

Plant homologs of mammalian MBT-domain protein-regulated KDM1 histone lysine demethylases do not interact with plant Tudor/PWWP/MBT-domain proteins.

PubMed

Sadiq, Irfan; Keren, Ido; Citovsky, Vitaly

2016-02-19

Histone lysine demethylases of the LSD1/KDM1 family play important roles in epigenetic regulation of eukaryotic chromatin, and they are conserved between plants and animals. Mammalian LSD1 is thought to be targeted to its substrates, i.e., methylated histones, by an MBT-domain protein SFMBT1 that represents a component of the LSD1-based repressor complex and binds methylated histones. Because MBT-domain proteins are conserved between different organisms, from animals to plants, we examined whether the KDM1-type histone lysine demethylases KDM1C and FLD of Arabidopsis interact with the Arabidopsis Tudor/PWWP/MBT-domain SFMBT1-like proteins SL1, SL2, SL3, and SL4. No such interaction was detected using the bimolecular fluorescence complementation assay in living plant cells. Thus, plants most likely direct their KDM1 chromatin-modifying enzymes to methylated histones of the target chromatin by a mechanism different from that employed by the mammalian cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiple-interactions among EMILIN1 and EMILIN2 N- and C-terminal domains.

PubMed

Bot, Simonetta; Andreuzzi, Eva; Capuano, Alessandra; Schiavinato, Alvise; Colombatti, Alfonso; Doliana, Roberto

2015-01-01

EMILIN1 and EMILIN2 belong to a family of extracellular matrix glycoproteins characterized by the N-terminal cysteine-rich EMI domain, a long segment with high probabilty for coiled-coil structure formation and a C-terminal gC1q domain. To study EMILIN1 and EMILIN2 interaction and assembly we have applied qualitative and quantitative two hybrid systems using constructs corresponding to the gC1q and EMI domains. The identified interactions were further confirmed in yeast extracts of co-transfected cells followed by co-immunoprecipitation. The data indicated that gC1q domains are able to self-interact as well as to interact one each other and with the EMI domains, but no self interactions were detected between the EMI domains. Furthermore EMILINs interactions were studied in 293-EBNA cells co-transfected with full lenght EMILIN1 and EMILIN2 constructs. Specific antibodies were able to co-immunoprecipitate EMILINs, indicating that also full-lenght proteins can give rise to non-covalent homo- and hetero-multimers even if reduced and alkylated before mixing. Immunofluorescence analysis on mouse cell cultures and tissues sections with specific antibodies showed co-distribution of EMILIN1 and EMILIN2. Thus, we can hypothesize that EMILINs multimers are formed by head-to-tail interaction between C-terminal and N-terminal domains of EMILIN1 and/or EMILIN2 but also by tail-to-tail interaction between gC1q domains. These multiple interactions may regulate homo-typic and/or hetero-typic linear and eventually lateral branching assemblies of EMILIN1 and EMILIN2 in tissues. Copyright © 2014. Published by Elsevier B.V.

Assignment of protein sequences to existing domain and family classification systems: Pfam and the PDB

PubMed Central

Dunbrack, Roland L.

2012-01-01

Motivation: Automating the assignment of existing domain and protein family classifications to new sets of sequences is an important task. Current methods often miss assignments because remote relationships fail to achieve statistical significance. Some assignments are not as long as the actual domain definitions because local alignment methods often cut alignments short. Long insertions in query sequences often erroneously result in two copies of the domain assigned to the query. Divergent repeat sequences in proteins are often missed. Results: We have developed a multilevel procedure to produce nearly complete assignments of protein families of an existing classification system to a large set of sequences. We apply this to the task of assigning Pfam domains to sequences and structures in the Protein Data Bank (PDB). We found that HHsearch alignments frequently scored more remotely related Pfams in Pfam clans higher than closely related Pfams, thus, leading to erroneous assignment at the Pfam family level. A greedy algorithm allowing for partial overlaps was, thus, applied first to sequence/HMM alignments, then HMM–HMM alignments and then structure alignments, taking care to join partial alignments split by large insertions into single-domain assignments. Additional assignment of repeat Pfams with weaker E-values was allowed after stronger assignments of the repeat HMM. Our database of assignments, presented in a database called PDBfam, contains Pfams for 99.4% of chains >50 residues. Availability: The Pfam assignment data in PDBfam are available at http://dunbrack2.fccc.edu/ProtCid/PDBfam, which can be searched by PDB codes and Pfam identifiers. They will be updated regularly. Contact: Roland.Dunbracks@fccc.edu PMID:22942020

Structural features of a close homologue of L1 (CHL1) in the mouse: a new member of the L1 family of neural recognition molecules.

PubMed

Holm, J; Hillenbrand, R; Steuber, V; Bartsch, U; Moos, M; Lübbert, H; Montag, D; Schachner, M

1996-08-01

We have identified a close homologue of L1 (CHL1) in the mouse. CHL1 comprises an N-terminal signal sequence, six immunoglobulin (Ig)-like domains, 4.5 fibronectin type III (FN)-like repeats, a transmembrane domain and a C-terminal, most likely intracellular domain of approximately 100 amino acids. CHL1 is most similar in its extracellular domain to chicken Ng-CAM (approximately 40% amino acid identity), followed by mouse L1, chicken neurofascin, chicken Nr-CAM, Drosophila neuroglian and zebrafish L1.1 (37-28% amino acid identity), and mouse F3, rat TAG-1 and rat BIG-1 (approximately 27% amino acid identity). The similarity with other members of the Ig superfamily [e.g. neural cell adhesion molecule (N-CAM), DCC, HLAR, rse] is 16-11%. The intracellular domain is most similar to mouse and chicken Nr-CAM, mouse and rat neurofascin (approximately 60% amino acid identity) followed by chicken neurofascin and Ng-CAM, Drosophila neuroglian and zebrafish L1.1 and L1.2 (approximately 40% amino acid identity). Besides the high overall homology and conserved modular structure among previously recognized members of the L1 family (mouse/human L1/rat NILE; chicken Ng-CAM; chicken/mouse Nr-CAM; Drosophila neuroglian; zebrafish L1.1 and L1.2; chicken/mouse neurofascin/rat ankyrin-binding glycoprotein), criteria characteristic of L1 were identified with regard to the number of amino acids between positions of conserved amino acid residues defining distances within and between two adjacent Ig-like domains and FN-like repeats. These show a collinearity in the six Ig-like domains and four adjacent FN-like repeats that is remarkably conserved between L1 and molecules containing these modules (designated the L1 family cassette), including the GPI-linked forms of the F3 subgroup (mouse F3/chicken F11/human CNTN1; rat BIG-1/mouse PANG; rat TAG-1/mouse TAX-1/chicken axonin-1). The colorectal cancer molecule (DCC), previously introduced as an N-CAM-like molecule, conforms to the L1 family

Exploring metazoan evolution through dynamic and holistic changes in protein families and domains

USDA-ARS?s Scientific Manuscript database

Understanding proteome evolution is important for deciphering processes that drive species diversity and adaptation. Herein, the dynamics of change in protein families and protein domains over the course of metazoan evolution was explored. Change, as defined by birth/death and duplication/deletion ...

Crystal structures of the coil 2B fragment and the globular tail domain of human lamin B1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruan, Jianbin; Xu, Chao; Bian, Chuanbing

2012-07-18

We present here the crystal structures of human lamin B1 globular tail domain and coiled 2B domain, which adopt similar folds to Ig-like domain and coiled-coil domain of lamin A, respectively. Despite the overall similarity, we found an extra intermolecular disulfide bond in the lamin B1 coil 2B domain, which does not exist in lamin A/C. In addition, the structural analysis indicates that interactions at the lamin B1 homodimer interface are quite different from those of lamin A/C. Thus our research not only reveals the diversely formed homodimers among lamin family members, but also sheds light on understanding the importantmore » roles of lamin B1 in forming the nuclear lamina matrix.« less

Molecular mechanism for the subversion of the retromer coat by the Legionella effector RidL

PubMed Central

Romano-Moreno, Miguel; Rojas, Adriana L.; Williamson, Chad D.; Lucas, María; Isupov, Michail N.; Bonifacino, Juan S.; Machner, Matthias P.; Hierro, Aitor

2017-01-01

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29–VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes. PMID:29229824

The Arabidopsis PLAT domain protein1 is critically involved in abiotic stress tolerance.

PubMed

Hyun, Tae Kyung; van der Graaff, Eric; Albacete, Alfonso; Eom, Seung Hee; Großkinsky, Dominik K; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

2014-01-01

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty.

The Arabidopsis PLAT Domain Protein1 Is Critically Involved in Abiotic Stress Tolerance

PubMed Central

Eom, Seung Hee; Großkinsky, Dominik K.; Böhm, Hannah; Janschek, Ursula; Rim, Yeonggil; Ali, Walid Wahid; Kim, Soo Young; Roitsch, Thomas

2014-01-01

Despite the completion of the Arabidopsis genome sequence, for only a relatively low percentage of the encoded proteins experimental evidence concerning their function is available. Plant proteins that harbour a single PLAT (Polycystin, Lipoxygenase, Alpha-toxin and Triacylglycerol lipase) domain and belong to the PLAT-plant-stress protein family are ubiquitously present in monocot and dicots. However, the function of PLAT-plant-stress proteins is still poorly understood. Therefore, we have assessed the function of the uncharacterised Arabidopsis PLAT-plant-stress family members through a combination of functional genetic and physiological approaches. PLAT1 overexpression conferred increased abiotic stress tolerance, including cold, drought and salt stress, while loss-of-function resulted in opposite effects on abiotic stress tolerance. Strikingly, PLAT1 promoted growth under non-stressed conditions. Abiotic stress treatments induced PLAT1 expression and caused expansion of its expression domain. The ABF/ABRE transcription factors, which are positive mediators of abscisic acid signalling, activate PLAT1 promoter activity in transactivation assays and directly bind to the ABRE elements located in this promoter in electrophoretic mobility shift assays. This suggests that PLAT1 represents a novel downstream target of the abscisic acid signalling pathway. Thus, we showed that PLAT1 critically functions as positive regulator of abiotic stress tolerance, but also is involved in regulating plant growth, and thereby assigned a function to this previously uncharacterised PLAT domain protein. The functional data obtained for PLAT1 support that PLAT-plant-stress proteins in general could be promising targets for improving abiotic stress tolerance without yield penalty. PMID:25396746

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

PubMed Central

Hong, Zebin; De Meulemeester, Laura; Jacobi, Annemarie; Pedersen, Jan Skov; Morth, J. Preben; Andreasen, Peter A.; Jensen, Jan K.

2016-01-01

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a type I transmembrane protein and inhibitor of several serine proteases, including hepatocyte growth factor activator and matriptase. The protein is essential for development as knock-out mice die in utero due to placental defects caused by misregulated extracellular proteolysis. HAI-1 contains two Kunitz-type inhibitor domains (Kunitz), which are generally thought of as a functionally self-contained protease inhibitor unit. This is not the case for HAI-1, where our results reveal how interdomain interactions have evolved to stimulate the inhibitory activity of an integrated Kunitz. Here we present an x-ray crystal structure of an HAI-1 fragment covering the internal domain and Kunitz-1. The structure reveals not only that the previously uncharacterized internal domain is a member of the polycystic kidney disease domain family but also how the two domains engage in interdomain interactions. Supported by solution small angle x-ray scattering and a combination of site-directed mutagenesis and functional assays, we show that interdomain interactions not only stabilize the fold of the internal domain but also stimulate the inhibitory activity of Kunitz-1. By completing our structural characterization of the previously unknown N-terminal region of HAI-1, we provide new insight into the interplay between tertiary structure and the inhibitory activity of a multidomain protease inhibitor. We propose a previously unseen mechanism by which the association of an auxiliary domain stimulates the inhibitory activity of a Kunitz-type inhibitor (i.e. the first structure of an intramolecular interaction between a Kunitz and another domain). PMID:27189939

Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains

PubMed Central

Cronin, Thomas C; DiNitto, Jonathan P; Czech, Michael P; Lambright, David G

2004-01-01

The pleckstrin homology (PH) domains of the homologous proteins Grp1 (general receptor for phosphoinositides), ARNO (Arf nucleotide binding site opener), and Cytohesin-1 bind phosphatidylinositol (PtdIns) 3,4,5-trisphosphate with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the β1/β2 loop exhibit dual specificity for PtdIns(3,4,5)P3 and PtdIns(4,5)P2. The structural basis for this dramatic specificity switch is not apparent from the known modes of phosphoinositide recognition. Here, we report crystal structures for dual specificity variants of the Grp1 and ARNO PH domains in either the unliganded form or in complex with the head groups of PtdIns(4,5)P2 and PtdIns(3,4,5)P3. Loss of contacts with the β1/β2 loop with no significant change in head group orientation accounts for the significant decrease in PtdIns(3,4,5)P3 affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PtdIns(4,5)P2 is explained by a novel binding mode, in which the glycine insertion alleviates unfavorable interactions with the β1/β2 loop. These observations are supported by a systematic mutational analysis of the determinants of phosphoinositide recognition. PMID:15359279

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

PubMed

Kükenshöner, Tim; Schmit, Nadine Eliane; Bouda, Emilie; Sha, Fern; Pojer, Florence; Koide, Akiko; Seeliger, Markus; Koide, Shohei; Hantschel, Oliver

2017-05-05

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

TIM-family proteins inhibit HIV-1 release

PubMed Central

Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

2014-01-01

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

OsBRI1 Activates BR Signaling by Preventing Binding between the TPR and Kinase Domains of OsBSK3 via Phosphorylation.

PubMed

Zhang, Baowen; Wang, Xiaolong; Zhao, Zhiying; Wang, Ruiju; Huang, Xiahe; Zhu, Yali; Yuan, Li; Wang, Yingchun; Xu, Xiaodong; Burlingame, Alma L; Gao, Yingjie; Sun, Yu; Tang, Wenqiang

2016-02-01

Many plant receptor kinases transduce signals through receptor-like cytoplasmic kinases (RLCKs); however, the molecular mechanisms that create an effective on-off switch are unknown. The receptor kinase BR INSENSITIVE1 (BRI1) transduces brassinosteroid (BR) signal by phosphorylating members of the BR-signaling kinase (BSK) family of RLCKs, which contain a kinase domain and a C-terminal tetratricopeptide repeat (TPR) domain. Here, we show that the BR signaling function of BSKs is conserved in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) and that the TPR domain of BSKs functions as a "phospho-switchable" autoregulatory domain to control BSKs' activity. Genetic studies revealed that OsBSK3 is a positive regulator of BR signaling in rice, while in vivo and in vitro assays demonstrated that OsBRI1 interacts directly with and phosphorylates OsBSK3. The TPR domain of OsBSK3, which interacts directly with the protein's kinase domain, serves as an autoinhibitory domain to prevent OsBSK3 from interacting with bri1-SUPPRESSOR1 (BSU1). Phosphorylation of OsBSK3 by OsBRI1 disrupts the interaction between its TPR and kinase domains, thereby increasing the binding between OsBSK3's kinase domain and BSU1. Our results not only demonstrate that OsBSK3 plays a conserved role in regulating BR signaling in rice, but also provide insight into the molecular mechanism by which BSK family proteins are inhibited under basal conditions but switched on by the upstream receptor kinase BRI1. © 2016 American Society of Plant Biologists. All Rights Reserved.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases

PubMed Central

Lambert, Abigail R.; Kuhar, Ryan; Jarjour, Jordan; Kulshina, Nadia; Parmeggiani, Fabio; Danaher, Patrick; Gano, Jacob; Baker, David; Stoddard, Barry L.; Scharenberg, Andrew M.

2012-01-01

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40–70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ∼70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications. PMID:22684507

Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1).

PubMed

Li, Huilin; Jin, Hui; Li, Yaqian; Liu, Dejian; Foda, Mohamed Frahat; Jiang, Yunbo; Luo, Rui

2017-09-01

Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Prioritisation of associations between protein domains and complex diseases using domain-domain interaction networks.

PubMed

Wang, W; Zhang, W; Jiang, R; Luan, Y

2010-05-01

It is of vital importance to find genetic variants that underlie human complex diseases and locate genes that are responsible for these diseases. Since proteins are typically composed of several structural domains, it is reasonable to assume that harmful genetic variants may alter structures of protein domains, affect functions of proteins and eventually cause disorders. With this understanding, the authors explore the possibility of recovering associations between protein domains and complex diseases. The authors define associations between protein domains and disease families on the basis of associations between non-synonymous single nucleotide polymorphisms (nsSNPs) and complex diseases, similarities between diseases, and relations between proteins and domains. Based on a domain-domain interaction network, the authors propose a 'guilt-by-proximity' principle to rank candidate domains according to their average distance to a set of seed domains in the domain-domain interaction network. The authors validate the method through large-scale cross-validation experiments on simulated linkage intervals, random controls and the whole genome. Results show that areas under receiver operating characteristic curves (AUC scores) can be as high as 77.90%, and the mean rank ratios can be as low as 21.82%. The authors further offer a freely accessible web interface for a genome-wide landscape of associations between domains and disease families.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

PubMed

Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

2016-03-27

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

An uncommon phenotype with familial central hypogonadism caused by a novel PROP1 gene mutant truncated in the transactivation domain.

PubMed

Reynaud, Rachel; Barlier, Anne; Vallette-Kasic, Sophie; Saveanu, Alexandru; Guillet, Marie-Pierre; Simonin, Gilbert; Enjalbert, Alain; Valensi, Paul; Brue, Thierry

2005-08-01

PROP1 gene mutations are usually associated with childhood onset GH and TSH deficiencies, whereas gonadotroph deficiency is diagnosed at pubertal age. We report a novel PROP1 mutation revealed by familial normosmic hypogonadotropic hypogonadism. We performed in vitro transactivation and DNA binding experiments to study functional consequences of this mutation. Three brothers were followed in the Department of Endocrinology of a French university hospital. These patients from a consanguineous kindred were referred for cryptorchidism and/or delayed puberty. Initial investigations revealed hypogonadotropic hypogonadism. One of the patients had psychomotor retardation, intracranial hypertension, and minor renal malformations. The brothers reached normal adult height and developed GH and TSH deficiencies after age 30. A novel homozygous nonsense mutation (W194X) was found in the PROP1 gene, indicating that the protein is truncated in its transactivation domain. Transfection studies confirmed the deleterious effect of this mutation, whose transactivation capacity was only 34.4% of that of the wild-type. Unexpectedly altered DNA-binding properties suggested that the C-terminal end of the factor plays a role in protein-DNA interaction. PROP1 mutations should be considered among the growing number of genetic causes of initially isolated hypogonadotropic hypogonadism. This report extends the phenotype variability associated with PROP1 mutations.

Enigmatic Moisture Effects on Al2O3 Scale and TBC Adhesion

NASA Technical Reports Server (NTRS)

Smialek, James L.

2008-01-01

Alumina scale adhesion to high temperature alloys is known to be affected primarily by sulfur segregation and reactive element additions. However adherent scales can become partially compromised by excessive strain energy and cyclic cracking. With time, exposure of such scales to moisture can lead to spontaneous interfacial decohesion, occurring while the samples are maintained at ambient conditions. Examples of this Moisture-Induced Delayed Spallation (MIDS) are presented for NiCrAl and single crystal superalloys, becoming more severe with sulfur level and cyclic exposure conditions. Similarly, delayed failure or Desk Top Spallation (DTS) results are reviewed for TBC s, culminating in the water drop failure test. Both phenomena are discussed in terms of moisture effects on bulk alumina and bulk aluminides. A mechanism is proposed based on hydrogen embrittlement and is supported by a cathodic hydrogen charging experiment. Hydroxylation of aluminum from the alloy interface appears to be the relevant basic reaction.

Akt1 binds focal adhesion kinase via the Akt1 kinase domain independently of the pleckstrin homology domain.

PubMed

Basson, M D; Zeng, B; Wang, S

2015-10-01

Akt1 and focal adhesion kinase (FAK) are protein kinases that play key roles in normal cell signaling. Individually, aberrant expression of these kinases has been linked to a variety of cancers. Together, Akt1/FAK interactions facilitate cancer metastasis by increasing cell adhesion under conditions of increased extracellular pressure. Pathological and iatrogenic sources of pressure arise from tumor growth against constraining stroma or direct perioperative manipulation. We previously reported that 15 mmHg increased extracellular pressure causes Akt1 to both directly interact with FAK and to phosphorylate and activate it. We investigated the nature of the Akt1/FAK binding by creating truncations of recombinant FAK, conjugated to glutathione S-transferase (GST), to pull down full-length Akt1. Western blots probing for Akt1 showed that FAK/Akt1 binding persisted in FAK truncations consisting of only amino acids 1-126, FAK(NT1), which contains the F1 subdomain of its band 4.1, ezrin, radixin, and moesin (FERM) domain. Using FAK(NT1) as bait, we then pulled down truncated versions of recombinant Akt1 conjugated to HA (human influenza hemagglutinin). Probes for GST-FAK(NT1) showed Akt1-FAK binding to occur in the absence of the both the Akt1 (N)-terminal pleckstrin homology (PH) domain and its adjacent hinge region. The Akt1 (C)-terminal regulatory domain was equally unnecessary for Akt1/FAK co-immunoprecipitation. Truncations involving the Akt1 catalytic domain showed that the domain by itself was enough to pull down FAK. Additionally, a fragment spanning from the PH domain to half way through the catalytic domain demonstrated increased FAK binding compared to full length Akt1. These results begin to delineate the Akt1/FAK interaction and can be used to manipulate their force-activated signal interactions. Furthermore, the finding that the N-terminal half of the Akt1 catalytic domain binds so strongly to FAK when cleaved from the rest of the protein may suggest a means

The PYRIN domain: A member of the death domain-fold superfamily

PubMed Central

Fairbrother, Wayne J.; Gordon, Nathaniel C.; Humke, Eric W.; O'Rourke, Karen M.; Starovasnik, Melissa A.; Yin, Jian-Ping; Dixit, Vishva M.

2001-01-01

PYRIN domains were identified recently as putative protein–protein interaction domains at the N-termini of several proteins thought to function in apoptotic and inflammatory signaling pathways. The ∼95 residue PYRIN domains have no statistically significant sequence homology to proteins with known three-dimensional structure. Using secondary structure prediction and potential-based fold recognition methods, however, the PYRIN domain is predicted to be a member of the six-helix bundle death domain-fold superfamily that includes death domains (DDs), death effector domains (DEDs), and caspase recruitment domains (CARDs). Members of the death domain-fold superfamily are well established mediators of protein–protein interactions found in many proteins involved in apoptosis and inflammation, indicating further that the PYRIN domains serve a similar function. An homology model of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1, a member of the Apaf-1/Ced-4 family of proteins, was constructed using the three-dimensional structures of the FADD and p75 neurotrophin receptor DDs, and of the Apaf-1 and caspase-9 CARDs, as templates. Validation of the model using a variety of computational techniques indicates that the fold prediction is consistent with the sequence. Comparison of a circular dichroism spectrum of the PYRIN domain of CARD7/DEFCAP/NAC/NALP1 with spectra of several proteins known to adopt the death domain-fold provides experimental support for the structure prediction. PMID:11514682

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCgamma1.

PubMed

Min, Lie; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

2009-12-15

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

TAIL1: an isthmin-like gene, containing type 1 thrombospondin-repeat and AMOP domain, mapped to ARVD1 critical region.

PubMed

Rossi, Valeria; Beffagna, Giorgia; Rampazzo, Alessandra; Bauce, Barbara; Danieli, Gian Antonio

2004-06-23

Isthmins represent a novel family of vertebrate secreted proteins containing one copy of the thrombospondin type 1 repeat (TSR), which in mammals is shared by several proteins with diverse biological functions, including cell adhesion, angiogenesis, and patterning of developing nervous system. We have determined the genomic organization of human TAIL1 (thrombospondin and AMOP containing isthmin-like 1), a novel isthmin-like gene encoding a protein that contains a TSR and a C-terminal AMOP domain (adhesion-associated domain in MUC4 and other proteins), characteristic of extracellular proteins involved in adhesion processes. TAIL1 gene encompasses more than 24.4 kb. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. Several consensus binding sites for the transcription factors Sp1 and MZF-1 were identified in this promoter region. In humans, TAIL1 gene is located on chromosome 14q24.3 within ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region; preliminary evidence suggests that it is expressed in several tissues, showing multiple alternative splicing.

The Sla2p/HIP1/HIP1R family: similar structure, similar function in endocytosis?

PubMed

Gottfried, Irit; Ehrlich, Marcelo; Ashery, Uri

2010-02-01

HIP1 (huntingtin interacting protein 1) has two close relatives: HIP1R (HIP1-related) and yeast Sla2p. All three members of the family have a conserved domain structure, suggesting a common function. Over the past decade, a number of studies have characterized these proteins using a combination of biochemical, imaging, structural and genetic techniques. These studies provide valuable information on binding partners, structure and dynamics of HIP1/HIP1R/Sla2p. In general, all suggest a role in CME (clathrin-mediated endocytosis) for the three proteins, though some differences have emerged. In this mini-review we summarize the current views on the roles of these proteins, while emphasizing the unique attributes of each family member.

A novel firmicute protein family related to the actinobacterial resuscitation-promoting factors by non-orthologous domain displacement.

PubMed

Ravagnani, Adriana; Finan, Christopher L; Young, Michael

2005-03-17

In Micrococcus luteus growth and resuscitation from starvation-induced dormancy is controlled by the production of a secreted growth factor. This autocrine resuscitation-promoting factor (Rpf) is the founder member of a family of proteins found throughout and confined to the actinobacteria (high G + C Gram-positive bacteria). The aim of this work was to search for and characterise a cognate gene family in the firmicutes (low G + C Gram-positive bacteria) and obtain information about how they may control bacterial growth and resuscitation. In silico analysis of the accessory domains of the Rpf proteins permitted their classification into several subfamilies. The RpfB subfamily is related to a group of firmicute proteins of unknown function, represented by YabE of Bacillus subtilis. The actinobacterial RpfB and firmicute YabE proteins have very similar domain structures and genomic contexts, except that in YabE, the actinobacterial Rpf domain is replaced by another domain, which we have called Sps. Although totally unrelated in both sequence and secondary structure, the Rpf and Sps domains fulfil the same function. We propose that these proteins have undergone "non-orthologous domain displacement", a phenomenon akin to "non-orthologous gene displacement" that has been described previously. Proteins containing the Sps domain are widely distributed throughout the firmicutes and they too fall into a number of distinct subfamilies. Comparative analysis of the accessory domains in the Rpf and Sps proteins, together with their weak similarity to lytic transglycosylases, provide clear evidence that they are muralytic enzymes. The results indicate that the firmicute Sps proteins and the actinobacterial Rpf proteins are cognate and that they control bacterial culturability via enzymatic modification of the bacterial cell envelope.

Comparative analysis of plant genomes allows the definition of the "Phytolongins": a novel non-SNARE longin domain protein family

PubMed Central

2009-01-01

Background Subcellular trafficking is a hallmark of eukaryotic cells. Because of their pivotal role in the process, a great deal of attention has been paid to the SNARE proteins. Most R-SNAREs, or "longins", however, also possess a highly conserved, N-terminal fold. This "longin domain" is known to play multiple roles in regulating SNARE activity and targeting via interaction with other trafficking proteins. However, the diversity and complement of longins in eukaryotes is poorly understood. Results Our comparative genome survey identified a novel family of longin-related proteins, dubbed the "Phytolongins" because they are specific to land plants. Phytolongins share with longins the N-terminal longin domain and the C-terminal transmembrane domain; however, in the central region, the SNARE motif is replaced by a novel region. Phylogenetic analysis pinpoints the Phytolongins as a derivative of the plant specific VAMP72 longin sub-family and allows elucidation of Phytolongin evolution. Conclusion "Longins" have been defined as R-SNAREs composed of both a longin domain and a SNARE motif. However, expressed gene isoforms and splice variants of longins are examples of non-SNARE motif containing longins. The discovery of Phytolongins, a family of non-SNARE longin domain proteins, together with recent evidence on the conservation of the longin-like fold in proteins involved in both vesicle fusion (e.g. the Trs20 tether) and vesicle formation (e.g. σ and μ adaptin) highlight the importance of the longin-like domain in protein trafficking and suggest that it was one of the primordial building blocks of the eukaryotic membrane-trafficking machinery. PMID:19889231

Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models.

PubMed

Khan, Sehrish Haider; Javed, Muhammad Rizwan; Qasim, Muhammad; Shahzadi, Samar; Jalil, Asma; Rehman, Shahid Ur

2014-01-01

Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the

Coiled-Coil Antagonism Regulates Activity of Venus Flytrap-Domain-Containing Sensor Kinases of the BvgS Family

PubMed Central

Lesne, Elodie; Dupré, Elian; Lensink, Marc F.; Locht, Camille

2018-01-01

ABSTRACT Bordetella pertussis controls the expression of its virulence regulon through the two-component system BvgAS. BvgS is a prototype for a family of multidomain sensor kinases. In BvgS, helical linkers connect periplasmic Venus flytrap (VFT) perception domains to a cytoplasmic Per-Arnt-Sim (PAS) domain and the PAS domain to the dimerization/histidine phosphotransfer (DHp) domain of the kinase. The two linkers can adopt coiled-coil structures but cannot do so simultaneously. The first linker forms a coiled coil in the kinase mode and the second in the phosphatase mode, with the other linker in both cases showing an increase in dynamic behavior. The intervening PAS domain changes its quaternary structure between the two modes. In BvgS homologues without a PAS domain, a helical “X” linker directly connects the VFT and DHp domains. Here, we used BvgS as a platform to characterize regulation in members of the PAS-less subfamily. BvgS chimeras of homologues with natural X linkers displayed various regulation phenotypes. We identified two distinct coiled-coil registers in the N- and C-terminal portions of the X linkers. Stable coil formation in the C-terminal moiety determines the phosphatase mode, similarly to BvgS; in contrast, coil formation in the N-terminal moiety along the other register leads to the kinase mode. Thus, antagonism between two registers in the VFT-DHp linker forms the basis for activity regulation in the absence of the PAS domain. The N and C moieties of the X linker play roles similar to those played by the two independent linkers in sensor kinases with a PAS domain, providing a unified mechanism of regulation for the entire family. PMID:29487240

Structural and Histone Binding Ability Characterizations of Human PWWP Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hong; Zeng, Hong; Lam, Robert

2013-09-25

The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members,more » implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical {beta}-barrel core, an insertion motif between the second and third {beta}-strands and a C-terminal {alpha}-helix bundle. Both the canonical {beta}-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones.« less

Resonance assignments for the substrate binding domain of Hsp70 chaperone Ssa1 from Saccharomyces cerevisiae.

PubMed

Hu, Wanhui; Wu, Huiwen; Zhang, Hong; Gong, Weibin; Perrett, Sarah

2015-10-01

Hsp70 chaperone proteins play crucial roles in the cell. Extensive structural and functional studies have been performed for bacterial and mammalian Hsp70s. Ssa1 from Saccharomyces cerevisiae is a member of the Hsp70 family. In vivo and biochemical studies on Ssa1 have revealed that it regulates prion propagation and the cell cycle. However, no structural data has been obtained for Ssa1 up to now. Here we report the almost complete (96 %) (1)H, (13)C, (15)N backbone and side chain NMR assignment of the 18.8 kDa Ssa1 substrate binding domain. The construct includes residues 382-554, which corresponds to the entire substrate binding domain and two following α-helices in homologous structures. The secondary structure predicted from the assigned chemical shifts is consistent with that of homologous Hsp70 substrate binding domains.

Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

PubMed Central

Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

2010-01-01

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

Systematic biochemical characterization of the SAM domains in Eph receptor family from Mus Musculus.

PubMed

Wang, Yue; Li, Qingxia; Zheng, Yunhua; Li, Gang; Liu, Wei

2016-05-13

The Eph receptor family is the largest subfamily of receptor tyrosine kinases and well-known for their pivotal roles in axon guidance, synaptogenesis, artery/venous differentiation and tumorigenesis, etc. Activation of the Eph receptor needs multimerization of the receptors. The intracellular C-terminal SAM domain of Eph receptor was reported to mediate self-association of Eph receptors via the homo SAM-SAM interaction. In this study, we systematically expressed and purified the SAM domain proteins of all fourteen Eph receptors of Mus musculus in Escherichia coli. The FPLC (fast protein liquid chromatography) results showed the recombinant SAM domains were highly homogeneous. Using CD (circular dichroism) spectrometry, we found that the secondary structure of all the SAM domains was typically alpha helical folded and remarkably similar. The thermo-stability tests showed that they were quite stable in solution. SEC-MALS (size exclusion chromatography coupled with multiple angle light scattering) results illustrated 200 μM Eph SAM domains behaved as good monomers in the size-exclusion chromatography. More importantly, DLS (dynamic light scattering) results revealed the overwhelming majority of SAM domains was not multimerized in solution either at 200 μM or 2000 μM protein concentration, which indicating the SAM domain alone was not sufficient to mediate the polymerization of Eph receptor. In summary, our studies provided the systematic biochemical characterizations of the Eph receptor SAM domains and implied their roles in Eph receptor mediated signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Proteomic differences with and without ozone-exposure in a smoking-induced emphysema lung model

PubMed Central

Uh, Soo-Taek; Koo, So-My; Jang, An Soo; Park, Sung Woo; Choi, Jae Sung; Kim, Yong-Hoon

2015-01-01

Background/Aims Acute exacerbations in chronic obstructive pulmonary disease may be related to air pollution, of which ozone is an important constituent. In this study, we investigated the protein profiles associated with ozone-induced exacerbations in a smoking-induced emphysema model. Methods Mice were divided into the following groups: group I, no smoking and no ozone (NS + NO); group II, no smoking and ozone (NS + O); group III, smoking and no ozone (S + NO); and group IV, smoking and ozone (S + O). Bronchoalveolar lavage, the mean linear intercept (MLI) on hematoxylin and eosin staining, nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS), and Western blotting analyses were performed. Results The MLIs of groups III (S + NO) and IV (S + O) (45 ± 2 and 44 ± 3 µm, respectively) were significantly higher than those of groups I (NS + NO) and II (NS + O) (26 ± 2 and 23 ± 2 µm, respectively; p < 0.05). Fourteen spots that showed significantly different intensities on image analyses of two-dimensional (2D) protein electrophoresis in group I (NS + NO) were identified by LC-MS/MS. The levels of six proteins were higher in group IV (S + O). The levels of vimentin, lactate dehydrogenase A, and triose phosphate isomerase were decreased by both smoking and ozone treatment in Western blotting and proteomic analyses. In contrast, TBC1 domain family 5 (TBC1D5) and lamin A were increased by both smoking and ozone treatment. Conclusions TBC1D5 could be a biomarker of ozone-induced lung injury in emphysema. PMID:25589837

AtRD22 and AtUSPL1, Members of the Plant-Specific BURP Domain Family Involved in Arabidopsis thaliana Drought Tolerance

PubMed Central

Harshavardhan, Vokkaliga Thammegowda; Van Son, Le; Seiler, Christiane; Junker, Astrid; Weigelt-Fischer, Kathleen; Klukas, Christian; Altmann, Thomas; Sreenivasulu, Nese; Bäumlein, Helmut; Kuhlmann, Markus

2014-01-01

Crop plants are regularly challenged by a range of environmental stresses which typically retard their growth and ultimately compromise economic yield. The stress response involves the reprogramming of approximately 4% of the transcriptome. Here, the behavior of AtRD22 and AtUSPL1, both members of the Arabidopsis thaliana BURP (BNM2, USP, RD22 and polygalacturonase isozyme) domain-containing gene family, has been characterized. Both genes are up-regulated as part of the abscisic acid (ABA) mediated moisture stress response. While AtRD22 transcript was largely restricted to the leaf, that of AtUSPL1 was more prevalent in the root. As the loss of function of either gene increased the plant's moisture stress tolerance, the implication was that their products act to suppress the drought stress response. In addition to the known involvement of AtUSPL1 in seed development, a further role in stress tolerance was demonstrated. Based on transcriptomic data and phenotype we concluded that the enhanced moisture stress tolerance of the two loss-of-function mutants is a consequence of an enhanced basal defense response. PMID:25333723

Novel Autophosphorylation Sites of Src Family Kinases Regulate Kinase Activity and SH2 Domain Binding Capacity

PubMed Central

Weir, Marion E.; Mann, Jacqueline E.; Corwin, Thomas; Fulton, Zachary W.; Hao, Jennifer M.; Maniscalco, Jeanine F.; Kenney, Marie C.; Roque, Kristal M. Roman; Chapdelaine, Elizabeth F.; Stelzl, Ulrich; Deming, Paula B.; Ballif, Bryan A.; Hinkle, Karen L.

2016-01-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

Association of Integrated Team-Based Care With Health Care Quality, Utilization, and Cost.

PubMed

Reiss-Brennan, Brenda; Brunisholz, Kimberly D; Dredge, Carter; Briot, Pascal; Grazier, Kyle; Wilcox, Adam; Savitz, Lucy; James, Brent

The value of integrated team delivery models is not firmly established. To evaluate the association of receiving primary care in integrated team-based care (TBC) practices vs traditional practice management (TPM) practices (usual care) with patient outcomes, health care utilization, and costs. A retrospective, longitudinal, cohort study to assess the association of integrating physical and mental health over time in TBC practices with patient outcomes and costs. Adult patients (aged ≥18 years) who received primary care at 113 unique Intermountain Healthcare Medical Group primary care practices from 2003 through 2005 and had yearly encounters with Intermountain Healthcare through 2013, including some patients who received care in both TBC and TPM practices. Receipt of primary care in TBC practices compared with TPM practices for patients treated in internal medicine, family practice, and geriatrics practices. Outcomes included 7 quality measures, 6 health care utilization measures, payments to the delivery system, and program investment costs. During the study period (January 2010-December 2013), 113,452 unique patients (mean age, 56.1 years; women, 58.9%) accounted for 163,226 person-years of exposure in 27 TBC practices and 171,915 person-years in 75 TPM practices. Patients treated in TBC practices compared with those treated in TPM practices had higher rates of active depression screening (46.1% for TBC vs 24.1% for TPM; odds ratio [OR], 1.91 [95% CI, 1.75 to 2.08), adherence to a diabetes care bundle (24.6% for TBC vs 19.5% for TPM; OR, 1.26 [95% CI, 1.11 to 1.42]), and documentation of self-care plans (48.4% for TBC vs 8.7% for TPM; OR, 5.59 [95% CI, 4.27 to 7.33]), lower proportion of patients with controlled hypertension (<140/90 mm Hg) (85.0% for TBC vs 97.7% for TPM; OR, 0.87 [95% CI, 0.80 to 0.95]), and no significant differences in documentation of advanced directives (9.6% for TBC vs 9.9% for TPM; OR, 0.97 [95% CI, 0.91 to 1.03]). Per 100 person

Miniature protein ligands for EVH1 domains: Interplay between affinity, specificity, and cell motility⊥

PubMed Central

Holtzman, Jennifer H.; Woronowicz, Kamil; Golemi-Kotra, Dasantila; Schepartz, Alanna

2008-01-01

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins--Mena, VASP, and Evl--are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. Our lab has previously reported a novel miniature protein, pGolemi, which binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven L. monocytogenes motility. Here, we use scanning mutagenesis to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation. PMID:17973491

Family Efficacy within Ethnically Diverse Families: A Qualitative Study.

PubMed

Kao, Tsui-Sui A; Caldwell, Cleopatra H

2017-03-01

Family efficacy, which refers to a family's belief in its ability to produce a desired outcome, has been shown to protect adolescents from risky health behaviors. Few studies have examined family efficacy within diverse populations, however, and understanding of how efficacy is framed and formed within the context of cultural and familial values is limited. This descriptive qualitative study examined sources of family efficacy within ethnically and socioeconomically diverse families, evaluating how such families develop and exercise family efficacy with the intent to protect adolescents from risky health behaviors (i.e., marijuana and alcohol use and early sexual activity). We collected qualitative data via two semi-structured interviews, 4-6 months apart, with 31 adolescents (ages 12-14) and their parent/s, for total of 148 one-on-one interviews. Thematic analysis identified three distinct domains of family efficacy: relational, pragmatic, and value-laden. Prior experiences and cultural background influenced the domain/s utilized by families. Significantly, families that consistently tapped into all three domains were able to effectively manage personal and family difficulties; these families also had family strategies in place to prevent adolescents from risky behaviors. Health professionals could utilize this concept of multidimensional family efficacy to promote health within culturally diverse families. © 2015 Family Process Institute.

Autoimmune Disease in a DFNA6/14/38 Family carrying a Novel Missense Mutation in WFS1

PubMed Central

Hildebrand, Michael S.; Sorensen, Jessica L.; Jensen, Maren; Kimberling, William J.; Smith, Richard J.H.

2008-01-01

Most familial cases of autosomal dominant low frequency sensorineural hearing loss (LFSNHL) are attributable to mutations in the Wolframin syndrome 1 (WFS1) gene at the DFNA6/14/38 locus. WFS1 mutations at this locus were first described in 2001 in six families segregating LFSNHL that was non-progressive below 2000 Hz; the causative mutations all clustered in the C-terminal domain of the wolframin protein. Mutations in WFS1 also cause Wolfram syndrome (WS), an autosomal recessive neurodegenerative disorder defined by diabetes mellitus, optic atrophy and often deafness, while numerous single nucleotide polymorphisms (SNPs) in WFS1 have been associated with increased risk for diabetes mellitus, psychiatric illnesses and Parkinson’s disease. This study was conducted in an American family segregating autosomal dominant LFSNHL. Two hearing impaired family members also had autoimmune diseases - Graves disease (GD) and Crohn’s disease (CD). Based on the low frequency audioprofile, mutation screening of WFS1 was completed and a novel missense mutation (c.2576G→A) that results in an arginine-to-glutamine substitution (p.R859Q) was identified in the C-terminal domain of the wolframin protein where most LFSNHL-causing mutations cluster. The family member with GD also carried polymorphisms in WFS1 that have been associated with other autoimmune diseases. PMID:18688868

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1.

PubMed

Prattes, Michael; Loibl, Mathias; Zisser, Gertrude; Luschnig, Daniel; Kappel, Lisa; Rössler, Ingrid; Grassegger, Manuela; Hromic, Altijana; Krieger, Elmar; Gruber, Karl; Pertschy, Brigitte; Bergler, Helmut

2017-03-17

AAA-ATPases fulfil essential roles in different cellular pathways and often act in form of hexameric complexes. Interaction with pathway-specific substrate and adaptor proteins recruits them to their targets and modulates their catalytic activity. This substrate dependent regulation of ATP hydrolysis in the AAA-domains is mediated by a non-catalytic N-terminal domain. The exact mechanisms that transmit the signal from the N-domain and coordinate the individual AAA-domains in the hexameric complex are still the topic of intensive research. Here, we present the characterization of a novel mutant variant of the eukaryotic AAA-ATPase Drg1 that shows dysregulation of ATPase activity and altered interaction with Rlp24, its substrate in ribosome biogenesis. This defective regulation is the consequence of amino acid exchanges at the interface between the regulatory N-domain and the adjacent D1 AAA-domain. The effects caused by these mutations strongly resemble those of pathological mutations of the AAA-ATPase p97 which cause the hereditary proteinopathy IBMPFD (inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia). Our results therefore suggest well conserved mechanisms of regulation between structurally, but not functionally related members of the AAA-family.

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

PubMed

Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

2016-04-01

Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. © 2016 Federation of European Biochemical Societies.

Sperm 1: a POU-domain gene transiently expressed immediately before meiosis I in the male germ cell.

PubMed Central

Andersen, B; Pearse, R V; Schlegel, P N; Cichon, Z; Schonemann, M D; Bardin, C W; Rosenfeld, M G

1993-01-01

Members of the POU-domain gene family encode for transcriptional regulatory molecules that are important for terminal differentiation of several organ systems, including anterior pituitary, sensory neurons, and B lymphocytes. We have identified a POU-domain factor, referred to as sperm 1 (Sprm-1). This factor is most related to the transactivator Oct-3/4, which is expressed in the early embryo, primordial germ cells, and the egg. However, in contrast with Oct-3/4, rat Sprm-1 is selectively expressed during a 36- to 48-hr period immediately preceding meiosis I in male germ cells. Although the POU-domain of Sprm-1 is divergent from the POU-domains of Oct-1 and Oct-2, random-site-selection assay reveals that Sprm-1 preferentially binds to a specific variant of the classic octamer DNA-response element in which the optimal sequence differs from that preferred by Oct-1 and Pit-1. These data suggest that the Sprm-1 gene encodes a DNA-binding protein that may exert a regulatory function in meiotic events that are required for terminal differentiation of the male germ cell. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7902581

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

PubMed Central

2009-01-01

The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold. PMID:19930717

Domain analyses of Usher syndrome causing Clarin-1 and GPR98 protein models

PubMed Central

Khan, Sehrish Haider; Javed, Muhammad Rizwan; Qasim, Muhammad; Shahzadi, Samar; Jalil, Asma; Rehman, Shahid ur

2014-01-01

Usher syndrome is an autosomal recessive disorder that causes hearing loss, Retinitis Pigmentosa (RP) and vestibular dysfunction. It is clinically and genetically heterogeneous disorder which is clinically divided into three types i.e. type I, type II and type III. To date, there are about twelve loci and ten identified genes which are associated with Usher syndrome. A mutation in any of these genes e.g. CDH23, CLRN1, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A and DFNB31 can result in Usher syndrome or non-syndromic deafness. These genes provide instructions for making proteins that play important roles in normal hearing, balance and vision. Studies have shown that protein structures of only seven genes have been determined experimentally and there are still three genes whose structures are unavailable. These genes are Clarin-1, GPR98 and Usherin. In the absence of an experimentally determined structure, homology modeling and threading often provide a useful 3D model of a protein. Therefore in the current study Clarin-1 and GPR98 proteins have been analyzed for signal peptide, domains and motifs. Clarin-1 protein was found to be without any signal peptide and consists of prokar lipoprotein domain. Clarin-1 is classified within claudin 2 super family and consists of twelve motifs. Whereas, GPR98 has a 29 amino acids long signal peptide and classified within GPCR family 2 having Concanavalin A-like lectin/glucanase superfamily. It was found to be consists of GPS and G protein receptor F2 domains and twenty nine motifs. Their 3D structures have been predicted using I-TASSER server. The model of Clarin-1 showed only α-helix but no beta sheets while model of GPR98 showed both α-helix and β sheets. The predicted structures were then evaluated and validated by MolProbity and Ramachandran plot. The evaluation of the predicted structures showed 78.9% residues of Clarin-1 and 78.9% residues of GPR98 within favored regions. The findings of present study has resulted in the

Unfurling of the band 4.1, ezrin, radixin, moesin (FERM) domain of the merlin tumor suppressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yogesha, S.D.; Sharff, Andrew J.; Giovannini, Marco

The merlin-1 tumor suppressor is encoded by the Neurofibromatosis-2 (Nf2) gene and loss-of-function Nf2 mutations lead to nervous system tumors in man and to several tumor types in mice. Merlin is an ERM (ezrin, radixin, moesin) family cytoskeletal protein that interacts with other ERM proteins and with components of cell-cell adherens junctions (AJs). Merlin stabilizes the links of AJs to the actin cytoskeleton. Thus, its loss destabilizes AJs, promoting cell migration and invasion, which in Nf2{sup +/-} mice leads to highly metastatic tumors. Paradoxically, the 'closed' conformation of merlin-1, where its N-terminal four-point-one, ezrin, radixin, moesin (FERM) domain binds tomore » its C-terminal tail domain, directs its tumor suppressor functions. Here we report the crystal structure of the human merlin-1 head domain when crystallized in the presence of its tail domain. Remarkably, unlike other ERM head-tail interactions, this structure suggests that binding of the tail provokes dimerization and dynamic movement and unfurling of the F2 motif of the FERM domain. We conclude the 'closed' tumor suppressor conformer of merlin-1 is in fact an 'open' dimer whose functions are disabled by Nf2 mutations that disrupt this architecture.« less

The interleukin (IL)-1 cytokine family--Balance between agonists and antagonists in inflammatory diseases.

PubMed

Palomo, Jennifer; Dietrich, Damien; Martin, Praxedis; Palmer, Gaby; Gabay, Cem

2015-11-01

The interleukin (IL)-1 family of cytokines comprises 11 members, including 7 pro-inflammatory agonists (IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ) and 4 defined or putative antagonists (IL-1R antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38) exerting anti-inflammatory activities. Except for IL-1Ra, IL-1 cytokines do not possess a leader sequence and are secreted via an unconventional pathway. In addition, IL-1β and IL-18 are produced as biologically inert pro-peptides that require cleavage by caspase-1 in their N-terminal region to generate active proteins. N-terminal processing is also required for full activity of IL-36 cytokines. The IL-1 receptor (IL-1R) family comprises 10 members and includes cytokine-specific receptors, co-receptors and inhibitory receptors. The signaling IL-1Rs share a common structure with three extracellular immunoglobulin (Ig) domains and an intracellular Toll-like/IL-1R (TIR) domain. IL-1 cytokines bind to their specific receptor, which leads to the recruitment of a co-receptor and intracellular signaling. IL-1 cytokines induce potent inflammatory responses and their activity is tightly controlled at the level of production, protein processing and maturation, receptor binding and post-receptor signaling by naturally occurring inhibitors. Some of these inhibitors are IL-1 family antagonists, while others are IL-1R family members acting as membrane-bound or soluble decoy receptors. An imbalance between agonist and antagonist levels can lead to exaggerated inflammatory responses. Several genetic modifications or mutations associated with dysregulated IL-1 activity and autoinflammatory disorders were identified in mouse models and in patients. These findings paved the road to the successful use of IL-1 inhibitors in diseases that were previously considered as untreatable. Copyright © 2015 Elsevier Ltd. All rights reserved.

Family, Employment, and Individual Resource-Based Antecedents of Maternal Work-Family Enrichment from Infancy through Middle Childhood

PubMed Central

Zhou, Nan; Buehler, Cheryl

2015-01-01

This study used data from the NICHD Study of Early Child Care and Youth Development (N = 1,019) to examine family, employment, and individual antecedents of maternal work-family enrichment from infancy through middle childhood. Work-family conflict and important confounding factors were controlled. From the family domain, higher income-to-needs ratio and social support were associated with higher work-family enrichment. From the employment domain, greater job rewards, benefits of employment for children, and work commitment were associated with higher work-family enrichment. From the individual domain, higher maternal education and extroversion were associated with higher work-family enrichment. No family, employment, and individual characteristics were associated with work-family conflict across time except for partner intimacy. In general, the results supported antecedents of work-family enrichment that supply needed resources. The present study contributed to the literature by identifying antecedents of maternal work-family enrichment across early child developmental stages, which goes beyond examinations of particular life stages and a work-family conflict perspective. Implications for theory and practice are discussed. PMID:26641483

New domains of neural cell-adhesion molecule L1 implicated in X-linked hydrocephalus and MASA syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jouet, M.; Kenwick, S.; Moncla, A.

1995-06-01

The neural cell-adhesion molecule L1 is involved in intercellular recognition and neuronal migration in the CNS. Recently, we have shown that mutations in the gene encoding L1 are responsible for three related disorders; X-linked hydrocephalus, MASA (mental retardation, aphasia, shuffling gait, and adducted thumbs) syndrome, and spastic paraplegia type I (SPG1). These three disorders represent a clinical spectrum that varies not only between families but sometimes also within families. To date, 14 independent L1 mutations have been reported and shown to be disease causing. Here we report nine novel L1 mutations in X-linked hydrocephalus and MASA-syndrome families, including the firstmore » examples of mutations affecting the fibronectin type III domains of the molecule. They are discussed in relation both to phenotypes and to the insights that they provide into L1 function. 39 refs., 5 figs., 3 tabs.« less

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

2000-01-01

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

PubMed

Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

2014-07-11

The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance

PubMed Central

Mulinta, Ras; Yao, Sylvia Y. M.; Ng, Amy M. L.; Cass, Carol E.; Young, James D.

2017-01-01

The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function. PMID:28385889

Characterization of a Gene Family Encoding SEA (Sea-urchin Sperm Protein, Enterokinase and Agrin)-Domain Proteins with Lectin-Like and Heme-Binding Properties from Schistosoma japonicum

PubMed Central

Mbanefo, Evaristus Chibunna; Kikuchi, Mihoko; Huy, Nguyen Tien; Shuaibu, Mohammed Nasir; Cherif, Mahamoud Sama; Yu, Chuanxin; Wakao, Masahiro; Suda, Yasuo; Hirayama, Kenji

2014-01-01

Background We previously identified a novel gene family dispersed in the genome of Schistosoma japonicum by retrotransposon-mediated gene duplication mechanism. Although many transcripts were identified, no homolog was readily identifiable from sequence information. Methodology/Principal Findings Here, we utilized structural homology modeling and biochemical methods to identify remote homologs, and characterized the gene products as SEA (sea-urchin sperm protein, enterokinase and agrin)-domain containing proteins. A common extracellular domain in this family was structurally similar to SEA-domain. SEA-domain is primarily a structural domain, known to assist or regulate binding to glycans. Recombinant proteins from three members of this gene family specifically interacted with glycosaminoglycans with high affinity, with potential implication in ligand acquisition and immune evasion. Similar approach was used to identify a heme-binding site on the SEA-domain. The heme-binding mode showed heme molecule inserted into a hydrophobic pocket, with heme iron putatively coordinated to two histidine axial ligands. Heme-binding properties were confirmed using biochemical assays and UV-visible absorption spectroscopy, which showed high affinity heme-binding (K D = 1.605×10−6 M) and cognate spectroscopic attributes of hexa-coordinated heme iron. The native proteins were oligomers, antigenic, and are localized on adult worm teguments and gastrodermis; major host-parasite interfaces and site for heme detoxification and acquisition. Conclusions The results suggest potential role, at least in the nucleation step of heme crystallization (hemozoin formation), and as receptors for heme uptake. Survival strategies exploited by parasites, including heme homeostasis mechanism in hemoparasites, are paramount for successful parasitism. Thus, assessing prospects for application in disease intervention is warranted. PMID:24416467

[Expression, crystallization and crystallographic study of the 1st IgV domain of human CD96].

PubMed

Jiang, Wenjing; Zhang, Shuijun; Yan, Jinghua; Guo, Ning

2013-05-01

CD96 (Tactile) is an adhesion receptor expressed mainly on activated T cells, NK cells. As a family member of the immunoglobulin-like cell receptor, CD96 consists of three immunoglobulin-like domains (V1, V2/C and C) in the extracellular region. Recent studies have shown that the 1st IgV domain of CD96 (CD96V1) plays an essential role in cell adhesion and NK cell-mediated killing. In this study, the 1st IgV domain of human CD96 (hCD96V1) was cloned and expressed in Escherichia coli (BL21). The soluble protein was obtained by refolding of the hCD96V1 inclusion bodies. From analytical ultracentrifugation, we could predict that CD96 V1 maily exists as dimer with approximate molecular weight of 26.9 kDa. The protein was then successfully crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.9 angstrom resolution and belonged to space group P21, with unit-cell parameters a = 35.1, b = 69.5, c = 49.6A, alpha=gamma=90 degrees, beta=105.4 degrees.

Functional, structural and phylogenetic analysis of domains underlying the Al sensitivity of the aluminum-activated malate/anion transporter, TaALMT1.

PubMed

Ligaba, Ayalew; Dreyer, Ingo; Margaryan, Armine; Schneider, David J; Kochian, Leon; Piñeros, Miguel

2013-12-01

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Gender, Ethnicity, Ethnic Identity, and Language Choices of Malaysian Youths: The Case of the Family Domain

ERIC Educational Resources Information Center

Granhemat, Mehdi; Abdullah, Ain Nadzimah

2017-01-01

This study examined the relationships between gender, ethnicity, ethnic identity, and language choices of Malaysian multilingual youths in the family domain of language use. Five hundred undergraduate students who belonged to different Malaysian ethnic groups were selected as participants of the study. The participant aged between 17 to 25 years…

Intensive hypermethylation of the CpG island of Ras association domain family 1A in hepatitis B virus-associated hepatocellular carcinomas.

PubMed

Zhong, Sheng; Yeo, Winnie; Tang, Mandy W; Wong, Nathalie; Lai, Paul B S; Johnson, Phillip J

2003-08-15

The human Ras association domain family 1A gene (RASSF1A) is a newly isolated tumor suppressor gene. In this study, we analyzed the methylation status of the promoter region of RASSF1A using bisulfite sequencing and PCR-RFLP in four liver cancer cell lines (Hep3B, HepG(2), SK-HEP-1, and Huh-7) and a cohort of 43 hepatitis B virus-associated hepatocellular carcinoma (HCC) tissues and their corresponding nontumor tissue specimens. The methylation of the CpG islands in the RASSF1A promoter was not detected in 4 samples of normal liver tissue or 10 samples of peripheral blood mononuclear cells from normal subjects. However, the CpG islands were completely methylated, and transcription of the RASSF1A was silenced in the four cell lines. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine reactivated the expression of RASSF1A in the Hep3B and HepG2 cells. In 41 of 43 (95%) HCC specimens studied, the promoter region of RASSF1A was intensively methylated at its CpG sites. Although heterogeneous methylation was also detected in 16 of the 23 (70%) corresponding nontumorous tissues analyzed, the level of methylation was significantly lower than in the corresponding tumor tissues. HCC has the highest incidence of promoter methylation of RASSF1A among all malignancies yet reported suggesting that hypermethylation of the CpG island promoter of RASSF1A may play an important pathological role in this tumor.

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

PubMed Central

Liang, H; Olejniczak, E T; Mao, X; Nettesheim, D G; Yu, L; Thompson, C B; Fesik, S W

1994-01-01

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins. Images PMID:7972119

ADAM disintegrin-like domain recognition by the lymphocyte integrins α4β1 and α4β7

PubMed Central

Bridges, Lance C.; Sheppard, Dean; Bowditch, Ron D.

2004-01-01

The ADAM (a disintegrin and metalloprotease) family of proteins possess both proteolytic and adhesive domains. We have established previously that the disintegrin domain of ADAM28, an ADAM expressed by human lymphocytes, is recognized by the integrin α4β1. The present study characterizes the integrin binding properties of the disintegrin-like domains of human ADAM7, ADAM28 and ADAM33 with the integrins α4β1, α4β7 and α9β1. Cell-adhesion assays demonstrated that, similar to ADAM28, the ADAM7 disintegrin domain supported α4β1-dependent Jurkat cell adhesion, whereas the ADAM33 disintegrin domain did not. The lymphocyte integrin α4β7 was also found to recognize both disintegrin domains of ADAM7 and ADAM28, but not of ADAM33. This is the first demonstration that mammalian disintegrins are capable of interacting with α4β7. All three disintegrin domains supported α9β1-dependent cell adhesion. Recognition by both α4β1 and α4β7 of ADAM7 and ADAM28 was activation-dependent, requiring either the presence of Mn2+ or an activating monoclonal antibody for cell attachment. Charge-to-alanine mutagenesis experiments revealed that the same residues within an individual ADAM disintegrin domain function in recognizing multiple integrins. However, the residues within a specific region of each ADAM disintegrin-like domain required for integrin binding were distinct. These results establish that ADAM7 and ADAM28 are recognized by the leucocyte integrins α4β1, α4β7 and α9β1. ADAM33 exclusively supported only α9β1-dependent adhesion. PMID:15504110

Family, employment, and individual resource-based antecedents of maternal work-family enrichment from infancy through middle childhood.

PubMed

Zhou, Nan; Buehler, Cheryl

2016-07-01

This study used data from the National Institute of Child Health and Human Development (NICHD) Study of Early Child Care and Youth Development (N = 1,019) to examine family, employment, and individual antecedents of maternal work-family enrichment from infancy through middle childhood. Work-family conflict and important confounding factors were controlled. From the family domain, higher income-to-needs ratio and social support were associated with higher work-family enrichment. From the employment domain, greater job rewards, benefits of employment for children, and work commitment were associated with higher work-family enrichment. From the individual domain, higher maternal education and extroversion were associated with higher work-family enrichment. No family, employment, and individual characteristics were associated with work-family conflict across time except for partner intimacy. In general, the results supported antecedents of work-family enrichment that supply needed resources. The present study contributed to the literature by identifying antecedents of maternal work-family enrichment across early child developmental stages, which goes beyond examinations of particular life stages and a work-family conflict perspective. Implications for theory and practice are discussed. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Interaction of the amyloid precursor protein-like protein 1 (APLP1) E2 domain with heparan sulfate involves two distinct binding modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahms, Sven O., E-mail: sdahms@fli-leibniz.de; Mayer, Magnus C.; Miltenyi Biotec GmbH, Robert-Koch-Strasse 1, 17166 Teterow

2015-03-01

Two X-ray structures of APLP1 E2 with and without a heparin dodecasaccharide are presented, revealing two distinct binding modes of the protein to heparan sulfate. The data provide a mechanistic explanation of how APP-like proteins bind to heparan sulfates and how they specifically recognize nonreducing structures of heparan sulfates. Beyond the pathology of Alzheimer’s disease, the members of the amyloid precursor protein (APP) family are essential for neuronal development and cell homeostasis in mammals. APP and its paralogues APP-like protein 1 (APLP1) and APP-like protein 2 (APLP2) contain the highly conserved heparan sulfate (HS) binding domain E2, which effects variousmore » (patho)physiological functions. Here, two crystal structures of the E2 domain of APLP1 are presented in the apo form and in complex with a heparin dodecasaccharide at 2.5 Å resolution. The apo structure of APLP1 E2 revealed an unfolded and hence flexible N-terminal helix αA. The (APLP1 E2){sub 2}–(heparin){sub 2} complex structure revealed two distinct binding modes, with APLP1 E2 explicitly recognizing the heparin terminus but also interacting with a continuous heparin chain. The latter only requires a certain register of the sugar moieties that fits to a positively charged surface patch and contributes to the general heparin-binding capability of APP-family proteins. Terminal binding of APLP1 E2 to heparin specifically involves a structure of the nonreducing end that is very similar to heparanase-processed HS chains. These data reveal a conserved mechanism for the binding of APP-family proteins to HS and imply a specific regulatory role of HS modifications in the biology of APP and APP-like proteins.« less

Work, Family and Community Support as Predictors of Work-Family Conflict: A Study of Low-Income Workers

ERIC Educational Resources Information Center

Griggs, Tracy Lambert; Casper, Wendy J.; Eby, Lillian T.

2013-01-01

This study examines relationships between support from work, family and community domains with time- and strain-based work-family conflict in a sample of low-income workers. Results reveal significant within-domain and cross-domain relationships between support from all three life domains with work--family conflict. With respect to family support,…

Identification of a novel mutation in the paired domain of PAX3 in an Iranian family with waardenburg syndrome type I.

PubMed

Sotirova, V N; Rezaie, T M; Khoshsorour, M M; Sarfarazi, M

2000-03-01

Waardenburg syndrome Type I (WS1) is an autosomal dominant disorder that has previously been associated with mutations in the PAX3 gene on the 2q35 region. In this study, we used an Iranian WS1 family with seven affected individuals in three generations. The phenotypic characteristics of the family include sensorineural deafness, dystopia canthorum, hypopigmented skin patches of the upper limbs, congenital white forelock, confluent white eyebrows, nonpigmented iris, poliosis, and hypopigmentation of the retina. Herein, we report a previously unidentified single-base substitution in exon II (C-->T at position 218) that results in a change of serine to leucine (S73L) in this family. This change was not observed in 100 chromosomes of healthy unrelated individuals. This mutation is within the PAX3 paired domain region, a structure that is highly conserved and implicated in DNA binding. This is the first identification of a PAX3 mutation for this phenotype in the Iranian population. This also provides additional confirmation for the involvement of this gene in the etiology of WS1.

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

A new family of StART domain proteins at membrane contact sites has a role in ER-PM sterol transport

PubMed Central

Gatta, Alberto T; Wong, Louise H; Sere, Yves Y; Calderón-Noreña, Diana M; Cockcroft, Shamshad; Menon, Anant K; Levine, Tim P

2015-01-01

Sterol traffic between the endoplasmic reticulum (ER) and plasma membrane (PM) is a fundamental cellular process that occurs by a poorly understood non-vesicular mechanism. We identified a novel, evolutionarily diverse family of ER membrane proteins with StART-like lipid transfer domains and studied them in yeast. StART-like domains from Ysp2p and its paralog Lam4p specifically bind sterols, and Ysp2p, Lam4p and their homologs Ysp1p and Sip3p target punctate ER-PM contact sites distinct from those occupied by known ER-PM tethers. The activity of Ysp2p, reflected in amphotericin-sensitivity assays, requires its second StART-like domain to be positioned so that it can reach across ER-PM contacts. Absence of Ysp2p, Ysp1p or Sip3p reduces the rate at which exogenously supplied sterols traffic from the PM to the ER. Our data suggest that these StART-like proteins act in trans to mediate a step in sterol exchange between the PM and ER. DOI: http://dx.doi.org/10.7554/eLife.07253.001 PMID:26001273

MYO15A (DFNB3) mutations in Turkish hearing loss families and functional modeling of a novel motor domain mutation.

PubMed

Kalay, Ersan; Uzumcu, Abdullah; Krieger, Elmar; Caylan, Refik; Uyguner, Oya; Ulubil-Emiroglu, Melike; Erdol, Hidayet; Kayserili, Hülya; Hafiz, Gunter; Başerer, Nermin; Heister, Angelien J G M; Hennies, Hans C; Nürnberg, Peter; Başaran, Seher; Brunner, Han G; Cremers, Cor W R J; Karaguzel, Ahmet; Wollnik, Bernd; Kremer, Hannie

2007-10-15

Myosin XVA is an unconventional myosin which has been implicated in autosomal recessive nonsyndromic hearing impairment (ARNSHI) in humans. In Myo15A mouse models, vestibular dysfunction accompanies the autosomal recessive hearing loss. Genomewide homozygosity mapping and subsequent fine mapping in two Turkish families with ARNSHI revealed significant linkage to a critical interval harboring a known deafness gene MYO15A on chromosome 17p13.1-17q11.2. Subsequent sequencing of the MYO15A gene led to the identification of a novel missense mutation, c.5492G-->T (p.Gly1831Val) and a novel splice site mutation, c.8968-1G-->C. These mutations were not detected in additional 64 unrelated ARNSHI index patients and in 230 Turkish control chromosomes. Gly1831 is a conserved residue located in the motor domains of the different classes of myosins of different species. Molecular modeling of the motor head domain of the human myosin XVa protein suggests that the Gly1831Val mutation inhibits the powerstroke by reducing backbone flexibility and weakening the hydrophobic interactions necessary for signal transmission to the converter domain. Copyright (c) 2007 Wiley-Liss, Inc.

Transcriptomic Signature of the SHATTERPROOF2 Expression Domain Reveals the Meristematic Nature of Arabidopsis Gynoecial Medial Domain1[OPEN

PubMed Central

Villarino, Gonzalo H.; Hu, Qiwen; Flores-Vergara, Miguel; Sehra, Bhupinder; Brumos, Javier; Stepanova, Anna N.; Sundberg, Eva; Heber, Steffen

2016-01-01

Plant meristems, like animal stem cell niches, maintain a pool of multipotent, undifferentiated cells that divide and differentiate to give rise to organs. In Arabidopsis (Arabidopsis thaliana), the carpel margin meristem is a vital meristematic structure that generates ovules from the medial domain of the gynoecium, the female floral reproductive structure. The molecular mechanisms that specify this meristematic region and regulate its organogenic potential are poorly understood. Here, we present a novel approach to analyze the transcriptional signature of the medial domain of the Arabidopsis gynoecium, highlighting the developmental stages that immediately proceed ovule initiation, the earliest stages of seed development. Using a floral synchronization system and a SHATTERPROOF2 (SHP2) domain-specific reporter, paired with FACS and RNA sequencing, we assayed the transcriptome of the gynoecial medial domain with temporal and spatial precision. This analysis reveals a set of genes that are differentially expressed within the SHP2 expression domain, including genes that have been shown previously to function during the development of medial domain-derived structures, including the ovules, thus validating our approach. Global analyses of the transcriptomic data set indicate a similarity of the pSHP2-expressing cell population to previously characterized meristematic domains, further supporting the meristematic nature of this gynoecial tissue. Our method identifies additional genes including novel isoforms, cis-natural antisense transcripts, and a previously unrecognized member of the REPRODUCTIVE MERISTEM family of transcriptional regulators that are potential novel regulators of medial domain development. This data set provides genome-wide transcriptional insight into the development of the carpel margin meristem in Arabidopsis. PMID:26983993

Structural insights into the regulation and the recognition of histone marks by the SET domain of NSD1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morishita, Masayo; Di Luccio, Eric, E-mail: eric.diluccio@gmail.com

2011-08-26

Highlights: {yields} NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 are histone methyltransferases linked to numerous cancers. {yields} Little is known about the NSD pathways and HMTase inhibitors are sorely needed in the epigenetic therapy of cancers. {yields} We investigate the regulation and the recognition of histone marks by the SET domain of NSD1. {yields} A unique and key mechanism is driven by a loop at the interface of the SET and postSET region. {yields} Implications for developing specific and selective HMTase inhibitors are presented. -- Abstract: The development of epigenetic therapies fuels cancer hope. DNA-methylation inhibitors, histone-deacetylase and histone-methyltransferase (HMTase) inhibitors are beingmore » developed as the utilization of epigenetic targets is emerging as an effective and valuable approach to chemotherapy as well as chemoprevention of cancer. The nuclear receptor binding SET domain (NSD) protein is a family of three HMTases, NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L1 that are critical in maintaining the chromatin integrity. A growing number of studies have reported alterations or amplifications of NSD1, NSD2, or NSD3 in numerous carcinogenic events. Reducing NSDs activity through specific lysine-HMTase inhibitors appears promising to help suppressing cancer growth. However, little is known about the NSD pathways and our understanding of the histone lysine-HMTase mechanism is partial. To shed some light on both the recognition and the regulation of epigenetic marks by the SET domain of the NSD family, we investigate the structural mechanisms of the docking of the histone-H4 tail on the SET domain of NSD1. Our finding exposes a key regulatory and recognition mechanism driven by the flexibility of a loop at the interface of the SET and postSET region. Finally, we prospect the special value of this regulatory region for developing specific and selective NSD inhibitors for the epigenetic therapy of cancers.« less

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology*

PubMed Central

Ramírez-Escudero, Mercedes; del Pozo, Mercedes V.; Marín-Navarro, Julia; González, Beatriz; Golyshin, Peter N.; Polaina, Julio; Ferrer, Manuel; Sanz-Aparicio, Julia

2016-01-01

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials. PMID:27679487

Family quality of life among families with a child who has a severe neurodevelopmental disability: Impact of family and child socio-demographic factors.

PubMed

Schertz, Mitchell; Karni-Visel, Yael; Tamir, Ada; Genizi, Jacob; Roth, Dana

2016-01-01

We aimed to examine family quality of life (FQOL) of Northern Israeli families having a child with a severe neurodevelopmental disability and its relation to socio-demographics. The cohort included caregivers of 70 children ages (mean ± standard deviation) 5.36 ± 3.53 years. Families were two-parent (85.7%), lived in the periphery (67.1%) and included Jews (60%), Muslims (18.6%), Druze (14.3%) and Christians (7.1%). Religiosity included: secular (38.6%), traditional (31.4%), religious (30%). Children's diagnosis included autistic spectrum disorder (41.4%), intellectual disability (21.4%), cerebral palsy (17.1%), genetic syndromes (17.1%) and sensorineural hearing loss (2.9%). Degree of support (1-minimal,5-greatest) required by the child was 3.67 ± 1.28 for physical and 3.49 ± 1.36 for communication. Primary caregivers completed the FQOL Survey. Domain scores were highest for family relations and lowest for financial well-being. Dimension scores were highest for importance and lowest for opportunities. Overall FQOL approximated average. Jewish families and residents of a major urban area reported higher and more religious families reported lower overall FQOL. Regression analysis found ethnicity contributing to overall FQOL and domain scores with residence contributing to support from services. Ethnicity and child dependence contributed to dimension scores. Northern Israeli families having a child with a severe neurodevelopmental disability report average FQOL scores. However, family and child dependence characteristics affect FQOL scores. Professionals working with these families should consider FQOL information when making recommendations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Microtubule-Actin Cross-Linking Factor 1: Domains, Interaction Partners, and Tissue-Specific Functions.

PubMed

Goryunov, Dmitry; Liem, Ronald K H

2016-01-01

The cytoskeleton of most eukaryotic cells is composed of three principal filamentous components: actin filaments, microtubules (MTs), and intermediate filaments. It is a highly dynamic system that plays crucial roles in a wide range of cellular processes, including migration, adhesion, cytokinesis, morphogenesis, intracellular traffic and signaling, and structural flexibility. Among the large number of cytoskeleton-associated proteins characterized to date, microtubule-actin cross-linking factor 1 (MACF1) is arguably the most versatile integrator and modulator of cytoskeleton-related processes. MACF1 belongs to the plakin family of proteins, and within it, to the spectraplakin subfamily. These proteins are characterized by the ability to bridge MT and actin cytoskeletal networks in a dynamic fashion, which underlies their involvement in the regulation of cell migration, axonal extension, and vesicular traffic. Studying MACF1 functions has provided insights not only into the regulation of the cytoskeleton but also into molecular mechanisms of both normal cellular physiology and cellular pathology. Multiple MACF1 isoforms exist, composed of a large variety of alternatively spliced domains. Each of these domains mediates a specific set of interactions and functions. These functions are manifested in tissue and cell-specific phenotypes observed in conditional MACF1 knockout mice. The conditional models described to date reveal critical roles of MACF1 in mammalian skin, nervous system, heart muscle, and intestinal epithelia. Complete elimination of MACF1 is early embryonic lethal, indicating an essential role for MACF1 in early development. Further studies of MACF1 domains and their interactions will likely reveal multiple new roles of this protein in various tissues. © 2016 Elsevier Inc. All rights reserved.

Higher risk of death among MEN1 patients with mutations in the JunD interacting domain: a Groupe d'etude des Tumeurs Endocrines (GTE) cohort study.

PubMed

Thevenon, Julien; Bourredjem, Abderrahmane; Faivre, Laurence; Cardot-Bauters, Catherine; Calender, Alain; Murat, Arnaud; Giraud, Sophie; Niccoli, Patricia; Odou, Marie-Françoise; Borson-Chazot, Françoise; Barlier, Anne; Lombard-Bohas, Catherine; Clauser, Eric; Tabarin, Antoine; Parfait, Béatrice; Chabre, Olivier; Castermans, Emilie; Beckers, Albert; Ruszniewski, Philippe; Le Bras, Morgane; Delemer, Brigitte; Bouchard, Philippe; Guilhem, Isabelle; Rohmer, Vincent; Goichot, Bernard; Caron, Philippe; Baudin, Eric; Chanson, Philippe; Groussin, Lionel; Du Boullay, Hélène; Weryha, Georges; Lecomte, Pierre; Penfornis, Alfred; Bihan, Hélène; Archambeaud, Françoise; Kerlan, Véronique; Duron, Françoise; Kuhn, Jean-Marc; Vergès, Bruno; Rodier, Michel; Renard, Michel; Sadoul, Jean-Louis; Binquet, Christine; Goudet, Pierre

2013-05-15

Multiple endocrine neoplasia syndrome type 1 (MEN1), which is secondary to mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Although genotype-phenotype studies have so far failed to identify any statistical correlations, some families harbor recurrent tumor patterns. The function of MENIN is unclear, but has been described through the discovery of its interacting partners. Mutations in the interacting domains of MENIN functional partners have been shown to directly alter its regulation abilities. We report on a cohort of MEN1 patients from the Groupe d'étude des Tumeurs Endocrines. Patients with a molecular diagnosis and a clinical follow-up, totaling 262 families and 806 patients, were included. Associations between mutation type, location or interacting factors of the MENIN protein and death as well as the occurrence of MEN1-related tumors were tested using a frailty Cox model to adjust for potential heterogeneity across families. Accounting for the heterogeneity across families, the overall risk of death was significantly higher when mutations affected the JunD interacting domain (adjusted HR = 1.88: 95%-CI = 1.15-3.07). Patients had a higher risk of death from cancers of the MEN1 spectrum (HR = 2.34; 95%-CI = 1.23-4.43). This genotype-phenotype correlation study confirmed the lack of direct genotype-phenotype correlations. However, patients with mutations affecting the JunD interacting domain had a higher risk of death secondary to a MEN1 tumor and should thus be considered for surgical indications, genetic counseling and follow-up.

The function of the yeast molecular chaperone Sse1 is mechanistically distinct from the closely related hsp70 family.

PubMed

Shaner, Lance; Trott, Amy; Goeckeler, Jennifer L; Brodsky, Jeffrey L; Morano, Kevin A

2004-05-21

The Sse1/Hsp110 molecular chaperones are a poorly understood subgroup of the Hsp70 chaperone family. Hsp70 can refold denatured polypeptides via a C-terminal peptide binding domain (PBD), which is regulated by nucleotide cycling in an N-terminal ATPase domain. However, unlike Hsp70, both Sse1 and mammalian Hsp110 bind unfolded peptide substrates but cannot refold them. To test the in vivo requirement for interdomain communication, SSE1 alleles carrying amino acid substitutions in the ATPase domain were assayed for their ability to complement sse1Delta yeast. Surprisingly, all mutants predicted to abolish ATP hydrolysis (D8N, K69Q, D174N, D203N) complemented the temperature sensitivity of sse1Delta and lethality of sse1Deltasse2Delta cells, whereas mutations in predicted ATP binding residues (G205D, G233D) were non-functional. Complementation ability correlated well with ATP binding assessed in vitro. The extreme C terminus of the Hsp70 family is required for substrate targeting and heterocomplex formation with other chaperones, but mutant Sse1 proteins with a truncation of up to 44 C-terminal residues that were not included in the PBD were active. Remarkably, the two domains of Sse1, when expressed in trans, functionally complement the sse1Delta growth phenotype and interact by coimmunoprecipitation analysis. In addition, a functional PBD was required to stabilize the Sse1 ATPase domain, and stabilization also occurred in trans. These data represent the first structure-function analysis of this abundant but ill defined chaperone, and establish several novel aspects of Sse1/Hsp110 function relative to Hsp70.

Crystal Structure of a Complex of the Intracellular Domain of Interferon λ Receptor 1 (IFNLR1) and the FERM/SH2 Domains of Human JAK1.

PubMed

Zhang, Di; Wlodawer, Alexander; Lubkowski, Jacek

2016-11-20

The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2. Published by Elsevier Ltd.

A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase.

PubMed

Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R; Yang, Shaoqing; Jiang, Zhengqiang

2015-12-16

Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions--a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins.

C-Terminal β9-Strand of the Cyclic Nucleotide-Binding Homology Domain Stabilizes Activated States of Kv11.1 Channels

PubMed Central

Ng, Chai Ann; Ke, Ying; Perry, Matthew D.; Tan, Peter S.; Hill, Adam P.; Vandenberg, Jamie I.

2013-01-01

Kv11.1 potassium channels are important for regulation of the normal rhythm of the heartbeat. Reduced activity of Kv11.1 channels causes long QT syndrome type 2, a disorder that increases the risk of cardiac arrhythmias and sudden cardiac arrest. Kv11.1 channels are members of the KCNH subfamily of voltage-gated K+ channels. However, they also share many similarities with the cyclic nucleotide gated ion channel family, including having a cyclic nucleotide-binding homology (cNBH) domain. Kv11.1 channels, however, are not directly regulated by cyclic nucleotides. Recently, crystal structures of the cNBH domain from mEAG and zELK channels, both members of the KCNH family of voltage-gated potassium channels, revealed that a C-terminal β9-strand in the cNBH domain occupied the putative cyclic nucleotide-binding site thereby precluding binding of cyclic nucleotides. Here we show that mutations to residues in the β9-strand affect the stability of the open state relative to the closed state of Kv11.1 channels. We also show that disrupting the structure of the β9-strand reduces the stability of the inactivated state relative to the open state. Clinical mutations located in this β9-strand result in reduced trafficking efficiency, which suggests that binding of the C-terminal β9-strand to the putative cyclic nucleotide-binding pocket is also important for assembly and trafficking of Kv11.1 channels. PMID:24204727

NMR Structure and Dynamics of the C-terminal Domain from Human Rev1 and its Complex with Rev1 Interacting Region of DNA Polymerase η

PubMed Central

Pozhidaeva, Alexandra; Pustovalova, Yulia; D'Souza, Sanjay; Bezsonova, Irina; Walker, Graham C.; Korzhnev, Dmitry M.

2013-01-01

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases. PMID:22691049

Family Satisfaction With Nursing Home Care.

PubMed

Shippee, Tetyana P; Henning-Smith, Carrie; Gaugler, Joseph E; Held, Robert; Kane, Robert L

2017-03-01

This article explores the factor structure of a new family satisfaction with nursing home care instrument and determines the relationship of resident quality of life (QOL) and facility characteristics with family satisfaction. Data sources include (1) family satisfaction interviews ( n = 16,790 family members), (2) multidimensional survey of resident QOL ( n = 13,433 residents), and (3) facility characteristics ( n = 376 facilities). We used factor analysis to identify domains of family satisfaction and multivariate analyses to identify the role of facility-level characteristics and resident QOL on facility-mean values of family satisfaction. Four distinct domains were identified for family satisfaction: "care," "staff," "environment," and "food." Chain affiliation, higher resident acuity, more deficiencies, and large size were all associated with less family satisfaction, and resident QOL was a significant (albeit weak) predictor of family satisfaction. Results suggest that family member satisfaction is distinct from resident QOL but is associated with resident QOL and facility characteristics.

A member of a new plant gene family encoding a meprin and TRAF homology (MATH) domain-containing protein is involved in restriction of long distance movement of plant viruses

PubMed Central

Cosson, Patrick; Sofer, Luc; Schurdi-Levraud, Valérie

2010-01-01

Restriction of long distance movement of several potyviruses in Arabidopsis thaliana is controlled by at least three dominant restricted TEV movement (RTM) genes, named RTM1, RTM2 and RTM3 and acts as a non-conventional resistance. RTM1 encodes a protein belonging to the jacalin family and RTM2 encodes a protein which has similarities to small heat shock proteins. The recent cloning of RTM3 which encodes a protein belonging to an unknown protein family of 29 members that has a meprin and TRAF homology (MATH) domain in its N-terminal region and a coiled-coil (CC) domain at its C-terminal end is an important breakthrough for a better understanding of this resistance process. Not only the third gene involved in this resistance has been identified and has allowed revealing a new gene family in plant but the discovery that the RTM3 protein interacts directly with RTM1 strongly suggests that the RTM proteins form a multimeric complex. However, these data also highlight striking similarities of the RTM resistance with the well known R-gene mediated resistance. PMID:20930558

Crystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus

PubMed Central

Asojo, Oluwatoyin A.; Loukas, Alex; Inan, Mehmet; Barent, Rick; Huang, Jicai; Plantz, Brad; Swanson, Amber; Gouthro, Mark; Meagher, Michael M.; Hotez, Peter J.

2005-01-01

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, purified and crystallized. Na-ASP-1 is a 406-amino-acid multi-domain ASP from the prevalent human hookworm parasite Necator americanus. Useful X-ray data to 2.2 Å have been collected from a crystal that belongs to the monoclinic space group P21 with unit-cell parameters a = 67.7, b = 74.27, c = 84.60 Å, β = 112.12°. An initial molecular-replacement solution has been obtained with one monomer in the asymmetric unit. PMID:16511050

Familial Blau syndrome without uveitis caused by a novel mutation in the nucleotide-binding oligomerization domain-containing protein 2 gene with good response to infliximab.

PubMed

Toral-López, Jaime; González-Huerta, Luz M; Martín-Del Campo, Mónica; Messina-Baas, Olga; Cuevas-Covarrubias, Sergio A

2018-05-01

The proband in this study was a 4-year-old Mexican girl with Blau syndrome. She and her affected family members had skin rash and arthritis but no uveitis. Exome sequencing and DNA direct sequencing from blood samples revealed a novel nucleotide-binding oligomerization domain-containing protein 2 gene mutation in the affected family members. This study is the first report of a Mexican family with Blau syndrome showing good infliximab treatment response. The novel mutation in the nucleotide-binding oligomerization domain-containing protein 2 gene (c.1808A>G) enriches the mutation spectrum in Blau syndrome. This family represents one of the few cases of autosomal Blau syndrome with no uveitis; because of phenotype variability, it is important to recognize Blau syndrome's clinical spectrum and recommend genetic consultation. © 2018 Wiley Periodicals, Inc.

Domain walls and ferroelectric reversal in corundum derivatives

NASA Astrophysics Data System (ADS)

Ye, Meng; Vanderbilt, David

Domain walls are the topological defects that mediate polarization reversal in ferroelectrics, and they may exhibit quite different geometric and electronic structures compared to the bulk. Therefore, a detailed atomic-scale understanding of the static and dynamic properties of domain walls is of pressing interest. In this work, we use first-principles methods to study the structures of 180° domain walls, both in their relaxed state and along the ferroelectric reversal pathway, in ferroelectrics belonging to the family of corundum derivatives. Our calculations predict their orientation, formation energy, and migration energy, and also identify important couplings between polarization, magnetization, and chirality at the domain walls. Finally, we point out a strong empirical correlation between the height of the domain-wall mediated polarization reversal barrier and the local bonding environment of the mobile A cations as measured by bond valence sums. Our results thus provide both theoretical and empirical guidance to further search for ferroelectric candidates in materials of the corundum derivative family. The work is supported by ONR Grant N00014-12-1-1035.

The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.

PubMed

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L; Levin, Henry L

2008-06-06

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.

CDD/SPARCLE: functional classification of proteins via subfamily domain architectures.

PubMed

Marchler-Bauer, Aron; Bo, Yu; Han, Lianyi; He, Jane; Lanczycki, Christopher J; Lu, Shennan; Chitsaz, Farideh; Derbyshire, Myra K; Geer, Renata C; Gonzales, Noreen R; Gwadz, Marc; Hurwitz, David I; Lu, Fu; Marchler, Gabriele H; Song, James S; Thanki, Narmada; Wang, Zhouxi; Yamashita, Roxanne A; Zhang, Dachuan; Zheng, Chanjuan; Geer, Lewis Y; Bryant, Stephen H

2017-01-04

NCBI's Conserved Domain Database (CDD) aims at annotating biomolecular sequences with the location of evolutionarily conserved protein domain footprints, and functional sites inferred from such footprints. An archive of pre-computed domain annotation is maintained for proteins tracked by NCBI's Entrez database, and live search services are offered as well. CDD curation staff supplements a comprehensive collection of protein domain and protein family models, which have been imported from external providers, with representations of selected domain families that are curated in-house and organized into hierarchical classifications of functionally distinct families and sub-families. CDD also supports comparative analyses of protein families via conserved domain architectures, and a recent curation effort focuses on providing functional characterizations of distinct subfamily architectures using SPARCLE: Subfamily Protein Architecture Labeling Engine. CDD can be accessed at https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Antibodies to B7.1 define the GFCC'C" face of the N-terminal domain as critical for co-stimulatory interactions.

PubMed

Wang, Suyue; Veldman, Geertruida M; Stahl, Mark; Xing, Yuzhe; Tobin, James F; Erbe, David V

2002-09-02

Antagonists of the B7 family of co-stimulatory molecules have the potential for altering immune responses therapeutically. To better define the requirements for such inhibitors, we have mapped the binding of an entire panel of blocking antibodies specific for human B7.1. By mutagenesis, each of the residues critical for blocking antibody binding appeared to fall entirely within the N-terminal V-set domain of B7.1. Thus, although antibody-antigen interacting surfaces can be quite large, these results indicate that a relatively small portion of the GFCC'C" face of this domain is crucial for further antagonist development.

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

A unique GCN5-related glucosamine N-acetyltransferase region exist in the fungal multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase

PubMed Central

Qin, Zhen; Xiao, Yibei; Yang, Xinbin; Mesters, Jeroen R.; Yang, Shaoqing; Jiang, Zhengqiang

2015-01-01

Glycoside hydrolase (GH) family 3 β-N-acetylglucosaminidases widely exist in the filamentous fungi, which may play a key role in chitin metabolism of fungi. A multi-domain GH family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (RmNag), exhibiting a potential N-acetyltransferase region, has been recently reported to show great potential in industrial applications. In this study, the crystal structure of RmNag was determined at 2.80 Å resolution. The three-dimensional structure of RmNag showed four distinctive domains, which belong to two distinguishable functional regions — a GH family 3 β-N-acetylglucosaminidase region (N-terminal) and a N-acetyltransferase region (C-terminal). From structural and functional analysis, the C-terminal region of RmNag was identified as a unique tandem array linking general control non-derepressible 5 (GCN5)-related N-acetyltransferase (GNAT), which displayed glucosamine N-acetyltransferase activity. Structural analysis of this glucosamine N-acetyltransferase region revealed that a unique glucosamine binding pocket is located in the pantetheine arm binding terminal region of the conserved CoA binding pocket, which is different from all known GNAT members. This is the first structural report of a glucosamine N-acetyltransferase, which provides novel structural information about substrate specificity of GNATs. The structural and functional features of this multi-domain β-N-acetylglucosaminidase could be useful in studying the catalytic mechanism of GH family 3 proteins. PMID:26669854

The methyltransferase NSD3 has chromatin-binding motifs, PHD5-C5HCH, that are distinct from other NSD (nuclear receptor SET domain) family members in their histone H3 recognition.

PubMed

He, Chao; Li, Fudong; Zhang, Jiahai; Wu, Jihui; Shi, Yunyu

2013-02-15

The NSD (nuclear receptor SET domain-containing) family members, consisting of NSD1, NSD2 (MMSET/WHSC1), and NSD3 (WHSC1L1), are SET domain-containing methyltransferases and aberrant expression of each member has been implicated in multiple diseases. They have specific mono- and dimethylase activities for H3K36, whereas play nonredundant roles during development. Aside from the well characterized catalytic SET domain, NSD proteins have multiple potential chromatin-binding motifs that are clinically relevant, including the fifth plant homeodomain (PHD5) and the adjacent Cys-His-rich domain (C5HCH) located at the C terminus. Herein, we report the crystal structures of the PHD5-C5HCH module of NSD3, in the free state and in complex with H3(1-7) (H3 residues 1-7), H3(1-15) (H3 residues 1-15), and H3(1-15)K9me3 (H3 residues 1-15 with trimethylation on K9) peptides. These structures reveal that the PHD5 and C5HCH domains fold into a novel integrated PHD-PHD-like structural module with H3 peptide bound only on the surface of PHD5 and provide the molecular basis for the recognition of unmodified H3K4 and trimethylated H3K9 by NSD3 PHD5. Structural studies and binding assays show that differences exist in histone binding specificity of the PHD5 domain between three members of the NSD family. For NSD2, the PHD5-C5HCH:H3 N terminus interaction is largely conserved, although with a stronger preference for unmethylated H3K9 (H3K9me0) than trimethylated H3K9 (H3K9me3), and NSD1 PHD5-C5HCH does not bind to H3 peptides. Our results shed light on how NSD proteins that mediate H3K36 methylation are localized to specific genomic sites and provide implications for the mechanism of functional diversity of NSD proteins.

[Coactivators in energy metabolism: peroxisome proliferator-activated receptor-gamma coactivator 1 family].

PubMed

Wang, Rui; Chang, Yong-sheng; Fang, Fu-de

2009-12-01

Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1) family is highly expressed in tissues with high energy metabolism. They coactivate transcription factors in regulating genes engaged in processes such as gluconeogenesis, adipose beta-oxydation, lipoprotein synthesis and secretion, mitochondrial biogenesis, and oxidative metabolism. Protein conformation studies demonstrated that they lack DNA binding domains and act as coactivators through physical interaction with transcription factors. PGC1 activity is regulated at transcription level or by multiple covalent chemical modifications such as phosphorylation, methylation and acetylation/deacetylation. Abnormal expression of PGC1 coactivators usually is closely correlated with diseases such as diabetes, obesity, hyperglycemia, hyperlipemia, and arterial and brain neuron necrosis diseases.

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family

PubMed Central

Dickson, Tracey C.; Mintz, C. David; Benson, Deanna L.; Salton, Stephen R.J.

2002-01-01

Ayeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane–cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM–actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis. PMID:12070130

Functional binding interaction identified between the axonal CAM L1 and members of the ERM family.

PubMed

Dickson, Tracey C; Mintz, C David; Benson, Deanna L; Salton, Stephen R J

2002-06-24

A yeast two-hybrid library was screened using the cytoplasmic domain of the axonal cell adhesion molecule L1 to identify binding partners that may be involved in the regulation of L1 function. The intracellular domain of L1 bound to ezrin, a member of the ezrin, radixin, and moesin (ERM) family of membrane-cytoskeleton linking proteins, at a site overlapping that for AP2, a clathrin adaptor. Binding of bacterial fusion proteins confirmed this interaction. To determine whether ERM proteins interact with L1 in vivo, extracellular antibodies to L1 were used to force cluster the protein on cultured hippocampal neurons and PC12 cells, which were then immunolabeled for ERM proteins. Confocal analysis revealed a precise pattern of codistribution between ERMs and L1 clusters in axons and PC12 neurites, whereas ERMs in dendrites and spectrin labeling remained evenly distributed. Transfection of hippocampal neurons grown on an L1 substrate with a dominant negative ERM construct resulted in extensive and abnormal elaboration of membrane protrusions and an increase in axon branching, highlighting the importance of the ERM-actin interaction in axon development. Together, our data indicate that L1 binds directly to members of the ERM family and suggest this association may coordinate aspects of axonal morphogenesis.

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx.

PubMed

Williams, N A; Holland, P W

1998-05-01

We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular phylogenetic analysis indicating that there was single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes.

A Missense Mutation in the Aggrecan C-type Lectin Domain Disrupts Extracellular Matrix Interactions and Causes Dominant Familial Osteochondritis Dissecans

PubMed Central

Stattin, Eva-Lena; Wiklund, Fredrik; Lindblom, Karin; Önnerfjord, Patrik; Jonsson, Björn-Anders; Tegner, Yelverton; Sasaki, Takako; Struglics, André; Lohmander, Stefan; Dahl, Niklas; Heinegård, Dick; Aspberg, Anders

2010-01-01

Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo. PMID:20137779

DOORS syndrome: phenotype, genotype and comparison with Coffin-Siris syndrome.

PubMed

Campeau, Philippe M; Hennekam, Raoul C

2014-09-01

DOORS syndrome (Deafness, Onychodystrophy, Osteodystrophy, mental Retardation, Seizures) is characterized mainly by sensorineural deafness, shortened terminal phalanges with small nails of hands and feet, intellectual deficiency, and seizures. Half of the patients with all clinical features have mutations in TBC1D24. We review here the manifestations of patients clinically diagnosed with DOORS syndrome. In this cohort of 32 families (36 patients) we detected 13 individuals from 10 families with TBC1D24 mutations. Subsequent whole exome sequencing in the cohort showed the same de novoSMARCB1 mutation (c.1130G>A), known to cause Coffin-Siris syndrome, in two patients. Distinguishing features include retinal anomalies, Dandy-Walker malformation, scoliosis, rocker bottom feet, respiratory difficulties and absence of seizures, and 2-oxoglutaric aciduria in the patients with the SMARCB1 mutation. We briefly discuss the heterogeneity of the DOORS syndrome phenotype and the differential diagnosis of this condition. © 2014 Wiley Periodicals, Inc.

ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

PubMed

Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

2014-01-01

ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

ABCA1, ABCG1, and ABCG4 Are Distributed to Distinct Membrane Meso-Domains and Disturb Detergent-Resistant Domains on the Plasma Membrane

PubMed Central

Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

2014-01-01

ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

Structural Basis for Endosomal Targeting by the Bro1 Domain

PubMed Central

Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

2010-01-01

Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family

PubMed Central

Kumar, Yellapu Nanda; Kumar, Pasupuleti Santhosh; Sowjenya, Gopal; Rao, Valasani Koteswara; Yeswanth, Sthanikam; Prasad, Uppu Venkateswara; Pradeepkiran, Jangampalli Adi; Sarma, PVGK; Bhaskar, Matcha

2012-01-01

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family. PMID:22829728

The GP(Y/F) Domain of TF1 Integrase Multimerizes when Present in a Fragment, and Substitutions in This Domain Reduce Enzymatic Activity of the Full-length Protein*S⃞

PubMed Central

Ebina, Hirotaka; Chatterjee, Atreyi Ghatak; Judson, Robert L.; Levin, Henry L.

2008-01-01

Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the γ-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining. PMID:18397885

Do Workers Who Experience Conflict between the Work and Family Domains Hit a "Glass Ceiling?": A Meta-Analytic Examination

ERIC Educational Resources Information Center

Hoobler, Jenny M.; Hu, Jia; Wilson, Morgan

2010-01-01

Based in Conservation of Resources (COR; Hobfoll, 1989) and self-verification (Swann, 1987) theories, we argue that when workers experience conflict between the work and family domains, this should have implications for evaluations of their work performance and ultimately affect more "objective" career outcomes such as salary and hierarchical…

Identification and Structural Characterization of the ALIX-Binding Late Domains of Simian Immunodeficiency Virus SIVmac239 and SIVagmTan-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Q.; Robinson, H.; Landesman, M. B.

2011-01-01

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX{sub n}L (where X{sub n} can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6{sup Gag} proteins of SIV{sub mac239} ({sub 40}SREK{und P}YKE{und VT}ED{und L}LHLNSLF{sub 59}) and SIV{sub agmTan-1} ({sub 24}AAG{und A}YDP{und AR}KL{und L}EQYAKK{sub 41}). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain,more » revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.« less

A single amino-acid substitution in the Ets domain alters core DNA binding specificity of Ets1 to that of the related transcription factors Elf1 and E74.

PubMed

Bosselut, R; Levin, J; Adjadj, E; Ghysdael, J

1993-11-11

Ets proteins form a family of sequence specific DNA binding proteins which bind DNA through a 85 aminoacids conserved domain, the Ets domain, whose sequence is unrelated to any other characterized DNA binding domain. Unlike all other known Ets proteins, which bind specific DNA sequences centered over either GGAA or GGAT core motifs, E74 and Elf1 selectively bind to GGAA corecontaining sites. Elf1 and E74 differ from other Ets proteins in three residues located in an otherwise highly conserved region of the Ets domain, referred to as conserved region III (CRIII). We show that a restricted selectivity for GGAA core-containing sites could be conferred to Ets1 upon changing a single lysine residue within CRIII to the threonine found in Elf1 and E74 at this position. Conversely, the reciprocal mutation in Elf1 confers to this protein the ability to bind to GGAT core containing EBS. This, together with the fact that mutation of two invariant arginine residues in CRIII abolishes DNA binding, indicates that CRIII plays a key role in Ets domain recognition of the GGAA/T core motif and lead us to discuss a model of Ets proteins--core motif interaction.

Relationship of work-family conflict with burnout and marital satisfaction: cross-domain or source attribution relations?

PubMed

Bagherzadeh, Razieh; Taghizadeh, Ziba; Mohammadi, Eesa; Kazemnejad, Anoshirvan; Pourreza, Abolghasem; Ebadi, Abbas

2016-01-01

The present study was conducted to examine the relationship between two dimensions of work-family conflict (WFC) with marital satisfaction and burnout in a society in which few studies have been done about the consequences of WFC. A cross-sectional study was conducted in 2015. Surveys were distributed to 420 employed married women with various jobs living in Bushehr province, Iran. Data were collected using a questionnaire for demographic characteristic, the Netmeyer's WFC questionnaire, Maslach Burnout Inventory: General Survey (MBI-GS), and Enrich maritalsatisfaction questionnaire. The data were analyzed using descriptive and inferential statistics. There was a negatively significant association between work interference with family(WIF) and overall burnout as well as emotional exhaustion (P < .01). Family interference with work (FIW) was significantly associated with depersonalization (P < .01). The overall marital satisfaction and its subscales were significantly associated with WIF (P < .01) and FIW (P < .01 for overall marital satisfaction and P < .05 for its subscales). In terms of practical implication, to avoid creating disadvantages of WIF and FIW,facilitation in two domains of improving work and family conditions can be a useful means to prevent WFC and its consequences.

Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects.

PubMed

Dixit, Radhika; Arakane, Yasuyuki; Specht, Charles A; Richard, Chad; Kramer, Karl J; Beeman, Richard W; Muthukrishnan, Subbaratnam

2008-04-01

A bioinformatics investigation of four insect species with annotated genome sequences identified a family of genes encoding chitin deacetylase (CDA)-like proteins, with five to nine members depending on the species. CDAs (EC 3.5.1.41) are chitin-modifying enzymes that deacetylate the beta-1,4-linked N-acetylglucosamine homopolymer. Partial deacetylation forms a heteropolysaccharide that also contains some glucosamine residues, while complete deacetylation produces the homopolymer chitosan, consisting exclusively of glucosamine. The genomes of the red flour beetle, Tribolium castaneum, the fruit fly, Drosophila melanogaster, the malaria mosquito, Anopheles gambiae, and the honey bee, Apis mellifera contain 9, 6, 5 and 5 genes, respectively, that encode proteins with a chitin deacetylase motif. The presence of alternative exons in two of the genes, TcCDA2 and TcCDA5, increases the protein diversity further. Insect CDA-like proteins were classified into five orthologous groups based on phylogenetic analysis and the presence of additional motifs. Group I enzymes include CDA1 and isoforms of CDA2, each containing in addition to a polysaccharide deacetylase-like catalytic domain, a chitin-binding peritrophin-A domain (ChBD) and a low-density lipoprotein receptor class A domain (LDLa). Group II is composed of CDA3 orthologs from each insect species with the same domain organization as group I CDAs, but differing substantially in sequence. Group III includes CDA4s, which have the ChBD domain but do not have the LDLa domain. Group IV comprises CDA5s, which are the largest CDAs because of a very long intervening region separating the ChBD and catalytic domains. Among the four insect species, Tribolium is unique in having four CDA genes in group V, whereas the other insect genomes have either one or none. Most of the CDA-like proteins have a putative signal peptide consistent with their role in modifying extracellular chitin in both cuticle and peritrophic membrane during

Structural and functional characterization of the NHR1 domain of the Drosophila neuralized E3 ligase in the notch signaling pathway.

PubMed

He, Fahu; Saito, Kohei; Kobayashi, Naohiro; Harada, Takushi; Watanabe, Satoru; Kigawa, Takanori; Güntert, Peter; Ohara, Osamu; Tanaka, Akiko; Unzai, Satoru; Muto, Yutaka; Yokoyama, Shigeyuki

2009-10-23

The Notch signaling pathway is critical for many developmental processes and requires complex trafficking of both Notch receptor and its ligands, Delta and Serrate. In Drosophila melanogaster, the endocytosis of Delta in the signal-sending cell is essential for Notch receptor activation. The Neuralized protein from D. melanogaster (Neur) is a ubiquitin E3 ligase, which binds to Delta through its first neuralized homology repeat 1 (NHR1) domain and mediates the ubiquitination of Delta for endocytosis. Tom, a Bearded protein family member, inhibits the Neur-mediated endocytosis through interactions with the NHR1 domain. We have identified the domain boundaries of the novel NHR1 domain, using a screening system based on our cell-free protein synthesis method, and demonstrated that the identified Neur NHR1 domain had binding activity to the 20-residue peptide corresponding to motif 2 of Tom by isothermal titration calorimetry experiments. We also determined the solution structure of the Neur NHR1 domain by heteronuclear NMR methods, using a (15)N/(13)C-labeled sample. The Neur NHR1 domain adopts a characteristic beta-sandwich fold, consisting of a concave five-stranded antiparallel beta-sheet and a convex seven-stranded antiparallel beta-sheet. The long loop (L6) between the beta6 and beta7 strands covers the hydrophobic patch on the concave beta-sheet surface, and the Neur NHR1 domain forms a compact globular fold. Intriguingly, in spite of the slight, but distinct, differences in the topology of the secondary structure elements, the structure of the Neur NHR1 domain is quite similar to those of the B30.2/SPRY domains, which are known to mediate specific protein-protein interactions. Further NMR titration experiments of the Neur NHR1 domain with the 20-residue Tom peptide revealed that the resonances originating from the bottom area of the beta-sandwich (the L3, L5, and L11 loops, as well as the tip of the L6 loop) were affected. In addition, a structural comparison

Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sunita,S.; Zhenxing, H.; Swaathi, J.

2006-01-01

Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine ({psi}) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E. coli RluF at 2.6 Angstroms resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. Themore » structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of {psi}-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.« less

Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

PubMed Central

Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

2008-01-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex▿

PubMed Central

Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R.; Elbing, Karin; Schmidt, Martin C.

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation. PMID:19897735

Differential roles of the glycogen-binding domains of beta subunits in regulation of the Snf1 kinase complex.

PubMed

Mangat, Simmanjeet; Chandrashekarappa, Dakshayini; McCartney, Rhonda R; Elbing, Karin; Schmidt, Martin C

2010-01-01

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic alpha subunit and regulatory beta and gamma subunits. In this study, the role of the beta subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (alpha), Snf4 (gamma), and one of three alternative beta subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three beta subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the beta subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.

Structure of the SPRY domain of the human RNA helicase DDX1, a putative interaction platform within a DEAD-box protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kellner, Julian N.; Meinhart, Anton, E-mail: anton.meinhart@mpimf-heidelberg.mpg.de

The structure of the SPRY domain of the human RNA helicase DDX1 was determined at 2.0 Å resolution. The SPRY domain provides a putative protein–protein interaction platform within DDX1 that differs from other SPRY domains in its structure and conserved regions. The human RNA helicase DDX1 in the DEAD-box family plays an important role in RNA processing and has been associated with HIV-1 replication and tumour progression. Whereas previously described DEAD-box proteins have a structurally conserved core, DDX1 shows a unique structural feature: a large SPRY-domain insertion in its RecA-like consensus fold. SPRY domains are known to function as protein–proteinmore » interaction platforms. Here, the crystal structure of the SPRY domain of human DDX1 (hDSPRY) is reported at 2.0 Å resolution. The structure reveals two layers of concave, antiparallel β-sheets that stack onto each other and a third β-sheet beneath the β-sandwich. A comparison with SPRY-domain structures from other eukaryotic proteins showed that the general β-sandwich fold is conserved; however, differences were detected in the loop regions, which were identified in other SPRY domains to be essential for interaction with cognate partners. In contrast, in hDSPRY these loop regions are not strictly conserved across species. Interestingly, though, a conserved patch of positive surface charge is found that may replace the connecting loops as a protein–protein interaction surface. The data presented here comprise the first structural information on DDX1 and provide insights into the unique domain architecture of this DEAD-box protein. By providing the structure of a putative interaction domain of DDX1, this work will serve as a basis for further studies of the interaction network within the hetero-oligomeric complexes of DDX1 and of its recruitment to the HIV-1 Rev protein as a viral replication factor.« less

Structural classification of small, disulfide-rich protein domains.

PubMed

Cheek, Sara; Krishna, S Sri; Grishin, Nick V

2006-05-26

Disulfide-rich domains are small protein domains whose global folds are stabilized primarily by the formation of disulfide bonds and, to a much lesser extent, by secondary structure and hydrophobic interactions. Disulfide-rich domains perform a wide variety of roles functioning as growth factors, toxins, enzyme inhibitors, hormones, pheromones, allergens, etc. These domains are commonly found both as independent (single-domain) proteins and as domains within larger polypeptides. Here, we present a comprehensive structural classification of approximately 3000 small, disulfide-rich protein domains. We find that these domains can be arranged into 41 fold groups on the basis of structural similarity. Our fold groups, which describe broader structural relationships than existing groupings of these domains, bring together representatives with previously unacknowledged similarities; 18 of the 41 fold groups include domains from several SCOP folds. Within the fold groups, the domains are assembled into families of homologs. We define 98 families of disulfide-rich domains, some of which include newly detected homologs, particularly among knottin-like domains. On the basis of this classification, we have examined cases of convergent and divergent evolution of functions performed by disulfide-rich proteins. Disulfide bonding patterns in these domains are also evaluated. Reducible disulfide bonding patterns are much less frequent, while symmetric disulfide bonding patterns are more common than expected from random considerations. Examples of variations in disulfide bonding patterns found within families and fold groups are discussed.

Topological domain walls in helimagnets

NASA Astrophysics Data System (ADS)

Schoenherr, P.; Müller, J.; Köhler, L.; Rosch, A.; Kanazawa, N.; Tokura, Y.; Garst, M.; Meier, D.

2018-05-01

Domain walls naturally arise whenever a symmetry is spontaneously broken. They interconnect regions with different realizations of the broken symmetry, promoting structure formation from cosmological length scales to the atomic level1,2. In ferroelectric and ferromagnetic materials, domain walls with unique functionalities emerge, holding great promise for nanoelectronics and spintronics applications3-5. These walls are usually of Ising, Bloch or Néel type and separate homogeneously ordered domains. Here we demonstrate that a wide variety of new domain walls occurs in the presence of spatially modulated domain states. Using magnetic force microscopy and micromagnetic simulations, we show three fundamental classes of domain walls to arise in the near-room-temperature helimagnet iron germanium. In contrast to conventional ferroics, the domain walls exhibit a well-defined inner structure, which—analogous to cholesteric liquid crystals—consists of topological disclination and dislocation defects. Similar to the magnetic skyrmions that form in the same material6,7, the domain walls can carry a finite topological charge, permitting an efficient coupling to spin currents and contributions to a topological Hall effect. Our study establishes a new family of magnetic nano-objects with non-trivial topology, opening the door to innovative device concepts based on helimagnetic domain walls.

Neuronal connexin36 association with zonula occludens-1 protein (ZO-1) in mouse brain and interaction with the first PDZ domain of ZO-1

PubMed Central

Li, Xinbo; Olson, Carl; Lu, Shijun; Kamasawa, Naomi; Yasumura, Thomas; Rash, John E.; Nagy, James I.

2007-01-01

Among the 20 members in the connexin family of gap junction proteins, only connexin36 (Cx36) is firmly established to be expressed in neurons and to form electrical synapses at widely distributed interneuronal gap junctions in mammalian brain. Several connexins have recently been reported to interact with the PDZ domain-containing protein zonula occludens-1 (ZO-1), which was originally considered to be associated only with tight junctions, but has recently been reported to associate with other structures including gap junctions in various cell types. Based on the presence of sequence corresponding to a putative PDZ binding motif in Cx36, we investigated anatomical relationships and molecular association of Cx36 with ZO-1. By immunofluorescence, punctate Cx36/ZO-1 colocalization was observed throughout the central nervous system of wild-type mice, whereas labelling for Cx36 was absent in Cx36 knockout mice, confirming the specificity of the anti-Cx36 antibodies employed. By freeze-fracture replica immunogold labelling, Cx36 and ZO-1 in brain were found colocalized within individual ultrastructurally identified gap junction plaques, although some plaques contained only Cx36 whereas others contained only ZO-1. Cx36 from mouse brain and Cx36-transfected HeLa cells was found to coimmunoprecipitate with ZO-1. Unlike other connexins that bind the second of the three PDZ domains in ZO-1, glutathione S-transferase-PDZ pull-down and mutational analyses indicated Cx36 interaction with the first PDZ domain of ZO-1, which required at most the presence of the four c-terminus amino acids of Cx36. These results demonstrating a Cx36/ZO-1 association suggest a regulatory and/or scaffolding role of ZO-1 at gap junctions that form electrical synapses between neurons in mammalian brain. PMID:15090040

The Classification of Protein Domains.

PubMed

Dawson, Natalie; Sillitoe, Ian; Marsden, Russell L; Orengo, Christine A

2017-01-01

The significant expansion in protein sequence and structure data that we are now witnessing brings with it a pressing need to bring order to the protein world. Such order enables us to gain insights into the evolution of proteins, their function and the extent to which the functional repertoire can vary across the three kingdoms of life. This has lead to the creation of a wide range of protein family classifications that aim to group proteins based upon their evolutionary relationships.In this chapter we discuss the approaches and methods that are frequently used in the classification of proteins, with a specific emphasis on the classification of protein domains. The construction of both domain sequence and domain structure databases is considered and we show how the use of domain family annotations to assign structural and functional information is enhancing our understanding of genomes.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

PubMed Central

Madsen, Kenneth L.; Eriksen, Jacob; Milan-Lobo, Laura; Han, Daniel S.; Niv, Masha Y.; Ammendrup-Johnsen, Ina; Henriksen, Ulla; Bhatia, Vikram K.; Stamou, Dimitrios; Sitte, Harald H.; McMahon, Harvey T.; Weinstein, Harel; Gether, Ulrik

2013-01-01

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment. PMID:18466293

Identification of the functional domain in the transcription factor RTEF-1 that mediates alpha 1-adrenergic signaling in hypertrophied cardiac myocytes.

PubMed

Ueyama, T; Zhu, C; Valenzuela, Y M; Suzow, J G; Stewart, A F

2000-06-09

Cardiac myocytes respond to alpha(1)-adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle alpha-actin. The skeletal muscle alpha-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the alpha(1)-adrenergic response of the skeletal muscle alpha-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the alpha(1)-adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the alpha(1)-adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of alpha(1)-adrenergic-dependent kinases. Mutation of a single serine residue, Ser-322, reduced the alpha(1)-adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the alpha(1)-adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of alpha(1)-adrenergic signaling in hypertrophied cardiac myocytes.

DNA-damage-inducible 1 protein (Ddi1) contains an uncharacteristic ubiquitin-like domain that binds ubiquitin

PubMed Central

Nowicka, Urszula; Zhang, Daoning; Walker, Olivier; Krutauz, Daria; Castañeda, Carlos A.; Chaturvedi, Apurva; Chen, Tony Y.; Reis, Noa; Glickman, Michael H.; Fushman, David

2015-01-01

SUMMARY Ddi1 belongs to a family of shuttle proteins targeting polyubiquitinated substrates for proteasomal degradation. Unlike the other proteasomal shuttles, Rad23 and Dsk2, Ddi1 remains an enigma: its function is not fully understood and structural properties are poorly characterized. We determined the structure and binding properties of the ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of Ddi1 from Saccharomyces cerevisiae. We found that, while Ddi1UBA forms a characteristic UBA:ubiquitin complex, Ddi1UBL has entirely uncharacteristic binding preferences. Despite having a ubiquitin-like fold, Ddi1UBL does not interact with typical UBL-receptors but, unexpectedly, binds ubiquitin, forming a unique interface mediated by hydrophobic contacts and by salt-bridges between oppositely-charged residues of Ddi1UBL and ubiquitin. In stark contrast with ubiquitin and other UBLs, the β-sheet surface of Ddi1UBL is negatively charged and, therefore, is recognized in a completely different way. The dual functionality of Ddi1UBL, capable of binding both ubiquitin and proteasome, suggests a novel mechanism for Ddi1 as a proteasomal shuttle. PMID:25703377

Biochemical reconstitution and phylogenetic comparison of human SET1 family core complexes involved in histone methylation.

PubMed

Shinsky, Stephen A; Monteith, Kelsey E; Viggiano, Susan; Cosgrove, Michael S

2015-03-06

Mixed lineage leukemia protein-1 (MLL1) is a member of the SET1 family of histone H3 lysine 4 (H3K4) methyltransferases that are required for metazoan development. MLL1 is the best characterized human SET1 family member, which includes MLL1-4 and SETd1A/B. MLL1 assembles with WDR5, RBBP5, ASH2L, DPY-30 (WRAD) to form the MLL1 core complex, which is required for H3K4 dimethylation and transcriptional activation. Because all SET1 family proteins interact with WRAD in vivo, it is hypothesized they are regulated by similar mechanisms. However, recent evidence suggests differences among family members that may reflect unique regulatory inputs in the cell. Missing is an understanding of the intrinsic enzymatic activities of different SET1 family complexes under standard conditions. In this investigation, we reconstituted each human SET1 family core complex and compared subunit assembly and enzymatic activities. We found that in the absence of WRAD, all but one SET domain catalyzes at least weak H3K4 monomethylation. In the presence of WRAD, all SET1 family members showed stimulated monomethyltransferase activity but differed in their di- and trimethylation activities. We found that these differences are correlated with evolutionary lineage, suggesting these enzyme complexes have evolved to accomplish unique tasks within metazoan genomes. To understand the structural basis for these differences, we employed a "phylogenetic scanning mutagenesis" assay and identified a cluster of amino acid substitutions that confer a WRAD-dependent gain-of-function dimethylation activity on complexes assembled with the MLL3 or Drosophila trithorax proteins. These results form the basis for understanding how WRAD differentially regulates SET1 family complexes in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

What’s for dinner? Types of food served at family dinner differ across parent and family characteristics

PubMed Central

Neumark-Sztainer, Dianne; MacLehose, Rich; Loth, Katie; Fulkerson, Jayne A.; Eisenberg, Marla E.; Berge, Jerica

2013-01-01

Objective To examine the types of food served at family dinner in the homes of adolescents and correlations with parent and family sociodemographic characteristics, psychosocial factors, and meal-specific variables. Design A cross-sectional population-based survey completed by mail or telephone by parents participating in Project F-EAT (Families and Eating and Activity in Teens) in 2009–2010. Setting Homes of families with adolescents in Minneapolis/St Paul urban area. Subjects Participants included 1,923 parents/guardians (90.8% female; 68.5% from ethnic/racial minorities) of adolescents who participated in EAT 2010. Results Less than a third (28%) of parents reported serving a green salad at family dinner on a regular basis, but 70% reported regularly serving vegetables (other than potatoes). About one-fifth (21%) of families had fast food at family dinners two or more times a week. Variables from within the sociodemographic domain (low educational attainment); psychosocial domain (high work-life stress, depressive symptoms, low family functioning); and meal-specific domain (low value of family meals, low enjoyment of cooking, low meal planning, high food purchasing barriers, and fewer hours in food preparation) were associated with lower healthfulness of foods served at family dinners, in analyses adjusted for sociodemographic characteristics. Conclusions There is a need for interventions to improve the healthfulness of food served at family meals. Interventions need to be suitable for parents with low levels of education; take parent and family psychosocial factors into account; promote more positive attitudes toward family meals; and provide skills to make it easier to plan and prepare healthful family meals. PMID:23083836

Deletion of uncharacterized domain from α-1,3-glucanase of Bacillus circulans KA-304 enhances heterologous enzyme production in Escherichia coli.

PubMed

Yano, Shigekazu; Suyotha, Wasana; Zanma, Sumika; Konno, Hiroyuki; Cherdvorapong, Vipavee; Wakayama, Mamoru

2018-05-08

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. k cat /K m values of AgΔUCD and Agl-KA were 11.4 and 11.1 s -1 mg -1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

PubMed

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Inorganic pyrophosphatases of Family II-two decades after their discovery.

PubMed

Baykov, Alexander A; Anashkin, Viktor A; Salminen, Anu; Lahti, Reijo

2017-10-01

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PP i ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (k cat ≈ 10 4 s -1 ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine β-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PP i levels, in response to changes in cell energy status (ATP/ADP ratio). © 2017 Federation of European Biochemical Societies.

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

NPAS1-ARNT and NPAS3-ARNT crystal structures implicate the bHLH-PAS family as multi-ligand binding transcription factors

DOE PAGES

Wu, Dalei; Su, Xiaoyu; Potluri, Nalini; ...

2016-10-26

Here, the neuronal PAS domain proteins NPAS1 and NPAS3 are members of the basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) family, and their genetic deficiencies are linked to a variety of human psychiatric disorders including schizophrenia, autism spectrum disorders and bipolar disease. NPAS1 and NPAS3 must each heterodimerize with the aryl hydrocarbon receptor nuclear translocator (ARNT), to form functional transcription complexes capable of DNA binding and gene regulation. Here we examined the crystal structures of multi-domain NPAS1-ARNT and NPAS3-ARNT-DNA complexes, discovering each to contain four putative ligand-binding pockets. Through expanded architectural comparisons between these complexes and HIF-1α-ARNT, HIF-2α-ARNT and CLOCK-BMAL1, we show the widermore » mammalian bHLH-PAS family is capable of multi-ligand-binding and presents as an ideal class of transcription factors for direct targeting by small-molecule drugs.« less

Development and testing of the Survey of Family Environment (SFE): a novel instrument to measure family functioning and needs for family support.

PubMed

Hohashi, Naohiro; Honda, Junko

2012-01-01

Hohashi's Concentric Sphere Family Environment Model (CSFEM; Hohashi & Honda, 2011) is a newly proposed family nursing theory for holistically understanding the family environment that acts on family well-being. The purpose of this article is to develop and psychometrically test the Japanese version of the Survey of Family Environment (SFE-J), grounded in the CSFEM, for measuring family's perceived family functioning and family's perceived needs for family support. The SFE-J is a 30-item self-administered instrument that assesses five domains (suprasystem, macrosystem, microsystem, family internal environment system, and chronosystem) and has been subjected to rigorous reliability and validity investigations among paired partners in child-rearing families (N of family = 1,990). Internal consistency reliability was high as measured by Cronbach's alpha coefficients. Temporal stability over a 2-week interval was supported by high (substantial or perfect) and significant intraclass correlation coefficients. The total score for the SFE-J was significantly correlated with the Japanese version of the Feetham Family Functioning Survey (FFFS-J), indicating an acceptable concurrent validity. Construct validity was supported by a confirmatory factor analysis that evaluated the five-factor structure to measure the concept of CSFEM. Results also demonstrate that the SFE-J family functioning scores show no significant differences between paired partners. The SFE-J is a reliable and valid instrument to assess not only intrafamily functioning but also interfamily functioning and, by identifying items/domains with high requirements for family support, serves to facilitate the providing of appropriate support to families.

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

PubMed

Lin, Xue; Yu, Ai-Qun; Zhang, Cui-Ying; Pi, Li; Bai, Xiao-Wen; Xiao, Dong-Guang

2017-11-09

Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

The 9aaTAD Transactivation Domains: From Gal4 to p53.

PubMed

Piskacek, Martin; Havelka, Marek; Rezacova, Martina; Knight, Andrea

2016-01-01

The family of the Nine amino acid Transactivation Domain, 9aaTAD family, comprises currently over 40 members. The 9aaTAD domains are universally recognized by the transcriptional machinery from yeast to man. We had identified the 9aaTAD domains in the p53, Msn2, Pdr1 and B42 activators by our prediction algorithm. In this study, their competence to activate transcription as small peptides was proven. Not surprisingly, we elicited immense 9aaTAD divergence in hundreds of identified orthologs and numerous examples of the 9aaTAD species' convergence. We found unforeseen similarity of the mammalian p53 with yeast Gal4 9aaTAD domains. Furthermore, we identified artificial 9aaTAD domains generated accidentally by others. From an evolutionary perspective, the observed easiness to generate 9aaTAD transactivation domains indicates the natural advantage for spontaneous generation of transcription factors from DNA binding precursors.

Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

PubMed

Monniot, Céline; Boisramé, Anita; Da Costa, Grégory; Chauvel, Muriel; Sautour, Marc; Bougnoux, Marie-Elisabeth; Bellon-Fontaine, Marie-Noëlle; Dalle, Frédéric; d'Enfert, Christophe; Richard, Mathias L

2013-01-01

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

Novel functions of CCM1 delimit the relationship of PTB/PH domains.

PubMed

Zhang, Jun; Dubey, Pallavi; Padarti, Akhil; Zhang, Aileen; Patel, Rinkal; Patel, Vipulkumar; Cistola, David; Badr, Ahmed

2017-10-01

Three NPXY motifs and one FERM domain in CCM1 makes it a versatile scaffold protein for tethering the signaling components together within the CCM signaling complex (CSC). The cellular role of CCM1 protein remains inadequately expounded. Both phosphotyrosine binding (PTB) and pleckstrin homology (PH) domains were recognized as structurally related but functionally distinct domains. By utilizing molecular cloning, protein binding assays and RT-qPCR to identify novel cellular partners of CCM1 and its cellular expression patterns; by screening candidate PTB/PH proteins and subsequently structurally simulation in combining with current X-ray crystallography and NMR data to defined the essential structure of PTB/PH domain for NPXY-binding and the relationship among PTB, PH and FERM domain(s). We identified a group of 28 novel cellular partners of CCM1, all of which contain either PTB or PH domain(s), and developed a novel classification system for these PTB/PH proteins based on their relationship with different NPXY motifs of CCM1. Our results demonstrated that CCM1 has a wide spectrum of binding to different PTB/PH proteins and perpetuates their specificity to interact with certain PTB/PH domains through selective combination of three NPXY motifs. We also demonstrated that CCM1 can be assembled into oligomers through intermolecular interaction between its F3 lobe in FERM domain and one of the three NPXY motifs. Despite being embedded in FERM domain as F3 lobe, F3 module acts as a fully functional PH domain to interact with NPXY motif. The most salient feature of the study was that both PTB and PH domains are structurally and functionally comparable, suggesting that PTB domain is likely evolved from PH domain with polymorphic structural additions at its N-terminus. A new β1A-strand of the PTB domain was discovered and new minimum structural requirement of PTB/PH domain for NPXY motif-binding was determined. Based on our data, a novel theory of structure, function and

Characterizing Functional Domains for TIM-Mediated Enveloped Virus Entry

PubMed Central

Moller-Tank, Sven; Albritton, Lorraine M.; Rennert, Paul D.

2014-01-01

ABSTRACT T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members were recently identified as phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance entry of Ebola virus (EBOV) and other viruses by binding PtdSer on the viral envelope, concentrating virus on the cell surface, and promoting subsequent internalization. The PtdSer-binding activity of the immunoglobulin-like variable (IgV) domain is essential for both virus binding and internalization by TIM-1. However, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry, indicating that other domains of TIM proteins are functionally important. Here, we investigate the domains supporting enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer-binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. IMPORTANCE T-cell immunoglobulin and mucin domain 1 (TIM-1) and other TIM family members are recently identified phosphatidylserine (PtdSer)-mediated virus entry-enhancing receptors (PVEERs). These proteins enhance virus entry by binding the phospholipid, PtdSer, present on the viral

Reciprocal Associations between Family and Peer Conflict in Adolescents’ Daily Lives1

PubMed Central

Chung, Grace H.; Fuligni, Andrew J.

2012-01-01

Using a daily diary method, this study assessed daily episodes of family and peer conflict among 578 adolescents in the ninth grade in order to examine potential bidirectional associations between the family and peer domains. Adolescents completed a daily diary checklist at the end of each day over a fourteen day period to report events of conflict and their emotional states for a given day. Overall, our within-person models provided evidence for the bidirectional nature of family-peer linkages across gender and ethnicity. Adolescents experienced more peer conflict on days in which they argued with parents or other family members, and vice versa. Effect of family conflict further spilled over into peer relationships the next day and two days later, whereas peer conflict predicted only the following day family conflict. Adolescents’ emotional distress partially explained these short term spillovers between family and peer conflict. PMID:21793820

Structural Basis of Membrane Targeting by the Dock180 Family of Rho Family Guanine Exchange Factors (Rho-GEFs)*

PubMed Central

Premkumar, Lakshmanane; Bobkov, Andrey A.; Patel, Manishha; Jaroszewski, Lukasz; Bankston, Laurie A.; Stec, Boguslaw; Vuori, Kristiina; Côté, Jean-Francois; Liddington, Robert C.

2010-01-01

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P3 head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180. PMID:20167601

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA domain Ubiquitination

PubMed Central

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K.; Keith, Amy L.; Razani, Babak; Weihl, Conrad C.

2017-01-01

Summary p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar to that seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT expressing cells increases ubiquitinated inclusion formation, p62’s association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62’s UBA domain at lysine 420 may regulate p62’s function and be disrupted in p62 associated disease. PMID:28380357

Crystal Structure of the Neuropilin-1 MAM Domain: Completing the Neuropilin-1 Ectodomain Picture.

PubMed

Yelland, Tamas; Djordjevic, Snezana

2016-11-01

Neuropilins (NRPs) are single-pass transmembrane receptors involved in several signaling pathways that regulate key physiological processes such as vascular morphogenesis and axon guidance. The MAM domain of NRP, which has previously been implicated in receptor multimerization, was the only portion of the ectopic domain of the NRPs for which the structure, until now, has been elusive. Using site-directed mutagenesis in the linker region preceding the MAM domain we generated a protein construct amenable to crystallization. Here we present the crystal structure of the MAM domain of human NRP1 at 2.24 Å resolution. The protein exhibits a jellyroll topology, with Ca 2+ ions bound at the inter-strand space enhancing the thermostability of the domain. We show that the MAM domain of NRP1 is monomeric in solution and insufficient to drive receptor dimerization, which leads us to propose a different role for this domain in the context of NRP membrane assembly and signaling. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Domain III of Cry1Ac Is Critical to Binding and Toxicity against Soybean Looper (Chrysodeixis includens) but Not to Velvetbean Caterpillar (Anticarsia gemmatalis).

PubMed

Mushtaq, Rubina; Shakoori, Abdul Rauf; Jurat-Fuentes, Juan Luis

2018-02-27

Insecticidal proteins Cry1Ac and Cry2Ac7 from the bacterium Bacillus thuringiensis (Bt) belong to the three-domain family of Bt toxins. Commercial transgenic soybean hybrids produce Cry1Ac to control the larvae of the soybean looper ( Chrysodeixis includens ) and the velvet bean caterpillar ( Anticarsia gemmatalis ). The specificity of Cry1Ac is determined by loops extending from domain II and regions of domain III in the three-dimensional structure of the toxin. In this study, we constructed a hybrid toxin (H1.2Ac) containing domains I and II of Cry1Ac and domain III of Cry2Ac7, in an attempt to obtain a protein with enhanced toxicity compared to parental toxins. Bioassays with H1.2Ac revealed toxicity against the larvae of A. gemmatalis but not against C. includens . Saturation binding assays with radiolabeled toxins and midgut brush border membrane vesicles demonstrated no specific H1.2Ac binding to C. includens , while binding in A. gemmatalis was specific and saturable. Results from competition binding assays supported the finding that Cry1Ac specificity against A. gemmatalis is mainly dictated by domain II. Taken together, these distinct interactions with binding sites may help explain the differential susceptibility to Cry1Ac in C. includens and A. gemmatalis , and guide the design of improved toxins against soybean pests.

Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1

PubMed Central

Hota, Prasanta K; Buck, Matthias

2009-01-01

Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation. PMID:19388051

BRCT-domain protein BRIT1 influences class switch recombination

PubMed Central

Yen, Wei-Feng; Chaudhry, Ashutosh; Vaidyanathan, Bharat; Yewdell, William T.; Pucella, Joseph N.; Sharma, Rahul; Li, Kaiyi; Rudensky, Alexander Y.; Chaudhuri, Jayanta

2017-01-01

DNA double-strand breaks (DSBs) serve as obligatory intermediates for Ig heavy chain (Igh) class switch recombination (CSR). The mechanisms by which DSBs are resolved to promote long-range DNA end-joining while suppressing genomic instability inherently associated with DSBs are yet to be fully elucidated. Here, we use a targeted short-hairpin RNA screen in a B-cell lymphoma line to identify the BRCT-domain protein BRIT1 as an effector of CSR. We show that conditional genetic deletion of BRIT1 in mice leads to a marked increase in unrepaired Igh breaks and a significant reduction in CSR in ex vivo activated splenic B cells. We find that the C-terminal tandem BRCT domains of BRIT1 facilitate its interaction with phosphorylated H2AX and that BRIT1 is recruited to the Igh locus in an activation-induced cytidine deaminase (AID) and H2AX-dependent fashion. Finally, we demonstrate that depletion of another BRCT-domain protein, MDC1, in BRIT1-deleted B cells increases the severity of CSR defect over what is observed upon loss of either protein alone. Our results identify BRIT1 as a factor in CSR and demonstrate that multiple BRCT-domain proteins contribute to optimal resolution of AID-induced DSBs. PMID:28724724

Cytoskeleton-interacting LIM-domain protein CRP1 suppresses cell proliferation and protects from stress-induced cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Latonen, Leena; Jaervinen, Paeivi M.; Haartman Institute, University of Helsinki, FIN-00014 Helsinki

2008-02-15

Members of the cysteine-rich protein (CRP) family are actin cytoskeleton-interacting LIM-domain proteins known to act in muscle cell differentiation. We have earlier found that CRP1, a founding member of this family, is transcriptionally induced by UV radiation in human diploid fibroblasts [M. Gentile, L. Latonen, M. Laiho, Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses, Nucleic Acids Res. 31 (2003) 4779-4790]. Here we show that CRP1 is induced by growth-inhibitory signals, such as increased cellular density, and cytotoxic stress induced by UV radiation or staurosporine. We found that high levels of CRP1more » correlate with differentiation-associated morphology towards the myofibroblast lineage and that expression of ectopic CRP1 suppresses cell proliferation. Following UV- and staurosporine-induced stresses, expression of CRP1 provides a survival advantage evidenced by decreased cellular death and increased cellular metabolic activity and attachment. Our studies identify that CRP1 is a novel stress response factor, and provide evidence for its growth-inhibitory and cytoprotective functions.« less

Ankyrin binding activity shared by the neurofascin/L1/NrCAM family of nervous system cell adhesion molecules.

PubMed

Davis, J Q; Bennett, V

1994-11-04

Neurofascin, L1, NrCAM, NgCAM, and neuroglian are membrane-spanning cell adhesion molecules with conserved cytoplasmic domains that are believed to play important roles in development of the nervous system. This report presents biochemical evidence that the cytoplasmic domains of these molecules associate directly with ankyrins, a family of spectrin-binding proteins located on the cytoplasmic surface of specialized plasma membrane domains. Rat neurofascin and NrCAM together comprise over 0.5% of the membrane protein in adult brain tissue. Linkage of these ankyrin-binding cell adhesion molecules to spectrin-based structures may provide a major class of membrane-cytoskeletal connections in adult brain as well as earlier stages of development.

Crystal structure of calpain-3 penta-EF-hand (PEF) domain - a homodimerized PEF family member with calcium bound at the fifth EF-hand

DOE Office of Scientific and Technical Information (OSTI.GOV)

Partha, Sarathy K.; Ravulapalli, Ravikiran; Allingham, John S.

2014-08-21

Calpains are Ca 2+dependent intracellular cysteine proteases that cleave a wide range of protein substrates to help implement Ca 2+ signaling in the cell. The major isoforms of this enzyme family, calpain-1 and calpain-2, are heterodimers of a large and a small subunit, with the main dimer interface being formed through their C-terminal penta-EF hand (PEF) domains. Calpain-3, or p94, is a skeletal muscle-specific isoform that is genetically linked to limb-girdle muscular dystrophy. Biophysical and modeling studies with the PEF domain of calpain-3 support the suggestion that full-length calpain-3 exists as a homodimer. Here, we report the crystallization of calpain-3'smore » PEF domain and its crystal structure in the presence of Ca 2+, which provides evidence for the homodimer architecture of calpain-3 and supports the molecular model that places a protease core at either end of the elongated dimer. Unlike other calpain PEF domain structures, the calpain-3 PEF domain contains a Ca 2+ bound at the EF5-hand used for homodimer association. Three of the four Ca 2+-binding EF-hands of the PEF domains are concentrated near the protease core, and have the potential to radically change the local charge within the dimer during Ca 2+ signaling. Examination of the homodimer interface shows that there would be steric clashes if the calpain-3 large subunit were to try to pair with a calpain small subunit.« less

26 CFR 1.262-1 - Personal, living, and family expenses.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 26 Internal Revenue 3 2011-04-01 2011-04-01 false Personal, living, and family expenses. 1.262-1... TAX (CONTINUED) INCOME TAXES (CONTINUED) Items Not Deductible § 1.262-1 Personal, living, and family... expressly provided in chapter 1 of the Code, for personal, living, and family expenses. (b) Examples of...

Signaling network of the Btk family kinases.

PubMed

Qiu, Y; Kung, H J

2000-11-20

The Btk family kinases represent new members of non-receptor tyrosine kinases, which include Btk/Atk, Itk/Emt/Tsk, Bmx/Etk, and Tec. They are characterized by having four structural modules: PH (pleckstrin homology) domain, SH3 (Src homology 3) domain, SH2 (Src homology 2) domain and kinase (Src homology 1) domain. Increasing evidence suggests that, like Src-family kinases, Btk family kinases play central but diverse modulatory roles in various cellular processes. They participate in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, cytokine receptors, G-protein coupled receptors, antigen-receptors and integrins. They are regulated by many non-receptor tyrosine kinases such as Src, Jak, Syk and FAK family kinases. In turn, they regulate many of major signaling pathways including those of PI3K, PLCgamma and PKC. Both genetic and biochemical approaches have been used to dissect the signaling pathways and elucidate their roles in growth, differentiation and apoptosis. An emerging new role of this family of kinases is cytoskeletal reorganization and cell motility. The physiological importance of these kinases was amply demonstrated by their link to the development of immunodeficiency diseases, due to germ-line mutations. The present article attempts to review the structure and functions of Btk family kinases by summarizing our current knowledge on the interacting partners associated with the different modules of the kinases and the diverse signaling pathways in which they are involved.

GNormPlus: An Integrative Approach for Tagging Genes, Gene Families, and Protein Domains

PubMed Central

Lu, Zhiyong

2015-01-01

The automatic recognition of gene names and their associated database identifiers from biomedical text has been widely studied in recent years, as these tasks play an important role in many downstream text-mining applications. Despite significant previous research, only a small number of tools are publicly available and these tools are typically restricted to detecting only mention level gene names or only document level gene identifiers. In this work, we report GNormPlus: an end-to-end and open source system that handles both gene mention and identifier detection. We created a new corpus of 694 PubMed articles to support our development of GNormPlus, containing manual annotations for not only gene names and their identifiers, but also closely related concepts useful for gene name disambiguation, such as gene families and protein domains. GNormPlus integrates several advanced text-mining techniques, including SimConcept for resolving composite gene names. As a result, GNormPlus compares favorably to other state-of-the-art methods when evaluated on two widely used public benchmarking datasets, achieving 86.7% F1-score on the BioCreative II Gene Normalization task dataset and 50.1% F1-score on the BioCreative III Gene Normalization task dataset. The GNormPlus source code and its annotated corpus are freely available, and the results of applying GNormPlus to the entire PubMed are freely accessible through our web-based tool PubTator. PMID:26380306

Synthesis of framework isomer MOFs containing zinc and 4-tetrazolyl benzenecarboxylic acid via a structure directing solvothermal approach

DOE PAGES

Ordonez, Carlos; Kinnibrugh, Tiffany L.; Xu, Hongwu; ...

2015-04-02

The solvothermal synthesis of framework isomers was carried out using the hybrid carboxylate and tetrazolate functional ligand, 4-tetrazolyl benzenecarboxylic acid (H₂TBC, TBC = 4-tetrazolyl benzenecarboxylate) and zinc. H₂TBC was also synthesized with the solvothermal approach, and is referred herein as structure 1. Using single-crystal X-ray diffraction, we found that the tetrazolate groups of TBC show an unusual “opposite-on” coordination mode with zinc. Three previously characterized metal-organic frameworks (MOFs) were obtained by systematically changing the solvents of the H₂TBC-Zn reaction, (1) ZnTBC, 2, which has a non-porous structure; (2) Zn₂(TBC)₂(H2O), 3, which has an amphiphilic pore structure and (3) Zn₂(TBC)₂{guest}, 4,more » which is porous and has channels containing uncoordinated N heteroatoms. Fluorescence spectra of 4 reveal a strong blue emission mainly from the TBC ligands.« less

The Nucleotide-Binding Oligomerization Domain-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome Regulates Bronchial Epithelial Cell Injury and Proapoptosis after Exposure to Biomass Fuel Smoke.

PubMed

Li, Chen; Zhihong, Huang; Wenlong, Li; Xiaoyan, Liu; Qing, Chen; Wenzhi, Luo; Siming, Xie; Shengming, Liu

2016-12-01

The number of individuals in the population exposed to biomass fuel smoke (BS) is far greater than the number of cigarette smokers. About 20% of cigarette smokers develop chronic obstructive pulmonary disease (COPD) due to smoke-induced irreversible damage and sustained inflammation of the airway epithelium. However, the role of BS in COPD pathogenesis remains to be elucidated. In this study, we investigated the expression of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing (NLRP) 3 and caspase-1 in the bronchial epithelium from patients with COPD, and further determined the specific role of the NLRP3 inflammasome in bronchial epithelium injury using two in vitro models (BS and cigarette smoke [CS]) in the human bronchial epithelial (HBE) cell line (16HBE). After exposure to BS and CS, the release of damage-associated molecular patterns, the transcriptional and translational up-regulation of NLRP3, and the activation of caspase-1 were observed in cells at different time points. Because IL-1β secretion was dependent on the NLRP3 inflammasome, we assessed CXCL-8 production in response to smoke. Using a transwell migration assay in which 16HBE cells and human alveolar macrophages were cocultured, we showed that smoke-induced NLRP3 activation in 16HBE cells increased the migration of human alveolar macrophages. When the NLRP3 expression was silenced, the average migration distance of 16HBE was increased in scratch assay, because the activation of NLRP3 induced apoptosis by the p53-Bax mitochondrial pathway in the smoke-induced response. These results demonstrate the importance of the NLRP3 inflammasome in mediating BS- and CS-induced HBE cell damage and proapoptosis.

The structure of the GemC1 coiled coil and its interaction with the Geminin family of coiled-coil proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caillat, Christophe; Fish, Alexander; Pefani, Dafni-Eleftheria

The GemC1 coiled-coil structure has subtle differences compared with its homologues Geminin and Idas. Co-expression experiments in cells and biophysical stability analysis of the Geminin-family coiled coils suggest that the GemC1 coiled coil alone is unstable. GemC1, together with Idas and Geminin, an important regulator of DNA-replication licensing and differentiation decisions, constitute a superfamily sharing a homologous central coiled-coil domain. To better understand this family of proteins, the crystal structure of a GemC1 coiled-coil domain variant engineered for better solubility was determined to 2.2 Å resolution. GemC1 shows a less typical coiled coil compared with the Geminin homodimer and themore » Geminin–Idas heterodimer structures. It is also shown that both in vitro and in cells GemC1 interacts with Geminin through its coiled-coil domain, forming a heterodimer that is more stable that the GemC1 homodimer. Comparative analysis of the thermal stability of all of the possible superfamily complexes, using circular dichroism to follow the unfolding of the entire helix of the coiled coil, or intrinsic tryptophan fluorescence of a unique conserved N-terminal tryptophan, shows that the unfolding of the coiled coil is likely to take place from the C-terminus towards the N-terminus. It is also shown that homodimers show a single-state unfolding, while heterodimers show a two-state unfolding, suggesting that the dimer first falls apart and the helices then unfold according to the stability of each protein. The findings argue that Geminin-family members form homodimers and heterodimers between them, and this ability is likely to be important for modulating their function in cycling and differentiating cells.« less

Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain.

PubMed

Hou, Tingjun; Zhang, Wei; Case, David A; Wang, Wei

2008-02-29

Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was

Interaction of a putative BH3 domain of clusterin with anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Dong-Hwa; Ha, Ji-Hyang; Kim, Yul

Highlights: {yields} Identification of a conserved BH3 motif in C-terminal coiled coil region of nCLU. {yields} The nCLU BH3 domain binds to BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. {yields} A conserved binding mechanism of nCLU BH3 and the other pro-apoptotic BH3 peptides with Bcl-X{sub L}. {yields} The absolutely conserved Leu323 and Asp328 of nCLU BH3 domain are critical for binding to Bcl-X{sub L.} {yields} Molecular understanding of the pro-apoptotic function of nCLU as a novel BH3-only protein. -- Abstract: Clusterin (CLU) is a multifunctional glycoprotein that is overexpressed in prostate and breast cancers. Although CLU is knownmore » to be involved in the regulation of apoptosis and cell survival, the precise molecular mechanism underlying the pro-apoptotic function of nuclear CLU (nCLU) remains unclear. In this study, we identified a conserved BH3 motif in C-terminal coiled coil (CC2) region of nCLU by sequence analysis and characterized the molecular interaction of the putative nCLU BH3 domain with anti-apoptotic Bcl-2 family proteins by nuclear magnetic resonance (NMR) spectroscopy. The chemical shift perturbation data demonstrated that the nCLU BH3 domain binds to pro-apoptotic BH3 peptide-binding grooves in both Bcl-X{sub L} and Bcl-2. A structural model of the Bcl-X{sub L}/nCLU BH3 peptide complex reveals that the binding mode is remarkably similar to those of other Bcl-X{sub L}/BH3 peptide complexes. In addition, mutational analysis confirmed that Leu323 and Asp328 of nCLU BH3 domain, absolutely conserved in the BH3 motifs of BH3-only protein family, are critical for binding to Bcl-X{sub L}. Taken altogether, our results suggest a molecular basis for the pro-apoptotic function of nCLU by elucidating the residue specific interactions of the BH3 motif in nCLU with anti-apoptotic Bcl-2 family proteins.« less

26 CFR 1.262-1 - Personal, living, and family expenses.

Code of Federal Regulations, 2010 CFR

2010-04-01

... trade or business expenses). (c) Cross references. Certain items of a personal, living, or family nature... 26 Internal Revenue 3 2010-04-01 2010-04-01 false Personal, living, and family expenses. 1.262-1... TAX (CONTINUED) INCOME TAXES Items Not Deductible § 1.262-1 Personal, living, and family expenses. (a...

Reconceptualizing the Domain and Boundaries of Family Life Education

ERIC Educational Resources Information Center

Myers-Walls, Judith A.; Ballard, Sharon M.; Darling, Carol Anderson; Myers-Bowman, Karen S.

2011-01-01

Many scholars have defined family life education (FLE), and some have differentiated it from other family-related fields. For example, Doherty (1995) provided a definition of the boundaries between FLE and family therapy; however, we believe those criteria can be improved. We explore the professions of family life education, family therapy, and…

The Pseudokinase Domain of Saccharomyces cerevisiae Tra1 Is Required for Nuclear Localization and Incorporation into the SAGA and NuA4 Complexes.

PubMed

Berg, Matthew D; Genereaux, Julie; Karagiannis, Jim; Brandl, Christopher J

2018-05-31

Tra1 is an essential component of the SAGA/SLIK and NuA4 complexes in S. cerevisiae , recruiting these co-activator complexes to specific promoters. As a PIKK family member, Tra1 is characterized by a C-terminal phosphoinositide 3-kinase (PI3K) domain. Unlike other PIKK family members ( e.g. , Tor1, Tor2, Mec1, Tel1), Tra1 has no demonstrable kinase activity. We identified three conserved arginine residues in Tra1 that reside proximal or within the cleft between the N- and C-terminal subdomains of the PI3K domain. To establish a function for Tra1's PI3K domain and specifically the cleft region, we characterized a tra1 allele where these three arginine residues are mutated to glutamine. The half-life of the Tra1[Formula: see text] protein is reduced but its steady state level is maintained at near wild-type levels by a transcriptional feedback mechanism. The tra1 [Formula: see text] allele results in slow growth under stress and alters the expression of genes also regulated by other components of the SAGA complex. Tra1[Formula: see text] is less efficiently transported to the nucleus than the wild-type protein. Likely related to this, Tra1[Formula: see text] associates poorly with SAGA/SLIK and NuA4. The ratio of Spt7 SLIK to Spt7 SAGA increases in the tra1 [Formula: see text] strain and truncated forms of Spt20 become apparent upon isolation of SAGA/SLIK. Intragenic suppressor mutations of tra1 [Formula: see text] map to the cleft region further emphasizing its importance. We propose that the PI3K domain of Tra1 is directly or indirectly important for incorporating Tra1 into SAGA and NuA4 and thus the biosynthesis and/or stability of the intact complexes. Copyright © 2018 Berg et al.

Keap1/Cullin3 Modulates p62/SQSTM1 Activity via UBA Domain Ubiquitination.

PubMed

Lee, YouJin; Chou, Tsui-Fen; Pittman, Sara K; Keith, Amy L; Razani, Babak; Weihl, Conrad C

2017-04-04

p62/SQSTM1 (p62) is a scaffolding protein that facilitates the formation and degradation of ubiquitinated aggregates via its self-interaction and ubiquitin binding domains. The regulation of this process is unclear but may relate to the post-translational modification of p62. In the present study, we find that Keap1/Cullin3 ubiquitinates p62 at lysine 420 within its UBA domain. Substitution of lysine 420 with an arginine diminishes p62 sequestration and degradation activity similar what is seen when the UBA domain is deleted. Overexpression of Keap1/Cullin3 in p62-WT-expressing cells increases ubiquitinated inclusion formation and p62's association with LC3 and rescues proteotoxicity. This effect is not seen in cells expressing a mutant p62 that fails to interact with Keap1. Interestingly, p62 disease mutants have diminished or absent UBA domain ubiquitination. These data suggest that the ubiquitination of p62's UBA domain at lysine 420 may regulate p62's function and be disrupted in p62-associated disease. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

PubMed Central

2009-01-01

Background Cloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases. Results Comprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed. Conclusion The identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family. PMID:19930663

Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

PubMed Central

Barral, José M.; Bauer, Christopher C.; Ortiz, Irving; Epstein, Henry F.

1998-01-01

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family. PMID:9832550

The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein.

PubMed

Barkan, Alice; Klipcan, Larik; Ostersetzer, Oren; Kawamura, Tetsuya; Asakura, Yukari; Watkins, Kenneth P

2007-01-01

The CRS1-YhbY domain (also called the CRM domain) is represented as a stand-alone protein in Archaea and Bacteria, and in a family of single- and multidomain proteins in plants. The function of this domain is unknown, but structural data and the presence of the domain in several proteins known to interact with RNA have led to the proposal that it binds RNA. Here we describe a phylogenetic analysis of the domain, its incorporation into diverse proteins in plants, and biochemical properties of a prokaryotic and eukaryotic representative of the domain family. We show that a bacterial member of the family, Escherichia coli YhbY, is associated with pre-50S ribosomal subunits, suggesting that YhbY functions in ribosome assembly. GFP fused to a single-domain CRM protein from maize localizes to the nucleolus, suggesting that an analogous activity may have been retained in plants. We show further that an isolated maize CRM domain has RNA binding activity in vitro, and that a small motif shared with KH RNA binding domains, a conserved "GxxG" loop, contributes to its RNA binding activity. These and other results suggest that the CRM domain evolved in the context of ribosome function prior to the divergence of Archaea and Bacteria, that this function has been maintained in extant prokaryotes, and that the domain was recruited to serve as an RNA binding module during the evolution of plant genomes.

Identification and Structural Characterization of the ALIX-Binding Late Domains of Simian Immunodeficiency Virus SIV mac239 and SIV agmTan-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Q Zhai; M Landesman; H Robinson

2011-12-31

Retroviral Gag proteins contain short late-domain motifs that recruit cellular ESCRT pathway proteins to facilitate virus budding. ALIX-binding late domains often contain the core consensus sequence YPX{sub n}L (where X{sub n} can vary in sequence and length). However, some simian immunodeficiency virus (SIV) Gag proteins lack this consensus sequence, yet still bind ALIX. We mapped divergent, ALIX-binding late domains within the p6{sup Gag} proteins of SIV{sub MAC239} ({sub 40}SREK{und P}YKE{und VT}ED{und L}LHLNSLF{sub 59}) and SIV{sub agmTan-1} ({sub 24}AAG{und A}YDP{und AR}KL{und L}EQYAKK{sub 41}). Crystal structures revealed that anchoring tyrosines (in lightface) and nearby hydrophobic residues (underlined) contact the ALIX V domain,more » revealing how lentiviruses employ a diverse family of late-domain sequences to bind ALIX and promote virus budding.« less

Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adams-Cioaba, Melanie A.; Guo, Yahong; Bian, ChuanBing

Expansion of the CGG trinucleotide repeat in the 5'-untranslated region of the FMR1, fragile X mental retardation 1, gene results in suppression of protein expression for this gene and is the underlying cause of Fragile X syndrome. In unaffected individuals, the FMRP protein, together with two additional paralogues (Fragile X Mental Retardation Syndrome-related Protein 1 and 2), associates with mRNA to form a ribonucleoprotein complex in the nucleus that is transported to dendrites and spines of neuronal cells. It is thought that the fragile X family of proteins contributes to the regulation of protein synthesis at sites where mRNAs aremore » locally translated in response to stimuli. Here, we report the X-ray crystal structures of the non-canonical nuclear localization signals of the FXR1 and FXR2 autosomal paralogues of FMRP, which were determined at 2.50 and 1.92 {angstrom}, respectively. The nuclear localization signals of the FXR1 and FXR2 comprise tandem Tudor domain architectures, closely resembling that of UHRF1, which is proposed to bind methylated histone H3K9. The FMRP, FXR1 and FXR2 proteins comprise a small family of highly conserved proteins that appear to be important in translational regulation, particularly in neuronal cells. The crystal structures of the N-terminal tandem Tudor domains of FXR1 and FXR2 revealed a conserved architecture with that of FMRP. Biochemical analysis of the tandem Tudor doamins reveals their ability to preferentially recognize trimethylated peptides in a sequence-specific manner.« less

RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

PubMed Central

Lin, Li; Tran, Thuy; Hu, Shuang; Cramer, Todd; Komuniecki, Richard; Steven, Robert M.

2012-01-01

RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth. PMID:22363657

Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

PubMed

Leiva, Natalia; Capmany, Anahí; Damiani, María Teresa

2013-01-01

Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

Beyond Conflict: Functional Facets of the Work-Family Interplay

ERIC Educational Resources Information Center

Wiese, Bettina S.; Seiger, Christine P.; Schmid, Christian M.; Freund, Alexandra M.

2010-01-01

The present paper deals with three positive facets of the work-family interplay, i.e., transfer of competencies, transfer of positive mood, and cross-domain compensation. The latter refers to the experience that engagement in one domain helps dealing with failures in the other domain. In two correlational studies (N[subscript 1] = 107 working…

Mutations of the RTEL1 Helicase in a Hoyeraal-Hreidarsson Syndrome Patient Highlight the Importance of the ARCH Domain.

PubMed

Jullien, Laurent; Kannengiesser, Caroline; Kermasson, Laetitia; Cormier-Daire, Valérie; Leblanc, Thierry; Soulier, Jean; Londono-Vallejo, Arturo; de Villartay, Jean-Pierre; Callebaut, Isabelle; Revy, Patrick

2016-05-01

The DNA helicase RTEL1 participates in telomere maintenance and genome stability. Biallelic mutations in the RTEL1 gene account for the severe telomere biology disorder characteristic of the Hoyeraal-Hreidarsson syndrome (HH). Here, we report a HH patient (P4) carrying two novel compound heterozygous mutations in RTEL1: a premature stop codon (c.949A>T, p.Lys317*) and an intronic deletion leading to an exon skipping and an in-frame deletion of 25 amino-acids (p.Ile398_Lys422). P4's cells exhibit short and dysfunctional telomeres similarly to other RTEL1-deficient patients. 3D structure predictions indicated that the p.Ile398_Lys422 deletion affects a part of the helicase ARCH domain, which lines the pore formed with the core HD and the iron-sulfur cluster domains and is highly specific of sequences from the eukaryotic XPD family members. © 2016 WILEY PERIODICALS, INC.

Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

PubMed

Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

2008-04-25

The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy

PubMed Central

Ren, Gang; Vajjhala, Parimala; Lee, Janet S.; Winsor, Barbara; Munn, Alan L.

2006-01-01

The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

Members of the LATERAL ORGAN BOUNDARIES DOMAIN Transcription Factor Family Are Involved in the Regulation of Secondary Growth in Populus[W

PubMed Central

Yordanov, Yordan S.; Regan, Sharon; Busov, Victor

2010-01-01

Regulation of secondary (woody) growth is of substantial economic and environmental interest but is poorly understood. We identified and subsequently characterized an activation-tagged poplar (Populus tremula × Populus alba) mutant with enhanced woody growth and changes in bark texture caused primarily by increased secondary phloem production. Molecular characterization of the mutation through positioning of the tag and retransformation experiments shows that the phenotype is conditioned by activation of an uncharacterized gene that encodes a novel member of the LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family of transcription factors. Homology analysis showed highest similarity to an uncharacterized LBD1 gene from Arabidopsis thaliana, and we consequently named it Populus tremula × Populus alba (Pta) LBD1. Dominant-negative suppression of Pta LBD1 via translational fusion with the repressor SRDX domain caused decreased diameter growth and suppressed and highly irregular phloem development. In wild-type plants, LBD1 was most highly expressed in the phloem and cambial zone. Two key Class I KNOTTED1-like homeobox genes that promote meristem identity in the cambium were downregulated, while an Altered Phloem Development gene that is known to promote phloem differentiation was upregulated in the mutant. A set of four LBD genes, including the LBD1 gene, was predominantly expressed in wood-forming tissues, suggesting a broader regulatory role of these transcription factors during secondary woody growth in poplar. PMID:21097711

GSDM family genes meet autophagy.

PubMed

Tamura, Masaru; Shiroishi, Toshihiko

2015-07-15

In the previous issue of Biochemical Journal, Shi et al. [(2015) 468, 325-336] report that Gasdermin (Gsdm) family proteins regulate autophagy activity, which is counter-balanced by the opposite functions of well-conserved N- and C-terminal domains of the proteins. The Gsdm family was originally identified as the causative gene of dominant skin mutations exhibiting alopecia. Each member of the Gsdm gene family shows characteristic expression patterns in the epithelium, which is tissue and differentiation stage-specific. Previous phenotype analyses of mutant mice, biochemical analyses of proteins and genome-wide association studies showed that the Gsdm gene family might be involved in epithelial cell development, apoptosis, inflammation, carcinogenesis and immune-related diseases. To date, however, their molecular function(s) remain unclear. Shi et al. found that mutations in the C-terminal domain of Gsdma3, a member of the Gsdm family, induce autophagy. Further studies revealed that the wild-type N-terminal domain has pro-autophagic activity and that the C-terminal domain conversely inhibits this N-terminal function. These opposite functions of the two domains were also observed in other Gsdm family members. Thus, their study provides a new insight into the function of Gsdm genes in epithelial cell lineage, causality of cancers and immune-related diseases including childhood-onset asthma. © 2015 Authors; published by Portland Press Limited.

Sequestosome 1/p62 links familial ALS mutant SOD1 to LC3 via an ubiquitin-independent mechanism.

PubMed

Gal, Jozsef; Ström, Anna-Lena; Kwinter, David M; Kilty, Renée; Zhang, Jiayu; Shi, Ping; Fu, Weisi; Wooten, Marie W; Zhu, Haining

2009-11-01

The p62/sequestosome 1 protein has been identified as a component of pathological protein inclusions in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). P62 has also been implicated in autophagy, a process of mass degradation of intracellular proteins and organelles. Autophagy is a critical pathway for degrading misfolded and/or damaged proteins, including the copper-zinc superoxide dismutase (SOD1) mutants linked to familial ALS. We previously reported that p62 interacted with ALS mutants of SOD1 and that the ubiquitin-association domain of p62 was dispensable for the interaction. In this study, we identified two distinct regions of p62 that were essential to its binding to mutant SOD1: the N-terminal Phox and Bem1 (PB1) domain (residues 1-104) and a separate internal region (residues 178-224) termed here as SOD1 mutant interaction region (SMIR). The PB1 domain is required for appropriate oligomeric status of p62 and the SMIR is the actual region interacting with mutant SOD1. Within the SMIR, the conserved W184, H190 and positively charged R183, R186, K187, and K189 residues are critical to the p62-mutant SOD1 interaction as substitution of these residues with alanine resulted in significantly abolished binding. In addition, SMIR and the p62 sequence responsible for the interaction with LC3, a protein essential for autophagy activation, are independent of each other. In cells lacking p62, the existence of mutant SOD1 in acidic autolysosomes decreased, suggesting that p62 can function as an adaptor between mutant SOD1 and the autophagy machinery. This study provides a novel molecular mechanism by which mutant SOD1 can be recognized by p62 in an ubiquitin-independent fashion and targeted for the autophagy-lysosome degradation pathway.

Crystal Structure of the Catalytic Domain of Drosophila [beta]1,4-Galactosyltransferase-7

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramakrishnan, Boopathy; Qasba, Pradman K.

2010-11-03

The {beta}1,4-galactosyltransferase-7 ({beta}4Gal-T7) enzyme, one of seven members of the {beta}4Gal-T family, transfers in the presence of manganese Gal from UDP-Gal to an acceptor sugar (xylose) that is attached to a side chain hydroxyl group of Ser/Thr residues of proteoglycan proteins. It exhibits the least protein sequence similarity with the other family members, including the well studied family member {beta}4Gal-T1, which, in the presence of manganese, transfers Gal from UDP-Gal to GlcNAc. We report here the crystal structure of the catalytic domain of {beta}4Gal-T7 from Drosophila in the presence of manganese and UDP at 1.81 {angstrom} resolution. In the crystalmore » structure, a new manganese ion-binding motif (HXH) has been observed. Superposition of the crystal structures of {beta}4Gal-T7 and {beta}4Gal-T1 shows that the catalytic pocket and the substrate-binding sites in these proteins are similar. Compared with GlcNAc, xylose has a hydroxyl group (instead of an N-acetyl group) at C2 and lacks the CH{sub 2}OH group at C5; thus, these protein structures show significant differences in their acceptor-binding site. Modeling of xylose in the acceptor-binding site of the {beta}4Gal-T7 crystal structure shows that the aromatic side chain of Tyr{sup 177} interacts strongly with the C5 atom of xylose, causing steric hindrance to any additional group at C5. Because Drosophila Cd7 has a 73% protein sequence similarity to human Cd7, the present crystal structure offers a structure-based explanation for the mutations in human Cd7 that have been linked to Ehlers-Danlos syndrome.« less

Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

PubMed Central

Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

2015-01-01

Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

A Genome-Wide Analysis of the LBD (LATERAL ORGAN BOUNDARIES Domain) Gene Family in Malus domestica with a Functional Characterization of MdLBD11

PubMed Central

Su, Ling; Liu, Xin; Hao, Yujin

2013-01-01

The plant-specific LBD (LATERAL ORGAN BOUNDARIES domain) genes belong to a major family of transcription factor that encode a zinc finger-like domain. It has been shown that LBD genes play crucial roles in the growth and development of Arabidopsis and other plant species. However, no detailed information concerning this family is available for apple. In the present study, we analyzed the apple (Malus domestica) genome and identified 58 LBD genes. This gene family was tested for its phylogenetic relationships with homologous genes in the Arabidopsis genome, as well as its location in the genome, structure and expression. We also transformed one MdLBD gene into Arabidopsis to evaluate its function. Like Arabidopsis, apple LBD genes also have a conserved CX2CX6CX3C zinc finger-like domain in the N terminus and can be divided into two classes. The expression profile indicated that apple LBD genes exhibited a variety of expression patterns, suggesting that they have diverse functions. At the same time, the expression analysis implied that members of this apple gene family were responsive to hormones and stress and that they may participate in hormone-mediated plant organogenesis, which was demonstrated with the overexpression of the apple LBD gene MdLBD11, resulting in an abnormal phenotype. This phenotype included upward curling leaves, delayed flowering, downward-pointing flowers, siliques and other abnormal traits. Based on these data, we concluded that the MdLBD genes may play an important role in apple growth and development as in Arabidopsis and other species. PMID:23468909

A genome-wide analysis of the LBD (LATERAL ORGAN BOUNDARIES domain) gene family in Malus domestica with a functional characterization of MdLBD11.

PubMed

Wang, Xiaofei; Zhang, Shizhong; Su, Ling; Liu, Xin; Hao, Yujin

2013-01-01

The plant-specific LBD (LATERAL ORGAN BOUNDARIES domain) genes belong to a major family of transcription factor that encode a zinc finger-like domain. It has been shown that LBD genes play crucial roles in the growth and development of Arabidopsis and other plant species. However, no detailed information concerning this family is available for apple. In the present study, we analyzed the apple (Malus domestica) genome and identified 58 LBD genes. This gene family was tested for its phylogenetic relationships with homologous genes in the Arabidopsis genome, as well as its location in the genome, structure and expression. We also transformed one MdLBD gene into Arabidopsis to evaluate its function. Like Arabidopsis, apple LBD genes also have a conserved CX2CX6CX3C zinc finger-like domain in the N terminus and can be divided into two classes. The expression profile indicated that apple LBD genes exhibited a variety of expression patterns, suggesting that they have diverse functions. At the same time, the expression analysis implied that members of this apple gene family were responsive to hormones and stress and that they may participate in hormone-mediated plant organogenesis, which was demonstrated with the overexpression of the apple LBD gene MdLBD11, resulting in an abnormal phenotype. This phenotype included upward curling leaves, delayed flowering, downward-pointing flowers, siliques and other abnormal traits. Based on these data, we concluded that the MdLBD genes may play an important role in apple growth and development as in Arabidopsis and other species.

Deletion at the SLC1A1 glutamate transporter gene co-segregates with schizophrenia and bipolar schizoaffective disorder in a 5-generation family.

PubMed

Myles-Worsley, Marina; Tiobech, Josepha; Browning, Sharon R; Korn, Jeremy; Goodman, Sarah; Gentile, Karen; Melhem, Nadine; Byerley, William; Faraone, Stephen V; Middleton, Frank A

2013-03-01

Growing evidence for genetic overlap between schizophrenia (SCZ) and bipolar disorder (BPD) suggests that causal variants of large effect on disease risk may cross traditional diagnostic boundaries. Extended multigenerational families with both SCZ and BPD cases can be a valuable resource for discovery of shared biological pathways because they can reveal the natural evolution of the underlying genetic disruptions and their phenotypic expression. We investigated a deletion at the SLC1A1 glutamate transporter gene originally identified as a copy number variant exclusively carried by members of a 5-generation Palauan family. Using an expanded sample of 21 family members, quantitative PCR confirmed the deletion in all seven individuals with psychosis, three "obligate-carrier" parents and one unaffected sibling, while four marry-in parents were non-carriers. Linkage analysis under an autosomal dominant model generated a LOD-score of 3.64, confirming co-segregation of the deletion with psychosis. For more precise localization, we determined the approximate deletion end points using alignment of next-generation sequencing data for one affected deletion-carrier and then designed PCR amplicons to span the entire deletion locus. These probes established that the deletion spans 84,298 bp, thus eliminating the entire promoter, the transcription start site, and the first 59 amino acids of the protein, including the first transmembrane Na(2+)/dicarboxylate symporter domain, one of the domains that perform the glutamate transport action. Discovery of this functionally relevant SLC1A1 mutation and its co-segregation with psychosis in an extended multigenerational pedigree provides further support for the important role played by glutamatergic transmission in the pathophysiology of psychotic disorders. Copyright © 2013 Wiley Periodicals, Inc.

Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain.

PubMed

Miyake, Zenshi; Takekawa, Mutsuhiro; Ge, Qingyuan; Saito, Haruo

2007-04-01

The mitogen-activated protein kinase (MAPK) module, composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK), is a cellular signaling device that is conserved throughout the eukaryotic world. In mammalian cells, various extracellular stresses activate two major subfamilies of MAPKs, namely, the Jun N-terminal kinases and the p38/stress-activated MAPK (SAPK). MTK1 (also called MEKK4) is a stress-responsive MAPKKK that is bound to and activated by the stress-inducible GADD45 family of proteins (GADD45alpha/beta/gamma). Here, we dissected the molecular mechanism of MTK1 activation by GADD45 proteins. The MTK1 N terminus bound to its C-terminal segment, thereby inhibiting the C-terminal kinase domain. This N-C interaction was disrupted by the binding of GADD45 to the MTK1 N-terminal GADD45-binding site. GADD45 binding also induced MTK1 dimerization via a dimerization domain containing a coiled-coil motif, which is essential for the trans autophosphorylation of MTK1 at Thr-1493 in the kinase activation loop. An MTK1 alanine substitution mutant at Thr-1493 has a severely reduced activity. Thus, we conclude that GADD45 binding induces MTK1 N-C dissociation, dimerization, and autophosphorylation at Thr-1493, leading to the activation of the kinase catalytic domain. Constitutively active MTK1 mutants induced the same events, but in the absence of GADD45.

Structure and function of the interacting domains of Spire and Fmn-family formins.

PubMed

Vizcarra, Christina L; Kreutz, Barry; Rodal, Avital A; Toms, Angela V; Lu, Jun; Zheng, Wei; Quinlan, Margot E; Eck, Michael J

2011-07-19

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

Identification of an Nav1.1 sodium channel (SCN1A) loss-of-function mutation associated with familial simple febrile seizures

PubMed Central

Mantegazza, Massimo; Gambardella, Antonio; Rusconi, Raffaella; Schiavon, Emanuele; Annesi, Ferdinanda; Cassulini, Rita Restano; Labate, Angelo; Carrideo, Sara; Chifari, Rosanna; Canevini, Maria Paola; Canger, Raffaele; Franceschetti, Silvana; Annesi, Grazia; Wanke, Enzo; Quattrone, Aldo

2005-01-01

Febrile seizures (FS) affect 5–12% of infants and children up to 6 years of age. There is now epidemiological evidence that FS are associated with subsequent afebrile and unprovoked seizures in ≈7% of patients, which is 10 times more than in the general population. Extensive genetic studies have demonstrated that various loci are responsible for familial FS, and the FEB3 autosomal-dominant locus has been identified on chromosome 2q23–24, where the SCN1A gene is mapped. However, gene mutations causing simple FS have not been found yet. Here we show that the M145T mutation of a well conserved amino acid in the first transmembrane segment of domain I of the human Nav1.1 channel α-subunit cosegregates in all 12 individuals of a large Italian family affected by simple FS. Functional studies in mammalian cells demonstrate that the mutation causes a 60% reduction of current density and a 10-mV positive shift of the activation curve. Thus, M145T is a loss-of-function mutant. These results show that monogenic FS should also be considered a channelopathy. PMID:16326807

Isolation and characterization of a J domain protein that interacts with ARC1 from ornamental kale (Brassica oleracea var. acephala).

PubMed

Lan, Xingguo; Yang, Jia; Cao, Mingming; Wang, Yanhong; Kawabata, Saneyuki; Li, Yuhua

2015-05-01

A novel J domain protein, JDP1, was isolated from ornamental kale. The C-terminus of JDP1 specifically interacted with ARC1, which has a conserved role in self-incompatibility signaling. Armadillo (ARM)-repeat containing 1 (ARC1) plays a conserved role in self-incompatibility signaling across the Brassicaceae and functions downstream of the S-locus receptor kinase. Here, we identified a J domain protein 1 (JDP1) that interacts with ARC1 using a yeast two-hybrid screen against a stigma cDNA library from ornamental kale (Brassica oleracea var. acephala). JDP1, a 38.4-kDa protein with 344 amino acids, is a member of the Hsp40 family. Fragment JDP1(57-344), originally isolated from a yeast two-hybrid cDNA library, interacted specifically with ARC1 in yeast two-hybrid assays. The N-terminus of JDP1 (JDP1(1-68)) contains a J domain, and the C-terminus of JDP1 (JDP1(69-344)) contains an X domain of unknown function. However, JDP1(69-344) was required and sufficient for interaction with ARC1 in yeast two-hybrid assays and in vitro binding assays. Moreover, JDP1(69-344) regulated the trafficking of ARC1 from the cytoplasm to the plasma membrane by interacting with ARC1 in Arabidopsis mesophyll protoplasts. Finally, Tyr(8) in the JDP1 N-terminal region was identified to be the specific site for regulating the interaction between JDP1 and BoARC1 in yeast two-hybrid assays. Possible roles of JDP1 as an interactor with ARC1 in Brassica are discussed.

Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

PubMed

Pessler, Frank; Hernandez, Nouria

2003-08-01