The mitochondrial protease ClpP is a druggable target that controls VSMC phenotype by a SIRT1-dependent mechanism.

Vascular smooth muscle cells (VSMCs), known for their remarkable lifelong phenotypic plasticity, play a pivotal role in vascular pathologies through their ability to transition between different phenotypes. Our group discovered that the deficiency of the mitochondrial protein Poldip2 induces VSMC differentiation both in vivo and in vitro. Further comprehensive biochemical investigations revealed Poldip2's specific interaction with the mitochondrial ATPase caseinolytic protease chaperone subunit X (CLPX), which is the regulatory subunit for the caseinolytic protease proteolytic subunit (ClpP) that forms part of the ClpXP complex - a proteasome-like protease evolutionarily conserved from bacteria to humans. This interaction limits the protease's activity, and reduced Poldip2 levels lead to ClpXP complex activation. This finding prompted the hypothesis that ClpXP complex activity within the mitochondria may regulate the VSMC phenotype. Employing gain-of-function and loss-of-function strategies, we demonstrated that ClpXP activity significantly influences the VSMC phenotype. Notably, both genetic and pharmacological activation of ClpXP inhibits VSMC plasticity and fosters a quiescent, differentiated, and anti-inflammatory VSMC phenotype. The pharmacological activation of ClpP using TIC10, currently in phase III clinical trials for cancer, successfully replicates this phenotype both in vitro and in vivo and markedly reduces aneurysm development in a mouse model of elastase-induced aortic aneurysms. Our mechanistic exploration indicates that ClpP activation regulates the VSMC phenotype by modifying the cellular NAD+/NADH ratio and activating Sirtuin 1. Our findings reveal the crucial role of mitochondrial proteostasis in the regulation of the VSMC phenotype and propose the ClpP protease as a novel, actionable target for manipulating the VSMC phenotype.

Coordinated adaptation of Staphylococcus aureus to calprotectin-dependent metal sequestration.

The host protein calprotectin inhibits the growth of a variety of bacterial pathogens through metal sequestration in a process known as "nutritional immunity." Staphylococcus aureus growth is inhibited by calprotectin in vitro, and calprotectin is localized in vivo to staphylococcal abscesses during infection. However, the staphylococcal adaptations that provide defense against nutritional immunity and the role of metal-responsive regulators are not fully characterized. In this work, we define the transcriptional response of S. aureus and the role of the metal-responsive regulators, Zur, Fur, and MntR, in response to metal limitation by calprotectin exposure. Additionally, we identified genes affecting the fitness of S. aureus during metal limitation through a Transposon sequencing (Tn-seq) approach. Loss of function mutations in clpP, which encodes a proteolytic subunit of the ATP-dependent Clp protease, demonstrate reduced fitness of S. aureus to the presence of calprotectin. ClpP contributes to pathogenesis in vivo in a calprotectin-dependent manner. These studies establish a critical role for ClpP to combat metal limitation by calprotectin and reveal the genes required for S. aureus to outcompete the host for metals. IMPORTANCE: Staphylococcus aureus is a leading cause of skin and soft tissue infections, bloodstream infections, and endocarditis. Antibiotic treatment failures during S. aureus infections are increasingly prevalent, highlighting the need for novel antimicrobial agents. Metal chelator-based therapeutics have tremendous potential as antimicrobials due to the strict requirement for nutrient metals exhibited by bacterial pathogens. The high-affinity transition metal-binding properties of calprotectin represents a potential therapeutic strategy that functions through metal chelation. Our studies provide a foundation to define mechanisms by which S. aureus combats nutritional immunity and may be useful for the development of novel therapeutics to counter the ability of S. aureus to survive in a metal-limited environment.

Knockout Mouse Studies Show That Mitochondrial CLPP Peptidase and CLPX Unfoldase Act in Matrix Condensates near IMM, as Fast Stress Response in Protein Assemblies for Transcript Processing, Translation, and Heme Production.

LONP1 is the principal AAA+ unfoldase and bulk protease in the mitochondrial matrix, so its deletion causes embryonic lethality. The AAA+ unfoldase CLPX and the peptidase CLPP also act in the matrix, especially during stress periods, but their substrates are poorly defined. Mammalian CLPP deletion triggers infertility, deafness, growth retardation, and cGAS-STING-activated cytosolic innate immunity. CLPX mutations impair heme biosynthesis and heavy metal homeostasis. CLPP and CLPX are conserved from bacteria to humans, despite their secondary role in proteolysis. Based on recent proteomic-metabolomic evidence from knockout mice and patient cells, we propose that CLPP acts on phase-separated ribonucleoprotein granules and CLPX on multi-enzyme condensates as first-aid systems near the inner mitochondrial membrane. Trimming within assemblies, CLPP rescues stalled processes in mitoribosomes, mitochondrial RNA granules and nucleoids, and the D-foci-mediated degradation of toxic double-stranded mtRNA/mtDNA. Unfolding multi-enzyme condensates, CLPX maximizes PLP-dependent delta-transamination and rescues malformed nascent peptides. Overall, their actions occur in granules with multivalent or hydrophobic interactions, separated from the aqueous phase. Thus, the role of CLPXP in the matrix is compartment-selective, as other mitochondrial peptidases: MPPs at precursor import pores, m-AAA and i-AAA at either IMM face, PARL within the IMM, and OMA1/HTRA2 in the intermembrane space.

ONC206 targeting ClpP induces mitochondrial dysfunction and protective autophagy in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for approximately 90 % of all cases. ONC201, a member of the imipridone drug family, has shown promising therapeutic potential and a good safety profile in both malignant pediatric central nervous system tumors (diffuse midline glioma [DMG]) and hematologic malignancies. ONC206 is a more potent analog of ONC201. However, the ONC206 potential and mechanism of action in HCC remain to be elucidated. We found that ONC206 hindered HCC growth by suppressing cell proliferation and inducing apoptosis. Moreover, ONC206 induced cytoprotective autophagy, and blocking autophagy enhanced the proapoptotic effect of ONC206. Additionally, ONC206 induced mitochondrial swelling, reduced the mitochondrial membrane potential (MMP), and led to the accumulation of mitochondrial ROS in HCC cells, ultimately resulting in mitochondrial dysfunction. The HCC patient samples exhibited notably elevated levels of caseinolytic protease proteolytic subunit (ClpP), which serves as a mediator of ONC206-induced mitochondrial dysfunction and the activation of protective autophagy. knockdown of ClpP reversed the cytotoxic effects of ONC206 on HCC cells. In summary, our results provide the first insight into the mechanism by which ONC206 exerts its anti-HCC effects and induces protective autophagy in HCC cells through ClpP.

RecA-dependent or independent recombination of plasmid DNA generates a conflict with the host EcoKI immunity by launching restriction alleviation.

Bacterial defence systems are tightly regulated to avoid autoimmunity. In Type I restriction-modification (R-M) systems, a specific mechanism called restriction alleviation (RA) controls the activity of the restriction module. In the case of the Escherichia coli Type I R-M system EcoKI, RA proceeds through ClpXP-mediated proteolysis of restriction complexes bound to non-methylated sites that appear after replication or reparation of host DNA. Here, we show that RA is also induced in the presence of plasmids carrying EcoKI recognition sites, a phenomenon we refer to as plasmid-induced RA. Further, we show that the anti-restriction behavior of plasmid-borne non-conjugative transposons such as Tn5053, previously attributed to their ardD loci, is due to plasmid-induced RA. Plasmids carrying both EcoKI and Chi sites induce RA in RecA- and RecBCD-dependent manner. However, inactivation of both RecA and RecBCD restores RA, indicating that there exists an alternative, RecA-independent, homologous recombination pathway that is blocked in the presence of RecBCD. Indeed, plasmid-induced RA in a RecBCD-deficient background does not depend on the presence of Chi sites. We propose that processing of random dsDNA breaks in plasmid DNA via homologous recombination generates non-methylated EcoKI sites, which attract EcoKI restriction complexes channeling them for ClpXP-mediated proteolysis.

Structural insights into the Clp protein degradation machinery.

The Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Target-locked: A mechanism for disaggregase binding to aggregated proteins.

ClpG is a novel autonomous disaggregase found in Pseudomonas aeruginosa that confers resistance to lethal heat stress. The mechanism by which ClpG specifically targets protein aggregates for disaggregation is unknown. In their recent work published in JBC, Katikaridis et al. (2023) identify an avidity-based mechanism by which four or more ClpG subunits, through specific N-terminal hydrophobic residues located on an exposed ß-sheet loop, interact with multiple hydrophobic patches on an aggregated protein substrate. This study establishes a model for substrate binding to a prokaryotic disaggregase that should inform further investigations into other autonomous disaggregases.

AmCBF1 activates the expression of GhClpR1 to mediate dark-green leaves in cotton (Gossypium hirsutum).

KEY MESSAGE: The transcription factor AmCBF1 deepens the leaf colour of transgenic cotton by binding to the promoter of the chloroplast development-related protein GhClpR1 to promote photosynthesis. The ATP-dependent caseinolytic protease (Clp protease) family plays a crucial role within chloroplasts, comprising several Clp proteins to maintain chloroplast homeostasis. At present, research on Clp proteins mainly focuses on Arabidopsis, leaving its function in other plants, particularly in crops, less explored. In this study, we overexpressed AmCBF1 from Ammopiptanthus mongolicus (A. mongolicus) in wild type (R15), and found a significant darkening of leaf colour in transgenic plants (L28 and L30). RNA-seq analysis showed an enrichment of pathways associated with photosynthesis. Subsequent screening of differentially expressed genes revealed a significant up-regulation of GhClpR1, a gene linked to chloroplast development, in the transgenic strain. In addition, GhClpR1 was consistently expressed in upland cotton, with the highest expression observed in leaves. Subcellular localization analysis revealed that the protein encoded by GhClpR1 was located in chloroplasts. Yeast one hybrid and dual luciferase experiments showed that the AmCBF1 transcription factor positively regulates the expression of GhClpR1. VIGs-mediated silencing of GhClpR1 led to a significant yellowing phenotype in the leaves. This was accompanied by a reduction in chlorophyll content, and microscopic examination of chloroplast ultrastructure revealed severe developmental impairment. Finally, yeast two-hybrid assays showed that GhClpR1 interacts with the Clp protease complex accessory protein GhClpT2. Our study provides a foundation for studying the function of the Clp protease complex and a new strategy for cultivating high-light-efficiency cotton resources.

Degron-Controlled Protein Degradation in Escherichia coli: New Approaches and Parameters.

Protein degron tags have proven to be uniquely useful for the characterization of gene function. Degrons can mediate quick depletion, usually within minutes, of a protein of interest, allowing researchers to characterize cellular responses to the loss of function. To develop a general-purpose degron tool in Escherichia coli, we sought to build upon a previously characterized system of SspB-dependent inducible protein degradation. For this, we created a family of expression vectors containing a destabilized allele of SspB, capable of a rapid and nearly perfect "off-to-on" induction response. Using this system, we demonstrated excellent control over several DNA metabolism enzymes. However, other substrates did not respond to degron tagging in such an ideal manner, indicating the apparent limitations of SspB-dependent systems. Several degron-tagged proteins were degraded too slowly to be completely depleted during active growth, whereas others appeared to be completely refractory to degron-promoted degradation. Thus, only a minority of our, admittedly biased, selection of degron substrates proved to be amenable to efficient SspB-catalyzed degradation. We also uncovered an apparent stalling and/or disengagement of ClpXP from a degron-tagged allele of beta-galactosidase (beta-gal). While a degron-containing fusion peptide attached to the carboxy-terminus of beta-gal was degraded quantitatively, no reductions in beta-gal activity or concentration were detected, demonstrating an apparently novel mechanism of protease resistance. We conclude that substrate-dependent effects of the SspB system present a continued challenge to the widespread adoption of this degron system. For substrates that prove to be degradable, we provide a series of titratable SspB-expression vehicles.

Mitochondrial Unfolded Protein Response Gene Clpp Is Required for Oocyte Function and Female Fertility.

Mitochondrial unfolded protein stress response (mtUPR) plays a critical role in regulating cellular and metabolic stress response and helps maintain protein homeostasis. Caseinolytic peptidase P (CLPP) is one of the key regulators of mtUPR and promotes unfolded protein degradation. Previous studies demonstrated that global deletion of Clpp resulted in female infertility, whereas no impairment was found in the mouse model with targeted deletion of Clpp in cumulus/granulosa cells. These results suggest the need to delineate the function of Clpp in oocytes. In this study, we aimed to further explore the role of mtUPR in female reproductive competence and senescence using a mouse model. Oocyte-specific targeted deletion of Clpp in mice resulted in female subfertility associated with metabolic and functional abnormalities in oocytes, thus highlighting the importance of CLPP-mediated protein homeostasis in oocyte competence and reproductive function.

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.

CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.

CLPB Deficiency Associated Neonatal Cavitating Leukoencephalopathy: A Potential Pathomechanism Underlying Neurologic Disorder.

Caseinolytic peptidase B homolog (CLPB) is a mitochondrial protein which is highly expressed in brain. Its deficiency may be associated with severe neonatal encephalopathy. This report describes a case of fatal neonatal encephalopathy associated with biallelic stop-gain mutation in CLPB (NM_001258392.3:c.1159C>T/p.Arg387*). Neurologic disorder encompasses pre- and post-natal features including polyhydramnios, intrauterine growth restriction, respiratory insufficiency, lethargy, excessive startle reflex, generalized hypertonia, and epileptic seizures. Brain macroscopic examination demonstrates frontal severe periventricular cystic leukoencephalopathy, along with mild ex-vacuo tri-ventricular dilatation. The most striking immunohistopathologic features are striato-thalamic neurodegeneration and deep white matter loss associated with strong reactive astrogliosis. This report supports that CLPB deficiency should be considered among the neurometabolic disorders associated with severe prenatal-onset neurologic impairment that may result from cystic leukoencephalopathy.

The ClpX chaperone and a hypermorphic FtsA variant with impaired self-interaction are mutually compensatory for coordinating Staphylococcus aureus cell division.

Bacterial cell division requires the coordinated assembly and disassembly of a large protein complex called the divisome; however, the exact role of molecular chaperones in this critical process remains unclear. We here provide genetic evidence that ClpX unfoldase activity is a determinant for proper coordination of bacterial cell division by showing the growth defect of a Staphylococcus aureus clpX mutant is rescued by a spontaneously acquired G325V substitution in the ATP-binding domain of the essential FtsA cell division protein. The polymerization state of FtsA is thought to control initiation of bacterial septum synthesis and, while restoring the aberrant FtsA dynamics in clpX cells, the FtsAG325V variant displayed reduced ability to interact with itself and other cell division proteins. In wild-type cells, the ftsAG325V allele shared phenotypes with Escherichia coli superfission ftsA mutants and accelerated the cell cycle, increased the risk of daughter cell lysis, and conferred sensitivity to heat and antibiotics inhibiting cell wall synthesis. Strikingly, lethality was mitigated by spontaneous mutations that inactivate ClpX. Taken together, our results suggest that ClpX promotes septum synthesis by antagonizing FtsA interactions and illuminates the critical role of a protein unfoldase in coordinating bacterial cell division.

Pathological mutations promote proteolysis of mitochondrial tRNA-specific 2-thiouridylase 1 (MTU1) via mitochondrial caseinolytic peptidase (CLPP).

MTU1 controls intramitochondrial protein synthesis by catalyzing the 2-thiouridine modification of mitochondrial transfer RNAs (mt-tRNAs). Missense mutations in the MTU1 gene are associated with life-threatening reversible infantile hepatic failure. However, the molecular pathogenesis is not well understood. Here, we investigated 17 mutations associated with this disease, and our results showed that most disease-related mutations are partial loss-of-function mutations, with three mutations being particularly severe. Mutant MTU1 is rapidly degraded by mitochondrial caseinolytic peptidase (CLPP) through a direct interaction with its chaperone protein CLPX. Notably, knockdown of CLPP significantly increased mutant MTU1 protein expression and mt-tRNA 2-thiolation, suggesting that accelerated proteolysis of mutant MTU1 plays a role in disease pathogenesis. In addition, molecular dynamics simulations demonstrated that disease-associated mutations may lead to abnormal intermolecular interactions, thereby impairing MTU1 enzyme activity. Finally, clinical data analysis underscores a significant correlation between patient prognosis and residual 2-thiolation levels, which is partially consistent with the AlphaMissense predictions. These findings provide a comprehensive understanding of MTU1-related diseases, offering prospects for modification-based diagnostics and novel therapeutic strategies centered on targeting CLPP.

Translation Fidelity and Respiration Deficits in CLPP-Deficient Tissues: Mechanistic Insights from Mitochondrial Complexome Profiling.

The mitochondrial matrix peptidase CLPP is crucial during cell stress. Its loss causes Perrault syndrome type 3 (PRLTS3) with infertility, neurodegeneration, and a growth deficit. Its target proteins are disaggregated by CLPX, which also regulates heme biosynthesis via unfolding ALAS enzymes, providing access for pyridoxal-5'-phosphate (PLP). Despite efforts in diverse organisms with multiple techniques, CLPXP substrates remain controversial. Here, avoiding recombinant overexpression, we employed complexomics in mitochondria from three mouse tissues to identify endogenous targets. A CLPP absence caused the accumulation and dispersion of CLPX-VWA8 as AAA+ unfoldases, and of PLPBP. Similar changes and CLPX-VWA8 co-migration were evident for mitoribosomal central protuberance clusters, translation factors like GFM1-HARS2, the RNA granule components LRPPRC-SLIRP, and enzymes OAT-ALDH18A1. Mitochondrially translated proteins in testes showed reductions to <30% for MTCO1-3, the mis-assembly of the complex IV supercomplex, and accumulated metal-binding assembly factors COX15-SFXN4. Indeed, heavy metal levels were increased for iron, molybdenum, cobalt, and manganese. RT-qPCR showed compensatory downregulation only for Clpx mRNA; most accumulated proteins appeared transcriptionally upregulated. Immunoblots validated VWA8, MRPL38, MRPL18, GFM1, and OAT accumulation. Co-immunoprecipitation confirmed CLPX binding to MRPL38, GFM1, and OAT, so excess CLPX and PLP may affect their activity. Our data mechanistically elucidate the mitochondrial translation fidelity deficits which underlie progressive hearing impairment in PRLTS3.

A Transient π-π or Cation-π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in Escherichia coli.

The Escherichia coli ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr91st residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP147th) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe143rd residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpYY91FQ, ClpYY91HQ, and ClpYY91WQ, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced ß-galactosidase activity of cpsB::lacZ, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulAF143H, SulAF143K and SulAF143W, harboring a ring structure or a cation side-group in 143rd residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpYY91FQ and ClpYY91HQ, were capable of effectively degrading SulAF143H and SulAF143K, highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π-π or cation-π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.

ClpP protease modulates bacterial growth, stress response, and bacterial virulence in Brucella abortus.

The process of intracellular proteolysis through ATP-dependent proteases is a biologically conserved phenomenon. The stress responses and bacterial virulence of various pathogenic bacteria are associated with the ATP-dependent Clp protease. In this study, a Brucella abortus 2308 strain, ΔclpP, was constructed to characterize the function of ClpP peptidase. The growth of the ΔclpP mutant strain was significantly impaired in the TSB medium. The results showed that the ΔclpP mutant was sensitive to acidic pH stress, oxidative stress, high temperature, detergents, high osmotic environment, and iron deficient environment. Additionally, the deletion of clpP significantly affected Brucella virulence in macrophage and mouse infection models. Integrated transcriptomic and proteomic analyses of the ΔclpP strain showed that 1965 genes were significantly affected at the mRNA and/or protein levels. The RNA-seq analysis indicated that the ΔclpP strain exhibited distinct gene expression patterns related to energy production and conversion, cell wall/membrane/envelope biogenesis, carbohydrate transport, and metabolism. The iTRAQ analysis revealed that the differentially expressed proteins primarily participated in amino acid transport and metabolism, energy production and conversion, and secondary metabolites biosynthesis, transport and catabolism. This study provided insights into the preliminary molecular mechanism between Clp protease to bacterial growth, stress response, and bacterial virulence in Brucella strains.

Assessment of the structure-activity relationship and antileukemic activity of diacylpyramide compounds as human ClpP agonists.

Human caseinolytic protease P (ClpP) is required for the regulatory hydrolysis of mitochondrial proteins. Allosteric ClpP agonists dysfunctionally activate mitochondrial ClpP in antileukemic therapies. We previously developed ZG111, a potent ClpP agonist derived from ICG-001, inhibits the proliferation of pancreatic ductal adenocarcinoma cell lines in vitro and in vivo by degrading respiratory chain complex proteins. Herein, we studied the structure-activity relationships of ICG-001 analogs as antileukemia agents. Compound ZG36 exhibited improved stabilization effects on the thermal stability of ClpP in acute myeloid leukemia (AML) cell lines compared with the stabilization effects of ZG111, indicating a direct binding between ZG36 and ClpP. Indeed, the resolved ZG36/ClpP structural complex reveals the mode of action of ZG36 during ClpP binding. Compound ZG36 nonselectively degrades respiratory chain complexes and decreases the mitochondrial DNA, eventually leading to the collapse of mitochondrial function and leukemic cell death. Finally, ZG36 treatment inhibited 3-D cell growth in vitro and suppressed the tumorigenesis of AML cells in xenografted mice models. Collectively, we developed a new class of human ClpP agonists that can be used as potential antileukemic therapies.

Comprehensive structural characterization of the human AAA+ disaggregase CLPB in the apo- and substrate-bound states reveals a unique mode of action driven by oligomerization.

The human AAA+ ATPase CLPB (SKD3) is a protein disaggregase in the mitochondrial intermembrane space (IMS) and functions to promote the solubilization of various mitochondrial proteins. Loss-of-function CLPB mutations are associated with a few human diseases with neutropenia and neurological disorders. Unlike canonical AAA+ proteins, CLPB contains a unique ankyrin repeat domain (ANK) at its N-terminus. How CLPB functions as a disaggregase and the role of its ANK domain are currently unclear. Herein, we report a comprehensive structural characterization of human CLPB in both the apo- and substrate-bound states. CLPB assembles into homo-tetradecamers in apo-state and is remodeled into homo-dodecamers upon substrate binding. Conserved pore-loops (PLs) on the ATPase domains form a spiral staircase to grip and translocate the substrate in a step-size of 2 amino acid residues. The ANK domain is not only responsible for maintaining the higher-order assembly but also essential for the disaggregase activity. Interactome analysis suggests that the ANK domain may directly interact with a variety of mitochondrial substrates. These results reveal unique properties of CLPB as a general disaggregase in mitochondria and highlight its potential as a target for the treatment of various mitochondria-related diseases.