ATP-dependent Protein Degradation Research Articles

Dissecting the mechanistic events in ATP-dependent degradation of membrane proteins mediated by the AAA+ protease FtsH

Dissecting the mechanistic events in ATP-dependent degradation of membrane proteins mediated by the AAA+ protease FtsH

Ubiquitin and its role in proteolisis: the 2004 Nobel prize in chemistry

In the early 1980-s, Aaron Ciechanover, Avram Hershko, and Irwin Rose discovered one of the most important cyclic cellular processes – a regulated ATP-dependent protein degradation, for which they were awarded the 2004 Nobel Prize in Chemistry. These scientists proved the existence of a non-lysosomal proteolysis pathway and completely changed the perception of intracellular protein degradation mechanisms. They demonstrated pre-labelling of a doomed protein in a cell with a biochemical marker called ubiquitin. Polyubiquitylation of a protein as a signal for its proteolysis was a new mechanism discovered as a result of collaborative efforts of three scientists on isolation of enzymes involved in this sequential process, clarification of the biochemical stages, and substantiating the energy dependence mechanism. The article contains biographical data of the Nobel laureates, the methods applied, and the history of the research resulted in the discovery of the phenomenon of proteasomal degradation of ubiquitin-mediated proteins. Keywords: PROTAC, regulated protein degradation, ubiquitin, І. Rose, А. Ciechanover, А. Hershko

Unstructured Biology of Proteins from Ubiquitin-Proteasome System: Roles in Cancer and Neurodegenerative Diseases.

The 26S proteasome is a large (~2.5 MDa) protein complex consisting of at least 33 different subunits and many other components, which form the ubiquitin proteasomal system (UPS), an ATP-dependent protein degradation system in the cell. UPS serves as an essential component of the cellular protein surveillance machinery, and its dysfunction leads to cancer, neurodegenerative and immunological disorders. Importantly, the functions and regulations of proteins are governed by the combination of ordered regions, intrinsically disordered protein regions (IDPRs) and molecular recognition features (MoRFs). The structure–function relationships of UPS components have not been identified completely; therefore, in this study, we have carried out the functional intrinsic disorder and MoRF analysis for potential neurodegenerative disease and anti-cancer targets of this pathway. Our report represents the presence of significant intrinsic disorder and disorder-based binding regions in several UPS proteins, such as extraproteasomal polyubiquitin receptors (UBQLN1 and UBQLN2), proteasome-associated polyubiquitin receptors (ADRM1 and PSMD4), deubiquitinating enzymes (DUBs) (ATXN3 and USP14), and ubiquitinating enzymes (E2 (UBE2R2) and E3 (STUB1) enzyme). We believe this study will have implications for the conformation-specific roles of different regions of these proteins. This will lead to a better understanding of the molecular basis of UPS-associated diseases.

Δ9-tetrahydrocannabinol modulates the proteasome system in the brain

Cannabis is the most consumed illicit drug worldwide. Its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), affects multiple brain functions, including cognitive performance, by modulating cannabinoid type-1 (CB1) receptors. These receptors are strongly enriched in presynaptic terminals, where they modulate neurotransmitter release. We analyzed, through a proteomic screening of hippocampal synaptosomal fractions, those proteins and pathways modulated 3 h after a single administration of an amnesic dose of THC (10 mg/kg, i.p.). Using an isobaric labeling approach, we identified 2040 proteins, 1911 of them previously reported in synaptic proteomes, confirming the synaptic content enrichment of the samples. Initial analysis revealed a significant alteration of 122 proteins, where 42 increased and 80 decreased their expression. Gene set enrichment analysis indicated an over-representation of mitochondrial associated functions and cellular metabolic processes. A second analysis focusing on extreme changes revealed 28 proteins with altered expression after THC treatment, 15 of them up-regulated and 13 down-regulated. Using a network topology-based scoring algorithm we identified those proteins in the mouse proteome with the greatest association to the 28 modulated proteins. This analysis pinpointed a significant alteration of the proteasome function, since top scoring proteins were related to the proteasome system (PS), a protein complex involved in ATP-dependent protein degradation. In this regard, we observed that THC decreases 20S proteasome chymotrypsin-like protease activity in the hippocampus. Our data describe for the first time the modulation of the PS in the hippocampus following THC administration under amnesic conditions that may contribute to an aberrant plasticity at synapses.

Folding-Degradation Relationship of a Membrane Protein Mediated by the Universally Conserved ATP-Dependent Protease FtsH.

ATP-dependent protein degradation mediated by AAA+ proteases is one of the major cellular pathways for protein quality control and regulation of functional networks. While a majority of studies of protein degradation have focused on water-soluble proteins, it is not well understood how membrane proteins with abnormal conformation are selectively degraded. The knowledge gap stems from the lack of an in vitro system in which detailed molecular mechanisms can be studied as well as difficulties in studying membrane protein folding in lipid bilayers. To quantitatively define the folding-degradation relationship of membrane proteins, we reconstituted the degradation using the conserved membrane-integrated AAA+ protease FtsH as a model degradation machine and the stable helical-bundle membrane protein GlpG as a model substrate in the lipid bilayer environment. We demonstrate that FtsH possesses a substantial ability to actively unfold GlpG, and the degradation significantly depends on the stability and hydrophobicity near the degradation marker. We find that FtsH hydrolyzes 380-550 ATP molecules to degrade one copy of GlpG. Remarkably, FtsH overcomes the dual-energetic burden of substrate unfolding and membrane dislocation with the ATP cost comparable to that for water-soluble substrates by robust ClpAP/XP proteases. The physical principles elucidated in this study provide general insights into membrane protein degradation mediated by ATP-dependent proteolytic systems.

Structure and Functional Properties of the Active Form of the Proteolytic Complex, ClpP1P2, from Mycobacterium tuberculosis

The ClpP protease complex and its regulatory ATPases, ClpC1 and ClpX, inMycobacterium tuberculosis(Mtb) are essential and, therefore, promising drug targets. TheMtbClpP protease consists of two heptameric rings, one composed of ClpP1 and the other of ClpP2 subunits. Formation of the enzymatically active ClpP1P2 complex requires binding of N-blocked dipeptide activators. We have found a new potent activator, benzoyl-leucine-leucine (Bz-LL), that binds with higher affinity and promotes 3-4-fold higher peptidase activity than previous activators. Bz-LL-activated ClpP1P2 specifically stimulates the ATPase activity ofMtbClpC1 and ClpX. The ClpC1P1P2 and ClpXP1P2 complexes exhibit 2-3-fold enhanced ATPase activity, peptide cleavage, and ATP-dependent protein degradation. The crystal structure of ClpP1P2 with bound Bz-LL was determined at a resolution of 3.07 Å and with benzyloxycarbonyl-Leu-Leu (Z-LL) bound at 2.9 Å. Bz-LL was present in all 14 active sites, whereas Z-LL density was not resolved. Surprisingly, Bz-LL adopts opposite orientations in ClpP1 and ClpP2. In ClpP1, Bz-LL binds with the C-terminal leucine side chain in the S1 pocket. One C-terminal oxygen is close to the catalytic serine, whereas the other contacts backbone amides in the oxyanion hole. In ClpP2, Bz-LL binds with the benzoyl group in the S1 pocket, and the peptide hydrogen bonded between parallel β-strands. The ClpP2 axial loops are extended, forming an open axial channel as has been observed with bound ADEP antibiotics. Thus occupancy of the active sites of ClpP allosterically alters sites on the surfaces thereby affecting the association of ClpP1 and ClpP2 rings, interactions with regulatory ATPases, and entry of protein substrates.

Engineered AAA+ Proteases Reveal Mechanisms of Degradation at the Mitochondrial Inner Membrane

The Yme1 protease is a hexameric membrane-anchored AAA+ machine that derives energy from ATP to control proteostasis in the inner membrane and intermembrane space of mitochondria. Substrates of Yme1 include soluble, peripheral membrane, and integral membrane proteins but the mechanisms of substrate recognition and degradation remain elusive. Detailed solution studies of this class of membrane-bound AAA+ enzyme have been hampered by the requirement for an insoluble transmembrane span to drive oligomerization and thus form the ATPase sites. By replacing the transmembrane span with a soluble coiled-coil analogue, we have developed a method for driving assembly of soluble active hexameric Yme1 proteases that are fully competent for ATP-dependent protein degradation in vitro.Using these engineered enzymes with model substrates we show that Yme1 requires substrates to present an accessible peptide signal to initiate degradation and displays clear preferences in the amino acid sequence of the degradation signal. Furthermore, the protease is capable of delivering a significant unfolding force to processively unravel and translocate stable protein substrates. These principles are extended to physiological substrates of Yme1 by examining the degradation of the intermembrane-space protein transport complex Tim9/10. We demonstrate the existence of a mitochondrial degradation signal that is necessary and sufficient to target proteins for proteolysis by Yme1.

PA28αβ: The Enigmatic Magic Ring of the Proteasome?

PA28αβ is a γ-interferon-induced 11S complex that associates with the ends of the 20S proteasome and stimulates in vitro breakdown of small peptide substrates, but not proteins or ubiquitin-conjugated proteins. In cells, PA28 also exists in larger complexes along with the 19S particle, which allows ATP-dependent degradation of proteins; although in vivo a large fraction of PA28 is present as PA28αβ-20S particles whose exact biological functions are largely unknown. Although several lines of evidence strongly indicate that PA28αβ plays a role in MHC class I antigen presentation, the exact molecular mechanisms of this activity are still poorly understood. Herein, we review current knowledge about the biochemical and biological properties of PA28αβ and discuss recent findings concerning its role in modifying the spectrum of proteasome’s peptide products, which are important to better understand the molecular mechanisms and biological consequences of PA28αβ activity.

Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase (936.1)

The 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation, yet the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP‐dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits a pronounced three‐fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C‐terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate‐opening, whereas the C‐terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases.Grant Funding Source: Supported by US National Institutes of Health grant R01‐GM094497‐01A1

Regulated protein turnover: snapshots of the proteasome in action

The ubiquitin proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and misfolded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures provide some surprising insights into the functional mechanism of the proteasome and will give invaluable guidance for genetic and biochemical studies of this key regulatory system.

Reconstitution of the 26S Proteasome Reveals Functional Asymmetries in its Heterohexameric AAA+ Unfoldase

The 26S proteasome is the major protease in eukaryotic cells responsible for selective protein degradation to mediate protein quality control and regulation. However, the detailed mechanisms by which the proteasomal heterohexameric AAA+ unfoldase drives ATP-dependent protein degradation remain poorly understood. Delineating the roles of the six distinct ATPase subunits in substrate processing has been hindered by limitations in working with endogenous proteasomes due to misassembly or lethal degradation defects. We therefore developed a heterologous expression system to produce the unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro to perform systematic mutational analyses of the individual ATPase subunits. Our studies demonstrate that the six ATPases have distinct functions in degradation, corresponding to their positions in the spiral staircases adopted by their large AAA+ domains in the absence or presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. Whereas the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits a pronounced three-fold symmetry. Only three ATPase subunits, arranged in alternate positions within the unfoldase ring, contain a conserved C-terminal hydrophobic/aromatic/unspecified (HbYX) motif that is critical for both peptidase binding and gate-opening, whereas the C-terminal tails of the interjacent ATPase subunits are dispensable. Our study provides an initial glimpse into the potential importance of the spiral staircase configurations of proteasomal ATPase subunits in substrate processing and highlights how the 26S proteasome may deviate from simpler, homomeric AAA+ proteases.

Ubiquitin and Drug Discovery: Challenges and Opportunities

In 1977 ATP-dependent protein degradation in reticulocytes was demonstrated by Alfred Goldberg. Beginning in 1978, the ATP-dependent ubiquitin system for proteolysis was described by Avram Hershko, Irwin Rose and Aaron Ciechanover and colleagues, with the proteasome being characterized soon thereafter. Before this, the prevailing concept was that whatever protein degradation was taking place in cells was through lysosomes.

Identification of a Region in the N-Terminus of Escherichia coli Lon That Affects ATPase, Substrate Translocation and Proteolytic Activity

Lon, also known as protease La, is an AAA+ protease machine that contains the ATPase and proteolytic domain within each enzyme subunit. Three truncated Escherichia coli Lon (ELon) mutants were generated based on a previous limited tryptic digestion result and hydrogen–deuterium exchange mass spectrometry analyses performed in this study. Using methods developed for characterizing wild-type (WT) Lon, we compared the ATPase, ATP-dependent protein degradation and ATP-dependent peptidase activities. With the exception of not degrading a putative structured substrate known as CcrM (cell-cycle-regulated DNA methyltransferase), the mutant lacking the first 239 residues behaved like WT ELon. Comparing the activity data of WT and ELon mutants reveals that the first 239 residues are not needed for minimal enzyme catalysis. The mutants lacking the first 252 residues or residues 232–252 displayed compromised ATPase, protein degradation and ATP-dependent peptide translocation abilities but retained WT-like steady-state peptidase activity. The binding affinities of WT and Lon mutants were evaluated by determining the concentration of λ N (KλN) needed to achieve 50% maximal ATPase stimulation. Comparing the KλN values reveals that the region encompassing 232–252 of ELon could contribute to λ N binding, but the effect is modest. Taken together, results generated from this study reveal that the region constituting residues 240–252 of ELon is important for ATPase activity, substrate translocation and protein degradation.

The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+) mouse.

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The ApcMin/+ mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the ApcMin/+ mouse is not known. Cachexia progression was studied in ApcMin/+ mice that were either weight stable (WS) or had initial (≤5%), intermediate (6–19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.

An Intrinsic Degradation Tag on the ClpA C-Terminus Regulates the Balance of ClpAP Complexes with Different Substrate Specificity

ATP-dependent protein degradation in bacteria is carried out by barrel-shaped proteases architecturally related to the proteasome. In Escherichia coli, ClpP interacts with two alternative ATPases, ClpA or ClpX, to form active protease complexes. ClpAP and ClpXP show different but overlapping substrate specificities. ClpXP is considered the primary recipient of ssrA-tagged substrates while ClpAP in complex with ClpS processes N-end rule substrates. Notably, in its free form, but not in complex with ClpS, ClpAP also degrades ssrA-tagged substrates and its own chaperone component, ClpA. To reveal the mechanism of ClpAP-mediated ClpA degradation, termed autodegradation, and its possible role in regulating ClpAP levels, we dissected ClpA to show that the flexible C-terminus of the second AAA module serves as the degradation signal. We demonstrate that ClpA becomes largely resistant to autodegradation in the absence of its C-terminus and, conversely, transfer of the last 11 residues of ClpA to the C-terminus of green fluorescent protein (GFP) renders GFP a substrate of ClpAP. This autodegradation tag bears similarity to the ssrA-tag in its degradation behavior, displaying similar catalytic turnover rates when coupled to GFP but a twofold lower apparent affinity constant compared to ssrA-tagged GFP. We show that, in analogy to the prevention of ssrA-mediated recognition, the adaptor ClpS inhibits autodegradation by a specificity switch as opposed to direct masking of the degradation signal. Our results demonstrate that in the presence of ssrA-tagged substrates, ClpA autodegradation will be competitively reduced. This simple mechanism allows for dynamic reallocation of free ClpAP versus ClpAPS in response to the presence of ssrA-tagged substrates.

Dissecting β-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.

The 26S proteasome of the lignin-degrading Basidiomycete Phlebia radiata

The 26S proteasome, a large multicatalytic protease complex is involved in highly selective ATP-dependent degradation of intracellular proteins in eukaryotes, typically those tagged with a polyubiquitin chain. The proteasome-mediated proteolysis plays a key role in the regulation of critical cellular processes such as transcriptional control, cell cycle progression, and stress response. Biochemical and structural properties of the 26S proteasomes described in this paper concern the white-rot fungus Phlebia radiata, which is interesting from the standpoint of biotechnological applications of ligninolytic enzymes. In addition, the characterization of 26S proteasomes from P. radiata which belongs to the class Basidiomycetes is the first one reported in the kingdom of fungi for an organism other than yeast representing the class Ascomycetes. All distinct peptidase activities: chymotrypsin-, trypsin-, and caspase-like attributed to proteasomes were detected in P. radiata proteasome preparations. Electrophoretic mobility, subunit composition, ATP-dependence, sensitivity to proteasome-specific inhibitors, and inactivation by low concentrations of SDS demonstrated for these preparations are typical features of 26S proteasome species. Moreover, findings from in vivo blocking of proteasome function strongly support the previous interpretation that the proteasomal pathway is involved in regulation of activity of some ligninolytic enzymes under nutrient deprivation [Staszczak M. Proteasomal degradation pathways in Trametes versicolor and Phlebia radiata. Enzyme Microb Technol 2002;30:537–41].

Human Mitochondrial ClpP Is a Stable Heptamer That Assembles into a Tetradecamer in the Presence of ClpX

The functional form of ClpP, the proteolytic component of ATP-dependent Clp proteases, is a hollow-cored particle composed of two heptameric rings joined face-to-face forming an aqueous chamber containing the proteolytic active sites. We have found that isolated human mitochondrial ClpP (hClpP) is stable as a heptamer and remains a monodisperse species (s(20,w) 7.0 S; M(app) 169, 200) at concentrations > or = 3 mg/ml. Heptameric hClpP has no proteolytic activity and very low peptidase activity. In the presence of ATP, hClpX interacts with hClpP forming a complex, which by equilibrium sedimentation measurements has a M(app) of 1 x 10(6). Electron microscopy confirmed that the complex consisted of a double ring of hClpP with an hClpX ring axially aligned on each end. The hClpXP complex has protease activity and greatly increased peptidase activity, indicating that interaction with hClpX affects the conformation of the hClpP catalytic active site. A mutant of hClpP, in which a cysteine residue was introduced into the handle region at the interface between the two rings formed stable tetradecamers under oxidizing conditions but spontaneously dissociated into two heptamers upon reduction. Thus, hClpP rings interact transiently but very weakly in solution, and hClpX must exert an allosteric effect on hClpP to promote a conformation that stabilizes the tetradecamer. These data suggest that hClpX can regulate the appearance of hClpP peptidase activity in mitochondria and might affect the nature of the degradation products released during ATP-dependent proteolytic cycles.

The Molecular Chaperone, ClpA, Has a Single High Affinity Peptide Binding Site per Hexamer

Substrate recognition by Clp chaperones is dependent on interactions with motifs composed of specific peptide sequences. We studied the binding of short motif-bearing peptides to ClpA, the chaperone component of the ATP-dependent ClpAP protease of Escherichia coli in the presence of ATPgammaS and Mg2+ at pH 7.5. Binding was measured by isothermal titration calorimetry (ITC) using the peptide, AANDENYALAA, which corresponds to the SsrA degradation motif found at the C terminus of abnormal nascent polypeptides in vivo. One SsrA peptide was bound per hexamer of ClpA with an association constant (K(A)) of 5 x 10(6) m(-1). Binding was also assayed by changes in fluorescence of an N-terminal dansylated SsrA peptide, which bound with the same stoichiometry of one per ClpA hexamer (K(A) approximately 1 x 10(7) m(-1)). Similar results were obtained when ATP was substituted for ATPgammaS at 6 degrees C. Two additional peptides, derived from the phage P1 RepA protein and the E. coli HemA protein, which bear different substrate motifs, were competitive inhibitors of SsrA binding and bound to ClpA hexamers with K(A)' > 3 x 10(7) m(-1). DNS-SsrA bound with only slightly reduced affinity to deletion mutants of ClpA missing either the N-terminal domain or the C-terminal nucleotide-binding domain, indicating that the binding site for SsrA lies within the N-terminal nucleotide-binding domain. Because only one protein at a time can be unfolded and translocated by ClpA hexamers, restricting the number of peptides initially bound should avoid nonproductive binding of substrates and aggregation of partially processed proteins.

Nobel Prize in Chemistry 2004. The mechanism of ATP-dependent degradation of proteins involving ubiquitin

Nobel Prize in Chemistry 2004. The mechanism of ATP-dependent degradation of proteins involving ubiquitin