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2.
J Am Chem Soc ; 132(23): 8010-9, 2010 Jun 16.
Article in English | MEDLINE | ID: mdl-20481530

ABSTRACT

Proteins have evolved to exploit long-range structural and dynamic effects as a means of regulating function. Understanding communication between sites in proteins is therefore vital to our comprehension of such phenomena as allostery, catalysis, and ligand binding/ejection. Double mutant cycle analysis has long been used to determine the existence of communication between pairs of sites, proximal or distal, in proteins. Typically, nonadditivity (or "thermodynamic coupling") is measured from global transitions in concert with a single probe. Here, we have applied the atomic resolution of NMR in tandem with native-state hydrogen exchange (HX) to probe the structure/energy landscape for information transduction between a large number of distal sites in a protein. Considering the event of amide proton exchange as an energetically quantifiable structural perturbation, m n-dimensional cycles can be constructed from mutation of n-1 residues, where m is the number of residues for which HX data is available. Thus, efficient mapping of a large number of couplings is made possible. We have applied this technique to one additive and two nonadditive double mutant cycles in a model system, eglin c. We find heterogeneity of HX-monitored couplings for each cycle, yet averaging results in strong agreement with traditionally measured values. Furthermore, long-range couplings observed at locally exchanging residues indicate that the basis for communication can occur within the native state ensemble, a conclusion not apparent from traditional measurements. We propose that higher-order couplings can be obtained and show that such couplings provide a mechanistic basis for understanding lower-order couplings via "spheres of perturbation". The method is presented as an additional tool for identifying a large number of couplings with greater coverage of the protein of interest.


Subject(s)
Hydrogen , Mutant Proteins/chemistry , Mutant Proteins/metabolism , Mutation , Feasibility Studies , Magnetic Resonance Spectroscopy , Models, Molecular , Mutant Proteins/genetics , Protein Conformation , Protein Denaturation , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Spectrometry, Fluorescence , Thermodynamics
3.
Mol Cell Biol ; 28(3): 977-87, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18039847

ABSTRACT

Canonical Wnt signaling is mediated by a molecular "switch" that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of beta-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of beta-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of beta-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.


Subject(s)
DNA-Binding Proteins/physiology , Nuclear Proteins/physiology , Proto-Oncogene Proteins/physiology , Repressor Proteins/genetics , TCF Transcription Factors/physiology , Transcription Factors/physiology , Transcription, Genetic , beta Catenin/metabolism , Animals , COS Cells , Chlorocebus aethiops , Cricetinae , DNA-Binding Proteins/genetics , Humans , Intestine, Small/chemistry , K562 Cells , Mice , Mice, Knockout , Nuclear Proteins/metabolism , Protein Binding , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myc/analysis , RUNX1 Translocation Partner 1 Protein , TCF Transcription Factors/genetics , Transcription Factor 7-Like 2 Protein , Transcription Factors/genetics , Transcription Factors/metabolism , Transfection , Xenopus Proteins
4.
Mol Biol Cell ; 18(9): 3340-50, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17567951

ABSTRACT

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.


Subject(s)
Cell Cycle Proteins/metabolism , DNA Damage , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Anaphase-Promoting Complex-Cyclosome , Catalysis , Cell Cycle , Cyclin-Dependent Kinases/metabolism , HeLa Cells , Humans , Phosphorylation , Polyubiquitin/metabolism , Protein Binding , Protein Processing, Post-Translational , Tumor Suppressor Proteins , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitin-Protein Ligases/chemistry
5.
Mol Cell Biol ; 26(20): 7667-81, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16908528

ABSTRACT

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G1, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.


Subject(s)
Cyclin-Dependent Kinases/metabolism , DNA Replication/genetics , Retinoblastoma/genetics , Retinoblastoma/metabolism , S Phase , Cell Cycle Proteins/genetics , Cell Line, Tumor , Chromosomes/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Gene Expression , Humans , Nuclear Proteins/genetics , Protein Binding , Signal Transduction
6.
Mol Cell Biol ; 25(21): 9576-85, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16227606

ABSTRACT

Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.


Subject(s)
Cell Lineage/physiology , Intestine, Small/cytology , Phosphoproteins/biosynthesis , Repressor Proteins/biosynthesis , Transcription, Genetic , Animals , Cell Line , Chlorocebus aethiops , Enteroendocrine Cells/cytology , Goblet Cells/cytology , Histone Deacetylases/metabolism , Humans , Intestine, Small/metabolism , Mice , Nuclear Proteins/metabolism , Nuclear Receptor Co-Repressor 1 , Paneth Cells/cytology , Phosphoproteins/genetics , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex
7.
Proc Natl Acad Sci U S A ; 100(6): 3257-62, 2003 Mar 18.
Article in English | MEDLINE | ID: mdl-12626745

ABSTRACT

Posttranslational modification by small ubiquitin-like modifier (SUMO) conjugation regulates the subnuclear localization of several proteins; however, SUMO modification has not been directly linked to nuclear export. The ETS (E-Twenty-Six) family member TEL (ETV6) is a transcriptional repressor that can inhibit Ras-dependent colony growth in soft agar and induce cellular aggregation of Ras-transformed cells. TEL is frequently disrupted by chromosomal translocations such as the t(12;21), which is associated with nearly one-fourth of pediatric B cell acute lymphoblastic leukemia. In the vast majority of t(12;21)-containing cases, the second allele of TEL is deleted, suggesting that inactivation of TEL contributes to the disease. Although TEL functions in the nucleus as a DNA-binding transcriptional repressor, it has also been detected in the cytoplasm. Here we demonstrate that TEL is actively exported from the nucleus in a leptomycin B-sensitive manner. TEL is posttranslationally modified by sumoylation at lysine 99 within a highly conserved domain (the "pointed" domain). Mutation of the sumo-acceptor lysine or mutations within the pointed domain that affect sumoylation impair nuclear export of TEL. Mutation of lysine 99 also results in an increase in TEL transcriptional repression, presumably because of decreased nuclear export. We propose that the ability of TEL to repress transcription and suppress growth is regulated by sumoylation and nuclear export.


Subject(s)
DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins , Repressor Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , 3T3 Cells , Active Transport, Cell Nucleus/drug effects , Animals , Burkitt Lymphoma/genetics , Burkitt Lymphoma/metabolism , COS Cells , Cell Line , Core Binding Factor Alpha 2 Subunit , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Fatty Acids, Unsaturated/pharmacology , HeLa Cells , Humans , Mice , Point Mutation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-ets , Repressor Proteins/chemistry , Repressor Proteins/genetics , Transcription Factors/metabolism , ETS Translocation Variant 6 Protein
8.
Mol Plant Microbe Interact ; 15(4): 380-7, 2002 Apr.
Article in English | MEDLINE | ID: mdl-12026177

ABSTRACT

To better understand the molecular regulation of defense responses in members of the genus Pinus, we tested the expression of various chitinase homologs in response to pathogen-associated signals. PSCHI4, a putative extracellular class II chitinase, was secreted into liquid medium by pine cells and was also secreted by transgenic tobacco cells that ectopically expressed pschi4. Extracellular proteins of pine were separated by isoelectric focusing; PSCHI4 was not associated with fractions containing detectable beta-N-acetylglucosaminidase or lysozyme activities. However, other fractions contained enzyme activities that increased markedly after elicitor treatment. The pschi4 transcript and protein accumulated in pine seedlings challenged with the necrotrophic pathogen Fusarium subglutinans f. sp. pini, with the protein reaching detectable levels in susceptible seedlings concomitant with the onset of visible disease symptoms. Additional chitinase transcripts, assigned to classes I and IV based on primary sequence analysis, were also induced by pathogen challenge. Jasmonic acid induced class I and class IV but not class II chitinase, whereas salicylic acid induced all three classes of chitinase. These results show that multiple chitinase homologs are induced after challenge by a necrotrophic pathogen and by potential signaling molecules identified in angiosperms. This suggests the potential importance of de novo pathogenesis-related (PR) gene expression in pathogen defense responses of pine trees.


Subject(s)
Chitinases/genetics , Cyclopentanes/pharmacology , Fungi/growth & development , Pinus/genetics , Salicylic Acid/pharmacology , Amino Acid Sequence , Chitinases/metabolism , Cloning, Molecular , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Genotype , Molecular Sequence Data , Multigene Family/genetics , Oxylipins , Pinus/drug effects , Pinus/microbiology , Plant Diseases/microbiology , Plant Growth Regulators/pharmacology , Sequence Homology , Nicotiana/cytology , Nicotiana/metabolism
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