ABSTRACT
Obesity has been linked to infertility through several mechanisms, including at a molecular level. Those living with obesity face additional barriers to accessing fertility treatments and achieving a successful pregnancy, which can contribute to their economic and psychosocial stressors. There is scope to further improve care for people living with obesity and infertility with empathy, via a multidisciplinary approach.
Subject(s)
Fertility , Infertility , Female , Humans , Pregnancy , Infertility/etiology , Infertility/therapy , Infertility/psychology , Obesity/complications , Obesity/therapy , Reproductive Techniques, AssistedABSTRACT
p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Apoptosis Regulatory Proteins , Calorimetry , Cryoelectron Microscopy , Protein Binding , Ultracentrifugation , Valosin Containing ProteinABSTRACT
The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.
Subject(s)
Adenosine Triphosphatases/metabolism , Models, Molecular , Nuclear Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphate/metabolism , Binding Sites , Cryoelectron Microscopy , Endoplasmic Reticulum-Associated Degradation , Humans , Metal Nanoparticles/chemistry , Microscopy, Atomic Force , Molecular Docking Simulation , Mutation , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly.
Subject(s)
Adenosine Triphosphatases/ultrastructure , Carrier Proteins/ultrastructure , Cell Cycle Proteins/ultrastructure , Models, Molecular , Multiprotein Complexes/ultrastructure , Protein Conformation , Proteins/ultrastructure , Cryoelectron Microscopy/methods , Escherichia coli , Image Processing, Computer-Assisted , Imaging, Three-Dimensional , Intercellular Signaling Peptides and Proteins , Intracellular Signaling Peptides and Proteins , Valosin Containing ProteinABSTRACT
p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is approximately 80 x 30 A in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways.
Subject(s)
Adenosine Triphosphatases/chemistry , Nuclear Pore Complex Proteins/chemistry , Nuclear Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Dimerization , Endoplasmic Reticulum/chemistry , Microscopy, Electron , Models, Molecular , Multiprotein Complexes , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/ultrastructure , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/ultrastructure , Nucleocytoplasmic Transport Proteins , Protein Binding , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/ultrastructure , Ubiquitin/metabolism , Vesicular Transport ProteinsABSTRACT
The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at approximately 20 Angstroms resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes.
Subject(s)
Adenosine Triphosphatases/ultrastructure , Nuclear Proteins/ultrastructure , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/ultrastructure , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/chemistry , Animals , Cryoelectron Microscopy , Models, Molecular , N-Ethylmaleimide-Sensitive Proteins/chemistry , N-Ethylmaleimide-Sensitive Proteins/ultrastructure , Nuclear Proteins/chemistry , Protein Conformation , Protein Structure, Tertiary , SNARE Proteins/chemistry , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/chemistryABSTRACT
p47 is a major adaptor molecule of the cytosolic AAA ATPase p97. The principal role of the p97-p47 complex is in regulation of membrane fusion events. Mono-ubiquitin recognition by p47 has also been shown to be crucial in the p97-p47-mediated Golgi membrane fusion events. Here, we describe the high-resolution solution structures of the N-terminal UBA domain and the central domain (SEP) from p47. The p47 UBA domain has the characteristic three-helix bundle fold and forms a highly stable complex with ubiquitin. We report the interaction surfaces of the two proteins and present a structure for the p47 UBA-ubiquitin complex. The p47 SEP domain adopts a novel fold with a betabetabetaalphaalphabeta secondary structure arrangement, where beta4 pairs in a parallel fashion to beta1. Based on biophysical studies, we demonstrate a clear propensity for the self-association of p47. Furthermore, p97 N binding abolishes p47 self-association, revealing the potential interaction surfaces for recognition of other domains within p97 or the substrate.
