Eri Sakata

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

Parkin, a product of the causative gene of autosomal‐recessive juvenile parkinsonism (AR‐JP), is a RING‐type E3 ubiquitin ligase and has an amino‐terminal ubiquitin‐like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR‐JP patients, the function of this domain is not clear. In this study, we determined the three‐dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15 N‐1 H residual dipolar‐coupling data. Inspection of chemical‐shift‐perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10‐binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR‐JP.

Near-atomic resolution structural model of the yeast 26S proteasome

The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits.

Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution

The structure of the 26S proteasome from Schizosaccharomyces pombe has been determined to a resolution of 9.1 Å by cryoelectron microscopy and single particle analysis. In addition, chemical cross-linking in conjunction with mass spectrometry has been used to identify numerous residue pairs in close proximity to each other, providing an array of spatial restraints. Taken together these data clarify the topology of the AAA-ATPase module in the 19S regulatory particle and its spatial relationship to the α-ring of the 20S core particle. Image classification and variance analysis reveal a belt of high “activity” surrounding the AAA-ATPase module which is tentatively assigned to the reversible association of proteasome interacting proteins and the conformational heterogeneity among the particles. An integrated model is presented which sheds light on the early steps of protein degradation by the 26S complex.

14‐3‐3η is a novel regulator of parkin ubiquitin ligase

Mutation of the parkin gene, which encodes an E3 ubiquitin‐protein ligase, is the major cause of autosomal recessive juvenile parkinsonism (ARJP). Although various substrates for parkin have been identified, the mechanisms that regulate the ubiquitin ligase activity of parkin are poorly understood. Here we report that 14‐3‐3η, a chaperone‐like protein present abundantly in neurons, could bind to parkin and negatively regulate its ubiquitin ligase activity. Furthermore, 14‐3‐3η could bind to the linker region of parkin but not parkin with ARJP‐causing R42P, K161N, and T240R mutations. Intriguingly, α‐synuclein (α‐SN), another familial Parkinsons disease (PD) gene product, abrogated the 14‐3‐3η‐induced suppression of parkin activity. α‐SN could bind tightly to 14‐3‐3η and consequently sequester it from the parkin–14‐3‐3η complex. PD‐causing A30P and A53T mutants of α‐SN could not bind 14‐3‐3η, and failed to activate parkin. Our findings indicate that 14‐3‐3η is a regulator that functionally links parkin and α‐SN. The α‐SN‐positive and 14‐3‐3η‐negative control of parkin activity sheds new light on the pathophysiological roles of parkin.

Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity.

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.

Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy

Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits—and all other non-ATPase subunits—in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome.

Crystal structure of a chaperone complex that contributes to the assembly of yeast 20S proteasomes

Eukaryotic 20S proteasomes are composed of two α-rings and two β-rings, which form an αββα stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1–Dmp2, in budding yeast. Dmp1–Dmp2 directly bound to the α5 subunit to facilitate α-ring formation. In Δdmp1 cells, α-rings lacking α4 and decreased formation of 20S proteasomes were observed. Dmp1–Dmp2 interacted with proteasome precursors early during proteasome assembly and dissociated from the precursors before the formation of half-proteasomes. Notably, the crystallographic structures of Dmp1 and Dmp2 closely resemble that of PAC3—a mammalian proteasome-assembling chaperone; nonetheless, neither Dmp1 nor Dmp2 showed obvious sequence similarity to PAC3. The structure of the Dmp1–Dmp2–α5 complex reveals how this chaperone functions in proteasome assembly and why it dissociates from proteasome precursors before the β-rings are assembled.

Structure of the human 26S proteasome at a resolution of 3.9 Å

Significance The 26S proteasome is a giant protease assembled from at least 32 different canonical subunits. In eukaryotic cells it is responsible for the regulated degradation of proteins marked for destruction by polyubiquitin tags. Mainly because of the conformational heterogeneity of the 26S holocomplex, its structure determination has been challenging. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the human 26S proteasome allowing us to put forward an essentially complete atomic model. This model provides insights into the proteasome’s mechanism of operation and could serve as a basis for structure-based drug discovery. Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.

Crystal Structure of UbcH5b∼Ubiquitin Intermediate: Insight into the Formation of the Self-Assembled E2∼Ub Conjugates

E2 ubiquitin-conjugating enzymes catalyze the attachment of ubiquitin to lysine residues of target proteins. The UbcH5b E2 enzyme has been shown to play a key role in the initiation of the ubiquitination of substrate proteins upon action of several E3 ligases. Here we have determined the 2.2 A crystal structure of an intermediate of UbcH5b~ubiquitin (Ub) conjugate, which is assembled into an infinite spiral through the backside interaction. This active complex may provide multiple E2 active sites, enabling efficient ubiquitination of substrates. Indeed, biochemical assays support a model in which the self-assembled UbcH5b~Ub can serve as a bridge for the gap between the lysine residue of the substrate and the catalytic cysteine of E2.

Cryo–electron tomography reveals a critical role of RIM1α in synaptic vesicle tethering

RIM1α-deficient synapses show structural defects in presynaptic vesicle distribution and tethering to the active zone that can be reversed by proteasome inhibition.