scholarly journals A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

1993 ◽  
Vol 13 (1) ◽  
pp. 487-495 ◽  
Author(s):  
L B Andersen ◽  
R Ballester ◽  
D A Marchuk ◽  
E Chang ◽  
D H Gutmann ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Type I ◽  
Gtpase Activating Protein ◽  
Nuclear Targeting ◽  
Protein Activity ◽  
Significant Similarity ◽  
Nf1 Gene

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

scholarly journals A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity

1993 ◽  
Vol 13 (1) ◽  
pp. 487-495
Author(s):  
L B Andersen ◽  
R Ballester ◽  
D A Marchuk ◽  
E Chang ◽  
D H Gutmann ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Amino Acid ◽  
Basic Amino Acid ◽  
Type I ◽  
Gtpase Activating Protein ◽  
Nuclear Targeting ◽  
Protein Activity ◽  
Significant Similarity ◽  
Nf1 Gene

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

scholarly journals The Topology of the l-Arginine Exporter ArgO Conforms to an Nin-CoutConfiguration in Escherichia coli: Requirement for the Cytoplasmic N-Terminal Domain, Functional Helical Interactions, and an Aspartate Pair for ArgO Function

2016 ◽  
Vol 198 (23) ◽  
pp. 3186-3199 ◽  
Author(s):  
Amit Pathania ◽  
Arvind Kumar Gupta ◽  
Swati Dubey ◽  
Balasubramanian Gopal ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Cytoplasmic Membrane ◽  
Basic Amino Acid ◽  
Intramolecular Interactions ◽  
Membrane Topology ◽  
Content Type ◽  
E Coli ◽  
Terminal Domain

ABSTRACTArgO and LysE are members of the LysE family of exporter proteins and ordinarily mediate the export ofl-arginine (Arg) inEscherichia coliandl-lysine (Lys) and Arg inCorynebacterium glutamicum, respectively. Under certain conditions, ArgO also mediates Lys export. To delineate the arrangement of ArgO in the cytoplasmic membrane ofE. coli, we have employed a combination of cysteine accessibilityin situ, alkaline phosphatase fusion reporters, and protein modeling to arrive at a topological model of ArgO. Our studies indicate that ArgO assumes an Nin-Coutconfiguration, potentially forming a five-transmembrane helix bundle flanked by a cytoplasmic N-terminal domain (NTD) comprising roughly its first 38 to 43 amino acyl residues and a short periplasmic C-terminal region (CTR). Mutagenesis studies indicate that the CTR, but not the NTD, is dispensable for ArgO functionin vivoand that a pair of conserved aspartate residues, located near the opposing edges of the cytoplasmic membrane, may play a pivotal role in facilitating transmembrane Arg flux. Additional studies on amino acid substitutions that impair ArgO functionin vivoand their derivatives bearing compensatory amino acid alterations indicate a role for intramolecular interactions in the Arg export mechanism, and some interactions are corroborated by normal-mode analyses. Lastly, our studies suggest that ArgO may exist as a monomerin vivo, thus highlighting the requirement for intramolecular interactions in ArgO, as opposed to interactions across multiple ArgO monomers, in the formation of an Arg-translocating conduit.IMPORTANCEThe orthologous proteins LysE ofC. glutamicumand ArgO ofE. colifunction as exporters of the basic amino acidsl-arginine andl-lysine and the basic amino acidl-arginine, respectively, and LysE can functionally substitute for ArgO when expressed inE. coli. Notwithstanding this functional equivalence, studies reported here show that ArgO possesses a membrane topology that is distinct from that reported for LysE, with substantial variation in the topological arrangement of the proximal one-third portions of the two exporters. Additional genetic andin silicostudies reveal the importance of (i) the cytoplasmic N-terminal domain, (ii) a pair of conserved aspartate residues, and (iii) potential intramolecular interactions in ArgO function and indicate that an Arg-translocating conduit is formed by a monomer of ArgO.

scholarly journals The Change P82L in the Rift Valley Fever Virus NSs Protein Confers Attenuation in Mice Not Related with a Type-I IFN Antagonistic Phenotype

2021 ◽  
Author(s):  
Belén Borrego ◽  
Sandra Moreno ◽  
Nuria de la Losa ◽  
Friedemann Weber ◽  
Alejandro Brun
Keyword(s):  
Amino Acid ◽  
Rift Valley ◽  
Rift Valley Fever ◽  
Rift Valley Fever Virus ◽  
Fever Virus ◽  
Single Amino Acid ◽  
Economic Losses ◽  
Type I ◽  
Valley Fever

Rift valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an IFN-competent cell line as well as the production of IFN-β did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results unveil a new RVFV virulence marker highlighting the multiple ways of NSs protein to modulate viral infectivity.

scholarly journals Dsk1p kinase phosphorylates SR proteins and regulates their cellular localization in fission yeast

2007 ◽  
Vol 405 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Zhaohua Tang ◽  
Amy Tsurumi ◽  
Sarah Alaei ◽  
Christopher Wilson ◽  
Cathleen Chiu ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Fission Yeast ◽  
Cellular Localization ◽  
Sr Proteins ◽  
Regulatory Function ◽  
General Mechanism ◽  
Nuclear Targeting ◽  
Sr Protein ◽  
Eukaryotic Organism

Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1+: Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.

scholarly journals Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

1996 ◽  
Vol 134 (6) ◽  
pp. 1455-1467 ◽  
Author(s):  
B Nikolic ◽  
E Mac Nulty ◽  
B Mir ◽  
G Wiche
Keyword(s):  
Amino Acid ◽  
Intermediate Filaments ◽  
Basic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

scholarly journals Molecular features of the viral and cellular Src kinases involved in interactions with the GTPase-activating protein.

1991 ◽  
Vol 11 (10) ◽  
pp. 5059-5067 ◽  
Author(s):  
B K Brott ◽  
S Decker ◽  
M C O'Brien ◽  
R Jove
Keyword(s):  
Amino Acid ◽  
Gap Analysis ◽  
Kinase Domain ◽  
Terminal Segment ◽  
Src Kinases ◽  
Gtpase Activating Protein ◽  
Amino Terminal ◽  
Molecular Features

GTPase-activating protein (GAP) enhances the rate of GTP hydrolysis by cellular Ras proteins and is implicated in mitogenic signal transduction. GAP is phosphorylated on tyrosine in cells transformed by Rous sarcoma virus and serves as an in vitro substrate of the viral Src (v-Src) kinase. Our previous studies showed that GAP complexes stably with normal cellular Src (c-Src), although its association with v-Src is less stable. To further investigate the molecular basis for interactions between GAP and the Src kinases, we examined GAP association with and phosphorylation by a series of c-Src and v-Src mutants. Analysis of GAP association with c-Src/v-Src chimeric proteins demonstrates that GAP associates stably with Src proteins possessing low kinase activity and poorly with activated Src kinases, especially those that lack the carboxy-terminal segment of c-Src containing the regulatory amino acid Tyr-527. Phosphorylated Tyr-527 is a major determinant of c-Src association with GAP, as demonstrated by c-Src point mutants in which Tyr-527 is changed to Phe. While the isolated amino-terminal half of the c-Src protein is insufficient for stable GAP association, analysis of point substitutions of highly conserved amino acid residues in the c-Src SH2 region indicate that this region also influences Src-GAP complex formation. Therefore, our results suggest that both Tyr-527 phosphorylation and the SH2 region contribute to stable association of c-Src with GAP. Analysis of in vivo phosphorylation of GAP by v-Src mutants containing deletions encompassing the SH2, SH3, and unique regions suggests that the kinase domain of v-Src contains sufficient substrate specificity for GAP phosphorylation. Even though tyrosine phosphorylation of GAP correlates to certain extent with the transforming ability of various c-Src and v-Src mutants, our data suggest that other GAP-associated proteins may also have roles in Src-mediated oncogenic transformation. These findings provide additional evidence for the specificity of Src interactions with GAP and support the hypothesis that these interactions contribute to the biological functions of the Scr kinases.

scholarly journals A yeast-based drug discovery platform to identify Plasmodium falciparum type II NADH dehydrogenase inhibitors

2021 ◽  
Author(s):  
Yu Cao ◽  
Chen Sun ◽  
Han Wen ◽  
Mengfei Wang ◽  
Pan Zhu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Specific Inhibitor ◽  
Type I ◽  
Type Ii ◽  
Protein Activity ◽  
Asexual Blood Stage

Conventional methods utilizing in vitro protein activity assay or in vivo parasite survival to screen for malaria inhibitors suffer from high experimental background and/or inconvenience. Here we introduce a yeast-based system to facilitate chemical screen for specific protein or pathway inhibitors. The platform comprises several isogeneic Pichia strains that only differ in the target of interest, so that a compound which inhibits one strain but not the other is implicated in working specifically against the target. We used Plasmodium falciparum NDH2(PfNDH2), a type II NADH dehydrogenase, as a proof of principle to show how well this works. Three isogenic Pichia strains harboring respectively exogeneously introduced PfNDH2, its own complex I (a type I NADH dehydrogenase), and PfNDH2 with its own complex I were constructed. In a pilot screen of more than2000 compounds, we identified a highly specific inhibitor that acts on PfNDH2. This compound poorly inhibit the parasites at the asexual blood stage, however, is highly effective in repressing oocyst maturation in the mosquito stage. Our results demonstrate that the yeast cell based screen platform is feasible, efficient, economical and with very low background noise. Similar strategies could be extended to the functional screen for interacting molecules of other targets.

scholarly journals Random Mutagenesis Defines a Domain of Theiler's Virus Leader Protein That Is Essential for Antagonism of Nucleocytoplasmic Trafficking and Cytokine Gene Expression

2009 ◽  
Vol 83 (21) ◽  
pp. 11223-11232 ◽  
Author(s):  
Céline Ricour ◽  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Stanleyson V. Hato ◽  
Frank J. M. van Kuppeveld ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Encephalomyocarditis Virus ◽  
Cytokine Gene Expression ◽  
Type I ◽  
Nucleocytoplasmic Trafficking ◽  
Protein Activity ◽  
Retroviral Transduction ◽  
L Protein ◽  
Leader Protein

ABSTRACT The leader protein of cardioviruses, Theiler's murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV), is a multifunctional protein known to antagonize type I interferon expression and to interfere with nucleocytoplasmic trafficking of host proteins and mRNA. This protein plays an important role in the capacity of TMEV to establish persistent infection of the central nervous system. Mutant forms of the TMEV leader protein were generated by random mutagenesis and selected after retroviral transduction on the basis of the loss of the highly toxic nature of this protein. Selected mutations define a short C-terminal domain of the leader conserved in TMEV and Saffold virus but lacking in the EMCV leader and thus called the Theilo domain. Mutations in this domain had a dramatic impact on TMEV L protein activity. Like the zinc finger mutation, Theilo domain mutations affected all of the activities of the L protein tested: interferon gene transcription and IRF-3 dimerization antagonism, alteration of nucleocytoplasmic trafficking, nucleoporin 98 hyperphosphorylation, and viral persistence in vivo. This suggests that the Zn finger and the Theilo domain of the protein cooperate for function. Moreover, the fact that all of the activities tested were affected by these mutations suggests that the various leader protein functions are somehow coupled.

scholarly journals The “Intracellular” Poly(3-Hydroxybutyrate) (PHB) Depolymerase of Rhodospirillum rubrum Is a Periplasm-Located Protein with Specificity for Native PHB and with Structural Similarity to Extracellular PHB Depolymerases

2004 ◽  
Vol 186 (21) ◽  
pp. 7243-7253 ◽  
Author(s):  
René Handrick ◽  
Simone Reinhardt ◽  
Philipp Kimmig ◽  
Dieter Jendrossek
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Chain Length ◽  
Rhodospirillum Rubrum ◽  
Catalytic Triad ◽  
Significant Similarity ◽  
Amino Acid Homology ◽  
Short Chain Length ◽  
Phb Depolymerase

ABSTRACT Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser42, Asp138, and His178 were identified as catalytic-triad amino acids, with Ser42 as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.

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