Transcriptional activation domains interact with ATPase subunits of yeast chromatin remodelling complexes SWI/SNF, RSC and INO80.

Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.

Single-molecule imaging of SWI/SNF chromatin remodelers reveals bromodomain-mediated and cancer-mutants-specific landscape of multi-modal DNA-binding dynamics.

Despite their prevalent cancer implications, the in vivo dynamics of SWI/SNF chromatin remodelers and how misregulation of such dynamics underpins cancer remain poorly understood. Using live-cell single-molecule tracking, we quantify the intranuclear diffusion and chromatin-binding of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), and resolve two temporally distinct stable binding modes for the fully assembled complex. Super-resolved density mapping reveals heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events (especially longer-lived ones) preferentially cluster. Importantly, we uncover distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner, pointing to a model where successive longer-lived binding within "hotspots" leads to sustained productive remodeling. Finally, systematic comparison of six common BRG1 mutants implicated in various cancers unveils alterations in chromatin-binding dynamics unique to each mutant, shedding insight into a multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers.

VirB11, a traffic ATPase, mediated flagella assembly and type IV pilus morphogenesis to control the motility and virulence of Xanthomonas albilineans.

Xanthomonas albilineans (Xal) is a gram-negative bacterial pathogen responsible for developing sugarcane leaf scald disease, which engenders significant economic losses within the sugarcane industry. In the current study, homologous recombination exchange was carried out to induce mutations within the virB/D4-like type IV secretion system (T4SS) genes of Xal. The results revealed that the virB11-deletion mutant (ΔvirB11) exhibited a loss in swimming and twitching motility. Application of transmission electron microscopy analysis further demonstrated that the ΔvirB11 failed to develop flagella formation and type IV pilus morphology and exhibited reduced swarming behaviour and virulence. However, these alterations had no discernible impact on bacterial growth. Comparative transcriptome analysis between the wild-type Xal JG43 and the deletion-mutant ΔvirB11 revealed 123 differentially expressed genes (DEGs), of which 28 and 10 DEGs were notably associated with flagellar assembly and chemotaxis, respectively. In light of these findings, we postulate that virB11 plays an indispensable role in regulating the processes related to motility and chemotaxis in Xal.

Novel phosphatidylinositol flippases contribute to phosphoinositide homeostasis in the plasma membrane.

Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane.

Platelets isolation and ectonucleotidase assay: Revealing functional aspects of the communication between the vasculature and the immune system.

Platelets are enucleated fragments of cells with a diversity of internal granules. They are responsible for functions related to hemostasis, coagulation, and inflammation. The activation of these processes depends on a cascade coordinated by cytokines, chemokines, and components of purinergic signaling, such as ATP, ADP, and adenosine. Platelets express distinct components of the purinergic system: P2X1, P2Y1, PY12, and P2Y14 receptors; and the ectonucleotidases NTPDase, NPP, and 5NTE (ecto-5'-nucleotidase). Except for P2Y14, which has not yet exhibited a known function, all other components relate to the biological processes mentioned before. Platelets are known to display specific responses to microorganisms, being capable of recognizing pathogen-associated molecular patterns (PAMPs), engulfing certain classes of viruses, and participating in NETosis. Platelet function dysregulation implicates various pathophysiological processes, including cardiovascular diseases (CVDs) and infections. In COVID-19 patients, platelets exhibit altered purinergic signaling and increased activation, contributing to inflammation. Excessive platelet activation can lead to complications from thrombosis, which can affect the circulation of vital organs. Therefore, controlling the activation is necessary to end the inflammatory process and restore homeostasis. Ectonucleotidases, capable of hydrolyzing ATP, ADP, and AMP, are of fundamental importance in activating platelets, promising pharmacological targets for clinical use as cardiovascular protective drugs. In this review, we revisit platelet biology, the purinergic receptors and ectonucleotidases on their surface, and their importance in platelet activity. Additionally, we describe methods for isolating platelets in humans and murine, as well as the main techniques for detecting the activity of ectonucleotidases in platelets. Considering the multitude of functions revealed by platelets and their potential use as potent bioreactors able to secrete and present molecules involved in the communication of the vasculature with the immune system, it is crucial to deeply understand platelet biology and purinergic signaling participation to contribute to the developing of therapeutic strategies in diseases of the cardiovascular, inflammatory, and immune systems.

Differential modulation of polycomb-associated histone marks by cBAF, pBAF, and gBAF complexes.

Chromatin regulators alter the physical properties of chromatin to make it more or less permissive to transcription by modulating another protein's access to a specific DNA sequence through changes in nucleosome occupancy or histone modifications at a particular locus. Mammalian SWI/SNF complexes are a group of ATPase-dependent chromatin remodelers. In mouse embryonic stem cells, there are three primary forms of mSWI/SNF: canonical BAF (cBAF), polybromo-associated BAF (pBAF), and GLTSCR-associated BAF (gBAF). Nkx2-9 is bivalent, meaning nucleosomes at the locus have active and repressive modifications. In this study, we used unique BAF subunits to recruit each of the three complexes to Nkx2-9 using dCas9-mediated inducible recruitment (FIRE-Cas9). We show that recruitment of cBAF complexes leads to a significant loss of the polycomb repressive-2 H3K27me3 histone mark and polycomb repressive-1 and repressive-2 complex proteins, whereas gBAF and pBAF do not. Moreover, nucleosome occupancy alone cannot explain the loss of these marks. Our results demonstrate that cBAF has a unique role in the direct opposition of polycomb-associated histone modifications that gBAF and pBAF do not share.

CDCA7 is an evolutionarily conserved hemimethylated DNA sensor in eukaryotes.

Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.

Asymmetric Dynamics Drive Catalytic Activation of the Hsp90 Chaperone.

The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended 19F NMR spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to show that within the context of heterodimeric Hsp90, the conformationally dynamic subunit stimulates the ATPase activity of the normal subunit. The contrasting dynamic properties of the subunits within heterodimeric Hsp90 provide a mechanistic framework to understand the molecular basis for asymmetric Hsp90 activation and its importance for the biological function of Hsp90.

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.

MksB is a novel mycobacterial condensin that orchestrates spatiotemporal positioning of replication machinery.

Condensins play important roles in maintaining bacterial chromatin integrity. In mycobacteria, three types of condensins have been characterized: a homolog of SMC and two MksB-like proteins, the recently identified MksB and EptC. Previous studies suggest that EptC contributes to defending against foreign DNA, while SMC and MksB may play roles in chromosome organization. Here, we report for the first time that the condensins, SMC and MksB, are involved in various DNA transactions during the cell cycle of Mycobacterium smegmatis (currently named Mycolicibacterium smegmatis). SMC appears to be required during the last steps of the cell cycle, where it contributes to sister chromosome separation. Intriguingly, in contrast to other bacteria, mycobacterial MksB follows replication forks during chromosome replication and hence may be involved in organizing newly replicated DNA.

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate. Our analysis reveals a continuum of structural snapshots that spans the entire translocation cycle. These data uncover elements of Shp1-Cdc48 interactions and support a 'hand-over-hand' mechanism in which the sequential movement of individual subunits is closely coordinated. D1 hydrolyzes ATP and disengages from substrate prior to D2, while D2 rebinds ATP and re-engages with substrate prior to D1, thereby explaining the dominant role played by the D2 motor in substrate translocation/unfolding.

Bidirectional pilus processing in the Tad pilus system motor CpaF.

The bacterial tight adherence pilus system (TadPS) assembles surface pili essential for adhesion and colonisation in many human pathogens. Pilus dynamics are powered by the ATPase CpaF (TadA), which drives extension and retraction cycles in Caulobacter crescentus through an unknown mechanism. Here we use cryogenic electron microscopy and cell-based light microscopy to characterise CpaF mechanism. We show that CpaF assembles into a hexamer with C2 symmetry in different nucleotide states. Nucleotide cycling occurs through an intra-subunit clamp-like mechanism that promotes sequential conformational changes between subunits. Moreover, a comparison of the active sites with different nucleotides bound suggests a mechanism for bidirectional motion. Conserved CpaF residues, predicted to interact with platform proteins CpaG (TadB) and CpaH (TadC), are mutated in vivo to establish their role in pilus processing. Our findings provide a model for how CpaF drives TadPS pilus dynamics and have broad implications for how other ancient type 4 filament family members power pilus assembly.

Prevalence of RNF213 rs112735431 Genetic Polymorphism in Non-Cardioembolic Ischemic Cerebrovascular Disease: A Cross-Sectional Study in Thai Patients.

INTRODUCTION: Moyamoya disease (MMD) and non-MMD intracranial cerebral artery stenosis (ICAS) have been linked to the RNF213 rs112735431 gene in Korean and Japanese populations. This cross-sectional study investigates the prevalence of the RNF213 rs112735431 gene in non-cardioembolic ischemic stroke (NCIS) among Thai patients. METHODS: A cross-sectional investigation was conducted on patients aged 18 years or older admitted to King Chulalongkorn Memorial Hospital between June 2015 and March 2016 with acute NCIS. ICAS and extracranial carotid artery stenosis (ECAS) were assessed through computer tomography angiography or magnetic resonance angiography. Blood samples were collected, and Sanger sequencing was performed. RESULTS: Among 234 acute NCIS cases, 113 exhibited ICAS, 12 had ECAS, 20 had both, and 89 had neither. The RNF213 rs112735431 gene variant was detected in 2 patients, both heterozygous A/G. The frequency of the RNF213 rs112735431 variant was 0.9% (2/234; 95% CI: 0-2.1%) in acute NCIS patients and 1.8% (2/113; 95% CI: 0-4.2%) in ICAS. All individuals with the RNF213 variant were males with hypertension, diabetes mellitus, dyslipidemia, and ICAS, without a family history of ischemic stroke. CONCLUSION: This study reveals that the RNF213 rs112735431 gene variant is uncommon among Thai NCIS patients, suggesting a discrepancy in the prevalence of this genetic variation between Thai and other Eastern Asian populations.

Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns.

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

Orientation-independent-DIC imaging reveals that a transient rise in depletion attraction contributes to mitotic chromosome condensation.

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells.

Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases.

Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape.

Targeting p97-Npl4 interaction inhibits tumor Treg cell development to enhance tumor immunity.

Targeting tumor-infiltrating regulatory T (TI-Treg) cells is a potential strategy for cancer therapy. The ATPase p97 in complex with cofactors (such as Npl4) has been investigated as an antitumor drug target; however, it is unclear whether p97 has a function in immune cells or immunotherapy. Here we show that thonzonium bromide is an inhibitor of the interaction of p97 and Npl4 and that this p97-Npl4 complex has a critical function in TI-Treg cells. Thonzonium bromide boosts antitumor immunity without affecting peripheral Treg cell homeostasis. The p97-Npl4 complex bridges Stat3 with E3 ligases PDLIM2 and PDLIM5, thereby promoting Stat3 degradation and enabling TI-Treg cell development. Collectively, this work shows an important role for the p97-Npl4 complex in controlling Treg-TH17 cell balance in tumors and identifies possible targets for immunotherapy.

The Role of Vitamin C on ATPases Activities in Monosodium Glutamate-Induced Oxidative Stress in Rat Striatum and Cerebellum.

Monosodium glutamate (MSG) is a silent excitotoxin used as a flavour enhancer but exerts serious health hazards to consumers. MSG plays a role in neuronal function as the dominant excitatory neurotransmitter. It is transferred into the blood and ultimately increases brain glutamate levels, causing functional disruptions notably via oxidative stress. The study evaluated the toxic effect of high consumption of MSG and the modulatory role of vitamin C on ATPase activities in the striatum and cerebellum of male Wistar rats for five weeks. Rats were grouped into four (A-D): group A was fed with rat's show only; Group B was fed with diet containing 15% MSG; Group C was treated with vitamin C (200 mg/kg b.wgt orally in 0.9% saline solution) only for 3 weeks; and group D rats were fed with MSG and vitamin C. The findings show that MSG does not affect body and cerebellum weights but increases striatal weight. MSG increases the malondialdehyde (MDA) level and significantly decreases catalase (CAT) and superoxide dismutase (SOD) activities and glutathione (GSH) levels. MSG significantly impaired striatal and cerebellar ATPases activities (Na+/K+-, Ca2+-, Mg2+- and total ATPases). Vitamin C treatment abolishes MSG-induced oxidative stress and improves ATPase activities. The findings show that vitamin C has beneficial effects in improving the functions of membrane-bound ATPases against MSG toxicity in rat's striatum and cerebellum.

Single-nucleosome imaging unveils that condensins and nucleosome-nucleosome interactions differentially constrain chromatin to organize mitotic chromosomes.

For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.

Proteolytic Regulation in the Biosynthesis of Natural Product Via a ClpP Protease System.

Protein degradation is a tightly regulated biological process that maintains bacterial proteostasis. ClpPs are a highly conserved family of serine proteases that associate with the AAA + ATPase (an ATPase associated with diverse cellular activities) to degrade protein substrates. Identification and biochemical characterization of protein substrates for the AAA + ATPase-dependent ClpP degradation systems are considered essential for gaining an understanding of the molecular operation of the complex ClpP degradation machinery. Consequently, expanding the repertoire of protein substrates that can be degraded in vitro and within bacterial cells is necessary. Here, we report that AAA + ATPase-ClpP proteolytic complexes promote degradation of the secondary metabolite surfactin synthetases SrfAA, SrfAB, and SrfAC in Bacillus subtilis. On the basis of in vitro and in-cell studies coupled with activity-based protein profiling of nonribosomal peptide synthetases, we showed that SrfAC is targeted to the ClpC-ClpP proteolytic complex, whereas SrfAA is hydrolyzed not only by the ClpC-ClpP proteolytic complex but also by different ClpP proteolytic complexes. Furthermore, SrfAB does not appear to be a substrate for the ClpC-ClpP proteolytic complex, thereby implying that other ClpP proteolytic complexes are involved in the degradation of this surfactin synthetase. Natural product biosynthesis is regulated by the AAA + ATPase-ClpP degradation system, indicating that protein degradation plays a role in the regulatory stages of biosynthesis. However, few studies have examined the regulation of protein degradation levels. Furthermore, SrfAA, SrfAB, and SrfAC were identified as protein substrates for AAA + ATPase-ClpP degradation systems, thereby contributing to a better understanding of the complex ClpP degradation machinery.