DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of ...DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.
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•The proteasome and SPRTN can each degrade DPCs during DNA replication•Efficient DPC ubiquitylation and proteasome targeting requires TRAIP•SPRTN can degrade DPCs that are not ubiquitylated•SPRTN-mediated DPC degradation is a post-replicative process
DNA-protein crosslinks (DPCs) pose major threats to genomic integrity. Using a cell-free system, Larsen et al. show that SPRTN and the proteasome operate as independent replication-coupled DPC proteases. The existence of two DPC proteases with distinct modes of action facilitates the degradation of chemically diverse DPCs.
•Using high-throughput screening to develop a simplified two-step downstream process for VLPs.•Using ATPS for product concentration and for the separation of DNA and cell debris.•Correlating VLP ...partitioning in ATPS with solubility curves.•Purifying VLPs with centrifugal partition chromatography.
The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.
DNA damage impedes replication fork progression and threatens genome stability. Upon encounter with most DNA adducts, the replicative CMG helicase (CDC45-MCM2-7-GINS) stalls or uncouples from the ...point of synthesis, yet eventually resumes replication. However, little is known about the effect on replication of single-strand breaks or “nicks,” which are abundant in mammalian cells. Using Xenopus egg extracts, we reveal that CMG collision with a nick in the leading strand template generates a blunt-ended double-strand break (DSB). Moreover, CMG, which encircles the leading strand template, “runs off” the end of the DSB. In contrast, CMG collision with a lagging strand nick generates a broken end with a single-stranded overhang. In this setting, CMG translocates along double-stranded DNA beyond the break and is then ubiquitylated and removed from chromatin by the same pathway used during replication termination. Our results show that nicks are uniquely dangerous DNA lesions that invariably cause replisome disassembly, and they suggest that CMG cannot be stored on dsDNA while cells resolve replication stress.
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•The structures of leading and lagging strand collapsed forks are different•CMG passively “runs off” the broken DNA end during leading strand fork collapse•CMG removal from duplex DNA after lag collapse depends on CRL2Lrr1 and p97•Nicks are uniquely toxic lesions that cause fork collapse and replisome disassembly
Vrtis et al. show that when a replisome encounters a single-strand DNA break in the leading or lagging strand template, the replication fork collapses, generating a double-strand break. Collapse is accompanied by loss of the replicative helicase from DNA, implying that fork restart would require recruitment of a new helicase.
Tensile forces regulate epithelial homeostasis, but the molecular mechanisms behind this regulation are poorly understood. Using structured illumination microscopy and proximity ligation assays, we ...show that the tight junction protein ZO-1 exists in stretched and folded conformations within epithelial cells, depending on actomyosin-generated force. We also show that ZO-1 and ZO-2 regulate the localization of the transcription factor DbpA and the tight junction membrane protein occludin in a manner that depends on the organization of the actin cytoskeleton, myosin-II activity, and substrate stiffness, resulting in modulation of gene expression, cell proliferation, barrier function, and cyst morphogenesis. Pull-down experiments show that interactions between N-terminal (ZPSG) and C-terminal domains of ZO-1 prevent binding of DbpA to the ZPSG, suggesting that force-dependent intra-molecular interactions regulate ZPSG binding to ligands within cells. In vivo and in vitro experiments also suggest that ZO-1 heterodimerization with ZO-2 promotes the stretched conformation and ZPSG interaction with ligands. Magnetic tweezers single-molecule experiments suggest that pN-scale tensions (∼2–4 pN) are sufficient to maintain the stretched conformation of ZO-1, while keeping its structured domains intact, and that 5–20 pN force is required to disrupt the interaction between the extreme C-terminal and the ZPSG domains of ZO-1. We propose that tensile forces regulate epithelial homeostasis by activating ZO proteins through stretching, to control the junctional recruitment and downstream signaling of their interactors.
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•The tight junction protein ZO-1 exists in stretched and folded conformations•Actin organization, myosin activity, and heterodimerization control ZO-1 conformation•The stretched conformation of ZO-1 recruits DbpA and occludin to junctions•Barrier function and proliferation are controlled by actomyosin through ZO proteins
Spadaro et al. use super-resolution microscopy to show that ZO-1, a protein that connects tight junction membrane proteins to the actin cytoskeleton, exists in either stretched or folded conformations, depending on actomyosin-dependent force, resulting in changes in the localization, stability, and downstream signaling of its interactors.
Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here, we ...determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at 3.3-Å resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, an N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and betacoronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alphacoronavirus S1-CTDs, but its structure differs from that of betacoronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the prefusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that deltacoronavirus spikes are structurally and evolutionally more closely related to alphacoronavirus spikes than to betacoronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by deltacoronaviruses as well as coronaviruses in general.
IMPORTANCE
In this study, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at a 3.3-Å resolution. This is the first atomic structure of a spike protein from the deltacoronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and betacoronavirus spike proteins. Here, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of PdCoV spike protein and provides many insights into its receptor recognition, membrane fusion, and immune evasion.
Multiple SARS-CoV-2 variants of concern (VOCs) have been emerging and some have been linked to an increase in case numbers globally. However, there is yet a lack of understanding of the molecular ...basis for the interactions between the human ACE2 (hACE2) receptor and these VOCs. Here we examined several VOCs including Alpha, Beta, and Gamma, and demonstrate that five variants receptor-binding domain (RBD) increased binding affinity for hACE2, and four variants pseudoviruses increased entry into susceptible cells. Crystal structures of hACE2-RBD complexes help identify the key residues facilitating changes in hACE2 binding affinity. Additionally, soluble hACE2 protein efficiently prevent most of the variants pseudoviruses. Our findings provide important molecular information and may help the development of novel therapeutic and prophylactic agents targeting these emerging mutants.
Acid-sensing ion channels (ASICs) are trimeric, proton-gated and sodium-selective members of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily of ion channels and are expressed ...throughout vertebrate central and peripheral nervous systems. Gating of ASICs occurs on a millisecond time scale and the mechanism involves three conformational states: high pH resting, low pH open and low pH desensitized. Existing X-ray structures of ASIC1a describe the conformations of the open and desensitized states, but the structure of the high pH resting state and detailed mechanisms of the activation and desensitization of the channel have remained elusive. Here we present structures of the high pH resting state of homotrimeric chicken (Gallus gallus) ASIC1a, determined by X-ray crystallography and single particle cryo-electron microscopy, and present a comprehensive molecular mechanism for proton-dependent gating in ASICs. In the resting state, the position of the thumb domain is further from the three-fold molecular axis, thereby expanding the 'acidic pocket' in comparison to the open and desensitized states. Activation therefore involves 'closure' of the thumb into the acidic pocket, expansion of the lower palm domain and an iris-like opening of the channel gate. Furthermore, we demonstrate how the β11-β12 linkers that demarcate the upper and lower palm domains serve as a molecular 'clutch', and undergo a simple rearrangement to permit rapid desensitization.
Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive ...goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.
Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription ...corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to β-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.
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•The chromodomain Y-like protein CDYL negatively regulates histone crotonylation•CDYL acts on crotonyl donor as a crotonyl-CoA hydratase•CDYL-regulated histone crotonylation is linked to its corepressor function•CDYL-regulated histone crotonylation is important for mammalian spermatogenesis
Liu et al. demonstrate that the chromodomain Y-like protein CDYL acts as a crotonyl-CoA hydratase to negatively regulate histone crotonylation. This activity is intrinsically linked to the transcription repression function of CDYL and is implemented in reactivation of sex chromosome-linked genes and histone replacement during spermatogenesis.
Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be ...further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.
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