Inhibition of 26S proteasome activity by α-synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1.

Parkinson's disease (PD) is characterized by aggregation of α-synuclein (α-syn) into protein inclusions in degenerating brains. Increasing amounts of aggregated α-syn species indicate significant perturbation of cellular proteostasis. Altered proteostasis depends on α-syn protein levels and the impact of α-syn on other components of the proteostasis network.

Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations.

Mono- and Intralink Filter (Mi-Filter) To Reduce False Identifications in Cross-Linking Mass Spectrometry Data.

Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow.

Allosteric control of Ubp6 and the proteasome via a bidirectional switch.

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect.

Molecular and cellular dynamics of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system serves to remove proteins that are either dysfunctional or no longer needed. The 26S proteasome is a 2.5 MDa multisubunit complex comprising the 20S core particle, where degradation is executed, and one or two regulatory particles which prepare substrates for degradation.

Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating.

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo-electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics.

Molecular Details Underlying Dynamic Structures and Regulation of the Human 26S Proteasome.

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells.

Structural insights into the functional cycle of the ATPase module of the 26S proteasome.

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA ATPase module, which unfolds and translocates substrates into the CP.

Recent advances in the structural biology of the 26S proteasome.

There is growing appreciation for the fundamental role of structural dynamics in the function of macromolecules. In particular, the 26S proteasome, responsible for selective protein degradation in an ATP dependent manner, exhibits dynamic conformational changes that enable substrate processing. Recent cryo-electron microscopy (cryo-EM) work has revealed the conformational dynamics of the 26S proteasome and established the function of the different conformational states.

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

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.

Structural characterization of the interaction of Ubp6 with the 26S proteasome.

In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function.

Mode of substrate recognition by the Josephin domain of ataxin-3, which has an endo-type deubiquitinase activity.

Ataxin-3, which is encoded by a gene that has been associated with Machado-Joseph disease, contains a catalytic N-terminal Josephin domain with deubiquitinase activity. Here, we show that the Josephin domain of ataxin 3 catalyzes endo-type cleavage of Lys48-linked polyubiquitin. Furthermore, NMR data obtained following site-specific paramagnetic spin labeling of Lys48-linked di-ubiquitin revealed that both ubiquitin units interact with the Josephin domain, with the C-terminal Gly76 of the proximal unit being situated in the vicinity of the catalytic triad of Josephin domain.

Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome.

The 26S proteasome is a 2.5-MDa multisubunit protease complex that degrades polyubiquitylated proteins. Although its functions and structure have been extensively characterized, little is known about its dynamics in living cells.

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

Synaptic vesicles are embedded in a complex filamentous network at the presynaptic terminal. Before fusion, vesicles are linked to the active zone (AZ) by short filaments (tethers). The identity of the molecules that form and regulate tethers remains unknown, but Rab3-interacting molecule (RIM) is a prominent candidate, given its central role in AZ organization.

Localization of the regulatory particle subunit Sem1 in the 26S proteasome.

The ubiquitin-proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1.

Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form.

A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer.

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.

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).

Backbone and side chain 1H, 13C, and 15N assignments of the ubiquitin-like domain of human HOIL-1L, an essential component of linear ubiquitin chain assembly complex.

HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date.

The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle.

The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP).

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.

Crystal structure of cyclic Lys48-linked tetraubiquitin.

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH.

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.

The proteasome's crown for destruction.

Three recent Molecular Cell papers from Zhang et al. (2009a, 2009b) and Djuranovic et al. (2009) provide new insights into how proteasomal ATPases recognize, unfold, and translocate their substrates, thus enhancing our understanding of regulated proteolysis.

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

Eukaryotic 20S proteasomes are composed of two alpha-rings and two beta-rings, which form an alphabetabetaalpha stacked structure. Here we describe a proteasome-specific chaperone complex, designated Dmp1-Dmp2, in budding yeast. Dmp1-Dmp2 directly bound to the alpha5 subunit to facilitate alpha-ring formation.