Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Cell Rep ; 30(2): 454-464.e5, 2020 01 14.
Article in English | MEDLINE | ID: mdl-31940489

ABSTRACT

Loss of heterozygosity (LOH) at 1p36 occurs in multiple cancers, including neuroblastoma (NBL). MYCN amplification and 1p36 deletions tightly correlate with markers of tumor aggressiveness in NBL. Although distal 1p36 losses associate with single-copy MYCN tumors, larger deletions correlate with MYCN amplification, indicating two tumor suppressor regions in 1p36, only one of which facilitates MYCN oncogenesis. To better define this region, we genome-edited the syntenic 1p36 locus in primary mouse neural crest cells (NCCs), a putative NBL cell of origin. In in vitro cell transformation assays, we show that Chd5 loss confers most of the MYCN-independent tumor suppressor effects of 1p36 LOH. In contrast, MYCN-driven tumorigenesis selects for NCCs with Arid1a deletions from a pool of NCCs with randomly sized 1p36 deletions, establishing Arid1a as the MYCN-associated tumor suppressor. Our findings reveal that Arid1a loss collaborates with oncogenic MYCN and better define the tumor suppressor functions of 1p36 LOH in NBL.


Subject(s)
Chromosome Disorders/genetics , DNA-Binding Proteins/metabolism , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/genetics , Transcription Factors/metabolism , Animals , Chromosome Deletion , Chromosomes, Human, Pair 1/genetics , Humans , Mice
2.
Mol Cell ; 72(1): 19-36.e8, 2018 10 04.
Article in English | MEDLINE | ID: mdl-30244836

ABSTRACT

Mutations in the tumor suppressor SPOP (speckle-type POZ protein) cause prostate, breast, and other solid tumors. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localizes to nuclear speckles. Although cancer-associated mutations in SPOP interfere with substrate recruitment to the ligase, mechanisms underlying assembly of SPOP with its substrates in liquid nuclear bodies and effects of SPOP mutations on assembly are poorly understood. Here, we show that substrates trigger phase separation of SPOP in vitro and co-localization in membraneless organelles in cells. Enzymatic activity correlates with cellular co-localization and in vitro mesoscale assembly formation. Disease-associated SPOP mutations that lead to the accumulation of proto-oncogenic proteins interfere with phase separation and co-localization in membraneless organelles, suggesting that substrate-directed phase separation of this E3 ligase underlies the regulation of ubiquitin-dependent proteostasis.


Subject(s)
Cell Compartmentation/genetics , Neoplasms/genetics , Nuclear Proteins/genetics , Proteostasis/genetics , Repressor Proteins/genetics , Cell Line, Tumor , Humans , Mutation , Neoplasms/pathology , Ubiquitin/genetics , Ubiquitin-Protein Ligases/genetics , Ubiquitination/genetics
3.
J Biol Chem ; 289(40): 27504-12, 2014 Oct 03.
Article in English | MEDLINE | ID: mdl-25143379

ABSTRACT

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates.


Subject(s)
Endoplasmic Reticulum/enzymology , HSP40 Heat-Shock Proteins/chemistry , HSP40 Heat-Shock Proteins/metabolism , Adenosine Triphosphatases/metabolism , Animals , COS Cells , Chlorocebus aethiops , Dimerization , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , HSP40 Heat-Shock Proteins/genetics , Humans , Kinetics , Protein Binding , Protein Folding , Proteins/metabolism
4.
J Biol Chem ; 287(11): 7969-78, 2012 Mar 09.
Article in English | MEDLINE | ID: mdl-22267725

ABSTRACT

Protein localization within cells regulates accessibility for interactions with co-factors and substrates. The endoplasmic reticulum (ER) BiP co-factor ERdj4 is up-regulated by ER stress and has been implicated in ER-associated degradation (ERAD) of multiple unfolded secretory proteins. Several other ERdj family members tend to interact selectively with nascent proteins, presumably because those ERdj proteins associate with the Sec61 translocon that facilitates entry of nascent proteins into the ER. How ERdj4 selects and targets terminally misfolded proteins for destruction remains poorly understood. In this study, we determined properties of ERdj4 that might aid in this function. ERdj4 was reported to retain its signal sequence and to be resistant to mild detergent extraction, suggesting that it was an integral membrane protein. However, live cell photobleaching analyses of GFP-tagged ERdj4 revealed that the protein exhibits diffusion coefficients uncommonly high for an ER integral membrane protein and more similar to the mobility of a soluble luminal protein. Biochemical characterization established that the ERdj4 signal sequence is cleaved to yield a soluble protein. Importantly, we found that both endogenous and overexpressed ERdj4 associate with the integral membrane protein, Derlin-1. Our findings now directly link ERdj4 to the ERAD machinery and suggest a model in which ERjd4 could help recruit clients from throughout the ER to ERAD sites.


Subject(s)
Endoplasmic Reticulum-Associated Degradation/physiology , Endoplasmic Reticulum/metabolism , Membrane Glycoproteins/metabolism , Models, Biological , Protein Sorting Signals/physiology , Animals , Cell Line , Dogs , Endoplasmic Reticulum/genetics , Intracellular Membranes/metabolism , Membrane Glycoproteins/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Protein Transport/physiology
5.
Semin Cell Dev Biol ; 21(5): 472-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20026282

ABSTRACT

BiP is the mammalian endoplasmic reticulum (ER) Hsp70 orthologue that plays a major role in all functions of this organelle including the seemingly opposing functions of aiding the maturation of unfolded nascent proteins and identifying and targeting chronically unfolded proteins for degradation. The recent identification of mammalian BiP co-factors combined with delineation of the ER degradation machinery and data suggesting that the ER is subdivided into unique regions helps explain how these different functions can occur in the same organelle and raises some unresolved issues.


Subject(s)
Endoplasmic Reticulum/metabolism , HSP70 Heat-Shock Proteins/metabolism , Animals , Humans
6.
Cell Cycle ; 9(1): 147-61, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-20016281

ABSTRACT

Int6/eIF3e is implicated in tumorigenesis, but its molecular functions remain unclear. We have studied its fission yeast homolog Yin6, reporting that it regulates proteolysis by controlling the assembly/localization of proteasomes, and binds directly to another conserved protein, Moe1. In the present study, we isolated Cdc48 as a Moe1-binding protein from a yeast two-hybrid screen, and confirmed biochemically that they form a stable complex in fission yeast. Overexpressing Moe1 or Yin6 partially rescued phenotypes of cdc48 mutants; conversely, overexpressing Cdc48 partially rescued phenotypes of moe1 or yin6 mutants. Mutants defective in both Cdc48 and the Yin6-Moe1 complex showed growth defects that were far more severe than either alone. These double mutants were severely deficient in endoplasmic reticulum associated degradation (ERAD), as they were hypersensitive to accumulation of misfolded proteins. In addition, their chromosomes showed frequent defects in spindle attachment and segregation--these mitotic defects correlated with Ase1 and Bir1/survivin mislocalization. These results suggest that Cdc48, Yin6 and Moe1 act in the same protein complex to concertedly control ERAD and chromosome segregation. Many of these properties are evolutionarily conserved in humans, since human Cdc48 rescued the lethality of the yeast cdc48Delta mutant, and Int6 and Moe1/eIF3d bind Cdc48 in human cells.


Subject(s)
Adenosine Triphosphatases/metabolism , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Chromosome Segregation/physiology , Eukaryotic Initiation Factors/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Adenosine Triphosphatases/genetics , Blotting, Far-Western , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Chromosome Segregation/genetics , Eukaryotic Initiation Factors/genetics , Humans , Immunoprecipitation , Protein Binding/physiology , Schizosaccharomyces pombe Proteins/genetics , Two-Hybrid System Techniques , Valosin Containing Protein
7.
Nat Struct Mol Biol ; 14(3): 208-14, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17293872

ABSTRACT

Cdc13, Stn1 and Ten1 are essential yeast proteins that both protect chromosome termini from unregulated resection and regulate telomere length. Cdc13, which localizes to telomeres through high-affinity binding to telomeric single-stranded DNA, has been extensively characterized, whereas the contribution(s) of the Cdc13-associated Stn1 and Ten1 proteins to telomere function have remained unclear. We show here that Stn1 and Ten1 are DNA-binding proteins with specificity for telomeric DNA substrates. Furthermore, Stn1 and Ten1 show similarities to Rpa2 and Rpa3, subunits of the heterotrimeric replication protein A (RPA) complex, which is the major single-stranded DNA-binding activity in eukaryotic cells. We propose that Cdc13, Stn1 and Ten1 function as a telomere-specific RPA-like complex. Identification of an RPA-like complex that is targeted to a specific region of the genome suggests that multiple RPA-like complexes have evolved, each making individual contributions to genomic stability.


Subject(s)
Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Replication Protein A/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Telomere-Binding Proteins/metabolism , Telomere/metabolism , Amino Acid Sequence , Base Sequence , Cell Cycle Proteins/chemistry , Chromosomal Proteins, Non-Histone/chemistry , DNA, Fungal/metabolism , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Tertiary , Replication Protein A/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Substrate Specificity , Telomere-Binding Proteins/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...