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1.
Elife ; 122023 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-37713320

RESUMEN

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.


Asunto(s)
Proteínas Nucleares , Proteína que Contiene Valosina , Humanos , Proteína que Contiene Valosina/genética , Proteínas Nucleares/metabolismo , Transporte Activo de Núcleo Celular
2.
Mol Biol Cell ; 34(13): ar126, 2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-37756124

RESUMEN

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.


Asunto(s)
Endosomas , Proteínas de la Membrana , Transporte Biológico , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Transferrina/metabolismo , Humanos
3.
Cell Rep ; 41(2): 111467, 2022 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-36223745

RESUMEN

In all domains of life, mechanisms exist that adjust translational capacity to nutrient restriction and other growth constraints. The mammalian target of rapamycin (mTOR) regulates the synthesis of ribosomal proteins and translation factors in mammalian cells via phosphorylation of the La-related protein 1 (LARP1). In the present model of starvation-induced translational silencing, LARP1 targets mRNAs carrying a 5' terminal oligopyrimidine (5'TOP) motif to shift these into subpolysomal ribonucleoprotein particles. However, how these mRNAs would be protected from degradation and rapidly made available to restore translation capacity when needed remained enigmatic. Here, to address this, we employ gradient profiling by sequencing (Grad-seq) and monosome footprinting. Challenging the above model, we find that 5'TOP mRNAs, instead of being translationally silenced during starvation, undergo low baseline translation with reduced initiation rates. This mode of regulation ensures a stable 5'TOP mRNA population under starvation and allows fast reversibility of the translational repression.


Asunto(s)
Biosíntesis de Proteínas , Serina-Treonina Quinasas TOR , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Ribosómicas/metabolismo , Serina-Treonina Quinasas TOR/metabolismo
4.
Mol Oncol ; 16(17): 3082-3106, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35364627

RESUMEN

Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a 'premalignant' state, and its inhibition synergizes with clinically established compounds used to target EGFRL858R -, BRAFV600E - or PI3KH1047R -driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours.


Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas B-raf , Carcinogénesis/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Receptores ErbB/genética , Humanos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción , Ubiquitina Tiolesterasa/genética
5.
Int J Mol Sci ; 23(7)2022 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-35408984

RESUMEN

Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin ('ubiquitylation'), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology.


Asunto(s)
Gránulos Citoplasmáticos , Ubiquitina , Gránulos Citoplasmáticos/metabolismo , Proteínas de Unión al ARN/metabolismo , Gránulos de Estrés , Estrés Fisiológico , Ubiquitina/metabolismo
6.
Trends Cell Biol ; 32(4): 278-280, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35058103

RESUMEN

The protein unfoldase Cdc48/p97 targets a wide variety of cellular substrates, but the molecular basis of substrate turnover remains incompletely understood. Two recent reports, by Ji et al. and van den Boom et al., provide detailed insights into the unfolding process and reveal pronounced flexibility of substrate handling by Cdc48/p97.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas , Proteínas de Ciclo Celular/metabolismo , Humanos , Pliegue de Proteína , Proteínas/metabolismo , Proteína que Contiene Valosina/química , Proteína que Contiene Valosina/metabolismo
7.
Life Sci Alliance ; 4(5)2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33687997

RESUMEN

Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslational modifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immunofluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Estrés Fisiológico/fisiología , Ubiquitinación/fisiología , Gránulos Citoplasmáticos/fisiología , Células HeLa , Respuesta al Choque Térmico/fisiología , Humanos , Presión Osmótica/fisiología , Estrés Oxidativo/fisiología , Estrés Fisiológico/genética , Ubiquitina/metabolismo , Ubiquitinación/genética
8.
Mol Cell ; 70(5): 906-919.e7, 2018 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-29804830

RESUMEN

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.


Asunto(s)
Arsenitos/toxicidad , Gránulos Citoplasmáticos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Compuestos de Sodio/toxicidad , Estrés Fisiológico , Factor 2 Asociado a Receptor de TNF/metabolismo , Autofagia/efectos de los fármacos , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/patología , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Complejo de la Endopetidasa Proteasomal/genética , Transporte de Proteínas , Proteolisis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Factor 2 Asociado a Receptor de TNF/genética
9.
FEBS Lett ; 589(19 Pt A): 2578-89, 2015 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-26320413

RESUMEN

p97 (also known as Cdc48, Ter94, and VCP) is an essential, abundant and highly conserved ATPase driving the turnover of ubiquitylated proteins in eukaryotes. Even though p97 is involved in highly diverse cellular pathways and processes, it exhibits hardly any substrate specificity on its own. Instead, it relies on a large number of regulatory cofactors controlling substrate specificity and turnover. The complexity as well as temporal and spatial regulation of the interactions between p97 and its cofactors is only beginning to be understood at the molecular level. Here, we give an overview on the structural framework of p97 interactions with its cofactors, the emerging principles underlying the assembly of complexes with different cofactors, and the pathogenic effects of disease-associated p97 mutations on cofactor binding.


Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Ciclo Celular/química , Estructura Terciaria de Proteína , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteína que Contiene Valosina
10.
Mol Cell ; 54(1): 3-4, 2014 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-24725593

RESUMEN

A novel autophagy pathway eliminates nonnative polytopic membrane proteins from the endoplasmic reticulum that evade degradation by the ubiquitin proteasome system.


Asunto(s)
Autofagia , Citosol/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Células Eucariotas/metabolismo , Pliegue de Proteína , Proteínas/metabolismo , Receptores LHRH/metabolismo , Animales , Humanos
11.
Genes Cells ; 19(3): 254-72, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24461064

RESUMEN

Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC) play an important role in NF-κB activation. However, the regulation of linear ubiquitin chain generation by LUBAC is not well characterized. Here, we identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear linkage specificity (OTULIN/Gumby/FAM105B) and cylindromatosis (CYLD) that can cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP interacts with both CYLD and OTULIN even in unstimulated cells. The interaction of CYLD and OTULIN with HOIP synergistically suppresses LUBAC-mediated linear polyubiquitination and NF-κB activation. Moreover, introduction of a HOIP mutant unable to bind either deubiquitinase into HOIP-null cells augments the activation of NF-κB by TNF-α stimulation. Thus, the interactions between these two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-α-induced NF-κB activation in cells by fine-tuning the generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling pathway activation by LUBAC. Our observations provide molecular insights into the roles of ligase-deubiquitinase interactions in regulating molecular events resulting from linear ubiquitin conjugation.


Asunto(s)
Endopeptidasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Enzima Desubiquitinante CYLD , Humanos , FN-kappa B/metabolismo , Subunidades de Proteína/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/genética , Vía de Señalización Wnt
12.
Subcell Biochem ; 66: 195-222, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23479442

RESUMEN

The chaperone-related, ubiquitin-selective AAA (ATPase associated with a variety of cellular activities) protein Cdc48 (also known as TER94, p97 and VCP) is a key regulator of intracellular proteolysis in eukaryotes. It uses the energy derived from ATP hydrolysis to segregate ubiquitylated proteins from stable assemblies with proteins, membranes and chromatin. Originally characterized as essential factor in proteasomal degradation pathways, Cdc48 was recently found to control lysosomal protein degradation as well. Moreover, impaired lysosomal proteolysis due to mutational inactivation of Cdc48 causes protein aggregation diseases in humans. This review introduces the major systems of intracellular proteolysis in eukaryotes and the role of protein ubiquitylation. It then discusses in detail structure, mechanism and cellular functions of Cdc48 with an emphasis on protein degradation pathways in yeast.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteolisis , Proteína que Contiene Valosina
13.
PLoS One ; 8(2): e56486, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23418575

RESUMEN

The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48(Shp1) complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48(Shp1) complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits.


Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Ciclo Celular/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína Fosfatasa 1/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina Trifosfatasas/metabolismo , Aurora Quinasas , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Núcleo Celular/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitosis/genética , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Mutación , Fosforilación , Unión Proteica , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína que Contiene Valosina
14.
J Biol Chem ; 286(44): 38670-38678, 2011 Nov 04.
Artículo en Inglés | MEDLINE | ID: mdl-21896481

RESUMEN

Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX(5)AAX(2)R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors.


Asunto(s)
Adenosina Trifosfatasas/química , Proteínas de Ciclo Celular/química , Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Relacionadas con la Autofagia , Sitios de Unión , Proteínas Portadoras/química , Biología Computacional/métodos , Células HEK293 , Humanos , Espectroscopía de Resonancia Magnética/métodos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos Híbridos , Ubiquitina/química , Proteína que Contiene Valosina
15.
Trends Biochem Sci ; 36(10): 515-23, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21741246

RESUMEN

Cdc48 is an essential, highly prominent ATP driven machine in eukaryotic cells. Physiological function of Cdc48 has been found in a multitude of cellular processes, for instance cell cycle progression, homotypic membrane fusion, chromatin remodeling, transcriptional and metabolic regulation, and many others. The molecular function of Cdc48 is arguably best understood in endoplasmic reticulum-associated protein degradation by the ubiquitin proteasome system. In this review, we summarize the general characteristics of Cdc48/p97 and the most recent results on the molecular function of Cdc48 in some of the above processes, which were found to finally end in proteolysis-connected pathways, either involving the proteasome or autophagocytosis-mediated lysosomal degradation.


Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas de Ciclo Celular/fisiología , Proteolisis , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Autofagia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Coenzimas/química , Degradación Asociada con el Retículo Endoplásmico , Humanos , Proteínas Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Proteínas Nucleares/metabolismo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Ubiquitinadas/metabolismo , Proteína que Contiene Valosina
16.
Structure ; 19(6): 833-43, 2011 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-21645854

RESUMEN

The hexameric AAA ATPase p97 is involved in several human proteinopathies and mediates ubiquitin-dependent protein degradation among other essential cellular processes. Via its N-terminal domain (N domain), p97 interacts with multiple regulatory cofactors including the UFD1/NPL4 heterodimer and members of the "ubiquitin regulatory X" (UBX) domain protein family; however, the principles governing cofactor selectivity remain to be deciphered. Our crystal structure of the FAS-associated factor 1 (FAF1)UBX domain in complex with the p97N domain reveals that the signature Phe-Pro-Arg motif known to be crucial for interactions of UBX domains with p97 adopts a cis-proline configuration, in contrast to a cis-trans mixture we derive for the isolated FAF1UBX domain. Biochemical studies confirm that binding critically depends on a proline at this position. Furthermore, we observe that the UBX proteins FAF1 and UBXD7 only bind to p97-UFD1/NPL4, but not free p97, thus demonstrating for the first time a hierarchy in p97-cofactor interactions.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Adenosina Trifosfatasas/química , Coenzimas/química , Proteínas Nucleares/química , Proteínas Adaptadoras del Transporte Vesicular , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Calorimetría , Proteínas Portadoras/química , Cromatografía en Gel , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Propiedades de Superficie , Volumetría , Ubiquitina/química , Proteínas Ubiquitinadas/química
17.
Mol Cell Biol ; 31(7): 1528-39, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21282470

RESUMEN

The chaperone-related AAA ATPase Cdc48 (p97/VCP in higher eukaryotes) segregates ubiquitylated proteins for subsequent degradation by the 26S proteasome or for nonproteolytic fates. The specific outcome of Cdc48 activity is controlled by the evolutionary conserved cofactors Ufd2 and Ufd3, which antagonistically regulate the substrates' ubiquitylation states. In contrast to the interaction of Ufd3 and Cdc48, the interaction between the ubiquitin chain elongating enzyme Ufd2 and Cdc48 has not been precisely mapped. Consequently, it is still unknown whether physiological functions of Ufd2 in fact require Cdc48 binding. Here, we show that Ufd2 binds to the C-terminal tail of Cdc48, unlike the human Ufd2 homologue E4B, which interacts with the N domain of p97. The binding sites for Ufd2 and Ufd3 on Cdc48 overlap and depend critically on the conserved residue Y834 but are not identical. Saccharomyces cerevisiae cdc48 mutants altered in residue Y834 or lacking the C-terminal tail are viable and exhibit normal growth. Importantly, however, loss of Ufd2 and Ufd3 binding in these mutants phenocopies defects of Δufd2 and Δufd3 mutants in the ubiquitin fusion degradation (UFD) and Ole1 fatty acid desaturase activation (OLE) pathways. These results indicate that key cellular functions of Ufd2 and Ufd3 in proteasomal protein degradation require their interaction with Cdc48.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Adenosina Trifosfatasas/química , Animales , Proteínas de Ciclo Celular/química , Secuencia Conservada/genética , Humanos , Mamíferos , Viabilidad Microbiana , Mutación/genética , Fosforilación , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Relación Estructura-Actividad , Tirosina/metabolismo , Proteína que Contiene Valosina
18.
Mol Cell ; 40(2): 238-52, 2010 Oct 22.
Artículo en Inglés | MEDLINE | ID: mdl-20965419

RESUMEN

In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfolding and aggregation. The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging, pathological states, and even cell death. To avert these dangers, cells have developed powerful quality control strategies that counteract protein damage in a compartment-specific way. Here, we compare the protein quality control systems of the eukaryotic cytosol and the endoplasmic reticulum, focusing on the principles of damage recognition, the triage decisions between chaperone-mediated refolding and proteolytic elimination of damaged proteins, the repair of misfolded and aggregated protein species, and the mechanisms by which perturbations of protein homeostasis are sensed to induce compartment-specific stress responses.


Asunto(s)
Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Células Eucariotas/metabolismo , Proteínas/metabolismo , Animales , Homeostasis , Humanos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteínas/química
19.
EMBO Rep ; 11(6): 479-85, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20414249

RESUMEN

The ubiquitin-selective chaperone p97 is involved in major proteolytic pathways of eukaryotic cells and has been implicated in several human proteinopathies. Moreover, mutations in p97 cause the disorder inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD). The molecular basis underlying impaired degradation and pathological aggregation of ubiquitinated proteins in IBMPFD is unknown. Here, we identify perturbed co-factor binding as a common defect of IBMPFD-causing mutant p97. We show that IBMPFD mutations induce conformational changes in the p97 N domain, the main binding site for regulatory co-factors. Consistently, mutant p97 proteins exhibit strongly altered co-factor interactions. Specifically, binding of the ubiquitin ligase E4B is reduced, whereas binding of ataxin 3 is enhanced, thus resembling the accumulation of mutant ataxin 3 on p97 in spinocerebellar ataxia type 3. Our results suggest that imbalanced co-factor binding to p97 is a key pathological feature of IBMPFD and potentially of other proteinopathies involving p97.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Demencia Frontotemporal/complicaciones , Demencia Frontotemporal/metabolismo , Miositis por Cuerpos de Inclusión/complicaciones , Miositis por Cuerpos de Inclusión/metabolismo , Osteítis Deformante/complicaciones , Osteítis Deformante/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular , Demencia Frontotemporal/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Mutación/genética , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas , Proteína que Contiene Valosina
20.
Subcell Biochem ; 54: 17-30, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-21222270

RESUMEN

Cdc48 (alias p97, VCP) is an important motor and regulator for the turnover of ubiquitylated proteins, both in proteasomal degradation and in nonproteolytic pathways. The diverse cellular tasks of Cdc48 are controlled by a large number of cofactors. Substrate-recruiting cofactors mediate the specific recognition of ubiquitylated target proteins, whereas substrate-processing cofactors often exhibit ubiquitin ligase or deubiquitylating activities that enable them to modulate the ubiquitylation state of substrates. This chapter introduces the major groups of Cdc48 cofactors and discusses the versatile options of substrate-processing cofactors to control the fate of Cdc48 substrates.


Asunto(s)
Saccharomyces cerevisiae , Ubiquitina , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinación
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