An internal GAP domain negatively regulates presynaptic dynamin in vivo

The mechanism by which the self-assembling GTPase dynamin functions in vesicle formation remains controversial. Point mutations in shibire, the Drosophila dynamin, cause temperature-sensitive (ts) defects in endocytosis. We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity. Three second-site suppressor mutations, one in the switch 1 region of the GTPase domain and two in the GTPase effector domain (GED), dynamin's putative GAP, fully rescue the shits2 defects in synaptic vesicle recycling. The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity. Based on these and other observations, we propose a two-step model for dynamin during vesicle formation in which an early regulatory GTPase-like function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.

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Point Mutations in Yeast CBF5 Can Abolish In Vivo Pseudouridylation of rRNA

Molecular and Cellular Biology ◽

10.1128/mcb.19.11.7461 ◽

1999 ◽

Vol 19 (11) ◽

pp. 7461-7472 ◽

Cited By ~ 133

Author(s):

Yeganeh Zebarjadian ◽

Tom King ◽

Maurille J. Fournier ◽

Louise Clarke ◽

John Carbon

Keyword(s):

Catalytic Domain ◽

Sequence Similarity ◽

Point Mutations ◽

Rrna Synthesis ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Sequence Motifs ◽

Conserved Sequence

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Ψ) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Ψ synthase, and shares the “KP” and “XLD” conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Ψ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Ψ synthase domain of Cbf5p. Yeast strains expressing these mutatedcbf5 genes in a cbf5Δ null background are viable at 25°C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Ψ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutantcbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Ψ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutantcbf5D95A, which lacks Ψ in rRNA. A subset of mutations in the Ψ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Ψ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

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Recombinant Respiratory Syncytial Viruses Lacking the C-Terminal Third of the Attachment (G) Protein Are Immunogenic and Attenuated In Vivo and In Vitro

Journal of Virology ◽

10.1128/jvi.78.11.5773-5783.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5773-5783 ◽

Cited By ~ 11

Author(s):

Matthew B. Elliott ◽

Karin S. Pryharski ◽

Qingzhong Yu ◽

Christopher L. Parks ◽

Todd S. Laughlin ◽

...

Keyword(s):

G Protein ◽

Reverse Genetics ◽

Point Mutations ◽

A549 Cells ◽

Temperature Sensitive ◽

Human Infants ◽

Attenuated Viruses ◽

Syncytial Virus

ABSTRACT The design of attenuated vaccines for respiratory syncytial virus (RSV) historically focused on viruses made sensitive to physiologic temperature through point mutations in the genome. These prototype vaccines were not suitable for human infants primarily because of insufficient attenuation, genetic instability, and reversion to a less-attenuated phenotype. We therefore sought to construct novel attenuated viruses with less potential for reversion through genetic alteration of the attachment G protein. Complete deletion of G protein was previously shown to result in RSV strains overly attenuated for replication in mice. Using reverse genetics, recombinant RSV (rRSV) strains were engineered with truncations at amino acid 118, 174, 193, or 213 and respectively designated rA2cpΔG118, rA2cpΔG174, rA2cpΔG193, and rA2cpΔG213. All rA2cpΔG strains were attenuated for growth in vitro and in the respiratory tracts of BALB/c mice but not restricted for growth at 37°C. The mutations did not significantly affect nascent genome synthesis in human lung epithelial (A549) cells, but infectious rA2cpΔG virus shed into the culture medium was dramatically diminished. Hence, the data suggested that a site within the C-terminal 85 amino acids of G protein is important for efficient genome packaging or budding of RSV from the infected cell. Vaccination with the rA2cpΔG strains also generated efficacious immune responses in mice that were similar to those elicited by the temperature-sensitive cpts248/404 strain previously tested in human infants. Collectively, the data indicate that the rA2cpΔG strains are immunogenic, not likely to revert to the less-attenuated phenotype, and thus candidates for further development as vaccines against RSV.

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Molecular Lesions Associated With Alleles of decapentuplegic Identify Residues Necessary for TGF-β/BMP Cell Signaling in Drosophila melanogaster

Genetics ◽

10.1093/genetics/142.2.493 ◽

1996 ◽

Vol 142 (2) ◽

pp. 493-505 ◽

Cited By ~ 3

Author(s):

Kristi Wharton ◽

Robert P Ray ◽

Seth D Findley ◽

Holly E Duncan ◽

William M Gelbart

Keyword(s):

Cell Signaling ◽

Low Temperatures ◽

Point Mutations ◽

Molecular Probes ◽

Genetic Interactions ◽

Temperature Sensitive ◽

In Vitro Mutagenesis ◽

Mutant Phenotypes

Abstract We have identified the molecular lesions associated with six point mutations in the Drosophila TGF-β homologue decapentaplegic (dpp). The sites of these mutations define residues within both the pro and ligand regions that are essential for dpp function in vivo. While all of these mutations affect residues that are highly conserved among TGF-β superfamily members, the phenotypic consequences of the different alleles are quite distinct. Through an analysis of these mutant phenotypes, both in cuticle preparations and with molecular probes, we have assessed the functional significance of specific residues that are conserved among the different members of the superfamily. In addition, we have tested for conditional genetic interactions between the different alleles. We show that two of the alleles are temperature sensitive for the embyronic functions of dpp, such that these alleles are not only embryonic viable as homozygotes but also partially complement other dpp hypomorphs at low temperatures. Our results are discussed with regard to in vitro mutagenesis data on other TGF-β-like molecules, as well as with regard to the regulation of dpp cell signaling in Drosophila.

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Probable Mechanisms Underlying Interallelic Complementation and Temperature-Sensitivity of Mutations at the shibire Locus of Drosophila melanogaster

Genetics ◽

10.1093/genetics/149.2.1019 ◽

1998 ◽

Vol 149 (2) ◽

pp. 1019-1030

Author(s):

Deanna Grant ◽

Shilpa Unadkat ◽

Alisa Katzen ◽

K S Krishnan ◽

Mani Ramaswami

Keyword(s):

Drosophila Melanogaster ◽

Class Ii ◽

Temperature Sensitive ◽

Missense Mutations ◽

Gtpase Domain ◽

Conserved Residues ◽

Middle Domain ◽

Interallelic Complementation

Abstract The shibire locus of Drosophila melanogaster encodes dynamin, a GTPase required for the fission of endocytic vesicles from plasma membrane. Biochemical studies indicate that mammalian dynamin is part of a complex containing multiple dynamin subunits and other polypeptides. To gain insight into sequences of dynamin critical for its function, we have characterized in detail a collection of conditional and lethal shi alleles. We describe a probable null allele of shi and show that its properties are distinct from those of two classes of lethal alleles (termed I and II) that show intergroup, interallelic complementation. Sequenced class I alleles, which display dominant properties, carry missense mutations in conserved residues in the GTPase domain of dynamin. In contrast, the sequenced class II alleles, which appear completely recessive, carry missense mutations in conserved residues of a previously uncharacterized “middle domain” that lies adjacent to the GTPase region. These data suggest that critical interactions mediated by this middle domain are severely affected by the class II lethal mutations; thus, the mutant sequences should be very useful for confirming the in vivo relevance of interactions observed in vitro. Viable heteroallelic combinations of shi lethals show rapid and reversible temperature-sensitive paralytic phenotypes hitherto only described for the ts alleles of shi. When taken together with the molecular analysis of shi mutations, these observations suggest that the GTPase domain of dynamin carries an intrinsically temperature-sensitive activity: hypomorphic mutations that reduce this activity at low temperatures result in conditional temperature-sensitive phenotype. These observations explain why screens for conditional paralytic mutants in Drosophila inevitably recover ts alleles of shi at high frequencies.

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A Well-Connected and Conserved Nucleoplasmic Helicase Is Required for Production of Box C/D and H/ACA snoRNAs and Localization of snoRNP Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.21.22.7731-7746.2001 ◽

2001 ◽

Vol 21 (22) ◽

pp. 7731-7746 ◽

Cited By ~ 83

Author(s):

Thomas H. King ◽

Wayne A. Decatur ◽

Edouard Bertrand ◽

E. Stuart Maxwell ◽

Maurille J. Fournier

Keyword(s):

Protein Complexes ◽

Early Stage ◽

Point Mutations ◽

Dna Helicase ◽

Nucleoside Triphosphate ◽

Temperature Sensitive ◽

Snornp Biogenesis ◽

Protein Components

ABSTRACT Biogenesis of small nucleolar RNA-protein complexes (snoRNPs) consists of synthesis of the snoRNA and protein components, snoRNP assembly, and localization to the nucleolus. Recently, two nucleoplasmic proteins from mice were observed to bind to a model box C/D snoRNA in vitro, suggesting that they function at an early stage in snoRNP biogenesis. Both proteins have been described in other contexts. The proteins, called p50 and p55 in the snoRNA binding study, are highly conserved and related to each other. Both have Walker A and B motifs characteristic of ATP- and GTP-binding and nucleoside triphosphate-hydrolyzing domains, and the mammalian orthologs have DNA helicase activity in vitro. Here, we report that theSaccharomyces cerevisiae ortholog of p50 (Rvb2, Tih2p, and other names) is required for production of C/D snoRNAs in vivo and, surprisingly, H/ACA snoRNAs as well. Point mutations in the Walker A and B motifs cause temperature-sensitive or lethal growth phenotypes and severe defects in snoRNA accumulation. Notably, depletion of p50 (called Rvb2 in this study) also impairs localization of C/D and H/ACA core snoRNP proteins Nop1p and Gar1p, suggesting a defect(s) in snoRNP assembly or trafficking to the nucleolus. Findings from other studies link Rvb2 orthologs with chromatin remodeling and transcription. Taken together, the present results indicate that Rvb2 is involved in an early stage of snoRNP biogenesis and may play a role in coupling snoRNA synthesis with snoRNP assembly and localization.

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Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

Communications Biology ◽

10.1038/s42003-021-02192-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Stéphane Perrier ◽

Eléonore Moreau ◽

Caroline Deshayes ◽

Marine El-Adouzi ◽

Delphine Goven ◽

...

Keyword(s):

Sodium Channel ◽

Anopheles Gambiae ◽

Calcium Concentration ◽

Mosquito Control ◽

Point Mutations ◽

Resting Membrane Potential ◽

Compensatory Mechanisms ◽

Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

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Characterization of LDD-2633 as a Novel RET Kinase Inhibitor with Anti-Tumor Effects in Thyroid Cancer

Pharmaceuticals ◽

10.3390/ph14010038 ◽

2021 ◽

Vol 14 (1) ◽

pp. 38

Author(s):

Hyo Jeong Lee ◽

Pyeonghwa Jeong ◽

Yeongyu Moon ◽

Jungil Choi ◽

Jeong Doo Heo ◽

...

Keyword(s):

Thyroid Cancer ◽

Thyroid Carcinoma ◽

Kinase Inhibitor ◽

Point Mutations ◽

Carcinoma Cells ◽

Medullary Thyroid ◽

Potent Inhibition ◽

Kinase Inhibitory Activity

Rearranged during transfection (RET), a receptor tyrosine kinase, is activated by glial cell line-derived neurotrophic factor family ligands. Chromosomal rearrangement or point mutations in RET are observed in patients with papillary thyroid and medullary thyroid carcinomas. Oncogenic alteration of RET results in constitutive activation of RET activity. Therefore, inhibiting RET activity has become a target in thyroid cancer therapy. Here, the anti-tumor activity of a novel RET inhibitor was characterized in medullary thyroid carcinoma cells. The indirubin derivative LDD-2633 was tested for RET kinase inhibitory activity. In vitro, LDD-2633 showed potent inhibition of RET kinase activity, with an IC50 of 4.42 nM. The growth of TT thyroid carcinoma cells harboring an RET mutation was suppressed by LDD-2633 treatment via the proliferation suppression and the induction of apoptosis. The effects of LDD-2633 on the RET signaling pathway were examined; LDD-2633 inhibited the phosphorylation of the RET protein and the downstream molecules Shc and ERK1/2. Oral administration of 20 or 40 mg/kg of LDD-2633 induced dose-dependent suppression of TT cell xenograft tumor growth. The in vivo and in vitro experimental results supported the potential use of LDD-2633 as an anticancer drug for thyroid cancers.

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Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

Virologica Sinica ◽

10.1007/s12250-021-00345-3 ◽

2021 ◽

Author(s):

Li-Nan Wang ◽

Xiang-Lei Peng ◽

Min Xu ◽

Yuan-Bo Zheng ◽

Yue-Ying Jiao ◽

...

Keyword(s):

Respiratory Syncytial Virus ◽

Gene Deletion ◽

Human Respiratory Syncytial Virus ◽

Temperature Sensitive ◽

T7 Promoter ◽

Vaccine Candidates ◽

Rsv Infection ◽

Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽

10.1093/genetics/142.3.661 ◽

1996 ◽

Vol 142 (3) ◽

pp. 661-672 ◽

Cited By ~ 5

Author(s):

Jodi L Vogel ◽

Vincent Geuskens ◽

Lucie Desmet ◽

N Patrick Higgins ◽

Ariane Toussaint

Keyword(s):

Binding Activity ◽

Temperature Sensitive ◽

Dna Binding Activity ◽

Heat Stable ◽

C Terminus ◽

Acid Domain ◽

Mutant Forms ◽

Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

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