ATPase Ring Research Articles

Exploring Metalloprotease from Dunaliella sp.: Production, Regulation, and Structural Insight

A green microalgal strain, identified as Dunaliella sp., was isolated from the Tunisian southern region. The enhancement of its protein and protease production was performed through culture condition optimization using the response surface methodology. The optimal conditions for protein and protease production were found to be, respectively, (i) NaCl concentrations of 135 and 45.55 g/L, (ii) NaHCO3 concentrations of 0.5 and 1.5 g/L, (iii) temperature of 28 °C for both, and (iv) light intensities of 400 and 100 µmol photons/m2/s. The optimization led to an increase in microalgae protein content from 11.98% ± 0.26 to 18.39% ± 0.10 and microalgae proteolytic activity from 7.36 ± 0.74 U/mg to 12.54 ± 0.86 U/mg. Specific focus was attributed to ATP-dependent metalloprotease, namely, FtsH2, which is involved in numerous cellular processes including cell division, cell differentiation, signal transduction, and stress response. Differential expression of the FtsH2 gene under various stress conditions showed that this expression was upregulated in response to salt stress, gibberellic acid, and Indole-3-butyric acid. A 3D modeling demonstrated two possible arrangements where the ATPase ring shows either a perfect six-fold symmetry with an open circular entrance covering the crucial pore residues, or a translocated model triggered by substrate binding inward movement of the aromatic pore residues.

Sec18 side-loading is essential for universal SNARE recycling across cellular contexts.

SNARE proteins drive membrane fusion as their core domains zipper into a parallel four-helix bundle1,2. After fusion, these bundles are disassembled by the AAA+ protein Sec18/NSF and its adaptor Sec17/ α-SNAP3,4 to make them available for subsequent rounds of membrane fusion. SNARE domains are often flanked by C-terminal transmembrane or N-terminal domains5. Previous structures of the NSF-α-SNAP-SNARE complex revealed SNARE domain threaded through the D1 ATPase ring6, posing a topological constraint as SNARE transmembrane domains would prevent complete substrate threading as suggested for other AAA+ systems7. Here, in vivo mass-spectrometry reveals N-terminal SNARE domain interactions with Sec18, exacerbating this topological issue. Cryo-EM structures of a yeast SNARE complex, Sec18, and Sec17 in a non-hydrolyzing condition shows SNARE Sso1 threaded through the D1 and D2 ATPase rings of Sec18, with its folded, N-terminal Habc domain interacting with the D2 ring. This domain does not unfold during Sec18/NSF activity. Cryo-EM structures under hydrolyzing conditions revealed substrate-released and substrate-free states of Sec18 with a coordinated opening in the side of the ATPase rings. Thus, Sec18/NSF operates by substrate side-loading and unloading topologically constrained SNARE substrates.

Identification of a new export signal that targets early subunits to the flagellar type III secretion export machinery.

Many bacterial pathogens utilize T3SS to inject virulence proteins (effectors) into host cells or to assemble flagella on the bacterial cell surface. Bacterial flagella present a paradigm for how cells build and operate complex cell-surface "nanomachines." Efficient subunit targeting from the bacterial cytosol to type III secretion systems is essential for rapid assembly and secretion by T3SSs. Subunits are thought to dock at the export machinery before being unfolded and translocated into the export channel. However, little is known about how subunits dock at the export machinery and the events that occur post docking. Here, we identified a new export signal within the C-termini of subunits that is essential for targeting of subunits to the type III export machinery. We show that this new export signal and previously identified export signals are recognized separately and sequentially, revealing a pathway for subunit transit through the type III export machinery in which sequential recognition events carry out different roles at major steps in the export pathway.

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-ATPase (PSMC) Gene Family in the Light of Proteasome Inhibitor Resistance in Multiple Myeloma.

For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM.

Purification of the Transmembrane Polypeptide Channel Complex of the Salmonella Flagellar Type III Secretion System.

Many motile bacteria employ the flagellar type III secretion system (fT3SS) to build the flagellum on the cell surface. The fT3SS consists of a transmembrane export gate complex, which acts as a proton/protein antiporter that couples proton flow with flagellar protein export, and a cytoplasmic ATPase ring complex, which works as an activator of the export gate complex. Three transmembrane proteins, FliP, FliQ, and FliR, form a core structure of the export gate complex, and this core complex serves as a polypeptide channel that allows flagellar structural subunits to be translocated across the cytoplasmic membrane. Here, we describe the methods for overproduction, solubilization, and purification of the Salmonella FliP/FliQ/FliR complex.

Two Distinct Modes of DNA Binding by an MCM Helicase Enable DNA Translocation.

A six-subunit ATPase ring forms the central hub of the replication forks in all domains of life. This ring performs a helicase function to separate the two complementary DNA strands to be replicated and drives the replication machinery along the DNA. Disruption of this helicase/ATPase ring is associated with genetic instability and diseases such as cancer. The helicase/ATPase rings of eukaryotes and archaea consist of six minichromosome maintenance (MCM) proteins. Prior structural studies have shown that MCM rings bind one encircled strand of DNA in a spiral staircase, suggesting that the ring pulls this strand of DNA through its central pore in a hand-over-hand mechanism where the subunit at the bottom of the staircase dissociates from DNA and re-binds DNA one step above the staircase. With high-resolution cryo-EM, we show that the MCM ring of the archaeal organism Saccharolobus solfataricus binds an encircled DNA strand in two different modes with different numbers of subunits engaged to DNA, illustrating a plausible mechanism for the alternating steps of DNA dissociation and re-association that occur during DNA translocation.

Genetic Alterations in Members of the Proteasome 26S Subunit, AAA-Atpase (PSMC) Gene Family Transmit Proteasome Inhibitor Resistance in Multiple Myeloma By Impacting the ADP/ATP Binding Pocket

Background For the treatment of Multiple Myeloma (MM), proteasome inhibitors (PIs) are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients e.g., mutations in the PI binding domain PSMB5. However, the proteasome is a large and complex machinery and its mechanistic understanding remains incomplete. In this study, we unravel the role of the six ATPases of the 19S proteasome regulator (PSMC1-6) in MM resistance. Methods As an example of cancer evolution and the acquisition of PI resistance, we conducted an in-depth analysis of an index patient applying deep sequencing of immunoglobulin rearrangements (IG), FISH, and WES in serial samples at MGUS, newly diagnosed MM, and five later timepoints including two relapses. Then we expand towards generic meta-analyses of PSMC alterations in our own and other published datasets comprising 1,499 newly diagnosed (NDMM) and 447 PI pretreated MM (PMM) patients for WGS/WES. For RNA sequencing, we analyzed 16 normal plasma cells (NPCs) and 360 NDMM patients. Furthermore, we explore the structural impact of PSMC single nucleotide variants on the proteasome and their relevance for PI resistance using 3D modeling with Chimera and Pymol. Results The WES analysis of our index patient uncovers a PSMC2 Y429S mutation acquired at the second relapse after intensive bortezomib-containing therapy. We functionally confirm this variant to mediate PI resistance in the cell line RPMI 8226. In the meta-analysis, a total of 36 mutations were identified (mutation incidence: 1.85%). The PSMC5 G152S mutation was found in three independent patients, and a G152R in a fourth patient, thus, representing a potential hotspot. Based on 3D structural projections, the identified mutations can be clustered into three main groups: first, mutations affecting the intrinsic conformation of the respective subunit; second, mutations perturbing the PSMC protomer interactions and the proper folding/functioning of the whole complex; and third, mutations impairing the ADP/ATP binding pocket (Figure). Nine mutations belong to the third category interacting with the adenine ring or the phosphate groups of the ATP. The expression analysis reveals a significant upregulation of PSMC3 (P=0.003), PSMC4 (P≤0.001), and PSMC5 (P=0.043) from NPC to NDMM. Conclusions The 19S hexameric AAA ATPase ring of the 26S proteasome is responsible for unfolding and introducing the substrates into the catalytic 20S complex. PSMC gene alterations are structurally often located in regulatory sites for activity e.g. impacting the ADP/ATP binding pocket. The following activation of the 26S proteasome increases the PI tolerance and represents a new PI resistance mechanism. Moreover, changes in the expression level of the PSMC members seem to play a role not only in the development of PI resistance but already in the development of MM, probably by increasing the protein turnover capacity of myeloma cells. This study opens the way to explore new classes of proteasome inhibitors avoiding this kind of mechanism of resistance. Figure:Mutations in the'PSMC' subunits of the 19S Base AAA-ATPase hexamer accumulate in proximity to the ATP/ADP binding pockets. The backbone of the PSMC hexamer is displayed as a ribbon diagram, mutations (red) and ADP (blue) as 3D spheres (PDB 5GJR). The mutations cluster in areas responsible for proper folding and functioning of the complex or in proximity to the ADP/ATP binding pockets, exemplarily shown for PSMC1, PSMC2, and PSMC5. ADP is presented as a wireframe stick in light green (PDB 5GJQ). The PSMC2Y429S mutation of the index patient is indicated in yellow. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteasome Regulatory Atpases PSMC2 and PSMC6 Modulate Proteosome Inhibitor Sensitivity and Correlate with Survival in Multiple Myeloma

Background: Multiple myeloma (MM) cells synthesize and secrete massive amounts of immunoglobulins and thus are exquisitely sensitive to therapeutics that disrupt proteostasis. The 26S proteasome complex is a 2.5-MDa holoenzyme comprised of >32 individual subunits responsible for the bulk of intracellular protein degradation. 26S proteasomes are comprised of barrel-shaped proteolytic core complex, the 20S core particle (CP), capped at either or both ends by 19S regulatory particles (RPs), which confer ATPase activity and specificity for ubiquitinated proteins. Six distinct ATPases (PSMC1-6) form a heterohexameric ring-like structure that rests atop the 20S CP's α-subunits and is responsible for unfolding and shuttling target proteins for degradation within the 20S CP. The proteasome inhibitor (PI) bortezomib (BTZ) is a potent, selective, and reversible drug that dramatically improved the prognosis of multiple myeloma (MM) patients and remains the backbone of induction therapy for transplant-eligible new diagnosis MM (NDMM), but response to therapy remains highly variable. Here, we predicted that gene expression analysis of publically available datasets could identify components within the ubiquitin-proteasome system (UPS) that areprognostic in patients treated with BTZ, suggesting potential new targets in MM. Methods: We interrogated gene expression profiles (GEP) from the randomized, phase 3 Assessment of Proteasome Inhibition for Extending Remissions (APEX) study that compared BTZ with high-dose dexamethasone (DEX) in RRMM patients who had received 1-3 prior therapies. APEX datasets served as a test cohort to identify genes that influenced survival in patients treated with BTZ. Patients consented to genomic analyses of pretreatment tumor samples. CD138+ cells were isolated from BM aspirates and analyzed by RNA microarrays to generate GEP datasets. We correlated gene expression with response to BTZ or DEX and overall survival (OS). We then validated individual genes identified from the APEX analysis using the MM Research Foundation (MMRF) CoMMpass study, a longitudinal observational analysis of the clinical and molecular data for >1000 NDMM patients receiving various standard approved treatments. Finally, we used shRNA to knockdown PSMC2 in RPMI8226 and ARH77 cells and tested their sensitivity to BTZ and carfilzomib (CFZ). Results: Analysis of the APEX dataset revealed that increased expression of genes PSMC2 and PSMC6, members of the heterohexameric ATPase ring of the 19S RP, correlated with poorer OS (PSMC2- Figure 1A, PSMC6- Median OS 320 versus 620 days for upper and lower tertile PSMC6, respectively). Using the CoMMpass database, increased PSMC2 expression also correlated with a worse OS (Figure 1B), while increased PSMC6 correlated with worse OS as well as progression free survival (PFS) (OS- HR= 2.29 p=0.0014, PFS- HR=1.63 p=0.0109). PSMC6 displayed greater expression in 50 patients above the median RNA expression (Fragments per Kb transcript per million mapped reads (FPKM)= 1 to 100) while PSMC2 expression was elevated in 400 patients above the median (FPKM= 25 to 100). shRNA-mediated PSMC2 knockdown (KD) increased PI susceptibility in ARH77 and the RPMI8226 MM cells. In ARH77 cells, the BTZ LD50 decreased from 11 nM to 2.5 nM and the CFZ LD50 decreased from 11 to 9 nM in control and KD cells, respectively. In RPMI8226 cells, the BTZ LD50 decreased from 30 nM to 20 nM and the CFZ LD50 decreased from 28 to 17 nM in control and KD cells, respectively. Conclusion: Taken together, our results indicate that increased PSMC2 and PSMC6 correlates with OS in MM in both patients treated with BTZ in the APEX and with a variety of standard therapies in the CoMMpass database. Analysis in this validation cohort extends the clinical significance of PSMC2/PSMC6 beyond the landmark APEX trial and suggests they may inform survival in patients treated with modern triplet anti-myeloma therapies. In addition to the prognostic importance across our test and validation datasets, point mutations in each ATPase subunit have been associated with PI resistance. Consistent with these findings, PSMC2 KD increased sensitivity to PI therapy. Controlled inhibition or degradation of proteasome 19S ATPase subunits using small molecules, PROTACs or RNA-based modalities now provides an opportunity to therapeutically target proteostasis in MM and other malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Structural basis of DNA packaging by a ring-type ATPase from an archetypal viral system.

Many essential cellular processes rely on substrate rotation or translocation by a multi-subunit, ring-type NTPase. A large number of double-stranded DNA viruses, including tailed bacteriophages and herpes viruses, use a homomeric ring ATPase to processively translocate viral genomic DNA into procapsids during assembly. Our current understanding of viral DNA packaging comes from three archetypal bacteriophage systems: cos, pac and phi29. Detailed mechanistic understanding exists for pac and phi29, but not for cos. Here, we reconstituted in vitro a cos packaging system based on bacteriophage HK97 and provided a detailed biochemical and structural description. We used a photobleaching-based, single-molecule assay to determine the stoichiometry of the DNA-translocating ATPase large terminase. Crystal structures of the large terminase and DNA-recruiting small terminase, a first for a biochemically defined cos system, reveal mechanistic similarities between cos and pac systems. At the same time, mutational and biochemical analyses indicate a new regulatory mechanism for ATPase multimerization and coordination in the HK97 system. This work therefore establishes a framework for studying the evolutionary relationships between ATP-dependent DNA translocation machineries in double-stranded DNA viruses.

Insight Into Distinct Functional Roles of the Flagellar ATPase Complex for Flagellar Assembly in Salmonella.

Most motile bacteria utilize the flagellar type III secretion system (fT3SS) to construct the flagellum, which is a supramolecular motility machine consisting of basal body rings and an axial structure. Each axial protein is translocated via the fT3SS across the cytoplasmic membrane, diffuses down the central channel of the growing flagellar structure and assembles at the distal end. The fT3SS consists of a transmembrane export complex and a cytoplasmic ATPase ring complex with a stoichiometry of 12 FliH, 6 FliI and 1 FliJ. This complex is structurally similar to the cytoplasmic part of the FOF1 ATP synthase. The export complex requires the FliH12-FliI6-FliJ1 ring complex to serve as an active protein transporter. The FliI6 ring has six catalytic sites and hydrolyzes ATP at an interface between FliI subunits. FliJ binds to the center of the FliI6 ring and acts as the central stalk to activate the export complex. The FliH dimer binds to the N-terminal domain of each of the six FliI subunits and anchors the FliI6-FliJ1 ring to the base of the flagellum. In addition, FliI exists as a hetero-trimer with the FliH dimer in the cytoplasm. The rapid association-dissociation cycle of this hetero-trimer with the docking platform of the export complex promotes sequential transfer of export substrates from the cytoplasm to the export gate for high-speed protein transport. In this article, we review our current understanding of multiple roles played by the flagellar cytoplasmic ATPase complex during efficient flagellar assembly.

USP14-regulated allostery of the human proteasome by time-resolved cryo-EM

Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels1–3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown4–7. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8–10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14–ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11–13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.

Activation mechanism of the bacterial flagellar dual-fuel protein export engine.

Bacteria employ the flagellar type III secretion system (fT3SS) to construct flagellum, which acts as a supramolecular motility machine. The fT3SS of Salmonella enterica serovar Typhimurium is composed of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. The transmembrane export gate complex is fueled by proton motive force across the cytoplasmic membrane and is divided into four distinct functional parts: a dual-fuel export engine; a polypeptide channel; a membrane voltage sensor; and a docking platform. ATP hydrolysis by the cytoplasmic ATPase complex converts the export gate complex into a highly efficient proton (H+)/protein antiporter that couples inward-directed H+ flow with outward-directed protein export. When the ATPase ring complex does not work well in a given environment, the export gate complex will remain inactive. However, when the electric potential difference, which is defined as membrane voltage, rises above a certain threshold value, the export gate complex becomes an active H+/protein antiporter to a considerable degree, suggesting that the export gate complex has a voltage-gated activation mechanism. Furthermore, the export gate complex also has a sodium ion (Na+) channel to couple Na+ influx with flagellar protein export. In this article, we review our current understanding of the activation mechanism of the dual-fuel protein export engine of the fT3SS. This review article is an extended version of a Japanese article, Membrane voltage-dependent activation of the transmembrane export gate complex in the bacterial flagellar type III secretion system, published in SEIBUTSU BUTSURI Vol. 62, p165-169 (2022).

A DNA packaging motor inchworms along one strand allowing it to adapt to alternative double-helical structures

Ring ATPases that translocate disordered polymers possess lock-washer architectures that they impose on their substrates during transport via a hand-over-hand mechanism. Here, we investigate the operation of ring motors that transport ordered, helical substrates, such as the bacteriophage ϕ29 dsDNA packaging motor. This pentameric motor alternates between an ATP loading dwell and a hydrolysis burst wherein it packages one turn of DNA in four steps. When challenged with DNA-RNA hybrids and dsRNA, the motor matches its burst to the shorter helical pitches, keeping three power strokes invariant while shortening the fourth. Intermittently, the motor loses grip on the RNA-containing substrates, indicating that it makes optimal load-bearing contacts with dsDNA. To rationalize these observations, we propose a helical inchworm translocation mechanism in which, during each cycle, the motor increasingly adopts a lock-washer structure during the ATP loading dwell and successively regains its planar form with each power stroke during the burst.

Atomistic basis of force generation, translocation, and coordination in a viral genome packaging motor.

Double-stranded DNA viruses package their genomes into pre-assembled capsids using virally-encoded ASCE ATPase ring motors. We present the first atomic-resolution crystal structure of a multimeric ring form of a viral dsDNA packaging motor, the ATPase of the asccφ28 phage, and characterize its atomic-level dynamics via long timescale molecular dynamics simulations. Based on these results, and previous single-molecule data and cryo-EM reconstruction of the homologous φ29 motor, we propose an overall packaging model that is driven by helical-to-planar transitions of the ring motor. These transitions are coordinated by inter-subunit interactions that regulate catalytic and force-generating events. Stepwise ATP binding to individual subunits increase their affinity for the helical DNA phosphate backbone, resulting in distortion away from the planar ring towards a helical configuration, inducing mechanical strain. Subsequent sequential hydrolysis events alleviate the accumulated mechanical strain, allowing a stepwise return of the motor to the planar conformation, translocating DNA in the process. This type of helical-to-planar mechanism could serve as a general framework for ring ATPases.

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

The Intrinsically Disordered Subunit Sem1 Enhances ATPase Activity to Drive Proteasomal Degradation

The 26S proteasome is an ~70 subunit ATP-dependent chambered protease responsible for most proteolysis in eukaryotes. Deciphering how its smallest subunit, Sem1 (DSS1 in metazoans), contributes to proteolysis has been challenging because Sem1 moonlights in other multisubunit complexes and is required for efficient proteasome biogenesis in vivo. To circumvent these limitations, we established a system to reconstitute Sem1-deficient proteasomes in vitro from purified components. We found that proteasomes lacking Sem1 displayed both compromised ATPase activity and substrate unfolding efficiency, whereas proteasomal substrate capture, conformational switching of the proteasome between inactive and activated states, removal of the substrate's ubiquitin targeting signal, and proteolysis were largely unchanged. Using targeted mutagenesis and protein engineering approaches, we found that Sem1 acts as a molecular brace between a key structural subunit and the ATPase ring. This work reveals how a small, unstructured subunit less than 1% the total size of the proteasome can control the efficiency of substrate degradation, and suggests that the function of the proteasomal ATPase ring is tightly regulated via exogenous intersubunit contacts.

A Unique Role for the Rpt3 Pore Loop in Proteasome Activation and Proteolysis

The 26S proteasome is a chambered protease that uses ATP-powered movements of pore loops within a ring of AAA+ ATPase subunits to drive unfolding and translocation of substrates into the peptidase core for proteolysis. Whereas most AAA+ family proteases contain a homohexameric ATPase ring in which each ATPase pore loop is thought to function identically and cooperatively, the proteasome contains six highly similar but non-identical ATPase subunits. Some limited evidence suggests their contributions to proteolysis are non-equivalent. We hypothesized that inactivation of each pore loop may distinctly impact substrate selection and/or processing by the proteasome. Using a collection of congenic pore loop mutant cells, we discovered many unique impacts on cell health, stress response, and ubiquitin homeostasis. Mutating the pore loop of one particular ATPase, Rpt3, led to a significant decrease in overall proteolytic activity. Genetic and biochemical analyses in yeast indicate that this pore loop transmits a signal to the peptidase component of the proteasome to trigger translocation of the substrate into the peptidase core for proteolysis. Together, our findings suggest an allosteric mechanism that couples pore loop movements with access to the peptidase centers for substrate processing.

Proteasome in action: substrate degradation by the 26S proteasome.

Ubiquitination is the major criteria for the recognition of a substrate-protein by the 26S proteasome. Additionally, a disordered segment on the substrate — either intrinsic or induced — is critical for proteasome engagement. The proteasome is geared to interact with both of these substrate features and prepare it for degradation. To facilitate substrate accessibility, resting proteasomes are characterised by a peripheral distribution of ubiquitin receptors on the 19S regulatory particle (RP) and a wide-open lateral surface on the ATPase ring. In this substrate accepting state, the internal channel through the ATPase ring is discontinuous, thereby obstructing translocation of potential substrates. The binding of the conjugated ubiquitin to the ubiquitin receptors leads to contraction of the 19S RP. Next, the ATPases engage the substrate at a disordered segment, energetically unravel the polypeptide and translocate it towards the 20S catalytic core (CP). In this substrate engaged state, Rpn11 is repositioned at the pore of the ATPase channel to remove remaining ubiquitin modifications and accelerate translocation. C-termini of five of the six ATPases insert into corresponding lysine-pockets on the 20S α-ring to complete 20S CP gate opening. In the resulting substrate processing state, the ATPase channel is fully contiguous with the translocation channel into the 20S CP, where the substrate is proteolyzed. Complete degradation of a typical ubiquitin-conjugate takes place over a few tens of seconds while hydrolysing tens of ATP molecules in the process (50 kDa/∼50 s/∼80ATP). This article reviews recent insight into biochemical and structural features that underlie substrate recognition and processing by the 26S proteasome.

Anti-HCMV activity by an irreversible p97 inhibitor LC-1310.

The AAA+ (ATPase associated with various cellular activities) protein p97, also called valosin-containing protein, is a hexameric ring ATPase and uses ATP hydrolysis to unfold or extract proteins from biological complexes. Many cellular processes are affected by p97 including ER-associated degradation, DNA damage response, cell signaling (NF-κB), cell cycle progression, autophagy, and others. Not surprisingly, with its role in many fundamental cellular processes, p97 function is important for the replication of many viruses. We tested irreversible p97-targeting compounds for their ability to inhibit the replication of multiple viruses compared to the known p97 inhibitors NMS-873 and CB-5083. Our results indicate that overall cellular toxicity for p97 compounds provides a challenge for antivirals targeting p97. However, we identified one compound with sub-micromolar activity against human cytomegalovirus and improved cell viability to provide evidence for the potential of irreversible p97 inhibitors as antivirals.

Biochemical and Biophysical Characterization of the dsDNA Packaging Motor from the Lactococcus lactis Bacteriophage Asccphi28.

Double-stranded DNA viruses package their genomes into pre-assembled protein procapsids. This process is driven by macromolecular motors that transiently assemble at a unique vertex of the procapsid and utilize homomeric ring ATPases to couple genome encapsidation to ATP hydrolysis. Here, we describe the biochemical and biophysical characterization of the packaging ATPase from Lactococcus lactis phage asccφ28. Size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), small angle X-ray scattering (SAXS), and negative stain transmission electron microscopy (TEM) indicate that the ~45 kDa protein formed a 443 kDa cylindrical assembly with a maximum dimension of ~155 Å and radius of gyration of ~54 Å. Together with the dimensions of the crystallographic asymmetric unit from preliminary X-ray diffraction experiments, these results indicate that gp11 forms a decameric D5-symmetric complex consisting of two pentameric rings related by 2-fold symmetry. Additional kinetic analysis shows that recombinantly expressed gp11 has ATPase activity comparable to that of functional ATPase rings assembled on procapsids in other genome packaging systems. Hence, gp11 forms rings in solution that likely reflect the fully assembled ATPases in active virus-bound motor complexes. Whereas ATPase functionality in other double-stranded DNA (dsDNA) phage packaging systems requires assembly on viral capsids, the ability to form functional rings in solution imparts gp11 with significant advantages for high-resolution structural studies and rigorous biophysical/biochemical analysis.