scholarly journals Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1

2000 ◽  
Vol 20 (3) ◽  
pp. 816-824 ◽  
Author(s):  
Zhaohua Tang ◽  
Tiffany Kuo ◽  
Jenny Shen ◽  
Ren-Jang Lin
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Specific Protein ◽  
Biochemical Properties ◽  
Growth Defect ◽  
Sr Protein ◽  
Kinase Gene ◽  
Control Circuits

ABSTRACT Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore,dsk1 + gene determines the phenotype ofprp2 + overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1 + andSRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.

scholarly journals The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

scholarly journals Cyclin-Dependent Kinase 9 (Cdk9) of Fission Yeast Is Activated by the CDK-Activating Kinase Csk1, Overlaps Functionally with the TFIIH-Associated Kinase Mcs6, and Associates with the mRNA Cap Methyltransferase Pcm1 In Vivo

2006 ◽  
Vol 26 (3) ◽  
pp. 777-788 ◽  
Author(s):  
Yi Pei ◽  
Hongyan Du ◽  
Juliet Singer ◽  
Courtney St. Amour ◽  
Selena Granitto ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Growth Defect ◽  
Transcriptional Elongation ◽  
Cyclin Dependent Kinase ◽  
Elongation Complex ◽  
Pol Ii

ABSTRACT Cyclin-dependent kinase 9 (Cdk9) of fission yeast is an essential ortholog of metazoan positive transcription elongation factor b (P-TEFb), which is proposed to coordinate capping and elongation of RNA polymerase II (Pol II) transcripts. Here we show that Cdk9 is activated to phosphorylate Pol II and the elongation factor Spt5 by Csk1, one of two fission yeast CDK-activating kinases (CAKs). Activation depends on Cdk9 T-loop residue Thr-212. The other CAK—Mcs6, the kinase component of transcription factor IIH (TFIIH)—cannot activate Cdk9. Consistent with the specificities of the two CAKs in vitro, the kinase activity of Cdk9 is reduced ∼10-fold by csk1 deletion, and Cdk9 complexes from csk1Δ but not csk1 + cells can be activated by Csk1 in vitro. A cdk9 T212A mutant is viable but phenocopies conditional growth defects of csk1Δ strains, indicating a role for Csk1-dependent activation of Cdk9 in vivo. A cdk9 T212A mcs6 S165A strain, in which neither Cdk9 nor Mcs6 can be activated by CAK, has a synthetic growth defect, implying functional overlap between the two CDKs, which have distinct but overlapping substrate specificities. Cdk9 forms complexes in vivo with the essential cyclin Pch1 and with Pcm1, the mRNA cap methyltransferase. The carboxyl-terminal region of Cdk9, through which it interacts with another capping enzyme, the RNA triphosphatase Pct1, is essential. Together, the data support a proposed model whereby Cdk9/Pch1—the third essential CDK-cyclin complex described in fission yeast—helps to target the capping apparatus to the transcriptional elongation complex.

scholarly journals Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

1999 ◽  
Vol 145 (3) ◽  
pp. 447-455 ◽  
Author(s):  
Joanne M. Yeakley ◽  
Hélène Tronchère ◽  
James Olesen ◽  
Jacqueline A. Dyck ◽  
Huan-You Wang ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Sr Proteins ◽  
Molecular Targeting ◽  
Splicing Factors ◽  
Binding Studies ◽  
Sr Protein ◽  
Domain Interactions

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

scholarly journals Isolated Mammalian and Schizosaccharomyces pombeRan-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

1999 ◽  
Vol 10 (7) ◽  
pp. 2175-2190 ◽  
Author(s):  
Isabel Novoa ◽  
Mark G. Rush ◽  
Peter D’Eustachio
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Wild Type ◽  
Ran Gtpase ◽  
Amino Terminal ◽  
Binding Domains ◽  
Charged Form ◽  
Wild Type Yeast

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import.sbp1 − yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1 −yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Specific Labeling of Protein Domains with Antibody Fragments

1981 ◽  
Vol 39 ◽  
pp. 416-417
Author(s):  
U. Aebi ◽  
L.E. Buhle ◽  
W.E. Fowler
Keyword(s):  
Image Processing ◽  
Specific Protein ◽  
Antibody Fragments ◽  
Supramolecular Organization ◽  
Two Dimensional ◽  
Ordered Structures ◽  
Virus Capsids ◽  
Processing Techniques

Many important supramolecular structures such as filaments, microtubules, virus capsids and certain membrane proteins and bacterial cell walls exist as ordered polymers or two-dimensional crystalline arrays in vivo. In several instances it has been possible to induce soluble proteins to form ordered polymers or two-dimensional crystalline arrays in vitro. In both cases a combination of electron microscopy of negatively stained specimens with analog or digital image processing techniques has proven extremely useful for elucidating the molecular and supramolecular organization of the constituent proteins. However from the reconstructed stain exclusion patterns it is often difficult to identify distinct stain excluding regions with specific protein subunits. To this end it has been demonstrated that in some cases this ambiguity can be resolved by a combination of stoichiometric labeling of the ordered structures with subunit-specific antibody fragments (e.g. Fab) and image processing of the electron micrographs recorded from labeled and unlabeled structures.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Cytological changes induced by platinum-thymine in sarcoma-180 cells: Electron microscopy study

1990 ◽  
Vol 48 (3) ◽  
pp. 290-291
Author(s):  
Gustav Ofosu
Keyword(s):  
Mammalian Cells ◽  
Antitumor Agent ◽  
Microscopy Study ◽  
Electron Microscopy Study ◽  
Limiting Factor ◽  
Sarcoma 180 ◽  
Electron Microscopic ◽  
Cytological Changes

Platinum-thymine has been found to be a potent antitumor agent, which is quite soluble in water, and lack nephrotoxicity as the dose-limiting factor. The drug has been shown to interact with DNA and inhibits DNA, RNA and protein synthesis in mammalian cells in vitro. This investigation was undertaken to elucidate the cytotoxic effects of piatinum-thymine on sarcoma-180 cells in vitro ultrastructurally, Sarcoma-180 tumor bearing mice were treated with intraperitoneal injection of platinum-thymine 40mg/kg. A concentration of 60μg/ml dose of platinum-thymine was used in in vitro experiments. Treatments were at varying time intervals of 3, 7 and 21 days for in vivo experiments, and 30, 60 and 120 min., 6, 12, and 24th in vitro. Controls were not treated with platinum-thymine.Electron microscopic analyses of the treated cells in vivo and in vitro showed drastic cytotoxic effect.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

The Role of nitro (NO2-), chloro (Cl), and fluoro (F) Substitution in the Design of Antileishmanial and Antichagasic Compounds

2020 ◽  
Vol 21 ◽  
Author(s):  
Boniface Pone ◽  
Ferreira Igne Elizabeth
Keyword(s):  
Chagas Disease ◽  
Mammalian Cells ◽  
Neglected Tropical Diseases ◽  
New Drugs ◽  
Pharmacokinetic Studies ◽  
Scientific Publications ◽  
Antitrypanosomal Activity ◽  
Chemical Groups

: Neglected tropical diseases (NTDs) are responsible for over 500,000 deaths annually and are characterized by multiple disabilities. Leishmaniasis and Chagas disease are among the most severe NTDs, and are caused by the Leishmania sp, and Trypanosoma cruzi, respectively. Glucantime, pentamidine and miltefosine are commonly used to treat leishmaniasis, whereas nifurtimox, benznidazole are current treatments for Chagas disease. However, these treatments are associated with drug resistance, and severe side effects. Hence, the development of synthetic products, especially those containing N02, F, or Cl, which chemical groups are known to improve the biological activity. The present work summarizes the information on the antileishmanial and antitrypanosomal activity of nitro-, chloro-, and fluoro-synthetic derivatives. Scientific publications referring to halogenated derivatives in relation to antileishmanial and antitrypanosomal activities were hand searched in databases such as SciFinder, Wiley, Science Direct, PubMed, ACS, Springer, Scielo, and so on. According to the literature information, more than 90 compounds were predicted as lead molecules with reference to their IC50/EC50 values in in vitro studies. It is worth to mention that only active compounds with known cytotoxic effects against mammalian cells were considered in the present study. The observed activity was attributed to the presence of nitro-, fluoro- and chloro-groups in the compound backbone. All in all, nitro and h0alogenated derivatives are active antileishmanial and antitrypanosomal compounds and can serve as baseline for the development of new drugs against leishmaniasis and Chagas disease. However, efforts on in vitro and in vivo toxicity studies of the active synthetic compounds is still needed. Pharmacokinetic studies, and the mechanism of action of the promising compounds need to be explored. The use of new catalysts and chemical transformation can afford unexplored halogenated compounds with improved antileishmanial and antitrypanosomal activity.

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents

2020 ◽  
Vol 16 ◽  
Author(s):  
Haicheng Liu ◽  
Yushi Futamura ◽  
Honghai Wu ◽  
Aki Ishiyama ◽  
Taotao Zhang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Antimalarial Activity ◽  
In Vitro Assays ◽  
Compound Library ◽  
Antimalarial Agents ◽  
Phenotypic Screen ◽  
Ic50 Values ◽  
Substituted Benzamides

Background: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. Objective: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3-cinnamamido-N-substituted benzamides. Method: In this study, a screening of our compound library was carried out against the multidrug-sensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters’ 4-day suppressive test. Results: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective which might be ascribed to the poor solubility of these compounds. Conclusion: In this study, phenotypic screen of our compound library resulted in the first report of 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.

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