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1.
Nat Cell Biol ; 15(4): 430-9, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23455478

ABSTRACT

Polo-like kinase 1 (PLK1) critically regulates mitosis through its dynamic localization to kinetochores, centrosomes and the midzone. The polo-box domain (PBD) and activity of PLK1 mediate its recruitment to mitotic structures, but the mechanisms regulating PLK1 dynamics remain poorly understood. Here, we identify PLK1 as a target of the cullin 3 (CUL3)-based E3 ubiquitin ligase, containing the BTB adaptor KLHL22, which regulates chromosome alignment and PLK1 kinetochore localization but not PLK1 stability. In the absence of KLHL22, PLK1 accumulates on kinetochores, resulting in activation of the spindle assembly checkpoint (SAC). CUL3-KLHL22 ubiquitylates Lys 492, located within the PBD, leading to PLK1 dissociation from kinetochore phosphoreceptors. Expression of a non-ubiquitylatable PLK1-K492R mutant phenocopies inactivation of CUL3-KLHL22. KLHL22 associates with the mitotic spindle and its interaction with PLK1 increases on chromosome bi-orientation. Our data suggest that CUL3-KLHL22-mediated ubiquitylation signals degradation-independent removal of PLK1 from kinetochores and SAC satisfaction, which are required for faithful mitosis.


Subject(s)
Cell Cycle Proteins/metabolism , Centrosome/metabolism , Chromosomes, Human/genetics , Kinetochores/metabolism , Microtubules/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Spindle Apparatus/metabolism , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Blotting, Western , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cullin Proteins/antagonists & inhibitors , Cullin Proteins/genetics , Cullin Proteins/metabolism , HeLa Cells , Humans , Immunoprecipitation , Microscopy, Fluorescence , Molecular Sequence Data , Phosphorylation , Protein Array Analysis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Sequence Homology, Amino Acid , Signal Transduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin/metabolism , Ubiquitination , Polo-Like Kinase 1
2.
Structure ; 20(3): 414-28, 2012 Mar 07.
Article in English | MEDLINE | ID: mdl-22405001

ABSTRACT

The endosomal sorting complexes required for transport (ESCRTs) facilitate endosomal sorting of ubiquitinated cargo, MVB biogenesis, late stages of cytokinesis, and retroviral budding. Here we show that ubiquitin associated protein 1 (UBAP1), a subunit of human ESCRT-I, coassembles in a stable 1:1:1:1 complex with Vps23/TSG101, VPS28, and VPS37. The X-ray crystal structure of the C-terminal region of UBAP1 reveals a domain that we describe as a solenoid of overlapping UBAs (SOUBA). NMR analysis shows that each of the three rigidly arranged overlapping UBAs making up the SOUBA interact with ubiquitin. We demonstrate that UBAP1-containing ESCRT-I is essential for degradation of antiviral cell-surface proteins, such as tetherin (BST-2/CD317), by viral countermeasures, namely, the HIV-1 accessory protein Vpu and the Kaposi sarcoma-associated herpesvirus (KSHV) ubiquitin ligase K5.


Subject(s)
Carrier Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Models, Molecular , Ubiquitin/metabolism , Amino Acid Sequence , Antigens, CD/metabolism , Carrier Proteins/genetics , Chromatography, Gel , Crystallography, X-Ray , GPI-Linked Proteins/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Humans , Immediate-Early Proteins/metabolism , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Viral Regulatory and Accessory Proteins/metabolism
3.
FEBS Lett ; 585(24): 3856-61, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-22044534

ABSTRACT

The two major antagonistic pathways of carbon metabolism in cells, glycolysis and gluconeogenesis, are tightly regulated. In the eukaryotic model organism Saccharomyces cerevisiae the switch from gluconeogenesis to glycolysis is brought about by proteasomal degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase. The ubiquitin ligase responsible for polyubiquitylation of fructose-1,6-bisphosphatase is the Gid complex. This complex consists of seven subunits of which subunit Gid2/Rmd5 contains a RING finger domain providing E3 ligase activity. Here we identify an additional subunit containing a degenerated RING finger, Gid9/Fyv10. This subunit binds to Gid2/Rmd5. A mutation in the degenerated RING finger of Gid9/Fyv10 abolishes polyubiquitylation and degradation of three enzymes specific for gluconeogenesis.


Subject(s)
Proteolysis , RING Finger Domains , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Protein Multimerization , Protein Structure, Quaternary , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Ubiquitination
4.
Stem Cells ; 29(5): 847-57, 2011 May.
Article in English | MEDLINE | ID: mdl-21394831

ABSTRACT

MicroRNAs (miRNAs) have been shown to play an important role in hematopoiesis. To elucidate the role of miRNAs in the early steps of hematopoiesis, we directly compared donor-matched CD133(+) cells with the more differentiated CD34(+) CD133(-) and CD34(-) CD133(-) cells from bone marrow on the miRNA and mRNA level. Using quantitative whole genome miRNA microarray and sequencing-based profiling, we found that between 109 (CD133(+) ) and 216 (CD34(-) CD133(-) ) miRNAs were expressed. Quantification revealed that the 25 highest expressed miRNAs accounted for 73% of the total miRNA pool. miR-142-3p was the highest expressed miRNA with up to 2,000 copies per cell in CD34(+) CD133(-) cells. Eighteen miRNAs were significantly differentially expressed between CD133(+) and CD34(+) CD133(-) cells. We analyzed their biological role by examining the coexpression of miRNAs and its bioinformatically predicted mRNA targets and luciferase-based reporter assays. We provide the first evidence for a direct regulation of CD133 by miR-142-3p as well as tropomyosin 1 and frizzled homolog 5 by miR-29a. Overexpression of miRNAs in CD133(+) cells demonstrated that miR-142-3p has a negative influence on the overall colony-forming ability. In conclusion, the miRNAs expressed differentially between the CD133(+) and CD34(+) CD133(-) cells are involved in inhibition of differentiation, prevention of apoptosis, and cytoskeletal remodeling. These results are highly relevant for stem cell-based therapies with CD133(+) cells and delineate for the first time how the stem cell character of CD133(+) cells is defined by the expression of specific miRNAs.


Subject(s)
Antigens, CD34/metabolism , Antigens, CD/metabolism , Cell Differentiation/physiology , Glycoproteins/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , MicroRNAs/genetics , Peptides/metabolism , RNA, Messenger/genetics , AC133 Antigen , Cell Differentiation/genetics , Cell Proliferation , Cells, Cultured , Humans , MicroRNAs/physiology , Oligonucleotide Array Sequence Analysis , Phylogeny , RNA, Messenger/physiology
5.
Mol Biosyst ; 5(12): 1797-808, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19734957

ABSTRACT

Ubiquitin specific proteases (USPs) are the largest family of deubiquitinating enzymes with approximately 56 members in humans. USPs regulate a wide variety of cellular processes by their ability to remove (poly)ubiquitin from target proteins. Their enzymatic activity is encoded in a common catalytic core of approximately 350 amino acids, however many USPs show significantly larger catalytic domains. Here we have analysed human and yeast USP domains, combining bioinformatics with structural information. We reveal that all USP domains can be divided into six conserved boxes, and we map the conserved boxes onto the USP domain core structure. The boxes are interspersed by insertions, some of which as large as the catalytic core. The two most common insertion points place inserts near the distal ubiquitin binding site, and in many cases ubiquitin binding domains or ubiquitin-like folds are found in these insertions, potentially directly affecting catalytic function. Other inserted sequences are unstructured, and removal of these might aid future structural and functional analysis. Yeast USP domains have a different pattern of inserted sequences, suggesting that the insertions are hotspots for evolutionary diversity to expand USP functionality.


Subject(s)
Computational Biology/methods , Endopeptidases/genetics , Endopeptidases/metabolism , Amino Acid Sequence , Catalytic Domain , Chromosome Mapping , Conserved Sequence/genetics , DNA Transposable Elements/genetics , Endopeptidases/chemistry , Genes, Fungal , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Sequence Alignment , Ubiquitin Thiolesterase/chemistry , Ubiquitin Thiolesterase/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Specific Proteases , Yeasts/enzymology , Yeasts/genetics , Zinc/chemistry , Zinc/metabolism
6.
EMBO Rep ; 9(10): 1034-40, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18704118

ABSTRACT

In budding yeast the cullin Rtt101 promotes replication fork progression through natural pause sites and areas of DNA damage, but its relevant subunits and molecular mechanism remain poorly understood. Here, we show that in budding yeast Mms1 and Mms22 are functional subunits of an Rtt101-based ubiquitin ligase that associates with the conjugating-enzyme Cdc34. Replication forks in mms1Delta, mms22Delta and rtt101Delta cells are sensitive to collisions with drug-induced DNA lesions, but not to transient pausing induced by nucleotide depletion. Interaction studies and sequence analysis have shown that Mms1 resembles human DDB1, suggesting that Rtt101(Mms1) is the budding yeast counterpart of the mammalian CUL4(DDB1) ubiquitin ligase family. Rtt101 interacts in an Mms1-dependent manner with the putative substrate-specific adaptors Mms22 and Crt10, the latter being a regulator of expression of ribonucleotide reductase. Taken together, our data suggest that the Rtt101(Mms1) ubiquitin ligase complex might be required to reorganize replication forks that encounter DNA lesions.


Subject(s)
Cullin Proteins/physiology , DNA Damage/physiology , DNA Replication/physiology , DNA-Binding Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/enzymology , Ubiquitin-Protein Ligases/physiology , Anaphase-Promoting Complex-Cyclosome , DNA-Binding Proteins/genetics , Humans , Protein Subunits/physiology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Ubiquitin-Conjugating Enzymes , Ubiquitin-Protein Ligase Complexes/physiology
7.
Mol Biol Cell ; 19(8): 3323-33, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18508925

ABSTRACT

Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.


Subject(s)
Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Fungal , Ubiquitin-Protein Ligases/chemistry , Carbohydrate Metabolism , Fructose-Bisphosphatase/chemistry , Gluconeogenesis , Glucose/metabolism , Models, Biological , Mutation , Phosphoenolpyruvate Carboxykinase (ATP)/metabolism , Plasmids/metabolism , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Vesicular Transport Proteins
8.
Mol Cell ; 29(4): 451-64, 2008 Feb 29.
Article in English | MEDLINE | ID: mdl-18313383

ABSTRACT

The tumor suppressor CYLD antagonizes NF-kappaB and JNK signaling by disassembly of Lys63-linked ubiquitin chains synthesized in response to cytokine stimulation. Here we describe the crystal structure of the CYLD USP domain, revealing a distinctive architecture that provides molecular insights into its specificity toward Lys63-linked polyubiquitin. We identify regions of the USP domain responsible for this specificity and demonstrate endodeubiquitinase activity toward such chains. Pathogenic truncations of the CYLD C terminus, associated with the hypertrophic skin tumor cylindromatosis, disrupt the USP domain, accounting for loss of CYLD catalytic activity. A small zinc-binding B box domain, similar in structure to other crossbrace Zn-binding folds--including the RING domain found in E3 ubiquitin ligases--is inserted within the globular core of the USP domain. Biochemical and functional characterization of the B box suggests a role as a protein-interaction module that contributes to determining the subcellular localization of CYLD.


Subject(s)
Lysine/metabolism , Polyubiquitin/metabolism , Protein Structure, Tertiary , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Crystallography, X-Ray , Deubiquitinating Enzyme CYLD , Genes, Tumor Suppressor , Humans , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Models, Molecular , Molecular Sequence Data , Mutation , NF-kappa B/metabolism , Neoplasms/genetics , Polyubiquitin/chemistry , Polyubiquitin/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Signal Transduction/physiology , Substrate Specificity , Tumor Suppressor Proteins/classification , Tumor Suppressor Proteins/genetics , Ubiquitin Thiolesterase/chemistry , Ubiquitin Thiolesterase/genetics , Ubiquitin-Specific Peptidase 7 , Zinc/metabolism
9.
Yeast ; 25(3): 199-217, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18260085

ABSTRACT

The great majority of proteasome substrates are marked for degradation by the attachment of polyubiquitin chains. Ornithine decarboxylase is degraded by the proteasome in the absence of this modification. We previously showed that this mechanism of degradation was conserved in eukaryotic cells. Here we use a reporter destabilized by mouse ornithine decarboxylase to screen non-essential Saccharomyces cerevisiae deletion mutants. We identified novel mutants that affect both ubiquitin-dependent and -independent proteasome degradation pathways. YLR021W (IRC25/POC3) and YPL144W (POC4) encode interacting proteins that function in proteasome assembly, with putative homologues widespread among eukaryotes. Several additional mutants suffered from defects in proteasome-mediated proteolysis. These included mutants in the urmylation pathway of protein modification (but not the Urm1 modifier itself) and the Reg1 regulatory subunit of protein phosphatase 1. Finally, we noted increased rates of ornithine decarboxylase turnover in an rpn10Delta mutant in which the degradation of certain ubiquitinated substrates is impaired. Together, these results highlight the utility of a ubiquitin-independent degron in uncovering novel factors affecting general and substrate-specific proteasome function.


Subject(s)
Ornithine Decarboxylase/metabolism , Proteasome Endopeptidase Complex/physiology , Saccharomyces cerevisiae/genetics , Sequence Deletion , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Mice , Molecular Chaperones/isolation & purification , Molecular Sequence Data , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Substrate Specificity
10.
J Biol Chem ; 282(44): 32414-23, 2007 Nov 02.
Article in English | MEDLINE | ID: mdl-17761670

ABSTRACT

Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.


Subject(s)
Carrier Proteins/metabolism , Proteasome Endopeptidase Complex/isolation & purification , Schizosaccharomyces pombe Proteins/isolation & purification , Amino Acid Sequence , COP9 Signalosome Complex , Carrier Proteins/chemistry , Carrier Proteins/genetics , HeLa Cells , Humans , Molecular Sequence Data , Multiprotein Complexes/metabolism , Peptide Hydrolases/metabolism , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Protein Structure, Tertiary , Protein Subunits/metabolism , Schizosaccharomyces/chemistry , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Sequence Alignment , Structure-Activity Relationship
11.
J Biol Chem ; 282(47): 34167-75, 2007 Nov 23.
Article in English | MEDLINE | ID: mdl-17728242

ABSTRACT

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.


Subject(s)
Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/physiology , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Ubiquitination/physiology , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Down-Regulation/physiology , HeLa Cells , Humans , Proteasome Endopeptidase Complex/genetics , Proteasome Inhibitors , SUMO-1 Protein/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Ubiquitin/genetics , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases
12.
FEBS Lett ; 581(17): 3189-96, 2007 Jul 10.
Article in English | MEDLINE | ID: mdl-17572409

ABSTRACT

The eukaryotic N-end rule pathway mediates ubiquitin- and proteasome-dependent turnover of proteins with a bulky amino-terminal residue. Arabidopsis locus At5g02310 shows significant similarity to the yeast N-end rule ligase Ubr1. We demonstrate that At5g02310 is a ubiquitin ligase and mediates degradation of proteins with amino-terminal Arg residue. Unlike Ubr1, the Arabidopsis protein does not participate in degradation of proteins with amino-terminal Phe or Leu. This modified target specificity coincides with characteristic differences in domain structure. In contrast to previous publications, our data indicate that At5g02310 is not identical to CER3, a gene involved in establishment of a protective surface wax layer. At5g02310 has therefore been re-designated PROTEOLYSIS 6 (PRT6), in accordance with its ubiquitin ligase function.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arginine/metabolism , Nuclear Proteins/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Carbon-Carbon Lyases , Models, Biological , Plants, Genetically Modified , Protein Processing, Post-Translational , Sequence Analysis, Protein , Signal Transduction , Substrate Specificity
13.
Curr Biol ; 15(13): 1217-21, 2005 Jul 12.
Article in English | MEDLINE | ID: mdl-16005295

ABSTRACT

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.


Subject(s)
Carrier Proteins/metabolism , Endopeptidases/metabolism , Multiprotein Complexes/metabolism , Peptide Hydrolases/metabolism , Zinc Fingers/genetics , Amino Acid Sequence , Blotting, Western , COP9 Signalosome Complex , DNA, Complementary/genetics , Endopeptidases/genetics , HeLa Cells , Humans , Microscopy, Electron , Molecular Sequence Data , Mucin-1/genetics , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/ultrastructure , Mutagenesis, Site-Directed , Mutation/genetics , Peptide Fragments/genetics , Peptide Hydrolases/ultrastructure , Phenanthrolines/pharmacology , Polyubiquitin/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ubiquitin/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Specific Proteases
14.
BMC Bioinformatics ; 6: 71, 2005 Mar 24.
Article in English | MEDLINE | ID: mdl-15790418

ABSTRACT

BACKGROUND: The 'lid' subcomplex of the 26S proteasome and the COP9 signalosome (CSN complex) share a common architecture consisting of six subunits harbouring a so-called PCI domain (proteasome, CSN, eIF3) at their C-terminus, plus two subunits containing MPN domains (Mpr1/Pad1 N-terminal). The translation initiation complex eIF3 also contains PCI- and MPN-domain proteins, but seems to deviate from the 6+2 stoichiometry. Initially, the PCI domain was defined as the region of detectable sequence similarity between the components mentioned above. RESULTS: During an exhaustive bioinformatical analysis of proteasome components, we detected multiple instances of tetratrico-peptide repeats (TPR) in the N-terminal region of most PCI proteins, suggesting that their homology is not restricted to the PCI domain. We also detected a previously unrecognized PCI domain in the eIF3 component eIF3k, a protein whose 3D-structure has been determined recently. By using profile-guided alignment techniques, we show that the structural elements found in eIF3k are most likely conserved in all PCI proteins, resulting in a structural model for the canonical PCI domain. CONCLUSION: Our model predicts that the homology domain PCI is not a true domain in the structural sense but rather consists of two subdomains: a C-terminal 'winged helix' domain with a key role in PCI:PCI interaction, preceded by a helical repeat region. The TPR-like repeats detected in the N-terminal region of PCI proteins most likely form an uninterrupted extension of the repeats found within the PCI domain boundaries. This model allows an interpretation of several puzzling experimental results.


Subject(s)
Computational Biology/methods , Eukaryotic Initiation Factor-3/chemistry , Multiprotein Complexes/chemistry , Peptide Hydrolases/chemistry , Proteasome Endopeptidase Complex/chemistry , Algorithms , Amino Acid Motifs , Amino Acid Sequence , Animals , COP9 Signalosome Complex , Cysteine Endopeptidases/chemistry , Databases, Protein , Genome , Humans , Molecular Sequence Data , Multienzyme Complexes , Peptides/chemistry , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Transcription Factors/chemistry , Ubiquitin/chemistry
15.
EMBO J ; 23(24): 4857-67, 2004 Dec 08.
Article in English | MEDLINE | ID: mdl-15538383

ABSTRACT

Polyamines are essential organic cations with multiple cellular functions. Their synthesis is controlled by a feedback regulation whose main target is ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis. In mammals, ODC has been shown to be inhibited and targeted for ubiquitin-independent degradation by ODC antizyme (AZ). The synthesis of mammalian AZ was reported to involve a polyamine-induced ribosomal frameshifting mechanism. High levels of polyamine therefore inhibit new synthesis of polyamines by inducing ODC degradation. We identified a previously unrecognized sequence in the genome of Saccharomyces cerevisiae encoding an orthologue of mammalian AZ. We show that synthesis of yeast AZ (Oaz1) involves polyamine-regulated frameshifting as well. Degradation of yeast ODC by the proteasome depends on Oaz1. Using this novel model system for polyamine regulation, we discovered another level of its control. Oaz1 itself is subject to ubiquitin-mediated proteolysis by the proteasome. Degradation of Oaz1, however, is inhibited by polyamines. We propose a model, in which polyamines inhibit their ODC-mediated biosynthesis by two mechanisms, the control of Oaz1 synthesis and inhibition of its degradation.


Subject(s)
Polyamines/metabolism , Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Animals , Base Sequence , Frameshifting, Ribosomal , Humans , Molecular Sequence Data , Proteasome Endopeptidase Complex/metabolism , Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Spermidine/metabolism , Ubiquitin/metabolism
17.
Hum Mol Genet ; 12(21): 2845-52, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-12944423

ABSTRACT

The spinocerebellar ataxias (SCAs) are a class of hereditary neurodegenerative diseases, which are caused by the pathological expansion of unstable CAG triplet repeats found in a number of apparently unrelated genes. The proteins encoded by the SCA genes typically translate this expanded (CAG)n repeat into an expanded poly(Q) stretch. Several pathological features are common to all SCAs, irrespective of the gene harbouring the expansion. The specific contributions of the mutated genes are currently hard to assess, as the physiological role of most of the so-called ataxins is not known. By combining the results of profile-based sequence analysis with genome-wide functional data available for model organisms, we have derived detailed predictions of the physiological function of two SCA gene products. Ataxin-3, the protein mutated in Machado Joseph Disease (SCA3), belongs to a novel group of cysteine-proteases and is predicted to be active against ubiquitin chains or related substrates. The catalytic site of this enzyme class is similar to that found in UBP and UCH type ubiquitin proteases. For ataxin-7, the gene product of the SCA7 gene, we have identified an orthology relationship to the yeast open reading frame Ygl066c. Recently published evidence from genome-wide studies suggests that Ygl066c is a component of the SAGA histone acetyltransferase complex. By analogy, a similar role for the mammalian ataxin-7 can be expected. The functional predictions reported here are sufficiently precise to allow a direct experimental verification. Moreover, both findings have implications for the general pathogenesis of spinocerebellar ataxias by providing a direct connection of these diseases with ubiquitin metabolism and histone acetylation.


Subject(s)
Computational Biology , Nerve Tissue Proteins/physiology , Acetylation , Amino Acid Sequence , Ataxin-3 , Ataxin-7 , Catalytic Domain/physiology , Histones/metabolism , Humans , Machado-Joseph Disease/genetics , Machado-Joseph Disease/metabolism , Molecular Sequence Data , Mutation , Nerve Tissue Proteins/genetics , Nuclear Proteins , Repressor Proteins , Sequence Homology , Ubiquitin/metabolism
19.
Hum Mol Genet ; 11(15): 1757-62, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12095917

ABSTRACT

Until recently, all genes found to be mutated in hereditary idiopathic epilepsies encoded subunits of ion channels, leading to the view of this class of diseases as channelopathies. Two apparent exceptions to this rule are the MASS1 gene, which is mutated in the Frings mouse model of audiogenic epilepsy, and the LGI1 gene, which is mutated in autosomal dominant partial epilepsy with auditory features (ADPEAF). Careful sequence analysis of the two protein products encoded by those genes shows a common feature: both sequences harbour a novel homology domain consisting of a 7-fold repeated 44-residue motif. The architecture and structural features of this new domain make it a likely member of the growing class of protein interaction domains with a seven-bladed beta-propeller fold. In the MASS1 gene product, which has recently been shown to be a fragment of the very large G-protein-coupled receptor VLGR1, this EAR domain (for epilepsy-associated repeat) is part of the ligand-binding ectodomain. LGI1, as well as a number of newly identified LGI1 relatives, is predicted to be a secreted protein, and consists of an N-terminal leucine-rich repeat region and a C-terminal EAR region. The known portion of the human genome encodes six EAR proteins, some of which map to chromosome regions associated with seizure disorders. The EAR domain is likely to play an important role in the pathogenesis of epilepsy, either by binding to an unknown anti-epileptic ligand, or more likely by interfering with axon guidance or synaptogenesis.


Subject(s)
Epilepsy/genetics , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Proteins/genetics , Receptors, G-Protein-Coupled , Sequence Homology , Amino Acid Sequence , Animals , Humans , Intracellular Signaling Peptides and Proteins , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Protein Structure, Tertiary , Proteins/metabolism , Sequence Alignment
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