scholarly journals Characterization of Schizosaccharomyces pombeHus1: a PCNA-Related Protein That Associates with Rad1 and Rad9

2000 ◽  
Vol 20 (4) ◽  
pp. 1254-1262 ◽  
Author(s):  
Thomas Caspari ◽  
Maria Dahlen ◽  
Gunilla Kanter-Smoler ◽  
Howard D. Lindsay ◽  
Kay Hofmann ◽  
...  
Keyword(s):  
Protein Complex ◽  
Small Proportion ◽  
S Phase ◽  
Dna Integrity ◽  
Related Protein ◽  
Transient Interaction ◽  
Two Hybrid

ABSTRACT Hus1 is one of six checkpoint Rad proteins required for allSchizosaccharomyces pombe DNA integrity checkpoints. MYC-tagged Hus1 reveals four discrete forms. The main form, Hus1-B, participates in a protein complex with Rad9 and Rad1, consistent with reports that Rad1-Hus1 immunoprecipitation is dependent on the rad9 + locus. A small proportion of Hus1-B is intrinsically phosphorylated in undamaged cells and more becomes phosphorylated after irradiation. Hus1-B phosphorylation is not increased in cells blocked in early S phase with hydroxyurea unless exposure is prolonged. The Rad1–Rad9–Hus1-B complex is readily detectable, but upon cofractionation of soluble extracts, the majority of each protein is not present in this complex. Indirect immunofluorescence demonstrates that Hus1 is nuclear and that this localization depends on Rad17. We show that Rad17 defines a distinct protein complex in soluble extracts that is separate from Rad1, Rad9, and Hus1. However, two-hybrid interaction, in vitro association and in vivo overexpression experiments suggest a transient interaction between Rad1 and Rad17.

scholarly journals Characterization of the interaction domains of Ure2p, a prion-like protein of yeast

1999 ◽  
Vol 338 (2) ◽  
pp. 403-407 ◽  
Author(s):  
Eric FERNANDEZ-BELLOT ◽  
Elisabeth GUILLEMET ◽  
Agnès BAUDIN-BAILLIEU ◽  
Sébastien GAUMER ◽  
Anton A. KOMAR ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Deletion Mutants ◽  
Loss Of Function ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Interaction Domains ◽  
Two Hybrid

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

scholarly journals Nep1, a Schizosaccharomyces pombe deneddylating enzyme

2005 ◽  
Vol 389 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Lihong Zhou ◽  
Felicity Z. Watts
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Cell Cycle Progression ◽  
Dna Integrity ◽  
Main Function ◽  
Cycle Progression ◽  
D Cells ◽  
Modified Forms

Nedd8 is a ubiquitin-like modifier that is attached to the cullin components of E3 ubiquitin ligases. More recently, p53 has also been shown to be Nedd8-modified. Nedd8 attachment occurs in a manner similar to that observed for other ubiquitin-like modifiers. In the present study, we report on the characterization of Nep1, a deneddylating enzyme in fission yeast (Schizosaccharomyces pombe). Unlike loss of ned8, deletion of the nep1 gene is not lethal, although nep1.d cells are heterogeneous in length, suggesting a defect in cell-cycle progression. Viability of nep1.d cells is dependent on a functional spindle checkpoint but not on the DNA integrity checkpoint. Deletion of a related gene (nep2), either alone or in combination with nep1.d, also has little effect on cell viability. We show that Nep1 can deneddylate the Pcu1, Pcu3 and Pcu4 cullins in vitro and that its activity is sensitive to N-ethylmaleimide, consistent with the idea that it is a member of the cysteine protease family. nep1.d cells accumulate Nedd8-modified proteins, although these do not correspond to modified forms of the cullins, suggesting that, although Nep1 can deneddylate cullins in vitro, this is not its main function in vivo. Nep1 can be co-precipitated with the signalosome subunit Csn5. Nep1 itself is present in a high-molecular-mass complex, but the presence of this complex is not dependent on the production of intact signalosomes. Our results suggest that, in vivo, Nep1 may be responsible for deneddylating proteins other than cullins.

scholarly journals A General RNA-Binding Protein Complex That Includes the Cytoskeleton-associated Protein MAP 1A

1998 ◽  
Vol 9 (7) ◽  
pp. 1695-1708 ◽  
Author(s):  
Christopher DeFranco ◽  
Marina E. Chicurel ◽  
Huntington Potter
Keyword(s):  
Protein Complex ◽  
Rna Binding ◽  
Fundamental Aspect ◽  
Dependent Manner ◽  
Size Dependent ◽  
Protein Map ◽  
Initial Characterization

Association of mRNA with the cytoskeleton represents a fundamental aspect of RNA physiology likely involved in mRNA transport, anchoring, translation, and turnover. We report the initial characterization of a protein complex that binds RNA in a sequence-independent but size-dependent manner in vitro. The complex includes a ∼160-kDa protein that is bound directly to mRNA and that appears to be either identical or highly related to a ∼1600-kDa protein that binds directly to mRNA in vivo. In addition, the microtubule-associated protein, MAP 1A, a cytoskeletal associated protein is a component of this complex. We suggest that the general attachment of mRNA to the cytoskeleton may be mediated, in part, through the formation of this ribonucleoprotein complex.

scholarly journals Nucleophosmin Is Required for DNA Integrity and p19Arf Protein Stability

2005 ◽  
Vol 25 (20) ◽  
pp. 8874-8886 ◽  
Author(s):  
Emanuela Colombo ◽  
Paola Bonetti ◽  
Eros Lazzerini Denchi ◽  
Paola Martinelli ◽  
Raffaella Zamponi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Genetic Alterations ◽  
Dna Integrity ◽  
Senescent Phenotype ◽  
Tumor Suppressive Function ◽  
Proliferation In Vitro

ABSTRACT Nucleophosmin (NPM) is a nucleolar phosphoprotein that binds the tumor suppressors p53 and p19Arf and is thought to be indispensable for ribogenesis, cell proliferation, and survival after DNA damage. The NPM gene is the most frequent target of genetic alterations in leukemias and lymphomas, though its role in tumorigenesis is unknown. We report here the first characterization of a mouse NPM knockout strain. Lack of NPM expression results in accumulation of DNA damage, activation of p53, widespread apoptosis, and mid-stage embryonic lethality. Fibroblasts explanted from null embryos fail to grow and rapidly acquire a senescent phenotype. Transfer of the NPM mutation into a p53-null background rescued apoptosis in vivo and fibroblast proliferation in vitro. Cells null for both p53 and NPM grow faster than control cells and are more susceptible to transformation by activated oncogenes, such as mutated Ras or overexpressed Myc. In the absence of NPM, Arf protein is excluded from nucleoli and is markedly less stable. Our data demonstrate that NPM regulates DNA integrity and, through Arf, inhibits cell proliferation and are consistent with a putative tumor-suppressive function of NPM.

scholarly journals Cloning and characterization of hELD/OSA1, a novel BRG1 interacting protein

2002 ◽  
Vol 364 (1) ◽  
pp. 255-264 ◽  
Author(s):  
Adam F.L. HURLSTONE ◽  
Ivan A. OLAVE ◽  
Nick BARKER ◽  
Mascha van NOORT ◽  
Hans CLEVERS
Keyword(s):  
Protein A ◽  
The Other ◽  
Dna Binding Domain ◽  
Related Protein ◽  
Related Gene ◽  
Human Homologue ◽  
Interacting Protein

A highly conserved multisubunit enzymic complex, SWI/SNF, participates in the regulation of eukaryote gene expression through its ability to remodel chromatin. While a single component of SWI/SNF, Swi2 or a related protein, can perform this function in vitro, the other components appear to modulate the activity and specificity of the complex in vivo. Here we describe the cloning of hELD/OSA1, a 189KDa human homologue of Drosophila Eld/Osa protein, a constituent of Drosophila SWI/SNF. By comparing conserved peptide sequences in Eld/Osa homologues we define three domains common to all family members. A putative DNA binding domain, or ARID (AT-rich DNA-interacting domain), may function in targetting SWI/SNF to chromatin. Two other domains unique to Eld/Osa proteins, EHD1 and EHD2, map to the C-teminus. We show that EHD2 mediates binding to Brahma-related gene 1 (BRG1), a human homologue of yeast Swi2. EHD1 and EHD2 also appear capable of interacting with each other. Using an antibody raised against EHD2 of hELD/OSA1, we detected Eld/Osa1 in endogenous SWI/SNF complexes derived from mouse brain.

scholarly journals Tyrosine Phosphorylation Regulates Alpha II Spectrin Cleavage by Calpain

2002 ◽  
Vol 22 (10) ◽  
pp. 3527-3536 ◽  
Author(s):  
Gaël Nicolas ◽  
Catherine M. Fournier ◽  
Colette Galand ◽  
Laurence Malbert-Colas ◽  
Odile Bournier ◽  
...  
Keyword(s):  
Sh3 Domain ◽  
Membrane Skeleton ◽  
Cellular Functions ◽  
Phosphatase Inhibitors ◽  
Recombinant Peptides ◽  
Two Hybrid ◽  
Hybrid Screening

ABSTRACT Spectrins, components of the membrane skeleton, are implicated in various cellular functions. Understanding the diversity of these functions requires better characterization of the interacting domains of spectrins, such as the SH3 domain. Yeast two-hybrid screening of a kidney cDNA library revealed that the SH3 domain of αII-spectrin binds specifically isoform A of low-molecular-weight phosphotyrosine phosphatase (LMW-PTP). The αII-spectrin SH3 domain does not interact with LMW-PTP B or C nor does LMW-PTP A interact with the αI-spectrin SH3 domain. The interaction of spectrin with LMW-PTP A led us to look for a tyrosine-phosphorylated residue in αII-spectrin. Western blotting showed that αII-spectrin is tyrosine phosphorylated in vivo. Using mutagenesis on recombinant peptides, we identified the residue Y1176 located in the calpain cleavage site of αII-spectrin, near the SH3 domain, as an in vitro substrate for Src kinase and LMW-PTP A. This Y1176 residue is also an in vivo target for kinases and phosphatases in COS cells. Phosphorylation of this residue decreases spectrin sensitivity to calpain in vitro. Similarly, the presence of phosphatase inhibitors in cell culture is associated with the absence of spectrin cleavage products. This suggests that the Y1176 phosphorylation state could modulate spectrin cleavage by calpain and may play an important role during membrane skeleton remodeling.

scholarly journals BUR1 and BUR2 Encode a Divergent Cyclin-Dependent Kinase–Cyclin Complex Important for Transcription In Vivo

2000 ◽  
Vol 20 (19) ◽  
pp. 7080-7087 ◽  
Author(s):  
Sheng Yao ◽  
Aaron Neiman ◽  
Gregory Prelich
Keyword(s):  
Protein Kinase ◽  
Genetic Selection ◽  
Cyclin Dependent Kinase ◽  
Selection For ◽  
Mutant Phenotypes ◽  
Two Hybrid ◽  
Putative Protein Kinase

ABSTRACT BUR1 and BUR2 were previously identified by a genetic selection for mutations that increase transcription from basal promoters in vivo. BUR1 encoded a putative protein kinase with greatest similarity to members of the cyclin-dependent kinase (CDK) family, although that similarity was not sufficient to classify it as a CDK. It was also not known whether Bur1 activity was cyclin dependent and, if so, which cyclins stimulated Bur1. The molecular cloning and characterization of BUR2 presented here sheds light on these issues. Genetic analysis indicates thatBUR2 function is intimately related to that ofBUR1: bur1 and bur2 mutations cause nearly identical spectra of mutant phenotypes, and overexpression ofBUR1 suppresses a bur2 null allele. Biochemical analysis has provided a molecular basis for these genetic observations. We find that BUR2 encodes a cyclin for the Bur1 protein kinase, based on the following evidence. First, the BUR2amino acid sequence reveals similarity to the cyclins; second, Bur1 and Bur2 coimmunoprecipitate from crude extracts and interact in the two-hybrid system; and third, BUR2 is required for Bur1 kinase activity in vitro. Our combined genetic and biochemical results therefore indicate that Bur1 and Bur2 comprise a divergent CDK-cyclin complex that has an important functional role during transcription in vivo.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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