scholarly journals The Cell Wall-Anchored Streptomyces reticuli Avicel-Binding Protein (AbpS) and Its Gene

1998 ◽  
Vol 180 (7) ◽  
pp. 1647-1654 ◽  
Author(s):  
Stefan Walter ◽  
Egbert Wellmann ◽  
Hildgund Schrempf
Keyword(s):  
Reverse Genetics ◽  
Binding Protein ◽  
Fluorescein Isothiocyanate ◽  
Proteinase K ◽  
Logarithmic Phase ◽  
C Terminus ◽  
Late Logarithmic Phase ◽  
In Vivo Labelling ◽  
Α Helix

ABSTRACT Streptomyces reticuli produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located α-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.

scholarly journals Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity

2017 ◽  
Vol 114 (36) ◽  
pp. E7516-E7525 ◽  
Author(s):  
Ying Zhang ◽  
Matthew J. Markert ◽  
Shayna C. Groves ◽  
Paul E. Hardin ◽  
Christine Merlin
Keyword(s):  
Danaus Plexippus ◽  
Monarch Butterfly ◽  
Constant Darkness ◽  
Transactivation Domain ◽  
C Terminus ◽  
Independent Mechanism ◽  
Reporter Assays ◽  
Cryptochrome 2 ◽  
Α Helix

Circadian repression of CLOCK-BMAL1 by PERIOD and CRYPTOCHROME (CRY) in mammals lies at the core of the circadian timekeeping mechanism. CRY repression of CLOCK-BMAL1 and regulation of circadian period are proposed to rely primarily on competition for binding with coactivators on an α-helix located within the transactivation domain (TAD) of the BMAL1 C terminus. This model has, however, not been tested in vivo. Here, we applied CRISPR/Cas9-mediated mutagenesis in the monarch butterfly (Danaus plexippus), which possesses a vertebrate-like CRY (dpCRY2) and an ortholog of BMAL1, to show that insect CRY2 regulates circadian repression through TAD α-helix–dependent and –independent mechanisms. Monarch mutants lacking the BMAL1 C terminus including the TAD exhibited arrhythmic eclosion behavior. In contrast, mutants lacking the TAD α-helix but retaining the most distal C-terminal residues exhibited robust rhythms during the first day of constant darkness (DD1), albeit with a delayed peak of eclosion. Phase delay in this mutant on DD1 was exacerbated in the presence of a single functional allele of dpCry2, and rhythmicity was abolished in the absence of dpCRY2. Reporter assays in Drosophila S2 cells further revealed that dpCRY2 represses through two distinct mechanisms: a TAD-dependent mechanism that involves the dpBMAL1 TAD α-helix and dpCLK W328 and a TAD-independent mechanism involving dpCLK E333. Together, our results provide evidence for independent mechanisms of vertebrate-like CRY circadian regulation on the BMAL1 C terminus and the CLK PAS-B domain and demonstrate the importance of a BMAL1 TAD-independent mechanism for generating circadian rhythms in vivo.

scholarly journals A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

1998 ◽  
Vol 18 (2) ◽  
pp. 872-879 ◽  
Author(s):  
Vered Ribon ◽  
John A. Printen ◽  
Noah G. Hoffman ◽  
Brian K. Kay ◽  
Alan R. Saltiel
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Binding Protein ◽  
Sequence Similarity ◽  
Peptide Hormone ◽  
Protein Product ◽  
Insulin Stimulation ◽  
C Terminus

ABSTRACT The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.

scholarly journals Topogenesis in membranes of the NTB–VPg protein of Tomato ringspot nepovirus: definition of the C-terminal transmembrane domain

2004 ◽  
Vol 85 (2) ◽  
pp. 535-545 ◽  
Author(s):  
Aiming Wang ◽  
Sumin Han ◽  
Hélène Sanfaçon
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Microsomal Membranes ◽  
Hydrophobic Amino Acids ◽  
Definition Of

The putative NTP-binding protein (NTB) of Tomato ringspot nepovirus (ToRSV) contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. In infected plants, the NTB–VPg polyprotein (containing the domain for the genome-linked protein) is associated with endoplasmic reticulum-derived membranes that are active in ToRSV replication. Recent results from proteinase K protection assays suggested a luminal location for the VPg domain in infected plants, providing support for the presence of a transmembrane domain at the C terminus of NTB. In this study, we have shown that NTB–VPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and adopts a topology similar to that observed in vivo in that the VPg is present in the lumen. Truncated proteins containing 60 amino acids at the C terminus of NTB and the entire VPg exhibited a similar topology, confirming that this region of the protein contains a functional transmembrane domain. Deletion of portions of the C-terminal hydrophobic region of NTB by mutagenesis and introduction of glycosylation sites to map the luminal regions of the protein revealed that only the first stretch of hydrophobic amino acids traverses the membrane, while the second stretch of hydrophobic amino acids is located in the lumen. Our results provide additional evidence supporting the hypothesis that the NTB–VPg polyprotein acts as a membrane-anchor for the replication complex.

scholarly journals WBP-2, a WW domain binding protein, interacts with the thyroid-specific transcription factor Pax8

2004 ◽  
Vol 377 (3) ◽  
pp. 553-560 ◽  
Author(s):  
Roberto NITSCH ◽  
Tina DI PALMA ◽  
Anna MASCIA ◽  
Mariastella ZANNINI
Keyword(s):  
Protein Interaction ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Interaction Domain ◽  
Ww Domain ◽  
Transcriptional Activation Domain ◽  
Culture Cells ◽  
C Terminus

The Pax gene family encodes transcription factors that are essential in organogenesis and in the differentiation of various organs in higher eukaryotes. Pax proteins have a DNA binding domain at the N-terminus, and a transcriptional activation domain at the C-terminus. How these domains interact with the transcriptional machinery of the cell is still unclear. In the present paper, we describe the identification by means of immunological screening of the WW domain binding protein WBP-2 as a biochemical interactor of Pax8 (a WW domain is a protein-interaction domain containing two conserved tryptophan residues). Pax8 is required for the morphogenesis of the thyroid gland and for the maintenance of the thyroid differentiated cellular phenotype. WBP-2 was identified originally as a WW domain binding protein, and its function is still unknown. WBP-2 binds to Pax8 in vitro in pulldown assays, and in vivo in tissue culture cells in co-immunoprecipitation assays. Interestingly, Pax8 does not contain a WW domain. Our results point to the identification of a new protein-interacting domain that is present in the C-terminal portion of Pax8 and that is required for protein–protein interaction with WBP-2. Our results demonstrate that WBP-2 is not a transcriptional co-activator of Pax8, but rather behaves as an adaptor molecule, as suggested in other studies.

scholarly journals Molecular determinants of Yellow Fever Virus pathogenicity in Syrian Golden Hamsters: one mutation away from virulence

2018 ◽  
Author(s):  
Raphaëlle Klitting ◽  
Laura Roth ◽  
Félix A. Rey ◽  
Xavier de Lamballerie
Keyword(s):  
Yellow Fever ◽  
Reverse Genetics ◽  
Yellow Fever Virus ◽  
Virulent Strain ◽  
Fever Virus ◽  
Viral Loads ◽  
Synonymous Mutations ◽  
E Protein ◽  
Α Helix

ABSTRACTYellow fever virus (Flavivirusgenus) is an arthropod-borne pathogen which can infect humans, causing a severe viscerotropic disease with a high mortality rate. Adapted viral strains allow the reproduction of yellow fever disease in hamsters with features similar to the human disease. Here, we used the Infectious Subgenomic Amplicons reverse genetics method to produce an equivalent to the hamster-virulent strain, Yellow FeverAp7, by introducing a set of 4 synonymous and 6 non-synonymous mutations into a single subgenomic amplicon, derived from the sequence of theAsibistrain. The resulting strain, Yellow FeverAp7M, induced a disease similar to that described forAp7in terms of symptoms, weight evolution, viral loads in the liver and lethality. Using the same methodology, we produced mutant strains derived from eitherAp7MorAsibiviruses and investigated the role of each ofAp7Mnon-synonymous mutations in itsin vivophenotype. This allowed identifying key components of the virulence mechanism in hamsters. InAp7Mvirus, the reversion of either E/Q27H or E/D155A mutations, led to an important reduction of both virulence andin vivoreplicative fitness. In addition, the introduction of the single D155AAp7Mmutation within the E protein of theAsibivirus was sufficient to drastically modify its phenotype in hamsters towards both a greater replication efficiency and virulence. Finally, inspection of theAsibistrain E protein structure combined toin vivotesting revealed the importance of an exposed α-helix in domain I, containing residues 154 and 155, forAp7Mvirulence in hamsters.

A topological study of the human γ-glutamyl carboxylase

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 973-978 ◽  
Author(s):  
Jianke Tie ◽  
Sheue-Mei Wu ◽  
Dayun Jin ◽  
Christopher V. Nicchitta ◽  
Darrel W. Stafford
Keyword(s):  
Transmembrane Domain ◽  
Membrane Topology ◽  
Proteinase K ◽  
In Vitro Translation ◽  
C Terminus ◽  
293 Cells ◽  
Leader Peptidase ◽  
Polytopic Membrane Protein

γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

scholarly journals A seven-residue deletion in PrP leads to generation of a spontaneous prion formed from C-terminal C1 fragment of PrP

2020 ◽  
Vol 295 (41) ◽  
pp. 14025-14039
Author(s):  
Carola Munoz-Montesino ◽  
Djabir Larkem ◽  
Clément Barbereau ◽  
Angélique Igel-Egalon ◽  
Sandrine Truchet ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cell Model ◽  
Prion Diseases ◽  
Electrophoretic Pattern ◽  
Cellular Prion Protein ◽  
Proteinase K ◽  
Rabbit Kidney ◽  
C Terminus ◽  
Α Helix ◽  
Unique Cell

Prions result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of β-sheet–rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)–resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190–196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.

A topological study of the human γ-glutamyl carboxylase

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 973-978 ◽  
Author(s):  
Jianke Tie ◽  
Sheue-Mei Wu ◽  
Dayun Jin ◽  
Christopher V. Nicchitta ◽  
Darrel W. Stafford
Keyword(s):  
Transmembrane Domain ◽  
Membrane Topology ◽  
Proteinase K ◽  
In Vitro Translation ◽  
C Terminus ◽  
293 Cells ◽  
Leader Peptidase ◽  
Polytopic Membrane Protein

Abstract γ-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K–dependent posttranslational modification of glutamate to γ-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

scholarly journals An α-Helical Extension of the ELMO1 Pleckstrin Homology Domain Mediates Direct Interaction to DOCK180 and Is Critical in Rac Signaling

2008 ◽  
Vol 19 (11) ◽  
pp. 4837-4851 ◽  
Author(s):  
David Komander ◽  
Manishha Patel ◽  
Mélanie Laurin ◽  
Nadine Fradet ◽  
Ariane Pelletier ◽  
...  
Keyword(s):  
Amino Acids ◽  
Direct Interaction ◽  
Pleckstrin Homology Domain ◽  
Ph Domain ◽  
Rac Gtpase ◽  
Interaction Sites ◽  
C Terminus ◽  
Evolutionarily Conserved ◽  
Α Helix

The mammalian DOCK180 protein belongs to an evolutionarily conserved protein family, which together with ELMO proteins, is essential for activation of Rac GTPase-dependent biological processes. Here, we have analyzed the DOCK180-ELMO1 interaction, and map direct interaction interfaces to the N-terminal 200 amino acids of DOCK180, and to the C-terminal 200 amino acids of ELMO1, comprising the ELMO1 PH domain. Structural and biochemical analysis of this PH domain reveals that it is incapable of phospholipid binding, but instead structurally resembles FERM domains. Moreover, the structure revealed an N-terminal amphiphatic α-helix, and point mutants of invariant hydrophobic residues in this helix disrupt ELMO1-DOCK180 complex formation. A secondary interaction between ELMO1 and DOCK180 is conferred by the DOCK180 SH3 domain and proline-rich motifs at the ELMO1 C-terminus. Mutation of both DOCK180-interaction sites on ELMO1 is required to disrupt the DOCK180-ELMO1 complex. Significantly, although this does not affect DOCK180 GEF activity toward Rac in vivo, Rac signaling is impaired, implying additional roles for ELMO in mediating intracellular Rac signaling.

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