RESUMO
DYT1 dystonia is a debilitating neurological movement disorder that arises upon Torsin ATPase deficiency. Nuclear envelope (NE) blebs that contain FG-nucleoporins (FG-Nups) and K48-linked ubiquitin are the hallmark phenotype of Torsin manipulation across disease models of DYT1 dystonia. While the aberrant deposition of FG-Nups is caused by defective nuclear pore complex assembly, the source of K48-ubiquitylated proteins inside NE blebs is not known. Here, we demonstrate that the characteristic K48-ubiquitin accumulation inside blebs requires p97 activity. This activity is highly dependent on the p97 adaptor UBXD1. We show that p97 does not significantly depend on the Ufd1/Npl4 heterodimer to generate the K48-ubiquitylated proteins inside blebs, nor does inhibiting translation affect the ubiquitin sequestration in blebs. However, stimulating global ubiquitylation by heat shock greatly increases the amount of K48-ubiquitin sequestered inside blebs. These results suggest that blebs have an extraordinarily high capacity for sequestering ubiquitylated protein generated in a p97-dependent manner. The p97/UBXD1 axis is thus a major factor contributing to cellular DYT1 dystonia pathology and its modulation represents an unexplored potential for therapeutic development.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Adenosina Trifosfatases , Proteínas Relacionadas à Autofagia , Distonia , Membrana Nuclear , Proteínas Nucleares , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Estruturas da Membrana Celular/metabolismo , Distonia/genética , Distonia/metabolismo , Distonia Muscular Deformante , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Membrana Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismoRESUMO
Valosin-containing protein/p97 is an ATP-driven protein segregase that cooperates with distinct protein cofactors to control various aspects of cellular homeostasis. Mutations at the interface between the regulatory N-domain and the first of two ATPase domains (D1 and D2) deregulate the ATPase activity and cause a multisystem degenerative disorder, inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia/amyotrophic lateral sclerosis. Intriguingly, the mutations affect only a subset of p97-mediated pathways correlating with unbalanced cofactor interactions and most prominently compromised binding of the ubiquitin regulatory X domain-containing protein 1 (UBXD1) cofactor during endolysosomal sorting of caveolin-1. However, how the mutations impinge on the p97-cofactor interplay is unclear so far. In cell-based endosomal localization studies, we identified a critical role of the N-terminal region of UBXD1 (UBXD1-N). Biophysical studies using NMR and CD spectroscopy revealed that UBXD1-N can be classified as intrinsically disordered. NMR titration experiments confirmed a valosin-containing protein/p97 interaction motif and identified a second binding site at helices 1 and 2 of UBXD1-N as binding interfaces for p97. In reverse titration experiments, we identified two distant epitopes on the p97 N-domain that include disease-associated residues and an additional interaction between UBXD1-N and the D1D2 barrel of p97 that was confirmed by fluorescence anisotropy. Functionally, binding of UBXD1-N to p97 led to a reduction of ATPase activity and partial protection from proteolysis. These findings indicate that UBXD1-N intercalates into the p97-ND1 interface, thereby modulating interdomain communication of p97 domains and its activity with relevance for disease pathogenesis. We propose that the polyvalent binding mode characterized for UBXD1-N is a more general principle that defines a subset of p97 cofactors.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Motivos de Aminoácidos , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Proteínas de Transporte/química , Caveolina 1/metabolismo , Linhagem Celular , Dicroísmo Circular , Endossomos/metabolismo , Epitopos/química , Polarização de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Homeostase , Humanos , Lisossomos/metabolismo , Espectroscopia de Ressonância Magnética , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ubiquitina/química , Proteína com ValosinaRESUMO
IMPORTANCE: Toxoplasma gondii (Tg) is a ubiquitous parasitic pathogen, infecting about one-third of the global population. Tg is controlled in immunocompetent people by mechanisms that are not fully understood. Tg infection drives the production of the inflammatory cytokine interferon gamma (IFNγ), which upregulates intracellular anti-pathogen defense pathways. In this study, we describe host proteins p97/VCP, UBXD1, and ANKRD13A that control Tg at the parasitophorous vacuole (PV) in IFNγ-stimulated endothelial cells. p97/VCP is an ATPase that interacts with a network of cofactors and is active in a wide range of ubiquitin-dependent cellular processes. We demonstrate that PV ubiquitination is a pre-requisite for recruitment of these host defense proteins, and their deposition directs Tg PVs to acidification in endothelial cells. We show that p97/VCP universally targets PVs in human cells and restricts Tg in different human cell types. Overall, these findings reveal new players of intracellular host defense of a vacuolated pathogen.
Assuntos
Parasitos , Toxoplasma , Animais , Humanos , Toxoplasma/metabolismo , Interferons/metabolismo , Vacúolos/metabolismo , Células Endoteliais , Interferon gama , Proteína com Valosina/metabolismoRESUMO
AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Relacionadas à Autofagia/química , Proteína com Valosina/química , Proteínas Adaptadoras de Transporte Vesicular/isolamento & purificação , Proteínas Relacionadas à Autofagia/isolamento & purificação , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Proteína com Valosina/isolamento & purificaçãoRESUMO
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.