The SPATA5-SPATA5L1 ATPase complex directs replisome proteostasis to ensure genome integrity.

Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.

Conservation and specialization of the Ycf2-FtsHi chloroplast protein import motor in green algae.

The protein import motor in chloroplasts plays a pivotal role in their biogenesis and homeostasis by driving the translocation of preproteins into chloroplasts. While the Ycf2-FtsHi complex serves as the import motor in land plants, its evolutionary conservation, specialization, and mechanisms across photosynthetic organisms are largely unexplored. Here, we isolated and determined the cryogenic electron microscopy (cryo-EM) structures of the native Ycf2-FtsHi complex from Chlamydomonas reinhardtii, uncovering a complex composed of up to 19 subunits, including multiple green-algae-specific components. The heterohexameric AAA+ ATPase motor module is tilted, potentially facilitating preprotein handover from the translocon at the inner chloroplast membrane (TIC) complex. Preprotein interacts with Ycf2-FtsHi and enhances its ATPase activity in vitro. Integrating Ycf2-FtsHi and translocon at the outer chloroplast membrane (TOC)-TIC supercomplex structures reveals insights into their physical and functional interplay during preprotein translocation. By comparing these findings with those from land plants, our study establishes a structural foundation for understanding the assembly, function, evolutionary conservation, and diversity of chloroplast protein import motors.

The Role of DEAD-Box ATPases in Gene Expression and the Regulation of RNA-Protein Condensates.

DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.

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.

Energy-driven genome regulation by ATP-dependent chromatin remodellers.

The packaging of DNA into chromatin in eukaryotes regulates gene transcription, DNA replication and DNA repair. ATP-dependent chromatin remodelling enzymes (re)arrange nucleosomes at the first level of chromatin organization. Their Snf2-type motor ATPases alter histone-DNA interactions through a common DNA translocation mechanism. Whether remodeller activities mainly catalyse nucleosome dynamics or accurately co-determine nucleosome organization remained unclear. In this Review, we discuss the emerging mechanisms of chromatin remodelling: dynamic remodeller architectures and their interactions, the inner workings of the ATPase cycle, allosteric regulation and pathological dysregulation. Recent mechanistic insights argue for a decisive role of remodellers in the energy-driven self-organization of chromatin, which enables both stability and plasticity of genome regulation - for example, during development and stress. Different remodellers, such as members of the SWI/SNF, ISWI, CHD and INO80 families, process (epi)genetic information through specific mechanisms into distinct functional outputs. Combinatorial assembly of remodellers and their interplay with histone modifications, histone variants, DNA sequence or DNA-bound transcription factors regulate nucleosome mobilization or eviction or histone exchange. Such input-output relationships determine specific nucleosome positions and compositions with distinct DNA accessibilities and mediate differential genome regulation. Finally, remodeller genes are often mutated in diseases characterized by genome dysregulation, notably in cancer, and we discuss their physiological relevance.

TRIM7 inhibits enterovirus replication and promotes emergence of a viral variant with increased pathogenicity.

To control viral infection, vertebrates rely on both inducible interferon responses and less well-characterized cell-intrinsic responses composed of "at the ready" antiviral effector proteins. Here, we show that E3 ubiquitin ligase TRIM7 is a cell-intrinsic antiviral effector that restricts multiple human enteroviruses by targeting viral 2BC, a membrane remodeling protein, for ubiquitination and proteasome-dependent degradation. Selective pressure exerted by TRIM7 results in emergence of a TRIM7-resistant coxsackievirus with a single point mutation in the viral 2C ATPase/helicase. In cultured cells, the mutation helps the virus evade TRIM7 but impairs optimal viral replication, and this correlates with a hyperactive and structurally plastic 2C ATPase. Unexpectedly, the TRIM7-resistant virus has a replication advantage in mice and causes lethal pancreatitis. These findings reveal a unique mechanism for targeting enterovirus replication and provide molecular insight into the benefits and trade-offs of viral evolution imposed by a host restriction factor.

Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.

An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission.

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity.

Rad54 Drives ATP Hydrolysis-Dependent DNA Sequence Alignment during Homologous Recombination.

Homologous recombination (HR) helps maintain genome integrity, and HR defects give rise to disease, especially cancer. During HR, damaged DNA must be aligned with an undamaged template through a process referred to as the homology search. Despite decades of study, key aspects of this search remain undefined. Here, we use single-molecule imaging to demonstrate that Rad54, a conserved Snf2-like protein found in all eukaryotes, switches the search from the diffusion-based pathways characteristic of the basal HR machinery to an active process in which DNA sequences are aligned via an ATP-dependent molecular motor-driven mechanism. We further demonstrate that Rad54 disrupts the donor template strands, enabling the search to take place within a migrating DNA bubble-like structure that is bound by replication protein A (RPA). Our results reveal that Rad54, working together with RPA, fundamentally alters how DNA sequences are aligned during HR.

Single Amino Acid Change Underlies Distinct Roles of H2A.Z Subtypes in Human Syndrome.

The developmental disorder Floating-Harbor syndrome (FHS) is caused by heterozygous truncating mutations in SRCAP, a gene encoding a chromatin remodeler mediating incorporation of histone variant H2A.Z. Here, we demonstrate that FHS-associated mutations result in loss of SRCAP nuclear localization, alter neural crest gene programs in human in vitro models and Xenopus embryos, and cause craniofacial defects. These defects are mediated by one of two H2A.Z subtypes, H2A.Z.2, whose knockdown mimics and whose overexpression rescues the FHS phenotype. Selective rescue by H2A.Z.2 is conferred by one of the three amino acid differences between the H2A.Z subtypes, S38/T38. We further show that H2A.Z.1 and H2A.Z.2 genomic occupancy patterns are qualitatively similar, but quantitatively distinct, and H2A.Z.2 incorporation at AT-rich enhancers and expression of their associated genes are both sensitized to SRCAP truncations. Altogether, our results illuminate the mechanism underlying a human syndrome and uncover selective functions of H2A.Z subtypes during development.

Physical and Functional Compartmentalization of Archaeal Chromosomes.

The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."

A Bacterial Effector Mimics a Host HSP90 Client to Undermine Immunity.

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.

Msp1/ATAD1 in Protein Quality Control and Regulation of Synaptic Activities.

Mitochondrial function depends on the efficient import of proteins synthesized in the cytosol. When cells experience stress, the efficiency and faithfulness of the mitochondrial protein import machinery are compromised, leading to homeostatic imbalances and damage to the organelle. Yeast Msp1 (mitochondrial sorting of proteins 1) and mammalian ATAD1 (ATPase family AAA domain-containing 1) are orthologous AAA proteins that, fueled by ATP hydrolysis, recognize and extract mislocalized membrane proteins from the outer mitochondrial membrane. Msp1 also extracts proteins that have become stuck in the import channel. The extracted proteins are targeted for proteasome-dependent degradation or, in the case of mistargeted tail-anchored proteins, are given another chance to be routed correctly. In addition, ATAD1 is implicated in the regulation of synaptic plasticity, mediating the release of neurotransmitter receptors from postsynaptic scaffolds to allow their trafficking. Here we discuss how structural and functional specialization imparts the unique properties that allow Msp1/ATAD1 ATPases to fulfill these diverse functions and also highlight outstanding questions in the field.

Disruption of the Na+/K+-ATPase-purinergic P2X7 receptor complex in microglia promotes stress-induced anxiety.

Na+/K+-ATPase (NKA) plays an important role in the central nervous system. However, little is known about its function in the microglia. Here, we found that NKAα1 forms a complex with the purinergic P2X7 receptor (P2X7R), an adenosine 5'-triphosphate (ATP)-gated ion channel, under physiological conditions. Chronic stress or treatment with lipopolysaccharide plus ATP decreased the membrane expression of NKAα1 in microglia, facilitated P2X7R function, and promoted microglia inflammatory activation via activation of the NLRP3 inflammasome. Accordingly, global deletion or conditional deletion of NKAα1 in microglia under chronic stress-induced aggravated anxiety-like behavior and neuronal hyperexcitability. DR5-12D, a monoclonal antibody that stabilizes membrane NKAα1, improved stress-induced anxiety-like behavior and ameliorated neuronal hyperexcitability and neurogenesis deficits in the ventral hippocampus of mice. Our results reveal that NKAα1 limits microglia inflammation and may provide a target for the treatment of stress-related neuroinflammation and diseases.

Genome folding through loop extrusion by SMC complexes.

Genomic DNA is folded into loops and topologically associating domains (TADs), which serve important structural and regulatory roles. It has been proposed that these genomic structures are formed by a loop extrusion process, which is mediated by structural maintenance of chromosomes (SMC) protein complexes. Recent single-molecule studies have shown that the SMC complexes condensin and cohesin are indeed able to extrude DNA into loops. In this Review, we discuss how the loop extrusion hypothesis can explain key features of genome architecture; cellular functions of loop extrusion, such as separation of replicated DNA molecules, facilitation of enhancer-promoter interactions and immunoglobulin gene recombination; and what is known about the mechanism of loop extrusion and its regulation, for example, by chromatin boundaries that depend on the DNA binding protein CTCF. We also discuss how the loop extrusion hypothesis has led to a paradigm shift in our understanding of both genome architecture and the functions of SMC complexes.

Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins.

As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

Ribosome-rescuer PELO catalyzes the oligomeric assembly of NOD-like receptor family proteins via activating their ATPase enzymatic activity.

NOD-like receptors (NLRs) are pattern recognition receptors for diverse innate immune responses. Self-oligomerization after engagement with a ligand is a generally accepted model for the activation of each NLR. We report here that a catalyzer was required for NLR self-oligomerization. PELO, a well-known surveillance factor in translational quality control and/or ribosome rescue, interacted with all cytosolic NLRs and activated their ATPase activity. In the case of flagellin-initiated NLRC4 inflammasome activation, flagellin-bound NAIP5 recruited the first NLRC4 and then PELO was required for correctly assembling the rest of NLRC4s into the NLRC4 complex, one by one, by activating the NLRC4 ATPase activity. Stoichiometric and functional data revealed that PELO was not a structural constituent of the NLRC4 inflammasome but a powerful catalyzer for its assembly. The catalytic role of PELO in the activation of cytosolic NLRs provides insight into NLR activation and provides a direction for future studies of NLR family members.

The molecular principles governing the activity and functional diversity of AAA+ proteins.

ATPases associated with diverse cellular activities (AAA+ proteins) are macromolecular machines that convert the chemical energy contained in ATP molecules into powerful mechanical forces to remodel a vast array of cellular substrates, including protein aggregates, macromolecular complexes and polymers. AAA+ proteins have key functionalities encompassing unfolding and disassembly of such substrates in different subcellular localizations and, hence, power a plethora of fundamental cellular processes, including protein quality control, cytoskeleton remodelling and membrane dynamics. Over the past 35 years, many of the key elements required for AAA+ activity have been identified through genetic, biochemical and structural analyses. However, how ATP powers substrate remodelling and whether a shared mechanism underlies the functional diversity of the AAA+ superfamily were uncertain. Advances in cryo-electron microscopy have enabled high-resolution structure determination of AAA+ proteins trapped in the act of processing substrates, revealing a conserved core mechanism of action. It has also become apparent that this common mechanistic principle is structurally adjusted to carry out a diverse array of biological functions. Here, we review how substrate-bound structures of AAA+ proteins have expanded our understanding of ATP-driven protein remodelling.

Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex.

The Cdc48 ATPase and its cofactors Ufd1/Npl4 (UN) extract polyubiquitinated proteins from membranes or macromolecular complexes, but how they perform these functions is unclear. Cdc48 consists of an N-terminal domain that binds UN and two stacked hexameric ATPase rings (D1 and D2) surrounding a central pore. Here, we use purified components to elucidate how the Cdc48 complex processes substrates. After interaction of the polyubiquitin chain with UN, ATP hydrolysis by the D2 ring moves the polypeptide completely through the double ring, generating a pulling force on the substrate and causing its unfolding. ATP hydrolysis by the D1 ring is important for subsequent substrate release from the Cdc48 complex. This release requires cooperation of Cdc48 with a deubiquitinase, which trims polyubiquitin to an oligoubiquitin chain that is then also translocated through the pore. Together, these results lead to a new paradigm for the function of Cdc48 and its mammalian ortholog p97/VCP.

The Logic of the 26S Proteasome.

The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation.