Disassembly Of Complex Research Articles

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.

Cover Image: Disassembly of the apical junctional complex during the transmigration of Leptospira interrogans across polarized renal proximal tubule epithelial cells (Cellular Microbiology 09/2021)

Focused ion beam-scanning electron microscopy image of renal proximal tubule epithelial cells infected for 24 hrs with Leptospira interrogans. Leptospires localized in the gap between two adjacent cells (red and blue). For further details, readers are referred to the article by Sebastián et al. on p. e13343 of this issue.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.

Next generation epigenetic modulators to target myeloid neoplasms.

Comprehensive sequencing studies aimed at determining the genetic landscape of myeloid neoplasms have identified epigenetic regulators to be among the most commonly mutated genes. Detailed studies have also revealed a number of epigenetic vulnerabilities. The purpose of this review is to outline these vulnerabilities and to discuss the new generation of drugs that exploit them. In addition to deoxyribonucleic acid-methylation, novel epigenetic dependencies have recently been discovered in various myeloid neoplasms and many of them can be targeted pharmacologically. These include not only chromatin writers, readers, and erasers but also chromatin movers that shift nucleosomes to allow access for transcription. Inhibitors of protein-protein interactions represent a novel promising class of drugs that allow disassembly of oncogenic multiprotein complexes. An improved understanding of disease-specific epigenetic vulnerabilities has led to the development of second-generation mechanism-based epigenetic drugs against myeloid neoplasms. Many of these drugs have been introduced into clinical trials and synergistic drug combination regimens have been shown to enhance efficacy and potentially prevent drug resistance.

NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth.

SUMMARYBAF chromatin remodeling complexes play important roles in chromatin regulation and cancer. Here, we report that Ewing sarcoma cells are dependent on the autocrine signaling mediated by NELL2, a secreted glycoprotein that has been characterized as an axon guidance molecule. NELL2 uses Robo3 as the receptor to transmit critical growth signaling. NELL2 signaling inhibits cdc42 and upregulates BAF complexes and EWS-FLI1 transcriptional output. We demonstrate that cdc42 is a negative regulator of BAF complexes, inducing actin polymerization and complex disassembly. Furthermore, we identify NELL2highCD133highEWS-FLI1high and NELL2lowCD133lowEWS-FLI1low populations in Ewing sarcoma, which display phenotypes consistent with high and low NELL2 signaling, respectively. We show that NELL2, CD133, and EWS-FLI1 positively regulate each other and upregulate BAF complexes and cell proliferation in Ewing sarcoma. These results reveal a signaling pathway regulating critical chromatin remodeling complexes and cancer cell proliferation.

Heat Stress-Dependent Association of Membrane Trafficking Proteins With mRNPs Is Selective.

The Arabidopsis AAA ATPase SKD1 is essential for ESCRT-dependent endosomal sorting by mediating the disassembly of the ESCRTIII complex in an ATP-dependent manner. In this study, we show that SKD1 localizes to messenger ribonucleoprotein complexes upon heat stress. Consistent with this, the interactome of SKD1 revealed differential interactions under normal and stress conditions and included membrane transport proteins as well as proteins associated with RNA metabolism. Localization studies with selected interactome proteins revealed that not only RNA associated proteins but also several ESCRTIII and membrane trafficking proteins were recruited to messenger ribonucleoprotein granules after heat stress.

Sumoylation Mediates the Disassembly of the Smad4 Nuclear Export Complex via RanGAP1*SUMO1 in Keloids

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, after SUMO1 inhibition was associated with RanGAP1. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction of the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.

Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

Disassembly of the apical junctional complex during the transmigration of Leptospira interrogans across polarized renal proximal tubule epithelial cells.

Bacterial pathogens have evolved multiple strategies to disassemble epithelial cell apical junctional complexes (AJCs) and infect epithelial cells. Leptospirosis is a widespread zoonotic infection, mainly caused by Leptospira interrogans, and its dissemination across host cell barriers is essential for its pathogenesis. However, the mechanism of bacterial dissemination across epithelial cell barriers remains poorly characterised. In this study, we analysed the interaction of L. interrogans with renal proximal tubule epithelial cells (RPTECs) and found that at 24 hr post‐infection, L. interrogans remain in close contact with the plasma membrane of the RPTEC by extracellularly adhering or crawling. Leptospira interrogans cleaved E‐cadherin and induced its endocytosis with release of the soluble N‐terminal fragment into the extracellular medium. Concomitantly, a gradual decrease in transepithelial electrical resistance (TEER), mislocalisation of AJC proteins (occludin, claudin‐10, ZO‐1, and cingulin) and cytoskeletal rearrangement were observed. Inhibition of clathrin‐mediated E‐cadherin endocytosis prevented the decrease in TEER. We showed that disassembly of AJCs in epithelial cells and transmigration of bacteria through the paracellular route are important for the dissemination of L. interrogans in the host.

Disease-associated dominant negative CARD11 mutations perturb the CARD11 Opening step during T cell receptor-induced activation of NF-κB and JNK2

Abstract CARD11 is an oligomeric, multidomain scaffold that mediates lymphocyte activation. Upon antigen receptor engagement, CARD11 becomes active and signals through NF-κB, JNK, and mTOR to elicit the adaptive immune response. The CARD11 signaling cycle consists of at least four stages beginning with the Opening step, followed by Cofactor Recruitment, Enzyme Activation, and Complex Disassembly. Primary Immunodeficiency Diseases (PIDDs) have been attributed to both autosomal dominant and recessive CARD11 variants. Combined Immunodeficiency with atopy is caused by the inheritance of a single dominant negative CARD11 allele, but the mechanistic basis for the dominant negative effects is not well understood. In this study, we investigated the mechanistic basis for dominant negative CARD11 mutations in T cells by assessing the activity of loss-of-function variants in the presence and absence of co-expressed wild-type CARD11. In the absence of wild-type CARD11, the variants perturbed the Opening step and downstream signaling steps, including recruitment of Bcl10, HOIP, and MALT1 to CARD11, Bcl10 polyubiquitinylation, MALT1 protease activity, NF-κB and JNK2 activation, and IL-2 production. When the variants were co-expressed with wild-type CARD11, a range of dominant negative effects on NF-κB activation was observed, which correlated with the ability of the CARD11 oligomer to open, recruit cofactors, and mediate IL-2 production. We conclude that dominant negative mutations interfere with the CARD11 Opening step and provide insights into the manifestation of PIDDs caused by the inheritance of dominant and recessive CARD11 alleles.

P97/VCP inhibition causes excessive MRE11-dependent DNA end resection promoting cell killing after ionizing radiation.

SummaryThe ATPase p97 is a central component of the ubiquitin-proteasome degradation system. p97 uses its ATPase activity and co-factors to extract ubiquitinated substrates from different cellular locations, including DNA lesions, thereby regulating DNA repair pathway choice. Here, we find that p97 physically and functionally interacts with the MRE11-RAD50-NBS1 (MRN) complex on chromatin and that inactivation of p97 blocks the disassembly of the MRN complex from the sites of DNA damage upon ionizing radiation (IR). The inhibition of p97 function results in excessive 5′-DNA end resection mediated by MRE11 that leads to defective DNA repair and radiosensitivity. In addition, p97 inhibition by the specific small-molecule inhibitor CB-5083 increases tumor cell killing following IR both in vitro and in vivo. Mechanistically, this is mediated via increased MRE11 nuclease accumulation. This suggests that p97 inhibitors might be exploited to improve outcomes for radiotherapy patients.

Conformational Dynamics of SNARE Proteins during NSF‐Mediated Disassembly

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins play key roles in cellular processes involved in vesicle trafficking and neurotransmitter release of synaptic vesicles. Neuronal SNAREs are composed of syntaxin-1A, SNAP-25A and synaptobrevin-2, forming a highly stable four-helix bundle to provide the energy necessary to drive membrane fusion between the synaptic vesicle and the plasma membrane. However, the mechanistic details underlying SNARE complex disassembly is largely unknown. NSF (N-ethylmaleimide-sensitive factor), a member of AAA+ ATPase family, together with αSNAP (alpha soluble NSF attachment protein), disassembles the post-fusion SNARE complex to maintain the pool of individual SNARE proteins for recurring rounds of vesicle fusion. Using smFRET (single-molecule fluorescence resonance energy transfer), we monitored the conformational dynamics of individual SNARE proteins during the NSF-mediated disassembly process. Interestingly, all three SNARE proteins underwent order-disorder transitions during disassembly and vice versa during re-assembly into SNARE complex. In addition, we discovered a unique intermediate conformational pathway of SNAP-25A during NSF-mediated disassembly process. This notion is in line with recent findings where the N-terminus of SNAP-25A engages through the D1 pore of NSF, likely involving conformational changes. Taken together, we suggest a novel conformational pathway essential for regulating SNARE complex disassembly as well as assembly during synaptic vesicle fusion.

A Non‐Encapsulated Polymorphous U‐Shaped Triboelectric Nanogenerator for Multiform Hydropower Harvesting

AbstractVarious structures for triboelectric nanogenerators (TENGs) have been designed to generate clean, low‐frequency hydropower, and provide solutions for next‐generation sustainable power supplies, including the solid–liquid contact electrification mode and full‐packaged solid–solid contact electrification mode. However, most of them can only adapt to a single form of hydropower. Moreover, electrostatic shielding in water and corrosion of the triboelectric material in the solid–liquid mode are inevitable. In addition, the devices in the fully enclosed mode have the disadvantages of complexity, low cost‐effectiveness, and complex disassembly, which hinder its development and practicality. Herein, a non‐encapsulated polymorphous U‐shaped TENG for multiform hydropower harvesting with effective robustness is designed, which contains two joinable parts: one U‐shaped multilayered TENG unit, and three trigger ends chosen according to actual requirements. The joinable design can directly reduce the waterproof treatment of U‐shaped multilayer section, performance degradation caused by humidity, and effectively avoids electrostatic shielding caused by water and corrosion of triboelectric materials. Furthermore, different forms of hydropower collection, such as the energy of surface water wave, underwater wave/flow, and water droplet can be achieved using the corresponding trigger end. This study provides a feasible scheme for efficient and economical multiform hydropower collection based on triboelectric technology.

The canonical α-SNAP is essential for gametophytic development in Arabidopsis.

The development of male and female gametophytes is a pre-requisite for successful reproduction of angiosperms. Factors mediating vesicular trafficking are among the key regulators controlling gametophytic development. Fusion between vesicles and target membranes requires the assembly of a fusogenic soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) complex, whose disassembly in turn ensures the recycle of individual SNARE components. The disassembly of post-fusion SNARE complexes is controlled by the AAA+ ATPase N-ethylmaleimide-sensitive factor (Sec18/NSF) and soluble NSF attachment protein (Sec17/α-SNAP) in yeast and metazoans. Although non-canonical α-SNAPs have been functionally characterized in soybeans, the biological function of canonical α-SNAPs has yet to be demonstrated in plants. We report here that the canonical α-SNAP in Arabidopsis is essential for male and female gametophytic development. Functional loss of the canonical α-SNAP in Arabidopsis results in gametophytic lethality by arresting the first mitosis during gametogenesis. We further show that Arabidopsis α-SNAP encodes two isoforms due to alternative splicing. Both isoforms interact with the Arabidopsis homolog of NSF whereas have distinct subcellular localizations. The presence of similar alternative splicing of human α-SNAP indicates that functional distinction of two α-SNAP isoforms is evolutionarily conserved.

Human-robot collaboration disassembly planning for end-of-life product disassembly process

• A method for human-robot collaboration disassembly planning is proposed to improve the disassembly process efficiency of end-of-life products. • New optimization parameters for human-robot collaboration based on the component target and operator change are defined and investigated. • Experiments are carried out to validate the task classification and allocation for human-robot collaboration. • An automotive case study is selected to test the proposed method, which results show a higher efficiency and reliability of the method in comparison with the particle swarm algorithm. The disassembly process is the main step of dealing with End-Of-Life (EOL) products. This process is carried out mostly manually so far. Manual disassembly is not efficient economically and the robotic systems are not reliable in dealing with complex disassembly operations as they have high-level uncertainty. In this research, a disassembly planning method based on human-robot collaboration is proposed. This method employs the flexibility and ability of humans to deal with complex tasks, alongside the repeatability and accuracy of the robot. Besides, to increase the efficiency of the process the components are targeted based on the remanufacturability parameters. First, human-robot collaboration tasks are classified, and using evaluation of components remanufacturability parameters, human-robot collaboration definition and characteristics are defined. To target the right components based on their remanufacturability factors, the PROMETHEE II method is employed to select the components based on Cleanability, Reparability, and Economy. Then, the disassembly process is represented using AND/OR representation and the mathematical model of the process is defined. New optimization parameters for human-robot collaboration are defined and the genetic algorithm was modified to find a near-optimal solution based on the defined parameters. To validate the task classification and allocation, a 6-DOF TECHMAN robot arm is used to test the peg-out-hole disassembly operation as a common disassembly task. The experiments confirm the task classification and allocation method. Finally, an automotive component was selected as a case study to validate the efficiency of the proposed method. The results in comparison with the Particle Swarm algorithm prove the efficiency and reliability of the method. This method produces a higher quality solution for the human-robot collaborative disassembly process.

Multiple, short protein binding motifs in ORC1 and CDC6 control the initiation of DNA replication

The initiation of DNA replication involves cell cycle-dependent assembly and disassembly of protein complexes, including the origin recognition complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short linear protein motifs (SLiMs) within intrinsically disordered regions (IDRs) in ORC1 and CDC6 mediate cyclin-CDK-dependent and independent protein-protein interactions, conditional on the cell cycle phase. A domain within the ORC1 IDR is required for interaction between the ORC1 and CDC6 AAA+ domains in G1, whereas the same domain prevents CDC6-ORC1 interaction during mitosis. Then, during late G1, this domain facilitates ORC1 destruction by a SKP2-cyclin A-CDK2-dependent mechanism. During G1, the CDC6 Cy motif cooperates with cyclin E-CDK2 to promote ORC1-CDC6 interactions. The CDC6 IDR regulates self-interaction by ORC1, thereby controlling ORC1 protein levels. Protein phosphatase 1 binds directly to a SLiM in the ORC1 IDR, causing ORC1 de-phosphorylation upon mitotic exit, increasing ORC1 protein, and promoting pre-RC assembly.

An experimental human-robot collaborative disassembly cell

Disassembly is the first operation in the remanufacture, repair and recycling of products that have reached the end of their service life. Both productivity and flexibility should be considered when using robots to carry out disassembly due to the complexities associated with the products returned for remanufacturing. Human-robot collaboration is a flexible semi-automated approach to mitigate against the effects of uncertainties in the frequency, quantity and quality of those End-of-Life (EoL) products.This paper presents a new experimental robotic disassembly cell comprising two collaborative robots and a human operator. The robots and the operator can work safely in tandem for individual, parallel or common disassembly tasks in a shared workspace. Active compliance control is employed by the collaborative robots to achieve complex disassembly tasks and safe human-robot interaction. The human operator communicates with the robots using a new method based on touch sensing combined with position control. The paper first provides a short literature review of robotic disassembly and human-robot collaboration focusing on disassembly. It then describes the collaborative disassembly demonstration cell. Finally, the paper details a case study highlighting the capabilities of the cell. The case study shows that the automation of complex disassembly tasks is feasible and has the potential to release people from tedious and repetitive work.

The mitochondrial AAA protease FTSH3 regulates Complex I abundance by promoting its disassembly.

ATP is generated in mitochondria by oxidative phosphorylation. Complex I (NADH:ubiquinone oxidoreductase or NADH dehydrogenase) is the first multisubunit protein complex of this pathway, oxidizing NADH and transferring electrons to the ubiquinone pool. Typically, Complex I mutants display a slow growth rate compared to wild-type plants. Here, using a forward genetic screen approach for restored growth of a Complex I mutant, we have identified the mitochondrial ATP-dependent metalloprotease, Filamentous Temperature Sensitive H 3 (FTSH3), as a factor that is required for the disassembly of Complex I. An ethyl methanesulfonate-induced mutation in FTSH3, named as rmb1 (restoration of mitochondrial biogenesis 1), restored Complex I abundance and plant growth. Complementation could be achieved with FTSH3 lacking proteolytic activity, suggesting the unfoldase function of FTSH3 has a role in Complex I disassembly. The introduction of the rmb1 to an additional, independent, and extensively characterized Complex I mutant, ndufs4, resulted in similar increases to Complex I abundance and a partial restoration of growth. These results show that disassembly or degradation of Complex I plays a role in determining its steady-state abundance and thus turnover may vary under different conditions.

Molecular mechanisms of assembly and TRIP13-mediated remodeling of the human Shieldin complex

The Shieldin complex, composed of REV7, SHLD1, SHLD2, and SHLD3, protects DNA double-strand breaks (DSBs) to promote nonhomologous end joining. The AAA+ ATPase TRIP13 remodels Shieldin to regulate DNA repair pathway choice. Here we report crystal structures of human SHLD3-REV7 binary and fused SHLD2-SHLD3-REV7 ternary complexes, revealing that assembly of Shieldin requires fused SHLD2-SHLD3 induced conformational heterodimerization of open (O-REV7) and closed (C-REV7) forms of REV7. We also report the cryogenic electron microscopy (cryo-EM) structures of the ATPγS-bound fused SHLD2-SHLD3-REV7-TRIP13 complexes, uncovering the principles underlying the TRIP13-mediated disassembly mechanism of the Shieldin complex. We demonstrate that the N terminus of REV7 inserts into the central channel of TRIP13, setting the stage for pulling the unfolded N-terminal peptide of C-REV7 through the central TRIP13 hexameric channel. The primary interface involves contacts between the safety-belt segment of C-REV7 and a conserved and negatively charged loop of TRIP13. This process is mediated by ATP hydrolysis-triggered rotatory motions of the TRIP13 ATPase, thereby resulting in the disassembly of the Shieldin complex.

Reaction Pathway Sampling and Free-Energy Analyses for Multimeric Protein Complex Disassembly by Employing Hybrid Configuration Bias Monte Carlo/Molecular Dynamics Simulation.

Physicochemical characterization of multimeric biomacromolecule assembly and disassembly processes is a milestone to understand the mechanisms for biological phenomena at the molecular level. Mass spectroscopy (MS) and structural bioinformatics (SB) approaches have become feasible to identify subcomplexes involved in assembly and disassembly, while they cannot provide atomic information sufficient for free-energy calculation to characterize transition mechanism between two different sets of subcomplexes. To combine observations derived from MS and SB approaches with conventional free-energy calculation protocols, we here designed a new reaction pathway sampling method by employing hybrid configuration bias Monte Carlo/molecular dynamics (hcbMC/MD) scheme and applied it to simulate the disassembly process of serum amyloid P component (SAP) pentamer. The results we obtained are consistent with those of the earlier MS and SB studies with respect to SAP subcomplex species and the initial stage of SAP disassembly processes. Furthermore, we observed a novel dissociation event, ring-opening reaction of SAP pentamer. Employing free-energy calculation combined with the hcbMC/MD reaction pathway trajectories, we moreover obtained experimentally testable observations on (1) reaction time of the ring-opening reaction and (2) importance of Asp42 and Lys117 for stable formation of SAP oligomer.